Aus dem Zentrum für Biochemie (Medizinische Fakultät) der Universität zu Köln
Institut für Biochemie I
Geschäftsführende Direktorin: Frau Universitätsprofessor Dr. rer. nat. A. A. Noegel
Interactions and subcellular distribution
of human SUN2
Inaugural-Dissertation zur Erlangung der Doktorwürde eines
Doctor rerum medicinalium
der Hohen Medizinischen Fakultät
der Universität zu Köln
vorgelegt von
Eva Mawina Vaylann
Promoviert am: 05.10.2011
Dekanin/Dekan:
Universitätsprofessor Dr. med.Th. Krieg
1. Berichterstatterin: Frau Universitätsprofessor Dr. rer. nat. A. A. Noegel
2. Berichterstatterin: Frau Universitätsprofessor Dr. rer. nat. B. Wirth
Erklärung
Ich erkläre hiermit, dass ich die vorliegende Dissertationsschrift ohne unzulässige Hilfe
Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe;
die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche
kenntlich gemacht. Bei der Auswahl und Auswertung des Materials sowie bei der
Herstellung des Manuskriptes habe ich keine Unterstützungsleistungen bzw.
Unterstützungsleistungen von folgenden Personen erhalten:
Frau Universitätsprofessor Dr. rer. nat. A. A. Noegel.
Weitere Personen waren an der geistigen Herstellung der vorliegenden Arbeit nicht
beteiligt. Insbesondere habe ich nicht die Hilfe einer Promotionsberaterin/eines
Promotionsberaters in Anspruch genommen. Dritte haben von mir weder unmittelbar
noch mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit
dem Inhalt der vorgelegten Dissertationsschrift stehen. Die Dissertationsschrift wurde
von mir bisher weder im Inland noch im Ausland in gleicher oder ähnlicher Form einer
anderen Prüfungsbehörde vorgelegt, und ist abgesehen von den angegebenen
Teilpublikationen noch nicht veröffentlicht worden.
Cologne/Köln: 24.02.2011 Signature/Unterschrift: Eva Vaylann
Dedicated to Munhu Mutema
Nyarara uzive ndini Mwari! Rwiyo 46:10
(Shona)
Danksagung
Ich möchte mich vor allem bei Frau Prof. Dr. A. A. Noegel für die Gelegenheit, an ihrem
renommierten Institut meine Dissertation anfertigen zu dürfen sowie für ihre Bereitschaft
meine Arbeit zu korrigieren, bedanken.
Größter Dank gilt meinen lieben Laborkollegen Tanja, Rashmi, Vivek, Karthic S., Xin
Napoleon und Ilknur, die dazu beigetragen haben, dass ich immer gerne an die Zeit im
Labor und die gemeinsam gemeisterten Höhen und Tiefen zurückdenken werde.
Insbesondere danke ich Sonja, Rosi, Berthold, Maria, Rolf und Martina für ihre
Unterstützung im Laboralltag. Ein herzlicher Dank geht auch an Budi und Gudrun für die
stetige und freundliche Hilfe bei organisatorischen und EDV-Angelegenheiten.
Ganz herzlich möchte ich mich auch bei allen meinen lieben aktuellen und ehemaligen
Kollegen bedanken: Kalle, Margit, Raphael, Sascha, Anja, Claudia, Sajid, Bhagyashri,
Karthic T., Liu, Sandra, Juliane, Christoph, Jan, Sze Man, Lin, Khalid, Mary, Georgia,
Verena und Surayya.
Allen anderen Mitgliedern des Instituts möchte ich für ihre Kollegialität und die wirklich
tolle Atmosphäre danken.
Ein besonderer Dank geht an meinen Mann Jens und an meine Freunde und Familie.
Table of contents
Abbreviations
1 Introduction ................................................................................................................. 1
1.1 The LINC complexes ....................................................................................... 1
1.1.1 Nesprins - ONM components of LINC complexes ...................................... 2
1.1.2 SUN proteins, emerin and lamins- INM components of LINC complexes ... 3
1.2 Cellular functions of LINC complexes ........................................................... 5
1.3 LINC complexes and human diseases .......................................................... 6
1.4 Aim of this study ........................................................................................... 10
2 Materials and methods ............................................................................................. 11
2.1 Materials ........................................................................................................ 11
2.2 Methods ......................................................................................................... 14
2.2.1 Molecular biological methods ................................................................... 14
2.2.1.1 Cloning strategies .................................................................................. 14
2.3 Protein chemical and immunological methods .......................................... 19
2.3.1 Protein extraction from E.coli and human cells ......................................... 19
2.3.2 Gel electrophoresis and immunoblotting .................................................. 20
2.3.4 Western blot stripping ............................................................................... 21
2.3.5 Cell fractionation ....................................................................................... 21
2.3.6 Preparation of GST fusion proteins .......................................................... 21
2.3.6 GST pull-down assay ............................................................................... 22
2.3.7 In vitro binding assay ................................................................................ 22
2.3.8 Immunofluorescence microscopy ............................................................. 22
2.4 Cell culture .................................................................................................... 24
2.4.1 Human cell lines and media ..................................................................... 24
2.4.2 Cultivation of mammalian cell lines ........................................................... 24
2.4.3 Freezing and thawing of mammalian cells ................................................ 25
2.5 Cell biological assays ................................................................................... 25
2.5. 1 Transient transfection by electroporation of mammalian cells ................. 25
2.5.2 Senescence-associated β-galactosidase assays ..................................... 26
2.5.3 Focal adhesion assay ............................................................................... 26
2.5.4 Cell synchronization ................................................................................. 27
2.6 Generation of a monoclonal antibody ......................................................... 27
2.6.1 Immunization of Balb/c mice ..................................................................... 27
2.6.2 Generation of hybridoma cells .................................................................. 27
2.6.3 Selection of monoclonal antibodies .......................................................... 28
2.6.4 Purification of IgG from hybridoma supernatant ....................................... 29
3 Results....................................................................................................................... 30
3.1 Human SUN2 protein .................................................................................... 30
3.2 Generation of a mouse monoclonal antibody against the N- terminal
region of human SUN2 ....................................................................................... 31
3.2.1 Determination of an epitope in the N-terminal sequence of human SUN2
suitable for antibody production ......................................................................... 31
3.2.2 Expression and detection of the SUN2NT protein .................................... 32
3.2.3 Identification of positive hybridoma clone K80-207-11 by immunoblot and
immunofluorescence analysis ........................................................................... 33
3.2.4 Transfection of POP10 cells with pJG129SUN2 full length, tagged with
V5*6xHis ............................................................................................................ 35
3.3 Distribution of endogenous SUN2 protein during the cell cycle .............. 38
3.4 Putative interaction partners of SUN2Nt ..................................................... 40
3.5 Direct interaction of SUN2Nt protein with LMNC polypeptides ................ 47
3.6 Characterization of fibroblasts from Duchenne muscular dystrophy
(DMD), Emery-Dreifuss muscular dystrophy/ Charcot-MarieTooth syndrome
(EDMD/CMT) and Stiff skin syndrome (SSS) patients ...................................... 50
3.6.1 Case report of Duchenne muscular dystrophy (DMD), and Emery-Dreifuss
muscular dystrophy/Charcot-Marie-Tooth syndrome (EDMD/CMT) and Stiff skin
syndrome (SSS) patients ................................................................................... 50
3.6.2 Patient fibroblasts show nuclear defects .................................................. 52
3.6.3 Proliferative ability of patient fibroblasts is restricted ................................ 54
3.6.4 Increased senescence is induced in patient fibroblasts ............................ 55
3.6.5 SUN2 gene expression is down-regulated in senescent patient cells ...... 56
3.6.6 Cell adhesion is altered in patient fibroblasts ........................................... 58
3.6.7 Distribution of nuclear envelope proteins in control fibroblasts and patient
fibroblasts .......................................................................................................... 61
3.6.8 Nucleus-centrosome distance is increased in EDMD/CMT, DMD and Stiff
skin syndrome fibroblasts .................................................................................. 68
3.6.9 Precipitation profile in EDMD/CMT fibroblast cells differ from control
fibroblast cells .................................................................................................... 70
4 Discussion ................................................................................................................ 73
4.1 Generation of a monoclonal antibody ......................................................... 73
4.2 Subcellular localization of endogenous SUN2 during the cell cycle ........ 73
4.3 Protein networks formed by SUN2 .............................................................. 74
4.4 Direct interactions of LMNA/C with the N-terminus of SUN2 in vitro ....... 78
4.5 Characterization of fibroblast from Stiff skin syndrome (SSS), Duchenne
muscular dystrophy (DMD) and Emery-Dreifuss muscular dystrophy /
Charcot-Marie-Tooth syndrome (EDMD/CMT) patients ................................... 80
4.5.1 Stiff skin syndrome (SSS) patients ........................................................... 80
4.5.2 Duchenne muscular dystrophy (DMD) and Emery-Dreifuss muscular
dystrophy / Charcot-Marie-Tooth syndrome (EDMD/CMT) ................................ 82
Summary ...................................................................................................................... 92
Zusammenfassung (deutsch) ..................................................................................... 88
References ................................................................................................................... 90
Preliminary Puplications ........................................................................................... 103
Curriculum vitae/Lebenslauf .................................................................................... 104
Abbreviations % aa Amp
APS
Aqua dest. ATP
bp
BSA
C
ca. CIP
Cm CoREST DMEM
DMSO
DNA DTT E. coli EDTA
ER
EtBr EtOH FCS
g
g
GAPDH GbM GST hr HEPES INM
KASH kb
kDa
λ M
mAb
MDa min
µg
ml µm
µl mM
mRNA NCoR
NE
ng
Percent Amino acid(s) Ampicilline Ammonium persulfate Aqua destillata, destilled water Adenosine triphosphat Base pair(s) Bovine serum albumine Celsius or nucleotide Cytosine Circa, approximately Calf intestine alcaline phosphatase Centimetre Corepressor for RE1 silencing transcription factor Dulbecco´s Modified Eagle Medium Dimethylsulfoxide Deoxyribonucleic acid Desoxyribonucleotidetriphosphat Dithiothreitol Escherichia coli Ethylen-Diamine-Tetra-acetate endoplasmatic reticulum Ethidiumbromide Ethanol Fetal calf serum Gramm Relative centrifugation force Glycerinaldehydephosphate dehydrogenase glioblastoma multiforme glutathione S-transferase Hour(s) N-(2-Hydroxyethyl)piperazin-N´-2-ethansulfonsäure Inner nuclear membrane Klarsicht/Anc-1/Syne homology kilo base kilo Dalton Wave length Molar Monoclonal antibody Mega Dalton Minute Microgramm Milliliter Micrometer Microliter Millimolar Messenger Ribonucleic acid nuclear receptor co-repressor Nuclear envelope Nanogramm
nm NURD ONM
ORF
pAb
PAGE
PBS
PCR
PNS
RNA
rpm
RT
SDS
sec SMRT
SUN
TAE
TE
Taq TEMED
Tris
U
UV
V
v/v w/v X-Gal
Nanometer Nucleosome remodeling and histone deactylation Outer nuclear membrane Open reading frame Polyclonal antibody Polyacrylamide gel electrophoresis Phosphat buffered saline Polymerase chain reaction Perinuclear space Ribonucleic acid Rounds per minute Room temperature Sodium dodecyl sulfate Second Silencing Mediator of Retinoid acid and Thyroid hormone receptor Sad1/UNC-84 homology Tris-Acetate-EDTA Tris-EDTA Thermus aquaticus N,N,N´,N´-Tetramethyl-ethylendiamin Trishydroxyaminomethan Unit Ultraviolet light Volt Volume per volume Weight per volume 5-Bromo-4-chlor-3-indolyl-β-D-galactopyranoside
Introduction
1
1 Introduction
1.1 The LINC complexes
The nucleus is separated from the cytoplasm by a double membrane, the outer
nuclear membrane (ONM) and the inner nuclear membrane (INM). The lumen
between both membranes is the perinuclear space (PNS). Linker of the
nucleoskeleton and cytoskeleton (LINC) complexes physically connect the nuclear
interior with the cytoskeleton. They consist of an INM transmembrane protein and an
ONM transmembrane protein which physically interact with each other in the PNS.
The INM LINC component interacts on the nucleoplasmic side with either the lamina,
a meshwork of intermediate filaments, or with an INM-associated protein. The ONM
LINC component on the other hand contacts on the cytoplasmatic side components
of the cytoskeleton. In mammals, the LINC complexes include nesprins NESPRIN1/2
(Nuclear envelope Spectrin repeat), SUN (Sad1p, UNC-84) domain proteins, emerin,
F-actin, microtubules, intermediate filaments, plectin, laminA/C (LMNA/C) and
chromatin (Fig. 1), (Crisp et al., 2006; Tzur and Gruenbaum, 2006).
Fig. 1: The LINC complex facilitates the coupling of the nuclear lamina to cytoplasmic cytoskeletal
systems comprised of SUN proteins in the INM that binds to the nuclear lamina via interactions with
laminA/C and potentially smaller nesprin isoforms and emerin. The KASH domain of the larger
isoforms of NESPRIN1/2 at the ONM associates with the SUN-domain of the SUN proteins within the
perinuclear space to tether the NE to either cytoplasmic actin, IFs or microtubule network and links the
MTOC. In the muscle sarcomere, nesprins are present in the sarcoplasmic reticulum, Z-line and A/I
junction and potentially link these structures and the actin cytoskeleton (Zhang et al, 2007). Question
marks indicate suggested but not proven interactions.
I-band
A-band
Z Z
emerin
LMNA/C
chromatin
actin- filaments/myosin
plasmamembrane
ONM
INM
NESPRIN 1/2
actin/myosin titin
SUN2
NESPRIN1α
α-actinin vinculin
talin
α/β intergrins
sarcoplasmatic reticulum
intermediate filaments
microtubules
MTOC
?
?
nucleus
Introduction
2
1.1.1 Nesprins - ONM components of LINC complexes
In mammalian cells, two giant (up to 1 MD) actin-binding proteins have been
identified (variously termed NUANCE, NESPRIN2 Giant [NESP2G], SYNE2,
NESPRIN1, ENAPTIN, SYNE1, and MYNE1) as integral proteins of the ONM. They
are encoded by the genes SYNE1 and SYNE2 and belong to type II membrane
proteins (Zhang et al., 2001; Mislow et al., 2002; Zhen et al., 2002; Padmakumar et
al., 2004). Both proteins are composed of an N-terminal alpha-actinin-like actin
binding domain, a long rod domain which harbors spectrin repeats and a highly
conserved C-terminal KASH (Klarsicht/ANC-1/Syne homologue) domain which is
sufficient for nuclear envelope targeting of these proteins. Via their N-terminal actin
binding domains NESPRIN1 and 2 bind to filamentous actin (F-actin) whereas the
KASH and transmembrane domains mediate their localization to the NE (Zhen et al.,
2002; Zhang et al., 2005). The KASH domains of NESPRIN1 and 2 directly bind to
SUN domain proteins at the INM stabilizing their interaction with the inner NE
(Padmakumar et al., 2005).
Mammalian NESPRIN1 and NESPRIN2 genes display enormous complexity,
generating a wide variety of transcripts that differ in length, domain composition,
expression pattern and probably in their functional properties (Zhang et al., 2002).
Multiple nesprin isoforms are produced by alternative splicing and transcription
initiation (Fig. 2). Moreover, the number of individual nesprin genes increased to four
with NESPRIN3 and 4 being significantly smaller proteins (Zhen et al., 2002;
Wilhelmsen et al., 2005; Warren et al., 2005; Roux et al., 2009).
Nesprins are also characterized by variable N-terminal motifs that enable interactions
with different components of the cytoskeleton. Giant isoforms of NESPRIN1 (~1
MDa) and NESPRIN2 (~800 kDa) have N-terminal calponin homology (CH) domains
that link to F-actin. NESPRIN3 (~110 kDa) contains a plectin-binding motif that
permits interactions with cytoplasmic intermediate filaments (IFs) while NESPRIN4
interacts indirectly with microtubules (Crisp et al, 2009). Via the KASH domain
nesprins localize to both the INM and ONM and are partitioned to these domains
depending on their size and binding partners. Thus, nesprins can form structural
connections on either face of the NE (Shanahan et al, 2010).
Introduction
3
Fig.2: Scheme of multiple nesprin isoforms of variable length and with different SR domains. Picture
was taken from Shanahan et al., 2010.
1.1.2 SUN proteins, emerin and lamins- INM components of LINC complexes
Mammalian SUN1 and SUN2 proteins were first identified by bioinformatic analysis
as homologues of C. elegans UNC84. Later, they have been confirmed in screens for
NE components (Dreger et al., 2001; Malone et al., 1999; Schirmer et al., 2003).
Based on shared homology between sad1 in Schizosaccharomyces pombe and
UNC84 in Caenorhabditis elegans the evolutionarily highly conserved sad1p-UNC84
SUN domain was identified suggesting that this family of proteins has crucial nuclear
and cellular roles.
Several SUN domain proteins have been identified in mammals, including SUN1,
SUN2, SUN3, SPAG4 and SPAG4L 46. SUN3 expression seems to be restricted to
testes and its localization is limited to the ER (Crisp et al., 2006). SPAG4 is only
Introduction
4
expressed in spermatids, pancreas and testes (Shao et al., 1999). SUN1 and 2 are
widely expressed. All SUN proteins are conserved type-II INM proteins and contain at
least one transmembrane domain and a C-terminal SUN domain localized inside the
lumen of the NE. KASH-domain proteins are recruited to the NE by binding to SUN
domain proteins SUN1 and SUN2 within the perinuclear space, forming the LINC
complex. Overexpression of dominant-negative KASH domain constructs and
knockdown of LINC components NESPRIN1/2, SUN1/2 or LMNA uncouples the INM
from the ONM, detaches the nucleus from the cytoskeleton and decreases
mechanical stiffness (Hodzic et al, 2009).
Emerin is together with LAP2 (lamina associated polypeptide) and MAN1 a LEM
(LAP2, Emerin, MAN1) domain-containing integral membrane protein of the nuclear
membrane in vertebrates. The LEM domain is composed of a motif of about 43
amino acids that is exposed to the nucleoplasm and interacts with BAF (barrier to
autointegration factor), an abundant chromatin-associated protein (Lin et al., 2000;
Laguri et al., 2001; Shumaker et al., 2001). Emerin is known to interact with nuclear
lamins and NESPRIN1α, and stabilizes and promotes the formation of a nuclear actin
cortical network. Emerin links centrosomes to the nuclear envelope via a microtubule
association. It is also reported to be involved in β-catenin inhibition by preventing its
accumulation in the nucleus (Holaska et al., 2004; Markiewicz et al., 2006;
Salpingidou et al., 2007).
Nuclear lamins are type V intermediate filament proteins containing a central α-
helical rod flanked by N- and C-terminal non-helical domains (Fig. 3). Most lamins,
except for LMNC, are farnesylated at their carboxy termini via a CaaX motif. LMNA
further contains a site for endoproteolytic cleavage that is recognised by ZMPSTE24-
FACE1 enzyme (P2) which cleaves the protein and removes the farnesylated
cysteine. LMNC, on the other hand does not undergo such post-translational
modifications (Hutchison et al., 2001).
Based on their primary sequences and biochemical features, lamins are subdivided
into A-type and B-type lamins (Gerace et al., 1978). Both major (A and C) and minor
(A∆10 and C2) A-type lamin species are encoded by a single developmentally
regulated gene (LMNA) and arise through alternative splicing (Fisher et al., 1986). By
contrast, the main B-type lamins (B1 and B2) are encoded by two separate genes
Introduction
5
LMNB1 and LMNB2 (Hoeger et al., 1990). A single minor B-type lamin (B3) is a
splice variant of LMNB2 (Furukawa and Hotta, 1993).
Fig.3: Basic structure of mammalian lamins; light blue: N-term. head domain (left); C. term. domain
(right), dark blue: coiled-coil domain; green: NLS (nuclear localisation signal); CaaX: posttranslational
modification motif
1.2 Cellular functions of LINC complexes
By linking the nuclear lamina with the cytoskeleton, LINC complexes play key roles in
many crucial cellular functions including cell proliferation, cytoskeleton organization
and organelle positioning. The conserved SUN domain proteins and the KASH
domain proteins of the nuclear envelope (NE) have been identified as molecular
linkers, which position the nucleus on actin filaments, intermediate filaments,
microtubules and the centrosome. Several studies revealed that SUN1 and SUN2
also form a physical interaction between the NE and the centrosome (Zhang et al.,
2009, Koizumi and Gleeson, 2009).
Proper nuclear positioning relative to the cell body is important for many cellular
processes during mammalian development. It has been shown that SUN-KASH
protein complexes function in synaptic and nonsynaptic nuclear anchorage.
Organization of synaptic and nonsynaptic nuclei and the localization of NESPRIN1 to
the NE of muscle cells are disrupted in Sun1/2 double-knockout mice. This indicates
prelamin A
mature LMNA
prelamin B
mature LMNB
prelamin C
mature LMNC
Introduction
6
critical functions for SUN1 and SUN2 in skeletal muscle cells for NESPRIN1
localization at the NE, which is essential for proper myonuclear positioning (Zhang et
al., 2008, Gundersen et al., 2011).
SUN-KASH protein complexes are also required for alignment of homologous
chromosomes, their pairing and recombination in meiosis. Mammalian SUN domain
proteins SUN1/2 and KASH-domain proteins NESPRIN1/2 are involved in nuclear-
centrosome coupling during cortical neuronal migration and interkinetic nuclear
migration during neurogenesis (Zhang et al., 2009). Beside this, certain roles in the
regulation of apoptosis and maturation and survival of the germline have been
proposed (Daboussi et al., 2005; Prasanth et al., 2004; Prasanth et al., 2002). SUN-
KASH-linkages contribute to the structural integrity of the NE in maintaining the
precise separation of the two membranes. Moreover, the SUN-KASH links provide
direct molecular connections between the actin cytoskeleton and the nuclear interior
due to the fact that giant nesprins are F-actin binding. This mechanical link not only
provides structural continuity within and between cells but it also allows for a direct
physical signaling pathway from the cell surface to the nucleus, potentially facilitating
rapid and regionalized gene regulation (Lifeng et al., 2007; Hassold et al., 2007;
Hassold and Hunt, 2001; Linge et al, 2001).
1.3 LINC complexes and human diseases
The centrosome-nucleus attachment is a prerequisite for faithful chromosome
segregation during mitosis, and centrosome abnormalities may therefore cause
chromosome missegregation promoting genome instability such as aneuploidy,
which are the hallmarks of all solid tumors. Recent studies reveal that centrosome
defects, including an excess number of centrioles, increased microtubule nucleation
capacity, and inappropriate phosphorylation of centrosomal proteins, are features of
malignant breast tumors and solid tumors in general. Presently two models for the
origin of centrosome defects in the development of cancer are being discussed. In
the first model, centrosome amplification arises through the failure of cytokinesis and
the consequent failure of equal partition of sister chromatids and spindle poles into
daughter cells. Therefore, a single 4N daughter cell inherits both spindle poles,
instead of just one, to yield two functional centrosomes. The two centrosomes double
again in the next cell cycle to yield four functional spindle poles and multipolar
Introduction
7
mitosis. In the second model, centrosome amplification arises through a deregulation
of the centriole duplication cycle leading to centrosomes with supernumerary
centrioles. Disruption of key cell and/or centrosome cycle regulators may play a
causative role. These models are not mutually exclusive and may operate
independently or sequentially in the development of cancer. Centrosome
amplification leads to an increased frequency of multipolar mitosis and consequent
chromosomal instability, and therefore, is one mechanism by which aneuploidy and
phenotypic variability arise in the development of cancer (Hassold et al., 2007; Lingle
et al., 2001; Salisbury et al., 2005; Fukasawa et al., 2005; Nigg et al, 2006).
Numerous mutations in the genes encoding the nuclear envelope proteins were
found to cause a wide range of human diseases, known collectively as nuclear
envelopathies or laminopathies. Examples are the autosomal-dominant form of
Emery-Dreifuss muscular dystrophy (AD-EDMD), dilated cardiomyopathy with
conduction system defects disease (DCMCD), Limb-girdle muscular dystrophy 1B
(LGMD1B), Dunnigan-type familial partial lipodystrophy (FPLD), atypical Werner
syndrome, Charcot-Marie-Tooth syndrome 2B (CMT2B) and Hutchinson-Gilford
progeria syndrome (HGPS). Muscle defects are common amongst the laminopathies
despite the large spectrum of affected tissues and disease phenotypes and more
than 80% of LMNA mutations lead to cardiac and/or skeletal muscle pathologies
(Muchir et al, 2000; Vytopil et al 2002; Verstraeten et al., 2007; Rankin et al 2008).
Laminopathies affecting specifically either striated muscles, the peripheral nerves, or
the adipose tissues are classified as “tissue-specific” laminopathies. In contrast, if
several tissues are affected concomitantly like in premature ageing syndromes, they
are tentatively classified as “systemic laminopathies”. Additionally to these two main
categories, a still expanding class of laminopathies corresponds to clinical
heterogeneous situations which are characterized by the coexistence of two or more
tissue involvements. These “overlapping laminopathies” suggest the existence of a
real continuum within all the different types of laminopathies (The UMD-LMNA
database: http://www.umd.be/LMNA/W_LMNA/).
For many of these disorders including EDMD, there is variable penetrance and
phenotypic heterogeneity which suggest that mutations in other, presently unknown
Introduction
8
modifier genes and their products, may contribute to the variable phenotypic
expression of the diseases (Politano et al, 2003). Also, approximately 60% of
patients with EDMD or EDMD-like phenotypes do not have mutations in either EMD
encoding for emerin, or LMNA suggesting the involvement of other genes and/or
gene products which are likely binding partners of emerin and LMNA/C at the INM,
particularly those highly expressed in muscle tissue (Zhang et al, 2007; Puckelwartz
et al., 2009; 2010).
EDMD is typically characterized by the clinical triad of 1) early contractures of the
achilles tendons, elbows and postcervical muscles (with subsequent limitation of
neck flexion, but later forward flexion of the entire spine becomes limited); 2)
progressive skeletal muscle weakness and wasting with a humero-peroneal
predominance at the onset of the disease (i.e. proximal in the upper limbs and distal
in the lower limbs) and 3) a life threatening cardiac disease where conduction defects
coexist with ventricular and supraventricular arrhythmias, chamber dilation and heart
failure.
Charcot-Marie-Tooth disease (CMT) constitutes a clinically and genetically
heterogeneous group of hereditary motor and sensory neuropathies. On the basis of
electrophysiological criteria, CMT are divided into two major types: type 1, the
demyelinating forms, characterized by a motor median nerve conduction velocity less
than 38 m/s; and type 2, the axonal form, with a normal or slightly reduced nerve
conduction velocity (The UMD-LMNA database).
Similar to mutations affecting the LINC complex proteins, disruption of signal
transmission pathways that occur upstream of the LINC complex in cytoskeletal
proteins can contribute to the wide spectrum of laminopathies. These pathways
reach from the extracellular matrix to the nuclear envelope. At the plasma membrane
cell-cell and cell-extracellular matrix interactions are mediated by integrin receptors
and dystroglycan which transduce signals coming from the matrix and link the
extracellular matrix to the actin cytoskeleton (Moore et al, 2010). The cytoskeleton is
connected to the internal nuclear envelope through the LINC complex which in turn is
connected with chromatin binding nuclear lamins (Fig. 1). Thus, mechanical forces
Introduction
9
can be transmitted directly from the extracellular matrix to the nuclear interior (Mejat
and Misteli, 2010).
Responsible for the connection of the cytoskeleton of each muscle fiber to the
dytsroglycan complex and the extracellular matrix is dystrophin. Mutations of the
dystrophin gene at locus Xp21 causes Duchenne muscular dystrophy, the most
common form of muscular dystrophies, which is an X-linked disorder characterized
by progressive wasting of skeletal muscles. First, limb-girdle muscles show
weakness by the age of 3 to 5 years, followed by an inability to walk by the age of 8
to 12 years. Other findings include elevated creatine kinase levels,
pseudohypertrophic calf muscles, and cognitive impairment in some patients.
Weakness of respiratory muscles leads to restrictive lung disease and eventual
respiratory failure in severe cases. Histopathological findings include absence of
dystrophin from the membrane of muscle fibers, increased adipose and connective
tissue between muscle fibers, increased variability in muscle fiber size, infiltration of
inflammatory cells, and centrally located nuclei, which are indicative of degenerating
and regenerating muscle fibers (Ehmsen et al., 2002; Lovering et al., 2004; Porter et
al., 2005).
Stiff Skin syndrome is characterized by an early onset of stony-hard skin, with
associated contracture like joint restriction, hypertrichosis, and postural and thoracic
wall abnormalities. Occasional findings include focal lipodystrophy and muscle
weakness. Histopathologic findings consist of either fascial sclerosis or increased
fibroblast cellularity with sclerotic collagen bundles in the deep reticular dermis and/or
subcutaneous septa (Liu et al., 2008). It is suggested that the defect is a heritable
disorder of the autosomal dominant type. The possibility of an autosomal recessive
pattern of inheritance can not be excluded since many of the reported cases are
progeny of consanguineous marriage (Esterly et al., 1971, Jablonska et al., 2000).
Cells from a patient suffering from the syndrome have been included in this study
based on findings that nuclear envelope proteins are involved in skin disease (Youn
et al., 2010).
Introduction
10
1.4 Aim of this study
To address the function and subcellular distribution of SUN2 and its possible role in
muscle dystrophies in different patient cell lines, a newly generated monoclonal
antibody against the N-terminal region was used. Its use in immunoblot and
immunofluorescence applications and the identification of interaction partners of
SUN2 should deliver novel insights into the function and subcellular distribution of
SUN2 during the cell cycle.
Furthermore, a characterization of three different primary cell lines from laminopathy
patients affected by either Stiff Skin syndrome (SSS), Duchenne muscular dystrophy
(DMD) which originally had been described as Emery-Dreifuss muscular dystrophy
(EDMD), and Emery-Dreifuss muscular dystrophy/Charcot-Marie-Tooth syndrome
(EDMD/CMT) was carried out.
In case of the DMD and EDMD/CMT patient cell lines it was initially reported that
they carry mutations in the LINC complex component NESPRIN1 which were then
claimed to be responsible for the disease (Zhang et al., 2007). Based on these
description components of the LINC complex and characteristics of the cells that are
associated with the complex were analysed. After completion of the project further
mutations were discovered and were taken into account in the discussion.
Materials and methods
11
2 Materials and methods
2.1 Materials
Standard laboratory reagents and materials were obtained from local suppliers, fine
chemicals from Sigma if not otherwise indicated and instruments were supplied by the
departmental facility.
Kits
M-MLV reverse transcriptase RNase H Minus-kit Promega
NucleoSpin Extraction Kit Macherey Nagel
pGEM-T easy Cloning Kit Promega
Pure YieldTM Plasmid System Promega
Qiagen RNeasy Mini Kit Qiagen
Enzymes
Calf intestinal alkaline phosphatase (CIAP) Boehringer
Lysozyme Sigma
Restriction endonucleases Life technologies, NEB
RNAse Boehringer
T4 DNA ligase Boehringer
Taq polymerase Boehringer
Thrombin Amersham
Trypsin Invitrogen
Inhibitors
Complete mini protease inhibitor cocktail Sigma
nocodazole Sigma
Antibiotics
Ampicillin Sigma
Kanamycin Sigma
Penicillin/Streptomycin Biochrom
Materials and methods
12
Primary antibodies
Rabbit-anti-GST pAb Institute of Biochemistry
Goat-anti-emerin pAb Santa Cruz
Mouse-anti-SUN2Nt mAb (K80-207-11) this study, E. Vaylann
Mouse-anti-LAP2 mAb BD Biosciences
Mouse-anti-tubulin WA3 mAb U. Euteneuer (thesis Y.
Lücke)
Mouse-anti-V5 mAb Invitrogen
Mouse-anti-vinculin mAb Sigma
Rabbit-anti-His mAb Invitrogen
Rabbit-anti-LMNB1 pAb Abcam
Rabbit-anti- LMNA/C pAb Santa Cruz
Rabbit-anti-NESPRIN1SpecII pAb S. Abraham, Thesis,
2004
Rabbit-anti-NESPRIN2abd pAb Libotte et al., 2005
Rabbit-anti-pericentrin pAb Abcam
Secondary antibodies
Anti-mouse IgG, peroxidase-coupled Sigma
Anti-rabbit IgG, peroxidase-coupled Sigma
Anti-goat IgG, peroxidase-coupled Sigma
Anti-mouse IgG, Alexa488-conjugated Sigma
Anti-mouse IgG, Alexa568-conjugated Sigma
Anti-goat IgG, Alexa568-conjugated Sigma
Bacterial host strains
E. coli XL1 Blue Bullock et al., 1987
E. coli BL21 (Hanahan, 1983)
Vectors and plasmids
pJG129SUN2 human FL Dr. J. Gotzmann
(Biocenter, Vienna).
pGEMTeasy Promega
pGEX-4T1 GE Healthcare
Materials and methods
13
pGST-LMNC-N-term/laminA (aa 1-127) Libotte et al., 2005
pGST-LMNC-coil1B-∆ (aa 128-218) Dreuillet et al., 2002
pGST-LMNC-coil2 (aa 243- 387) Dreuillet et al., 2002
pGST-LMNC-tail (aa 384-566) Dreuillet et al., 2002
pGST-∆LMNC (aa 128-572) Dreuillet et al., 2002
Oligonucleotides
Oligonucleotides for PCR were purchased from Roth GmbH (Karlsruhe), Germany
SUN2Nt 1-139.3´ CGCGAATTCATGTCCCGAAGAAGCCAGCGC-3´
SUN2Nt 1-139.5´ CGCCTCGAGGTCGTCCTCAGAGGAGTAGCC-5´
GAPDH3’ GCCGTCTAGAAAAACCTGCCAAATATGATG-3’
GAPDH5’ GTGAGGGTCTCTCTCTTCCTCTTGTGCTCT-5’
Anesthetics
Isoflurane (2-chloro-2-(difluoromethoxy)-1, 1, 1-trifluoro-ethane)
Materials and methods
14
2.2 Methods
2.2.1 Molecular biological methods
Standard molecular biology techniques were performed as described in "Laboratory
Manual", Cold Spring Harbor Laboratory Press, NY, Vol. 1-3 (Sambrook et al., 1989).
All media, solutions and reagents that have been used are given in the corresponding
sections. Media and buffers were prepared using deionized water, filtered through an
ion-exchange unit (Membrane Pure). All media and buffers were sterilized by
autoclaving at 120 °C; the antibiotics were added to the media after cooling to approx.
50 °C. Agar plates were prepared using a semi-automatic plate-pouring machine
(Technomat).
2.2.1.1 Cloning strategies
Plasmid pJG129 containing V5·6xHis-tagged full-length human SUN2 was kindly
provided by Dr. Josef Gotzmann (Biocenter, Vienna). It was used for SUN2Nt
amplification by SUN2Nt 1-139.3´ and SUN2Nt 1-139.5´ primers designed from
published human SUN2 DNA sequences (accession No. AY682988) purified by gel-
extraction and cloned into pGEMTeasy vector. After verification by sequencing SUN2Nt
was cloned into EcoRI and XhoI cut pGEX4T-1 vector.
2.2.1.2 Polymerase chain reaction (PCR)
Reaction mixture: PCR buffer:
× µl Template DNA (10 ng cDNA or plasmid DNA) 100 mM Tris/HCl; pH 8.3
1 µl Oligonucleotides (primer) A (10 pmol/ml) 500 mM KCl
1 µl Oligonucleotides (primer) B (10 pmol/ml) 20 mM MgCl2
1 µl dNTP mix (10mM)
5 µl 10 × PCR buffer
1 µl Taq polymerase (3-4U)
Add aqua dest. to 50 µl
Materials and methods
15
2.2.1.3 Ligation of vector and DNA fragments
Ligation buffer Ligation reaction
150 mM Tris/HCl; pH 7,8 Linearized vector (200-400 ng)
50 mM MgCl2 DNA-fragment (1-2-µg)
50 mM DTT 4 µl 5x Ligation buffer
5 mM ATP 1µl T4-ligase (1U/µl)
25% PEG-6000 Add H2O to 10 µl
Ligation reaction of vector/SUN2Nt with a ratio of 5:1 was catalyzed by T4-DNA-ligase
incubated overnight 12-16 hours at 8 °C.
2.2.1.4 Dephosphorylation of DNA fragments
10x CIAP-Puffer (pH 9.0)
5 M Tris/HCl (pH 9.0)
10 mM MgCl2
10 mM MgCl2
10 mM spermidin
To prevent religation, 5´-ends of the linearized plasmid were dephosphorylated by calf
intestinal alkaline phosphatase (CIAP). Therefore, 1-5 µg of the vector were incubated
with 1 U of CIAP in a 50 µl reaction volume (37 °C, 10 min). The enzyme was removed
by extraction with phenol/chloroform and precipitated with 1/10 volume sodium acetate,
pH 5.2 and 2.5 volume of 96% ethanol.
2.2.1.5 Restriction digestion of DNA
Digestion of DNA with restriction endonucleases was performed in buffer systems
provided by the manufacturers at the recommended temperatures.
2.2.1.6 Plasmid DNA preparation from E. coli
2.2.1.6.1 DNA-Mini-preparation (Birnboim et al., 1979)
With this DNA isolation method plasmid DNA from small amounts of bacterial cultures
was prepared.
An overnight E. coli culture was centrifuged for 2 minutes (5000 g). The pellet was
suspend in 300 µl B1, 300 µl B2 was added, mixed, incubated (5 min, RT), 300 µl B3
Materials and methods
16
was added, mixed again and centrifuged for 20 minutes (14.000g, 4 °C). The
supernatant was precipitated with 0.8 ml isopropanol. Then, the DNA was pelleted by
centrifugation (14.000 g, 4 °C, 20 min). Finally, the precipitate was washed with 200 µl
of 70% ethanol. After a further centrifugation step the ethanol was discarded and the
plasmid DNA was dried on air and suspend in 10-20 µl Tris/HCl; pH 8.0-8.5.
B1 B2 B3
50 mM Tris/HCl; pH 8,0 0,2 N NaOH 3 M KAc; pH 5,5
10 mM EDTA 1% SDS
100 µg/ml RNAse
2.2.1.6.1 DNA-Midi/Maxi preparation – Pure YieldTM Plasmid System
Buffer and Solutions:
Cell Suspension Solution Cell Lysis Solution
50 mM Tris/HCl; pH 7.5 10 mM EDTA; 8.0 0.2 M NaOH 1% SDS
5 10 mM EDTA; 8.0
100 µg/ml RNase A
neutralization solution column wash
4.09 M Guanidinium hydrochloride 60% ethanol
759 mM Potassium acetate 60 mM Potassium acetate
2.12 M Glacial acetic acid 8.3 mM Tris/HCl; pH 7.5
0.04 mM EDTA
endotoxin removal wash
nuclease free water
Instruments/Materials:
clearing column, binding column, vacuum station
High amounts of plasmid DNA were needed (10-15 µg plasmid DNA/transfection) for the
transfection of eukaryotic cells by electroporation. Therefore, 50-100 ml midi/250 ml
maxi, of an overnight E. coli culture were centrifuged (5000 g, 5 min) and subsequently
Materials and methods
17
the pellet was suspend in 3 ml/6ml cell suspension solution. After the addition of 3 ml/6
ml cell lysis solution the mixture was carefully mixed followed by an incubation step at
room temperature for 2 minutes. The reaction was then stopped by adding 5 ml/10 ml
neutralization solution. Furthermore, the mixture was incubated at room temperature for
3 minutes, and subsequently centrifuged (15.000g, 10 min). DNA was bound to the resin
of a binding-column, washed with 5 ml endotoxin removal wash followed by a washing
step with 20 ml column wash solution and eluted with 600 µl nuclease free water.
2.2.1.7 Phenol/chloroform extraction and ethanol precipitation of DNA
In order to separate DNA from proteins in a DNA-containing solution phenol-chloroform
extraction was used. To the aqueous solution one volume phenol was added, shaken
and centrifuged at 14.000 g for 5 min. One volume of chloroform was added to the DNA
containing aqueous phase, mixed and centrifuged at 14.000 × g for 5 minutes. DNA
precipitation was performed by adding 1/10 volume of sodium acetate, subsequent short
shaking, and further addition of 2.5 volume of 96% ethanol. The solution was mixed,
incubated (−70 °C, 15 min) and centrifuged (16.000 g, 4 °C, 15 min). The pellet was
washed with 50-100 µl 70% ethanol and after renewed pelleting at 16.000 g, 4 °C for 5
min the DNA was suspended in 20-30 µl de-ionized and sterile H2O.
2.2.1.8 Isolation of DNA fragments from agarose gels
Bands containing DNA fragments of interest were excised from the agarose gel and
subsequently purified using a gel elution kit (NucleoSpin Exctraction kit); the DNA was
bound to a silica matrix, washed several times and eluted with a low salt solution.
2.2.1.9 Measurement of DNA and RNA concentrations
Concentrations of DNA and RNA were estimated by determining the absorbance at a
wavelength of 260 nm. A ratio of OD260/OD280 >2 higher than 2.2 indicates that a
contamination with phenol could have happened whereas a ratio under 1.8 suggests a
contamination with proteins.
2.2.1.10 Total RNA isolation and cDNA generation for RT-PCR analysis
Total RNA was extracted from cells grown in a monolayer in cell culture dishes with a kit
following the instructions. The concentration of the RNA was measured with a UV
spectrophotometer. First-strand cDNA synthesis was performed using M-MLV reverse
Materials and methods
18
transcriptase RNase H Minus-kit from Promega. Briefly, total RNA was reverse
transcribed using oligo (dT) primers and reverse transcriptase (Promega) according to
the manufacture’s instructions. The cDNA (2 µg) was amplified with sets of specific
primers for human SUN2 for 36 cycles using the following conditions: 94°C for 30
seconds, 71, 5°C for 45 seconds and 72°C for 45 seconds, resulting in a 417-bp cDNA
coding for human SUN2. GAPDH was amplified as control using GAPDH specific
primers. Different concentrations of Annexin DNA were used as internal calibration
alignment.
2.2.1.11 Transformation of plasmids in E. coli
SOC-medium
2% Bacto-Trypton
0.5% Yeast extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
20 mM Glucose
5 ng of plasmid DNA were mixed with 200 µl competent E. coli cells and incubated on
ice for 5 minutes. The cells were then heat-shocked (42 °C, 60 sec) and immediately
transferred to ice for 5 minutes. 1 ml SOC-medium was added and the cells were
shaken (600 rpm, 37 °C, 1 h). Finally, 50-150 µl of the transformation mix was plated
onto selection plates and the transformants were grown overnight (37 °C).
2.2.1.12 Blue-white selection
If plasmid vectors with a multiple cloning site located in the ß-galactosidase ORF were
used such as pGEMTeasy, a blue-white selection could be performed. For this selection
plates were coated with 30 µl of the substrate X-Gal and 10 µl of the lac-operon inductor
IPTG (isopropyl-thiogalactosid) in addition to the according antibiotics. In cells carrying
the original vector the enzyme ß-galactosidase was expressed and could subsequently
convert the substrate X-Gal (Br-Cl-Indoxyl-ß-D-Galactoside) into Br-Cl-Indoxyl that
shows a blue colouring. In contrast, colonies carrying plasmids with insert appeared
white.
Materials and methods
19
2.2.1.13 Stocks of E. coli cultures
Glycerol stocks of all bacterial strains/transformants were prepared for long-term
storage. The colony of interest was grown in LB-medium containing the selective
antibiotic. 850 µl of an overnight culture was added to 150 µl of sterile glycerol and
stored at −80 °C.
2.3 Protein chemical and immunological methods
2.3.1 Protein extraction from E.coli and human cells
E.coli cells were lysed with prokaryotic lysis buffer (PLB). Human cells were rinsed with
icecold PBS and lysed either in hypotonic lysis buffer (HLB) or RIPA buffer. To obtain
cell lysates for subsequent incubation with GST-Sepharose 4B, 1% Triton X-100 was
added to the respective lysis buffer. After sonification the soluble fraction of the lysate
was obtained by centrifugation (13000 g, 4 °C, 20 min).
RIPA buffer HLB PLB
1M Tris/HCl; pH7.5 1 M HEPES, pH7.9 50 mM Tris-HCl, pH 7.5
5 M NaCl 1 M MgCl2 150 mM NaCl
10% NP 40 2.5 M KCl lysozyme
10% deoxycholate 1 M DTT 1% Sarcosyl
Protease inhibitor cocktail
PIC (Sigma)
Protease inhibitor cocktail
PIC (Sigma)
Protease inhibitor cocktail
PIC (Sigma)
PBS (pH 7.2)
10 mM KCl
10 mM NaCl
16 mM Na2HPO4
32 mM KH2PO4
Materials and methods
20
2.3.2 Gel electrophoresis and immunoblotting
SDS-loading buffer was added to protein samples and the proteins separated on 10%
polyacrylamide gels (SDS-PAGE), then either stained with Coomassie Brillant Blue
R250 and subsequent destained using Destain solution, or transferred onto
nitrocellulose membranes (Schleicher and Schuell) for semi-dry or wet blotting transfer,.
After transfer, the membranes were blocked with 5% (w/v) milk powder in 1x NCP prior
to the appropriate antibody detections. The primary antibodies were detected using the
according peroxidase-conjugated secondary antibodies and visualized by enhanced
chemiluminescence (ECL) reactions. ECL reactions on the nitrocellulose membranes
were documented on X-ray films.
5 × SDS loading buffer 10 × SDS-PAGE running buffer
2.5 ml 1M Tris/HCl; pH 6.5 0.25 M Tris
4.0 ml 10% SDS 1.9 M Glycin
2.0 ml Glycerol 1% SDS
1.0 ml 14.3 M ß-Mercaptoethanol
200 µl 10% Bromophenol blue
Coomassie Blue R 250 Destain solution
0,1% Coomassie brilliant blue R 250 10% Ethanol
50% Ethanol 7% Acetic acid
10% Acetic acid
Transfer buffer
39 mM Glycine
48 mM Tris/HCl; pH 8.0
0.0375% SDS
20% Ethanol
Ponceau staining solution NCP buffer (pH 8.0)
2 g Ponceau S 100 mM Tris/HCl
100 ml 3% Trichloroacetic acid 1.5 M NaCl
Materials and methods
21
0.04% Tween 20 5 ml Tween 20
2.0 g sodium azide
ECL solution:
2 ml 1 M Tris/HCl (pH 8.5)
200 µl Luminol (0.25 M in DMSO) 3-aminonaphthylhydrazide
89 µl (0.1 M in DMSO) p-coumaric acid
18 ml dH2O
6.1 µl 30% H2O2
2.3.4 Western blot stripping
Western blot stripping allows an already immunolabelled nitrocellulose membrane to be
repeatedly treated with antibodies. Primary and secondary antibodies were removed by
shaking the membrane in 1% SDS for 1 hour. Prior to new immunolabelling, the
membrane was washed in NCP for 10 minutes and unspecific binding sites were
blocked with 4% milk powder in NCP for 1 hour.
2.3.5 Cell fractionation
For nuclei preparation, cells were lysed in HLB and nuclei were sedimented (1,000 g,
4°C, 15 min.). For further fractionation the supernatant was centrifuged at (100,000 g,
30 min. 4°C). Both fractions were resuspended in HLB and analyzed on immunoblots
with the according antibodies.
2.3.6 Preparation of GST fusion proteins
Recombinant GST-SUN2Nt and GST-LMNC polypeptides expression was induced in E.
coli strain BL21 (0.5 mM IPTG, 4h, 37°C). Cells were lysed as described and the
proteins were isolated from the supernatant by incubating with glutathione agarose
beads (4 h, 4°C). Glutathione agarose beads coupled with GST-SUN2Nt was washed
five times with PBS (500 g, 4°C, 10 min) before performing thrombin cleavage (4°C, 24-
48 h) or incubating with the according human total cell lysate.
Materials and methods
22
LB-medium
10 g Bacto-Trypton
5 g Yeast extract
5 g NaCl
Add H2O to 1l
Thrombin cleavage buffer
20 mM Tris/HCl; pH 7.4
10 mM CaCl2
2.3.6 GST pull-down assay
Total cell lysate of human cells was prepared as described above. Glutathione agarose
beads coupled with GST-SUN2Nt (see above) were incubated with total cell lysate
(100000 g supernatant) at 4°C for 6 h. Beads were washed three times with PBS (500 g,
4°C, 1 min) and boiled in SDS sample buffer (95°C, 5 min). Samples were analyzed
using 12% SDS polyacrylamide gels and stained with Coomassie Brilliant Blue. Protein
bands of interest were excised from the gels and subjected to LCMS analysis.
2.3.7 In vitro binding assay
Total cell lysate of cultured human cells was prepared as described above. Glutathione
agarose beads coupled with GST-LMNC polypeptides (see above) were incubated with
total cell lysate (100000 g supernatant, 4°C, 6 h). Beads were washed three times with
PBS (500 g, 4°C, 1min) and then analyzed by SDS-PAGE using 15% SDS
polyacrylamide gels and concomitant western blot analysis.
2.3.8 Immunofluorescence microscopy
If not mentioned otherwise, standard immunofluorescence stainings were carried out
using 3% paraformaldehyde (PFA) as fixative (5 min, RT) prior to lysis using 1% Triton-
X100 in PBS (5 min, RT) and subsequent incubation with blocking solution (30 min, RT).
The appropriate antibodies were diluted in the blocking solution to the working
concentration and applied (12 h, 4°C for mAb K80-207-11; RT for remaining antibodies).
The excess of antibodies was removed by washing with PBS prior to the incubation with
Materials and methods
23
the according secondary antibodies (1 h, RT). Nuclear DNA was stained with 4’-6-
diamidino-2-phenylindole (DAPI). Coverslips were embedded in Gelvatol and left at
room temperature for polymerization overnight. Images of immunolabelled cells were
acquired by a confocal laser scanning microscope TCS-SP (Leica) equipped with
TCSNT software. A 488-nm argon-ion laser for excitation of GFP and Alexa 488
fluorescence and a 568-nm krypton-ion laser for excitation of TRITC, Cy3 and Alexa 568
fluorescence were simultaneously used. For the proper acquisition of both signals the
emission signals for green and red fluorophores were separated by using appropriate
wavelength settings for each photo multipler. Image processing was done with Leica
LAS AF Lite software and Microsoft office picture manager, respectively.
Gelvatol PBG (pH 7.4):
4.8 g Polyvinyl alcohol (87%-89%, Sigma P 8136) 0.5% BSA
12 g Glycerol 0.045% fish gelatine
Add 12 ml de-ionized water, stir (RT, 10h)
24 ml 0.2 M Tris/HCl; pH 8.5; stir (50 °C, 20-40 min)
cenrifugation (15 min, 5000 g)
2.5% Diazabicyclooktan (DABCO)
Aliquot-storage: – 20°C
Materials and methods
24
2.4 Cell culture
2.4.1 Human cell lines and media
name species tissue
HeLa Homo sapiens Human cervical cancer
HaCaT Homo sapiens Human keratinocytes
Pop10 Homo sapiens Human hepatocellular carcinoma
U373 Homo sapiens Human glioblastoma-astrocytoma
SSS Homo sapiens Human primary fibroblasts
DMD Homo sapiens Human primary fibroblasts
EDMD/CMT Homo sapiens Human primary fibroblasts
control Homo sapiens Human primary fibroblasts
Media:
Dulbecco`s modified Eagle`s medium (DMEM): 4.5 g/l Glucose, 10% Fetale Bovine
Serum (FBS), 2 mM L-glutamine, 1 mM pyruvate, 100 U/ml Penicillin G, 100 µg/ml
Streptomycin, Non Essential Amino Acids (see table 2.2)
Composition of Non Essential Amino Acids (mg/l):
L-Alanine 890
L-Asparagin 1500
L-Asparatic acid 1330
L-Glutamic acide 1470
Glycien 750
L-Proline 750
L-Serine 1050
2.4.2 Cultivation of mammalian cell lines
Trypsin/EDTA PBS pH 7.4
0.05%/0.02% 137 mM NaCl
2.7 mM KCl
8.1 mM Na2HPO4
1.5 mM KH2PO4
Materials and methods
25
Mammalian cells were cultivated in petri dishes kept in an incubator at 5% CO2 and
water-satured atmosphere at 37°C in the corresponding medium. To passage
subconfluent cell cultures, cells were incubated with 0.05% Trypsin/EDTA to detach
cells from the plates after rinsing with PBS. Trypsin reaction was stopped by adding
serum containing media, diluted in ratios of 1:2-1:10 according to their growth, and
plated onto new petri dishes.
2.4.3 Freezing and thawing of mammalian cells
Freezing medium
80% DMEM or RPMI-1640
10% FBS
10% DMSO
To store cells as DMSO stocks in liquid nitrogen, cells were pelleted at 250 g for 5
minutes and transferred to the corresponding freezing medium. The cells were quickly
aliquoted into cryotubes and placed into a styropor box at −80 °C. After 24 hours
cryotubes were transferred to liquid nitrogen. Cryopreserved cells were thawed rapidly
and plated at a relatively high density to optimize recovery.
2.5 Cell biological assays
2.5. 1 Transient transfection by electroporation of mammalian cells
Media: DMEM
HEPES (Biochrom, L1613): 1 M; pH 7.2
Hanks, 10 × (Biochrom, L2023) Electroporation medium
137 mM NaCl 1 ml Hepes
5 mM KCl 5 ml Hanks (10 × )
0.8 mM MgSO4 44 ml de-ionized water
0.33 mM Na2HPO4
0.44 mM KH2PO4
0.25 mM CaCl2
Materials and methods
26
1 mM MgCl2
1 mM Sodium butyrate
0.15 mM Tris-HCl; pH7.4
Approximately 107 cells were pelleted at 1000 g at 4 °C, resuspended in 800 µl
electroporation medium and transferred to an electroporation cuvette containing 10-12
µg plasmid DNA. After incubated on ice for 10 minutes, cells were electroporated using
a Gene pulser (Bio-Rad) set at 975 µF and 200 V. Finally, cells were seeded on petri
dishes in fresh medium.
2.5.2 Senescence-associated β-galactosidase assays
Cells were seeded on cover slips; the next day cover slips were washed with PBS and
fixed with 3% PFA (5 min, RT). Cells were washed twice with PBS and incubated at
37°C with freshly prepared senescence-associated β-Gal (SA-β-Gal) staining solution.
Examination for staining was done after 4-8 hours under bright field microscopy at 40x
magnification.
SA-β-Gal staining solution
1 mg/ml 5-bromo-4-chloro-3-indolyl β-D-galactoside (X-Gal
40 mM citric acid/sodium phosphate pH 6.0
5 mM potassium ferrocyanide K4Fe(CN)6
5 mM potassium ferricyanide K3Fe(CN)6
150 mM NaCl
2 mM MgCl2).
2.5.3 Focal adhesion assay
Trypsinized cells were seeded on coverslips in culture dishes with an initial cell number
of 1x103 and subjected to immunofluorescence as described above. Analysis was
carried out with a confocal laser scanning microscope TCS-SP (Leica) equipped with
TCSNT software. All pictures were taken in the same z-plane so that the spreading of
focal adhesions on the surface of the coverslip is comparable. LAS-AF Lite Application
Suite software from Leica was used to quantify the spread area in µm2.
Materials and methods
27
2.5.4 Cell synchronization
At 40% confluency HaCaT cells were incubated with 2 mM thymidine for 24 h. Cells
were released from the thymidine block by washing the culture plates with PBS and
adding fresh medium for 3 h. Then 100 ng/ml nocodazole was added to the media. After
the 12 h nocodazole block, cells were harvested and lysed as described above and the
supernatant was used for subsequent GST pull down assays.
2.6 Generation of a monoclonal antibody
2.6.1 Immunization of Balb/c mice
Immunization-solutions:
FC: Freund`s adjuvant complete (Sigma, F-5881)
FCI: Freund`s adjuvant incomplete (Sigma, F-5506)
For the generation of a monoclonal antibody four female Balb/c mice were immunized
with the according antigen. The antigen was suspended in PBS, so that a concentration
of 1 µg/µl was achieved. The antigen was injected 6 × in a time interval of 3 days. For
the immunization 50 µl of the antigen were added to 50 µl of FC-solution in the first
immunization, to 50 µl FCI-solution in the second immunization and to 50 µl PBS in the
last four immunizations. Isolated macrophages were fused with the myeloma cell lines
AG8 and PAI.
2.6.2 Generation of hybridoma cells
Cultivation solution Fusion solution; pH 7.4
RPMI 1640 (PAA) PEG-solution (SIGMA)
4 mM L-Glutamine
10% FBS
1 mM ß-mercaptoethanol
Bri: Hybridoma cloning medium (NICB)
Materials and methods
28
Prior to the fusion, macrophages were extracted from 12 male Balb/c mice aged 10
weeks. Mice were anesthetized with Isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-
trifluoro-ethane) and killed by neck translocation, fixed and after removal of the
abdominal wall 10 ml of cold cultivation medium was injected into the peritoneum
without injuring the internal organs in order to solubilise the macrophages. Macrophage
containing medium was centrifuged twice (400 g, 4 °C, 5 min) and the pellet was
suspended in cultivation medium and transferred onto 40 × 24 well plates (500 µl/well)
and cultivated at 37 °C and 5% CO2 in the incubator.
The four female immunized mice were anesthetized and killed by neck translocation at
the day of fusion and lymph nodes localized at the back of the knee joint were removed
and transferred into cultivation medium. After removal of lymph tissue, single
lymphocytes were transferred into fresh medium, subdivided into four fractions and
pelleted (200 g, 5 min), added to Ag8 and Pai myeloma cells and centrifuged (200 g, 5
min). The resulting pellet was warmed to 37 °C and 1 ml PEG-solution (37 °C) was
added slowly within 60 seconds at room temperature. The mixture was again incubated
for 60 seconds at 37 °C and subsequently 20 ml cultivation medium (37 °C) were
applied within 2 minutes by light shaking; 1 ml within 30 seconds, further 3 ml within 30
seconds and the residual 16 ml within 60 seconds. After further incubation (37 °C, 5
min) the pellet was suspended in 125 ml cultivation medium and distributed onto 24 well
plates (500 µl/well). This procedure was done for all four fractions.
2.6.3 Selection of monoclonal antibodies
Cultivation medium Selection-medium HAT Selection-medium HT
RPIM-1640 medium Cultivation medium Cultivation medium
4 mM L-Glutamine 0.1 mM Sodium hypoxanthin 0.1 mM Sodium hypoxanthin
10% FBS 0.4 µM Aminopterin 16 µM Thymidine
1 mM ß-mercaptoethanol 16 µM Thymidine
Applying of the HAT selection medium after the fusion of lymphocytes and myeloma
cells led to the survival of only hybridoma cells. Hybridoma cells were cultivated in
selection medium HAT which was changed every third day for three times, at the fourth
time selection medium HT was used and afterwards only Bri-containing culturing
Materials and methods
29
medium was used. Western blot strips of E.coli cell lysates containing GST-SUN2Nt
were incubated with collected hybridoma supernatants followed by horseradish-
peroxidase (POD) conjugated secondary antibody and chemiluminescence analysis. As
a negative control the strips were incubated with RPMI medium. Positive clones were
selected according to the detection of a signal at ~45 kDa and subcloned by dilutions of
the desired hybridoma clones. Using a thin capillary tube, single cells were distributed
onto 96 well plates and macrophages containing Bri medium was added. Supernatant of
positive subclones was collected according to the detection of GST-SUN2Nt in E.coli
lysates, recombinant SUN2Nt protein after thrombin cleavage and the protein in whole
HeLa cell lysates transferred onto a nitrocellulose membrane. Positive tested mother-
and subclones were stored as DMSO stocks in liquid nitrogen:
2.6.4 Purification of IgG from hybridoma supernatant
For the purification of the antibody 500 ml hybridoma supernatant was subjected to a
Protein-A-Sepharose-column (1 ml Protein-A-Sepharose) equilibrated with 50 ml PBS /
2 mM sodium azide and circulated for 24-36 hours at 4°C with a pumping system
(BioRad). After incubation the column was washed with 50 ml PBS / 2 mM sodium azide
until the eluate exhibited a constant OD280 < 0.01. The elution of the bound antibody
was performed by adding 10 × 1 ml 0, 2 M glycine (pH 2.7) and immediate neutralization
with NaHCO3. The OD280 of the collected eluate was checked against glycine-buffer.
Antibody containing fractions identified by SDS-PAGE were dialyzed against PBS.
Results
30
3 Results
3.1 Human SUN2 protein
According to the UniGene program, the SUN2 ( also termed KIAA0668) gene
(LocusLink 25777) is located on the human chromosome 22 (cytoband: 22q13.1),
chromosome location 39130730- 39190148 and could be found in the following cDNA
sources (tissue expression): adipose tissue, adrenal gland, ascites, bladder, blood,
bone, bone marrow, brain, cervix, connective tissue, embryonic tissue, eye, heart,
intestine, kidney, larynx, liver, lung, lymph node, mammary gland, mouth, muscle, nerve,
ovary, pancreas, parathyroid, pharynx, placenta, prostate, skin, spleen, stomach, testis,
thymus, thyroid, tonsil, trachea, uterus, vascular adrenal tumor, bladder, carcinoma
breast (mammary gland) tumor, cervical tumor, chondrosarcoma, colorectal tumor,
gastrointestinal tumor, germ cell tumor, glioma, head and neck tumor, kidney tumor,
leukemia, liver tumor, lung tumor, non-neoplasia, normal ovarian tumor, pancreatic
tumor, primitive neuroectodermal tumor of the CNS, prostate cancer, retinoblastoma
skin tumor, soft tissue/muscle tissue tumor, uterine tumor, embryoid body blastocyst,
whole embryo (The Human Protein Atlas). KIAA0668 is expressed in all tissues at a
moderately high level, and at a particularly high level in heart, brain, testis and ovary.
Homo sapiens SUN2 consists of 717 amino acids and contains according to SMART
one predicted transmembrane (TM) domain (213-233 aa), three potential coiled-coil
regions and one C-terminal SUN domain (601-717 aa) (Fig. 4). SUN2 also has one
serine-rich region, one poly-arginine region and two poly-glycine regions. The human
SUN2 shares 66% identity and 71% similarity with its mouse homolog (NP_919323).
The identity of the SUN domain between these two species of SUN2 protein is 95%.
Fig.4: structural domains of human SUN2 protein: TM domain (dark blue), C-terminal SUN domain
(yellow) serine-rich (turquoise), poly-arginine (orange), coiled-coil (light blue) and poly-glycine (green)
region. Antibody epitope is indicated by black bar.
coiled-coil
poly-gly
poly-arg
ser-rich
TM SUN
1 717
K80 207-11
Results
31
3.2 Generation of a mouse monoclonal antibody against the N-
terminal region of human SUN2
3.2.1 Determination of an epitope in the N-terminal sequence of human SUN2
suitable for antibody production
For the generation of a mouse monoclonal antibody directed against the N-terminus of
human SUN2 protein, a short sequence stretch specific for SUN2 protein was
determined. Alignment of the N-terminal region of human SUN2, murin Sun2, human
SUN1 and murin Sun1 by protein BLAST (Basic Local Alignment Search Tools)
revealed a unique sequence localized in the N-terminus of the SUN2 protein with a
homology of 76% between the human and the murin sequence. This specific region is
located in the nucleoplasm and consists of 414 base pairs or 138 amino acids (aa 1-
138), respectively (Fig 5). The corresponding peptide has a molecular mass of 15 kDa
and an isoelectric point of 9.4. In the following, this epitope region will be named as
SUN2Nt.
HsSUN2 --------------------------------------------------MSRRSQRLTR 10
MmSun2 --------------------------------------------------MSRRSQRLTR 10
HsSUN1 MDFSRLHMYSPPQCVPENTGYTYALSSSYSSDALDFETEHKLDPVFDSPRMSRRSLRLAT 60
MmSun1 MDFSRVHTYTPPQCVPENTGYTYALSSSYSSDALDFETEHKLEPVFDSPRMSRRSLRLVT 60
***** **.
HsSUN2 YSQGDDDG-SSSSGGSSVAGSQSTLFKDSPLRTLKRKSSNMKRLSPAPQLGPSSDAHTSY 69
MmSun2 YSQDDNDGGSSSSGASSVAGSQGTVFKDSPLRTLKRKSSNMKHLSPAPQLGPSSDSHTSY 70
HsSUN1 TA-CTLGD--GEAVGADSGTSSAVSLKNRAARTTKQRRSTNKSAFSINHVSRQVTSSGVS 117
MmSun1 TASYSSGD--SQAIDSHISTSRATPAKGRETRTVKQRRSASKPAFSINHLSGKGLSSSTS 118
: .. ..: : . * .. *. ** *:: * * . ::. . :
HsSUN2 YSESLVHESWFPP-------RSSL--EELHGDANWGEDLRVRRRRGTGGSESSRASGLVG 120
MmSun2 YSESVVRESYIGSPRAVSLARSALLDDHLHSEPYWSGDLRGRRRRGTGGSESSKANGLTA 130
HsSUN1 YGGTVSLQDAVTRRP--PVLDESWIREQTTVDHFWGLDDDGDLKGGNKAAIQGNGDVGVA 175
MmSun1 HDSSCSLRSATVLRH--PVLDESLIREQTKVDHFWGLDDDGDLKGGNKAATQGNGELAAE 176
:. : .. .: :. : *. * : *. .: ..... .
HsSUN2 R-KATEDFLGSSSGYSSE------------------------------------------ 137
MmSun2 ESKASEDFFGSSSGYSSE------------------------------------------ 148
HsSUN1 AATAHNGFSCSNCSMLSERKDVLTAHPAAPGPVSRVYSRDRNQK---------------- 219
MmSun1 VASS-NGYTCRDCRMLSARTDALTAHSAIHGTTSRVYSRDRTLKPPHLGHCGRMTAGELS 235
.: :.: .. *
HsSUN2 --------DDYVGYSDVDQQSS---------------------------------SSRLR 156
MmSun2 --------DDLAG----------------------------------------------- 153
HsSUN1 -------CDDCKGKRHLDAHPG----------RAGTLWHIWACAGYFLLQILRRIGAVGQ 262
MmSun1 RVDGESLCDDCKGKKHLEIHTATHSQLPQPHRVAGAMGRLCIYTGDLLVQALRRTRAAGW 295
** *
Results
32
HsSUN2 SAVSRAGSLLWMVATSPGRLFRLLYWWAGTTWYRLTTAASLLDVFVLTR--RFSS-LKTF 213
MmSun2 ------------------RLFGLLYWWIGTTWYRLTTAASLLDVFVLTRSRHFSLNLKSF 195
HsSUN1 AVSRTAWSALWLAVVAPGKAASGVFWWLGIGWYQFVTLISWLNVFLLTR------CLRNI 316
MmSun1 SVAEAVWSVLWLAVSAPGKAASGTFWWLGSGWYQFVTLISWLNVFLLTR------CLRNI 349
: :** * **::.* * *:**:*** *:.:
HsSUN2 LWFLLPLLLLTCLTYGAWYFYPYGLQTFHPALVSWWAAKDSRRPDEGWEARDSSPHFQAE 273
MmSun2 LWFLLLLLLLTGLTYGAWHFYPLGLQTLQPAVVSWWAAKESRKQPEVWESRDASQHFQAE 255
HsSUN1 CKFLVLLIPLFLLL-AGLSLRGQGNFFSFLPVLNWASMHRTQRVDDPQDVFKPTTSRLKQ 375
MmSun1 CKVFVLLLPLLLLLGAGVSLWGQGNLFSLLPVLNWTAMQPTQRVDDSKGMHRPGPLPPSP 409
.:: *: * * .. : * .::.* : : ::: : .
Fig.5: Sequence homology of SUN-domain proteins: HsSUN2: human SUN2 (AAT905001); MmSun2:
murin Sun2 (NP919323); HsSUN1: human SUN1 (NP079430); MmSun1: murin Sun1 NP077771. Grey
arrows mark the region used for antibody-generation. Colours are according to the physiochemical
characteristics of the amino acids: nonpolar, neutral (red); polar, neutral, noncharged (green); polar, acid,
charged (blue); polar, basic, charged (pink). Identical residues are shown with an ‘*’, conserved with ‘:’
and semi-conserved with a ‘.’ .
3.2.2 Expression and detection of the SUN2NT protein
The DNA sequence encoding the N-terminal domain (SUN2Nt) was cloned into the
bacterial expression vector pGEX-4T1. The 45 kDa fusion protein GST-SUN2Nt (Fig.6A)
confirmed by western blot using GST specific antibodies (Fig.6B) was purified from the
Escherichia coli strain XL1-Blue and the GST tag was subsequently removed by
thrombin cleavage and a concentration of 1 µg/µl was achieved (Fig.6C). To confirm the
peptide identity prior to immunization, the 15 kDa gel band and the smaller products
suspected to be degradation products of the SUN2Nt peptide were subjected to PMF
(Peptide Mass Fingerprinting) analysis and the identity of SUN2Nt protein and its
degradation products were confirmed.
Results
33
Fig. 6: A: Coomassie Blue stained polyacrylamide (PAA) gels (12% acrylamide) of uninduced (T0) and
induced (T1) E.coli lysates expressing GST-SUN2Nt fusion protein; supernatant (SN) and pellet (P)
fraction of E.coli lysates. B: immunoblot using GST-specific antibodies to detect the GST-SUN2Nt fusion
protein. C: Coomassie Blue stained PAA gel (12%) of decreasing BSA concentrations (1µg; 0 8µg; 0, 6
µg; 0,4 µg; 0,2 µg/µl ) for calibration; 1µl input of recombinant SUN2Nt protein after thrombin cleavage.
Black arrow: main SUN2Nt protein product; grey arrows: degradations products of SUN2Nt.
3.2.3 Identification of positive hybridoma clone K80-207-11 by immunoblot and
immunofluorescence analysis
Antibody producing hybridoma clones were identified by westernblot analysis.
Supernatant of more than 800 growing mother clones were tested for the successful
production of monoclonal antibody against human SUN2Nt. Subclones K80-207-4, K80-
207-11, K80- 739-3, K80-650-3 and K80-845-4 were able to recognize the recombinant
SUN2Nt peptide and the GST-SUN2Nt protein expressed in E.coli (Fig. 7A) as well as
the endogenous SUN2 in HeLa cell lysates. Subclone K80-207-11 was chosen due to
specific staining observed in immunofluorescence experiments as described in the
A B T0 T1 SN P
97
66
45
30
14
66
45
30
anti- GST
C
kDa
kDa
1,0 0,8 0,6 0,4 0,2
BSA µg/µl
SUN2Nt degradation products
SUN2Nt:
1µl
Results
34
following. Subclones K80-207-4, K80-207-11, K80-739-3, K80-650-3 and K80-845-4
recognized the 15 kDa recombinant SUN2Nt peptide and its degradation products (Fig.
7A). Subclones K80-207-4 and K80-207-11 additionally detected endogenous SUN2 in
HeLa cell lysates (Fig. 7B). Staining of two bands of approximately 80 kDa (Fig. 7B,
upper band) and 70 kDa (Fig. 7B, lower band) occurred of which the smaller one might
be a degradation product or a splice variant. Subclone K80-207-11 was chosen for
further studies as it recognized the protein also in immunofluorescence analysis as
described in the following. The antibodies were purified from the hybridoma supernatant
by affinity chromatography using Protein A Sepharose beads and by subsequent salt
removal through dialysis against PBS. A dilution range test of K80-207-11 indicated that
a dilution of 1:50 for western blot analysis is applicable.
Fig.7: Detection of recombinant SUN2Nt protein and its degradation products (grey arrows) blotted on
nitrocellulose membrane by hybridoma supernatant of different mother clones and their subclones. A: E.
coli lysate expressing recombinant SUN2Nt. B: HeLa lysate expressing endogenous SUN2. Detection:
hybridoma supernatant as primary antibody of different mother- and subclones; secondary antibody:
peroxidase (POD), detection was done by enhanced chemiluminescence (ECL). Negative control: RPMI
medium.
Since endogenous SUN2 is reported as inner nuclear envelope component (Hodzic et
al., 2004; Turgay et al., 2010), cellular components of HeLa cell lysates were isolated by
fractionation. Samples were mixed with sample buffer and heated for five minutes at 37,
42, 56, 72, 82 and 98°C and analyzed on western blots. Using various heating
temperatures to achieve different denaturing levels of the protein components, the ability
K80-207- 11 4
ctrl 739- 3
207- 4 11
650- 3
15
11
kDa
845- 4
A B
72
kDa
Results
35
of K80-207-11 to recognize mild to fully denatured endogenous SUN2 was tested. To
verify the accuracy of the fractionation procedure, samples from the cytoplasmic fraction
and the nuclear fraction were incubated with antibodies against emerin and α- tubulin
(Fig.8).
K80-207-11 detected the endogenous SUN2 protein in fractionated cell lysates heated
at 37, 42, 56, 72, 82 and 98°C at the same intensity levels. Again, two bands differing in
approximately 5 kDa were detected by mAb K80-207-11.
As a marker for the inner nuclear envelope, the majority of endogenous emerin was
predominantly localized to the nuclear fractions. Comparable to emerin endogenous
SUN2 was recovered by K80-207-11 in the nuclear fractions and was not detected in
the cytoplasmic fraction. α-tubulin was predominantly seen in the cytoplasmic fraction
but also to some extend in the nuclear fraction.
Fig.8: Western blot analysis of the nuclear and cytoplasmic fraction of HeLa cell lysates using K80-207-
11 antibody and WA3 (anti−tubulin) and anti-emerin as control for the separation of the cellular fractions.
3.2.4 Transfection of POP10 cells with pJG129SUN2 full length, tagged with
V5*6xHis
To further verify the selected antibody produced by the subclone K80-207-11, Pop10
cells, which are derived from hepatocellular carcinoma, were transfected with pJG129
encoding V5*6xHis tagged SUN2 full length and subjected to immunoblotting and
α-tubulin
nuclear fraction
cytoplasmic fraction
37°C 42°C 56°C 72°C 82°C 98°C
72
37°C 42°C 56°C 72°C 82°C 98°C
55
36
emerin
K80-207-11
Results
36
immunofluorescence. Primary antibodies anti-His, anti-V5 and K80-207-11 hybridoma
supernatant detected the V5*6xHis- fusion protein SUN2 in cell lysates from transfected
cells, mAb K80-207-11 detected two bands which corresponds to endogenous and
tagged SUN2 which should be ~2kDa larger than the endogenouse protein. α-Tubulin
was used as loading control. In confocal immunofluorescence analysis K80-207-11
staining colocalized with anti-His-tag antibody staining in transfected cells (Fig. 9B,
white arrows). Because of the low SUN2 expression in hepatocytes (annotated by The
Human Protein Atlas), non transfected cells showed a week nuclear envelope staining.
B
Fig.9: A: Immunoblotting of Pop10-cells transfected with pJG129SUN2 full length V5*6xHis; anti- His,
anti-V5 and K80-207-11 hybridoma supernatant as primary antibody, POD as secondary antibody,
detection by ECL. B: Confocal immunofluorescence of Pop10-cells transfected with pJG129SUN2 full
length V5*6xHis; K80-207-11, anti-His–tag as primary antibody, Alexa Flour 568, 488 as second antibody,
DAPI for nuclear staining; scale bar 10µm. White arrows indicate successfully transfected cells.
merge
DAPI
K80-207-11
anti- His-tag
V5*6xHis ctrl
95
kDa
A
V5*6xHis ctrl ctrl V5*6xHis
anti-tubulin
anti- His anti-V5 K80-207-11
55
Results
37
In immunofluorescence analysis the subclone K80-207-11 localized the endogenous
SUN2 to the nuclear envelope and to some extent also in the cytoplasm in Pop10 (Fig.
9) and HeLa cells (Fig. 10). To further confirm the nuclear envelope localization, HeLa
cells were stained with DAPI to visualize the DNA, with anti-emerin as inner nuclear
envelope marker and with K80-207-11. Anti-emerin localized emerin exclusively to the
nuclear envelope. In a merged image, SUN2 can be observed surrounding the nucleus
in a rim-like pattern, clearly colocalizing with emerin, demonstrating that K80-207-11
recognizes specifically endogenous SUN2. The strongly stained spots inside the
nucleus might represent nucleoplasmic reticulum (Malhas et al., 2011) (Fig. 10).
Subsequently, mAb K80-207-11 was used for all cell biological analyses.
Fig.10: Confocal immunofluorescence of HeLa cells, anti-emerin and K80-207-11 as primary antibody,
Alexa Flour 568, 488 as conjugated secondary antibodies, DAPI for nuclear staining; scale bar 5µm.
merge DAPI K80-207-11 emerin
Results
38
3.3 Distribution of endogenous SUN2 protein during the cell cycle
During interphase endogenous SUN2 protein was associated with the nuclear envelope
and colocalized with lamins. By contrast, in mitotic cells no colocalisation with LMNA/C
was observed (Fig. 11A).
At the onset of mitosis, the nuclear envelope becomes disrupted by spindle
microtubules during mid-late prophase and intranuclear contents are released. The
lamina depolymerizes and the nuclear membranes disperse into the endoplasmic
reticulum network during prometaphase. Nuclear rim staining by K80-207-11 persisted
during prophase. At metaphase, when the nuclear envelope is completely
disassembled, SUN2 associated with the condensed chromosomes. SUN2 staining
could also be detected in two dot-like structures presumably the centrosomes. In
Schizosaccharomyces pombe endogenous sad1 (homolog of mammalian SUN2)
localizes to the spindle pole body (SPB) (Hagan et al., 1995). Costaining of HeLa cells
with pericentrin (an integral component of the pericentriolar material) revealed
colocalisation with SUN2 and confirmed its centrosomal localization (Fig. 11 C).
In anaphase the K80-207-11 antibody localized the SUN2 protein still associated with
the condensed chromosomes but in a more distributed and in a vesicular manner (Fig.
11A; B, panel four). The SUN2 protein appeared to accumulate at distinct chromosome
regions of the condensed chromatids which might be telomeres as described in meiotic
cells for sad1 in S. pombe (Hagan et al., 1995; Alsheimer et al., 2006).
The envelope reassembles onto chromosomes during late anaphase and telophase and
K80- 207-11 detected the SUN2 protein reconstituted in a rim like pattern.
A
sun2
merge
DAPI K80-207-11 LMNA/C merge
merge
non-mitotic
mitotic
Results
39
B
DAPI K80-207-11 merge
interphase
prophase
metaphase
telophase
DAPI K80-207-11
anaphase
metaphase
Results
40
C
Fig.11: Distribution of SUN2 during mitosis: A: Immunofluorescence of non- mitotic (upper panel) and
mitotic (bottom panel) HeLa cells stained with mAb K80-207-11 and anti-LMNA/C as primary antibodies,
Alexa Flour 568, 488 as conjugated secondary antibodies, DAPI for nuclear staining; scale bar 2 µm. B:
Immunofluorescence analysis of HeLa cells during mitosis (interphase-telophase), mAb K80-207-11 as
primary antibody, Alexa Flour 568 as conjugated secondary antibodies, DAPI for nuclear staining; scale
bar 5 µm. C: localization of SUN2 and pericentrin in telophase: Immunofluorescence analysis of HeLa
cells stained with mAb K80-207-11 and anti-pericentrin as primary antibody, Alexa Flour 568, 488 as
conjugated secondary antibodies, DAPI for nuclear staining; scale bar 5 µm.
3.4 Putative interaction partners of SUN2Nt
To identify protein interaction partners of SUN2Nt, pull down experiments with GST-
SUN2Nt and HaCaT cells lysates and lysates from HaCaT cells arrested in
prometaphase (mHaCaT) were performed. To analyze possibly changing interaction
partners of SUN2 during nuclear envelope breakdown, pulldown experiments were
performed using GST-SUN2Nt as bait and incubated with either normal grown,
untreated HaCaT total cell lysates or mitotically arrested HaCaT total cell lysates
(mHaCaT).
For the mitotic arrest cells were treated with nocodazole and arrested with a G2- or M-
phase DNA content. Microscopic analysis of nocodazole-treated cells showed that they
do enter mitosis but can not form metaphase spindles because microtubules cannot
polymerize. The absence of microtubule attachment to kinetochores activates the
spindle assembly checkpoint, causing the cells to arrest in prometaphase. Therefore,
lysates of HaCaT cells arrested in prometaphase were used to perform a pulldown
experiment. DAPI staining and microscopic analysis confirmed the prometaphase arrest
of more than 90% of the cells (Fig. 12).
pericentrin DAPI merge K80-207-11 merge
Results
41
Using recombinant GST-SUN2Nt immobilized on Sepharose beads several proteins
have been isolated from HaCaT total cell lysates, that bind either directly or indirectly to
SUN2Nt. In control reactions, HaCaT cell lysates were incubated with GST immobilized
on GST-Sepharose beads. GST-SUN2Nt immobilized on Sepharose beads was loaded
for comparison. The cell lysates were obtained from asynchronously growing (HaCaT)
and mitotically arrested HaCaT cells (mHaCaT). Analysis was done by separating the
proteins by SDS-PAGE (12% acrylamide) (Fig. 13), cutting out protein bands and
characterization by LC-MS (Liquid chromatography-mass spectrometry). Protein
separation was performed by nano-liquid chromatography and the proteins were
introduced into a mass spectrometer via an ionization interface. Identification of the
proteins by mass spectrometry after reduction and alkylation of Cys residues by
proteolysis with trypsin, V8, or other endopeptidase, is coupled online (HCT ESI-ion
trap) or offline (UltrafleXtreme MALDI-TOF-TOF) with MS1 detection (molecular mass)
and MS2 fragmentation. Combined information from molecular mass and the
corresponding fragment spectra is used for Mascot searches in virtual digests and MS
fragmentation libraries.
Fig.12: HaCaT cell arrested in
prometaphase; DNA visualized with DAPI
Results
42
Fig. 13: A: Coomassie Blue stained PAA-gel (12%) loaded with GST-SUN2Nt Sepharose beads
incubated with PBS as first control (first lane); GST-SUN2Nt Sepharose beads incubated with HaCaT cell
lysates (second lane); GST-Sepharose beads incubated with HaCaT cell lysates as second control (third
lane). B: Coomassie Blue stained PAA-gel (12%) probed with GST-SUN2Nt Sepharose beads incubated
with PBS as first control (first lane); GST-Sepharose beads incubated with mHaCaT cell lysates as
second control (second lane); GST-SUN2Nt Sepharose beads incubated with mHaCaT cell lysates (third
lane). Grey arrows and brackets indicate areas of interest for protein analysis.
The mass spectrometry results were further analysed according to the following criteria
(Table 1 A-D): The size of the detected protein matched the size of the band in the
Coomassie stained SDS-gel; the protein has a similar cellular localization like SUN2; the
Probability Based MOWSE (MOlecular Weight SEarch) Score are >32 indicating identity
or extensive homology (significant threshold p<0.05). MOWSE is a method for
identification of proteins from the molecular weight of peptides created by proteolytic
digestion and measured with mass spectrometry. Mascot is a software search engine
that uses mass spectrometry data to identify proteins from primary sequence databases.
Normally, the protein length is taken into account. In Mascot, a protein can be correctly
scored even though it is substantially shorter than the database entry because in any
database entry which exceeds the full mass of the protein, the code searches for the
A B GST+ mHaCaT -lysate
GST SUN2Nt +mHaCaT - lysate
GST-SUN2Nt +PBS
95
130
72
250
55
kDa
97
66
30
GST- SUN2Nt +PBS
GST-SUN2Nt + HaCaT -lysate
GST + HaCaT -lysate
45
kDa
Results
43
highest scoring set of matches which occur within the full mass window of the protein.
Therefore, also proteins have been added to the list which had sequence coverage less
than 10%.
Tab.1: Proteins detected in total HaCaT or mHaCaT cell lysates precipitated by GST-SUN2Nt categorized
in gene regulatory (A), RNA processing (B), architectural complex (C) and signaling (D) proteins. Identifier
indicates the SwissProt accession number if not declared otherwise.
Identifier Name Category HaCaT mHaCaT
Q16576.1 RBBP7 A + +
Q9Y265.1 RuvBL1 A + +
Q9Y230.3 RuvBL 2 A + -
Q9NVE4 (UniProtKB) CCDC87 A + +
P12956.2 XRCC A + -
P62805.2 H4 A + -
Q09028.3 RBBP4 A + -
9606 (NCBI) H3-like A + -
P19338.3 nucleolin A + +
Q13547.1 HDAC1 A - +
O94776.1 MTA2 A - +
YLBM1_HUMAN YLB-motif containing protein1 A - +
P56545 CTB2 A - +
Q8N7H5.2 PAF1 A, B - +
Q14498.2 RNA-binding protein 39 A, B - +
O15371.1 EIF3D B + -
NP_991247.1 HNRNPH1 B + -
O00571.3 RNA helicase 3X B + -
O00148.2 RNA helicase 39 B + -
P17844.1 RNA helicase 5 B + -
P52272 HNRNPM B + -
O00148.2 HNRNPK B + +
P52597 HNRNPF B - +
Q8N9N8 EIF1G B - +
P60842 (UniProtKB) EIF4A1 B - +
P60228.1 EIF3E B - +
NP_060145.2 protocadherin C + -
O43707.2 α-actinin1 C - +
Results
44
P12814.2 α-actinin4 C - +
P41351 tubulin a C + +
P05219 tubulin ß C + +
P46940 Ras-GTPase activating like protein C - +
Q9ULV4.1 coronin1C C - +
P02549 spectrin α C, D - +
P11277 spectrin β C, D - +
P68032 actin C, D - +
P35579 myosin9 C, D - +
P35221.1 β-catenin C, D - +
P16144 integrin β 4 C, D - +
P02545.1 laminA/C C, D + +
P67775.1 ser/thr protein phosphatase 2A C, D + +
P35222.1 α-catenin C, D + +
CAI12454.1 coatomer subunit α D + -
P53618.3 coatomer subunit β D + +
AAH20498.2 coatomer subunit γ D + +
NP_001646.2 coatomer subunit δ D + +
Q2NKX8.1 ERCCL6 D + -
Q9NZM1 myoferlin D - +
Q05655 PRKCD D - +
B5BU72 PICALM D - +
Table 1 presents a list of proteins which are categorized in proteins taking part in gene
regulatory processes e.g. DNA-transcription and chromatin remodeling (A) and in
proteins contained in RNA processing complexes (B). Furthermore they can be
classified in proteins of architectural complexes (C) and proteins participating in
signaling (D). Figure 14 summarizes in a diagram the distribution of proteins in either
total HaCaT or mHaCaT cell lysates precipitated by GST-SUN2Nt as bait. Proteins
found in both lysates are illustrated in the intersection.
Proteins contributing to gene regulation e.g. DNA replication and chromatin remodeling
can be found in almost equal amounts in HaCaT and mHaCaT cell lysates.
Approximately half of these proteins appeared in HaCaT and in mHaCaT cell lysates as
Results
45
well. Both lysates contained predominantly histone modification and transcription
repressing proteins such as RBBP7, RuvBL1 and nucleolin.
In the protein pool participating in RNA processing, an equal number of proteins such as
several translation factors and RNA-Pol II associated factor1, could be detected. Only
HNRNPK was detected in both cell lysates. Lysates of mHaCaT cells contained three
translation factors absent from the HaCaT cell lysates. Translation initiation factors were
listed in this category as they appear to have diverse roles i.e. in RNA biogenesis
(Alexandrov et al., 2011).
In mHaCaT cell lysates more than twice the number of proteins contributing to the cell
architecture could be detected which were absent from the HaCaT cell lysates. Proteins
found only in the mHaCaT cell lysates participate mainly in the actin cytoskeleton e.g.
actin, myosin and intergrinβ4, but also take part in signaling events. Accordingly, the
number of proteins involved in signaling events is increased in mHaCaT cell lysates
compared to lysates from HaCaT cells.
Fig. 14: Distribution of proteins in either total HaCaT or mHaCaT cell lysates precipitated by GST-SUN2Nt
as bait. Proteins found in both lysates are illustrated in the intersection.
protocadherin RuvBL2, XRCC,
H4, RBBP4, H3-like, EIF3D, HNRNPH1, RNA helicase 3X, RNA helicase 39,
HNRNPK, RNA- helicase 5,
HNRNPM, ERCCL6
HDAC1, MTA2, YLB-motif containing protein1, CTB2,
PAF1, RNA-binding protein 39, HNRNPK,
HNRNPF, EIF1G, EIF4A1,
EIF3E, α-actinin1,
α-actinin4, spectrinα,
spectrinβ, Ras-GTPase activating like protein,
integrinβ4, β- catenin, coronin1C, ser/thr protein
kinaseβ, myoferlin, actin, myosin9, PRKCD,
PICALM, β-catenin
RuvBL1, nucleolin, RBBP7,
CCDC87, ser/thr protein
phosphatase 2A,
α-catenin,
coatomerβ,
coatomerδ,
coatomerγ,
laminA/C,
tubulinα,
tubulinß
HaCaT
mHaCaT
Results
46
The F-actin binding protein coronin2A has been identified as a component of the co-
repressor complex NcoR and recent studies revealed that it is required for recruiting
further factors to the co-repressor complex such as the transrepressor LXRα/β. For this,
its actin binding activity is required (Huang et al., 2011). In previous studies coronin1C,
also termed coronin3 or CRN2, was detected in the nucleus (Spoerl et al., 2002). In the
present study, coronin1C was detected in pull down assays of mHaCaT cell lysates
using SUN2Nt-GST. Since coronin1C expression is altered in diffuse glioblastomas
(Roadcap et al., 2008; Xavier et al., 2009), the human glioblastoma-astrocytoma cell line
U373 was stained with K80-207-11. SUN2 staining was significantly increased in U373
cells compared to the moderate staining in HeLa cells and the typical rim staining of
SUN2 was expanded and significantly broadened, in some cells appearing in lobulations
of the NE reaching into the cytoplasm (Fig. 15, third picture from the left). These findings
are consistent with strong SUN2 staining of malignant glioma tissue annotated by The
Human Protein Atlas.
Fig. 15: Immunofluorescence analysis of HeLa and U373 cells stained with mAb K80-207-11 as primary
antibody, Alexa Flour 568 as conjugated secondary antibodies, DAPI for nuclear staining, scale bar 5 µm.
U373
K80-207-11
HeLa U373 U373
Results
47
3.5 Direct interaction of SUN2Nt protein with LMNC polypeptides
LMNA/C was present in the pulldowns from asynchronously growing and mitotically
arrested cells. A direct interaction with the N-termini of SUN1 and SUN2 has been
reported recently (Haque et al., 2010).The LMNA gene codes for four proteins: LMNA,
LMNC, LMNA_10, testis-specific LMNC2, which are generated through alternative
splicing. Lamins A and C differ in that LMNA possesses additional 90 amino acids at its
C-terminus. To map the respective LMNA/C interacting domains, five GST-LMNC
fragments have been tested for interaction with the N-terminus of recombinant SUN2 by
GST pulldown assays: GST-LMNC N-term/LMNA (aa 1-127), GST-LMNC coil1B-∆ (aa
128-218), GST-LMNC coil2 (aa 243- 387), GST-LMNC tail (aa 384-566), GST-∆LMNC
(aa 128-572). Fig. 16 B shows a schematic overview of the GST-LMNC constructs used
for the in vitro pull down assay.
Fig. 16 A: Overview of the domain architecture of the LMNA/C protein. B: Schematic overview of LMNA/C
protein structure and the GST-lamin fusion proteins used for the in vitro GST-pull down assays: GST-
LMNC N-term/LMNA (aa 1-127), GST-LMNC coil1B-∆ (aa 128-218), GST-LMNC coil2 (aa 243- 387),
GST-LMNC tail (aa 384-566), GST-∆LMNC (aa 128-572).
N-term. globular domain central rod domain (dimerization)
C-term. globular domain
N-term/LMNA (1-127)
coil1B∆ (128−218)
coil2 (243-387)
tail (384-566)
∆LMNC (128-572)
LMNC 1
34 70 80 218 242 383 574
LMNA 1
34 70 80 218 242 383 574 664
Results
48
Equal amounts of uninduced (T0) and induced (T1) E. coli cell lysates expressing each
one of the five different GST-LMNC fusion proteins were subjected to SDS-PAGE. The
expression of the correct fusion proteins and their degradation products was verified by
western blot analysis using anti-GST antibodies (Fig.17 A and B). For all proteins there
was some expression already in uninduced cells. The amount of the proteins increased
after IPTG induction. The antibody detected also degradation products.
Approximately equal amounts of five different GST-LMNC fusion proteins were
immobilized on sepharose beads and incubated with equal amounts of recombinant
human SUN2 protein after thrombin cleavage. The samples were centrifuged and the
supernatant and the pellet fractions were subjected to SDS-PAGE followed by
Coomassie blue staining to detect the GST-LMNC fusion proteins (Fig. 17 C, top panel)
and western blot analysis to detect the recombinant SUN2 protein (Fig. 17 C, bottom
panel).
The N-terminus of SUN2 was coprecipitated by all five GST-LMNC fusion proteins with
variable binding affinities. The GST-fusion constructs LMNNt and coil1B∆ which are
contained within ∆LMNC could precipitate high amounts of SUN2Nt whereas in the
supernatant of ∆LMNC decreased amounts of SUN2Nt could be detected. GST alone
as control was not able to precipitate SUN2Nt. These observed differences in the
binding affinity for ∆LMNC compared with LMNNt might be due to folding and
dimerization processes of overexpressed recombinant LMNC polypeptides in E. coli.
kDa
36
55
72
kDa
LMNNt coil1B∆ coil2 ∆LMNC tail
anti-GST
T0 T1 T0 T1 T0 T1 T0 T1 T0 T1
28
T0 T1 GST
anti-GST
A B
Results
49
Fig. 17: A: Western blot analysis of uninduced (T0) and induced (T1) E. coli XL2 blue cell lysates
expressing the five different GST-LMNC fusion proteins GST-LMNNt, GST- coil1B∆, GST- coil2, GST- tail,
GST-∆LMNC using anti- GST antibody. B: Western blot of uninduced (T0) and induced (T1) E. coli cell
lysates expressing GST alone as control. Anti-GST as primary antibody, POD as secondary antibody,
detection by ECL. C: Coomassie blue stained PAA gel (15%) shows the GST-LMNC fusion proteins (top
panel); western blot using mAb K80-207-11 detecting the recombinant SUN2Nt protein (bottom panel),
POD as secondary antibody, detection by ECL. D: recombinant SUN2Nt protein as control.
17 207-11
SN P SN P SN P SN P SN P
LMNNt coil1B∆ coil2 ∆LMNC tail
SN P
GST
72
55
28
36
kDa
SUN2Nt
207-11
17
kDa
C
D
Results
50
3.6 Characterization of fibroblasts from Duchenne muscular
dystrophy (DMD), Emery-Dreifuss muscular dystrophy/ Charcot-
MarieTooth syndrome (EDMD/CMT) and Stiff skin syndrome (SSS)
patients
In this study, control fibroblasts from a healthy donor individual and three patients
suffering from three different phenotypes related to laminopathies were subjected to a
basic characterization. At the start of this study the patients have been diagnosed by
their symptoms as Emery-Dreifuss muscular dystrophy (EDMD), Emery-Dreifuss
muscular dystrophy/ Charcot Marie-Tooth syndrome (EDMD/CMT) or Stiff skin
syndrome (SSS). In the EDMD and EDMD/CMT patients mutations in the NESPRIN1
gene SYNE1 had been identified and were thought to cause the disease (Zhang et al.,
2007). After the completion of this study, new genetic information resulted in a
redefinition of previous Emery-Dreifuss muscular dystrophy for one patient to Duchenne
muscular dystrophy (DMD) as a mutation in the dystrophin gene was found. Further, for
the EDMD/CMT case the mutation in NESPRIN1 also may not be responsible for the
disease.
3.6.1 Case report of Duchenne muscular dystrophy (DMD), and Emery-Dreifuss
muscular dystrophy/Charcot-Marie-Tooth syndrome (EDMD/CMT) and Stiff skin
syndrome (SSS) patients
Following information (current stage) concerning the patients was obtained from Prof.
Dr. M. Wehnert, Institute for Human Molecular Genetics, Greifswald; Germany.
Patient 1: This patient harbors a pathogenic nonsense E1137X mutation in the DMD
gene coding for dystrophin (c.3409C>T, p. E1137X). Based on this data, the phenotype
of this patient is now classified as Duchenne muscular dystrophy. Prior to genetic
background information the phenotype of this patient was recognized as Emery-Dreifuss
muscular dystrophy.
Notably, this patient harbors a 29A>G mutation in the 5’UTR of the NESPRIN1α2
isoform. As 4% of an unaffected asian reference population reveal these 29A>G
mutation in the 5’UTR of the NESPRIN1α2 isoform, these described nesprin mutation is
probably not responsible for the clinical phenotype but might contribute to the clinical
outcome.
Results
51
Patient 2: The second patient is suffering from EDMD and Charchot Marie Tooth
syndrome (CMT) and has a N323H mutation in a spectrin repeat of NESPRIN1α1
isoform inherited from a clinical healthy asian mother. Similar to the first described
patient, this nesprin mutation seems not to be responsible for the clinical phenotype but
might contribute to another, presently unknown mutation in these patient cells.
Patient 3: This patient is suffering from a phenotype recognized as Stiff skin syndrome
(SSS) (Jablonska et al., 1984; 2000).
Recently it was shown that a mutation in the fibrillin gene FBN1 causes SSS (Loeys et
al., 2010). Fibrillin is a glycoprotein, which is essential for the formation of elastic fibers
found in connective tissue (Kielty et al., 2002). Based on this Dr. Robinson, Charitè,
Berlin, sequenced all exons of the FBN1 gene of the patient with the exception of the
last exon. As no pathogenic mutations were detected an analysis of the coding exons
and intron-exon boundaries of the ZMPSTE24 (FACE1) and the LMNA gene were done.
None of these genes carried disease associated mutations either. ZMPSTE24 (FACE1)
encodes a zinc metalloproteinase involved in the two step post-translational proteolytic
cleavage of carboxy terminal residues of farnesylated prelamin A to form mature LMNA.
Both genes are frequently affected in restrictive dermopathy (RD), a condition which
overlaps with SSS (Youn et al., 2010).
Therefore, underlying mutations in this patient are presently unknown, yet the patient’s
phenotype is categorized as SSS. In addition to a recent publication where a disease in
dogs resembling SSS was due to a mutation in the metalloprotease ADAMTSL2 (Bader
et al., 2010), mutations in FBN1 were also not detected in human SSS patients
(personal communication Dr. P. Robinson, Charité, Berlin) indicating that this condition
may be caused by mutations in different genes as well.
Since laminopathies exhibit variable penetrance and phenotypic heterogeneity and the
fact that about 60% of patients suffering from EDMD or EDMD-like phenotypes do not
have mutations in either emerin or lamin, the involvement of mutations in other,
presently unknown genes and their products is suggested (Politano et al, 2003; Zhang
et al, 2007). It is conceivable that the here discussed NESPRIN1 mutations may
contribute to the disease state in the patients although individuals carrying the
NESPRIN1 mutations are phenotypically normal. Therefore, the patient’s clinical
Results
52
phenotype might diverge from the one expected for the corresponding genotype,
explaining the difficulty to clearly classify the patient’s phenotype.
3.6.2 Patient fibroblasts show nuclear defects
Several abnormalities affecting nuclear shape and distribution of nuclear envelope
proteins could be observed in nuclei from patient cells. To quantify these observations
100 nuclei of control and patient fibroblasts with the EDMD/CMT and DMD phenotype
and the Stiff skin syndrome cells were evaluated at passage 8, 12 and 16. Each
experiment was carried out twice.
Nuclei of control cell generally had a round or ovoid shape. In patient fibroblasts the
number of cells with nuclear defects was increased compared to control cells and
micronuclei and a variety of nuclear shape defects including folds, lobulations and blebs
were observed (Fig. 18). Remarkably, the DMD patient cells exhibited the most
significant nuclear shape alterations. At an average, 30% of DMD cells showed
misshapen nuclei whit a slight increase from passage 8 to passage 16, while in Stiff skin
syndrome cells ~20% of the nuclei were misshapen and no significant increase of
altered nuclei shape was observed in higher passages. In contrast, EDMD/CMT
fibroblasts exhibited 15 to 19% misshapen cells in passage 8 and passage 12,
respectively, and at passage 16 increase to 32 % of altered shaped cells were
observed.
The relation between micronuclei and misshapen cells correlated and remained almost
constant.
Results
53
D
Fig.18: A-C: 100 nuclei per control and patient cells were evaluated at passage 8, 12 and 16. Each
experiment was carried out twice. D: Immunofluorescence of DMD and EDMD/CMT patient fibroblasts.
Nuclei stained with DAPI. Arrows indicate the observed defects.
EDMD/CMT EDMD/CMT DMD
A
C
B
DMD
Results
54
3.6.3 Proliferative ability of patient fibroblasts is restricted
To evaluate the effect of the nuclear envelope defects observed in the EDMD/CMT,
DMD and stiff skin syndrome patient fibroblast on cellular functions the proliferative
ability was analyzed. To determine cell growth rates 1x105 cells of each patient cell line
and the control cells were seeded and counted every 48 hours for a period of 6 days
(Fig. 19). For the DMD patient cells the cell number in the beginning was 1x104 due to
very slow growth rate and therefore the limited availability of these cells. The experiment
was carried out twice.
Patient cell lines exhibited decreased proliferation when compared with control cells.
The growth curve of the patient cell line with the EDMD/CMT shows a remarkably
reduced growth compared to the control whereas the stiff skin cells revealed only a
slightly reduced growth. In contrast, fibroblasts from the patient suffering from DMD
showed significantly reduced growth compared to control cells and to the other two
patient cells.
-1
0
1
2
3
4
5
6
7
8
1 2 3 4 5 6
cell
num
ber
x 1
05
time (d)
ctrlEDMD/CMTstiff skinDMD
Fig. 19: Growth curves of control and patient fibroblasts.
Results
55
3.6.4 Increased senescence is induced in patient fibroblasts
A decline in the growth rate is often associated with cell senescence. To examine
whether cellular senescence was induced in patient fibroblasts the expression of a
specific isoenzyme of β galactosidase, which is referred to as senescence-associated β-
galactosidase (SA-β-gal) was analysed. Cells fixed in 4% PFMA were incubated with
freshly prepared SA-β-Gal staining solution at 37°C for 12 hours and subsequently the
percentage of stained cells under bright field microscopy determined. The experiment
was carried out twice with 200 cells each. In control fibroblasts less than 1% of the cells
were positive for β-galactosidase. In contrast, 8% fibroblasts from the patient with the
Stiff skin syndrome expressed β-galactosidase and a blue staining was observed. Cells
from EDMD/CMT patient revealed a positive staining in 11% of the cells whereas in
DMD patient fibroblasts 14% of the cells were stained (Fig. 20).
0
2
4
6
8
10
12
14
16
ctrl DMD EDMD/CMT stiff skin
% o
f se
nescen
t cells
Fig. 20: Percentage of senescent cells in control and patient cells. The experiment was done in duplicate, 200 cells were counted each).
Results
56
3.6.5 SUN2 gene expression is down-regulated in senescent patient cells
To evaluate the gene expression level of the SUN2 protein in the patient fibroblast in
comparison to the control fibroblasts, total RNA was isolated (control fibroblasts at
passage 8 and 22, EDMD/CMT fibroblasts at passage 4 and 16, DMD fibroblasts at
passage 6 and 8, and Stiff skin syndrome fibroblasts at passage 5 and 17). The cDNA
was used for quantitative Real Time PCR (qRT-PCR) experiments. To normalize the
expression of the targeted gene the stably expressed housekeeping gene GAPDH was
chosen to correct possible differences in RNA quantity or quality across experimental
samples. Each experiment was performed in triplicate and repeated 3 times.
At lower passages transcript level of SUN2 is not up- or down- regulated in a statistically
significant way in the patient cells with the DMD and EDMD/CMT phenotype. Patient
fibroblasts with the Stiff skin syndrome show a regulation factor of 1.1 and therefore an
increased gene expression of SUN2 (Fig. 21). In the senescence stadium of the cells
the transcript level of SUN2 is remarkably decreased in the DMD and EDMD/CMT
patient cells. Compared to the control cells the transcript level of SUN2 is not
significantly decreased in the stiff skin fibroblasts at passage 17 but in comparison with
the expression levels measured in lower passages the gene expression of SUN2 is
remarkably decreased.
Fig. 21: SUN2 transcript levels in control and patient fibroblasts as determined by qRT-PCR. A: The
SUN2 mRNA level in control fibroblasts at passage 8 were taken for reference (1), EDMD/CMT fibroblasts
at passage 4, DMD fibroblasts at passage 6, Stiff skin syndrome fibroblasts at passage 5. B: control
fibroblasts at passage 22, EDMD/CMT fibroblasts at passage 16, DMD fibroblasts at passage 8, Stiff skin
syndrome fibroblasts at passage 17.
0,6
0,68
0,76
0,84
0,92
1
1,08
EMD/CMT p16 DMD p8 stiff skin p17
0,94
0,96
0,98
1
1,02
1,04
1,06
1,08
1,1
1,12
EMD/CMT p4 DMD p6 stiff skin p5
A B
Results
57
Immunoblot analysis of control and patient fibroblast using mAb K80-207-11 confirmed
the results obtained by quantitative PCR. Tubulin was used as loading control. Patient
fibroblast with the Stiff skin syndrome showed a higher amount of SUN2 protein at lower
passages compared to the control cells, and a decreased expression level in higher
passages. The tubulin-loading control was slightly decreased in DMD and EDMD/CMT
cell lysates; nevertheless, a decrease in the protein levels of SUN2 in the DMD and
EDMD/CMT patient cells at higher passages was observed.
Fig.22: Immunoblot analysis of lysates from control and patient fibroblasts at different passages: A:
control fibroblasts at passage 8, EDMD/CMT fibroblasts at passage 4, DMD fibroblasts at passage 6, Stiff
skin syndrome fibroblasts at passage 5. B: control fibroblasts at passage 22, EDMD/CMT fibroblasts at
passage 16, DMD fibroblasts at passage 8, Stiff skin syndrome fibroblasts at passage 17. mAb K80-207-
11 and WA3 (anti-tubulin) as loading control were used as primary antibodies, POD as secondary
antibody, detection was done by ECL.
anti-tubulin
SUN2
ctrl p22
SSSp17
EDMD/ CMTp16
ctrlp8
SSSp6
EDMD/ CMTp6
DMDp6
DMD p8
A B
Results
58
3.6.6 Cell adhesion is altered in patient fibroblasts
Cell adhesion makes an important contribution to the maintenance of tissue structure,
the promotion of cell migration, and the transduction of information about the cell
microenvironment across the plasma membrane. To evaluate the adhesion ability of the
patient cells to a substratum compared to the control fibroblasts, trypsinized cells were
seeded on coverslips in culture dishes with an initial cell number of 1x103. Every 15
minutes three coverslips of each cell line were rinsed with PBS and cells remaining
attached on the coverslips were subjected to immunofluorescence analysis with vinculin
specific antibodies (Fig. 23 A). Vinculin is a membrane-associated cytoskeletal protein in
focal adhesion plaques that is involved in the linkage of integrin adhesion molecules to
the actin cytoskeleton. The individual immunofluorescences shown in Fig. 23 A have the
same magnification (scale bar, 76.4 µm) and were taken in the same z-plane so that the
spreading of focal adhesions on the surface of the coverslip is comparable. To quantify
the observed differences in spreading, the area measured in µm2 was evaluated using
LAS-AF Lite Application Suite software from Leica (Fig.23B (b)).
As shown in the table in Fig. 23 B (a), all cell lines attached and the adhesions
increased progressively. Control cells had at every measured time point the largest area
of spreading on the substratum. Fibroblasts from the Stiff skin syndrome affected patient
exhibited the lowest spreading at all analysed time points. Notably, when settling of the
cells was completed after 75 min, spreading on the substratum of patient cells was
approximately two fold lower than the spreading observed for the control cells.
A statistic evaluation of the adhesion ability is shown in Fig. 23 C. After 15 minutes less
than 20% cells off the EDMD/CMT, DMD and Stiff skin syndrome patient cell lines were
attached to the surface of the coverslips. In comparison, 30% of the control cells were
attached to the coverslips after 15 minutes. Attachment to the substratum was
significantly increased in EDMD/CMT and DMD patient cells in comparison to the
control cells after 30 and 45 minutes. After 45 minutes the number of attached
EDMD/CMT and DMD patient cells was close to the number of attached control cells.
After 45 minutes the number of attached stiff skin cells was two fold lower than the
number of attached control cells. Attachment to the surface of Stiff skin syndrome
patient cells have been remarkably decreased at all evaluated time points. 90 minutes
after initial seeding the cells of all cell lines have been completely attached to the
surface of the coverslips.
Results
59
A
B (a)
15 min 30 min 45 min 60 min 75 min
ctrl 1277.5 2385.51 2389.04 3882.86 7165.07
SSS 722.37 865.16 834.83 1002.25 2865.36
EDMD/CMT 978.02 936.09 1453.69 3058.94 3193.63
DMD 989.18 1126.05 1800.52 2691.37 4012.26
B (b)
ctrl
SSS
EDMD/CMT
DMD
15‘ 30‘ 45‘ 60‘ 75‘
Results
60
C
0
20
40
60
80
100
120
15 30 45 60 75 90
min
% o
f a
tta
ch
ed
ce
lls
ctrl
stiff skin
EDMD/CMT
DMD
Fig. 23: A: Immunofluorescence analysis of control and patient fibroblasts 15, 30, 45, 60 and 75 min after
seedin stained for vinculin. Alexa Flour 488 conjugated secondary antibody was used, DAPI for nuclear
staining; scale bar 76.4µm. B (a): vinculin stained area on the surface of coverslips of control and patient
fibroblasts 15, 30, 45, 60 and 75 min after seeding, measured in µm2. (b): polygon drawn with LAS-AF
Lite Application Suite software from Leica indicates the encircled area in µm2. C: Adhesion ability of
control and patient fibroblasts 15, 30, 45, 60 and 75 min after seeding. After 90 min seeding attachment
was completed.
Results
61
3.6.7 Distribution of nuclear envelope proteins in control fibroblasts and patient
fibroblasts
The distribution of several nuclear envelope proteins was assessed by
immunofluorescence analysis. Control fibroblasts show a continuous nuclear rim
staining for SUN2 with mAb K80-207-11. EDMD/CMT, DMD and Stiff skin syndrome
fibroblasts staining for SUN2 with mAb K80-207-11 exhibit a similar staining pattern.
SUN2 was localized to the nuclear envelope but was also present in micro nuclei as well
as in blebs and foldings (Fig. 24-28, second column, arrowhead). Additionally, in
EDMD/CMT and Stiff skin syndrome cells the SUN2 staining appeared discontinuously
distributed and the rim staining was weaker than in the control cells. Especially for stiff
skin patient fibroblasts a broadened rim staining for SUN2 was observed. The same
observations could be made for emerin which was localized at the nuclear envelope but
also in blebs and folds and enriched in micronuclei in EDMD/CMT patient cells.
Especially EDMD/CMT and stiff skin patient fibroblasts exhibit more of an overall
nuclear emerin staining and a weaker rim staining compared to the control cells (Fig.
24).
Results
62
Fig. 24: Distribution of nuclear envelope proteins in control and patient fibroblasts. Control, EDMD/CMT,
DMD and Stiff skin syndrome fibroblasts were stained for SUN2 and emerin using mAb K80-207-11 and
anti-emerin as first antibodies; Alexa Flour 568 and 488 as conjugated secondary antibodies; DAPI for
nuclear staining. Arrowheads indicate SUN2 staining in nuclear shape defects; arrows indicate the
staining in nuclear shape defects described in the text. Scale bar 5 µm.
LMNA/C staining was localized to the nuclear envelope and to some extend distributed
within the nuclei in control cells and in the EDMD/CMT patient cells. SUN2 staining was
slightly diminished in these cells compared to the control cells.
Severely misshapen nuclei of DMD and stiff skin patient cells exhibited localization of
LMNA/C into foldings of the nuclear envelope (arrows). The strong cytoplasmic staining
in the bottom row is most probably unspecific staining due to cell fragments from dead
cells that have not been completely removed during the immunofluorescence
preparation (Fig. 25).
FR
Wt
emerin
-11
merge emerin DAPI 207-11
SSS
FR
Wt
emerin
-11
merge emerin DAPI
ctrl
DMD
207-11
EDMD/CMT
Results
63
Fig. 25: Distribution of nuclear envelope proteins in control and patient fibroblasts. Control, EDMD/CMT,
DMD and Stiff skin syndrome fibroblasts were stained for SUN2 and LMNA/C using mAb K809-207-11
and anti-LMNA/C as first antibodies; Alexa Flour 568 and 488 as conjugated secondary antibodies; DAPI
for nuclear staining. Arrowheads indicate SUN2 staining in nuclear shape defects; arrows indicate the
staining in nuclear shape defects described in the text. Scale bar 5 µm.
LMNB1 was localized to the nuclear envelope in control cells and in all patient fibroblast.
Like SUN2, emerin and LMNA/C, LMNB1 is located in all regions of the nuclear surface,
extending into clefts and protuberances of the nuclear envelope in EDMD/CMT and
DMD patient cells (arrows). Compared to control cells SUN2 staining was slightly
weaker in EDMD/CMT cells.
LMNB1 was found in stiff skin patient cells colocalizing with SUN2. Note the protrusion
stained by LMNB1 observed in the lower area of the stiff skin fibroblasts (dotted arrow)
(Fig. 26).
WT
merge LMNA/C
DMD
ctrl.
SSS
K80-207-11 DAPI
EDMD/CMT
Results
64
Fig. 26: Distribution of nuclear envelope proteins in control and patient fibroblasts. Control, EDMD/CMT,
DMD and Stiff skin syndrome fibroblasts were stained for SUN2 and LMNB1 using mAb K809-207-11 and
anti-LMNB1 as first antibodies; Alexa Flour 568 and 488 as conjugated secondary antibodies; DAPI for
nuclear staining. Arrowheads indicate SUN2 staining in nuclear shape defects; arrows indicate the
staining in nuclear shape defects described in the text. Scale bar 5 µm.
To detect NESPRIN1 polyclonal antibodies directed against C-terminal spectrin repeats
of the protein (NESPRIN1SpecII) were used. In all cells, the NESPRIN1 staining was
week. In control cells NESPRIN1 was localized to the nuclear envelope. In EDMD/CMT
cells diminished staining for NESPRIN1 was detected in comparison to the control cells.
In DMD fibroblasts NESPRIN1 was localized to the nuclear envelope but also distributed
in the nuclei and found in the cytoplasm to some extend. In stiff skin patient fibroblasts
the arrow indicates an accumulation of NESPRIN1 staining (Fig. 27).
laminB w
20
-1
LMNB1
DMD
ctrl
l
SSS
K80-207-11 DAPI merge
EDMD/CMT
Results
65
Fig. 27: Distribution of nuclear envelope proteins in control and patient fibroblasts. Control, EDMD/CMT,
DMD and Stiff skin syndrome fibroblasts were stained for SUN2 and NESPRIN1 using mAb K809-207-11
and anti-NES1SpecII as first antibodies; Alexa Flour 568 and 488 as conjugated secondary antibodies;
DAPI for nuclear staining. Arrowheads indicate SUN2 staining in nuclear shape defects; arrows indicate
the staining defects described in the text. Scale bar 5 µm.
To investigate the localization pattern of NESPRIN2, polyclonal antibodies directed
against the last two C-terminal spectrin repeats of human NESPRIN2 were used. The
patchy and distributed staining of NESPRIN2 in the nucleus that can be observed for
control cells could also be seen in all patient cell lines. Additionally, NESPRIN2
appeared to be enriched and evenly distributed in micro nuclei found in DMD patient
fibroblasts. Week cytoplasmic staining of NESPRIN2 was seen in control cells as well as
in all patient cells. Extended locations of NESPRIN2 into the cytoplasm were seen in
wt
merge
EDMD/CMT
DMD
ctrl.
NES1SpecII
SSS
DAPI K80-207-11
Results
66
Stiff skin syndrome cells. The strong cytoplasmic staining in the second row is
presumably an artefact (Fig. 28).
Fig. 28: Distribution of nuclear envelope proteins in control and patient fibroblasts. Control, EDMD/CMT,
DMD and Stiff skin syndrome fibroblasts were stained for SUN2 and NESPRIN2 using mAb K809-207-11
and anti-NES2 as first antibodies; Alexa Flour 568 and 488 as conjugated secondary antibodies; DAPI for
nuclear staining. Arrowheads indicate SUN2 staining in nuclear shape defects; arrows indicate the
staining in nuclear shape defects described in the text. Scale bar 5 µm.
The lamina-associated polypeptide-2 (LAP2) is also an inner nuclear membrane protein,
which has been shown to bind to A-type lamins and BAF-DNA complexes (Dechat et al.,
2000; Shumaker et al., 2001). The distribution of LAP2 was assessed using LAP2
antibodies. LAP2 distribution was seen at the nuclear envelope and to some extent to
the nucleoplasm in control cells as well as in all patient cells. In dysmorphic patient cell
merge NES2
DMD
EDMD/CMT
ctrl
SSS
DAPI K80-207-11
Results
67
nuclei LAP2 was present on the nuclear surface and in blebs and increased staining can
be seen in micro nuclei of DMD patient fibroblasts (arrow) (Fig. 29). Rim staining of
LAP2 was reduced in EDMD/CMT cells relative to control cells. Since LAP2 and K80-
207-11 are both mouse monoclonal antibodies, costaining for SUN2 was not possible.
Fig. 29: Distribution of nuclear envelope proteins in control and patient fibroblasts. Control, EDMD/CMT,
DMD and Stiff skin syndrome fibroblasts were stained for SUN2 and LAP2 using mAb K809-207-11 and
anti-LAP2 as first antibodies; Alexa Flour 568 and 488 as conjugated secondary antibodies; DAPI for
nuclear staining. Arrow indicates the staining in nuclear shape defects described in the text. Scale bar 5
µm.
merge
ctrl
DAPI LAP2
SSS
DMD
EDMD/CMT
Results
68
3.6.8 Nucleus-centrosome distance is increased in EDMD/CMT, DMD and Stiff skin
syndrome fibroblasts
The centrosome plays a key role in cellular architecture by determining the position of
several associated organelles, including the nucleus. Previous published data indicate
that nuclear envelope proteins like LINC- proteins and emerin are participating in
centrosome-nucleus juxtaposition and mediate shuttling of nuclear and centrosomal
proteins between these organelles (Hutchison et al., 2007, Xiong et al., 2008).
Therefore, the localization of the centrosome relative to the nucleus was investigated
using antibodies against pericentrin (Fig. 30 A, B). Nucleus-centrosome distance was
measured for 50 cells for each patient cell line using Leica LAS AF Lite software. The
experiment was carried out once (Fig. 30 C).
In control cells each nucleus was located in close proximity of one centrosome during
the interphase. In control cells the centrosome was positioned adjacent to the
membrane or within 1.30 µm of the nuclear envelope. Misshapen nuclei from patient
cells as well as normal shaped nuclei exhibit a slightly increased distance of the
centrosome from the nuclei. The mean distance of the nucleus to the centrosome that
was measured for DMD cells was 3.25 µm, the centrosome was positioned between 0
µm and 8.67 µm and this cells showed therefore an increased nucleus-centrosome
distance in comparison to the control cells. In EDMD/CMT fibroblasts also an increased
distance of 4.01 µm was exhibited. The maximum distance that was measured was 9.05
µm. In stiff skin patient cells the mean centrosome-nucleus distance was 2.37 µm, with
a maximum distance of 8.42 µm (Fig. 30 C).
In regular-shaped control cells, the nucleus number correlated with that of the
centrosome. These observations were also true for regular-shaped and misshapen
nuclei from patient cells. For EDMD/CMT cells one cell with two centrosomes was seen.
The presence of micronuclei had no influence on the nucleus-centrosome distance (Fig.
30 B).
Results
69
C
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
ctrl 0-1.30 EDMD/CMT 0-9.05 DMD 0-8.67 stiff skin 0-8.42
mean d
ista
nce (
µm
)
Fig.30 A, B: Immunofluorescence of control, EDMD/CMT, DMD and stiff skin syndrome fibroblasts, anti-
pericentrin as primary antibody, Alexa Flour 488 as conjugated secondary antibodies, DAPI for nuclear
staining. Scale bar: 5µm. C: Nucleus–centrosome mean distances measured in µm with Leica LAS AF
Lite software, 50 cells for control, EDMD/CMT, DMD and Stiff skin syndrome fibroblasts were examined.
Minimal- and maximal values measured for each patient cell line are listed in the x-axis.
ctrl DMD SSS
A
2,62 µm
5,43 µm
8,67 µm
8,30 µm
2,32 µm
EDMD/CMT
616 nm
2.09µm
EDMD/CMT DMD
B
Results
70
3.6.9 Precipitation profile in EDMD/CMT fibroblast cells differ from control
fibroblast cells
As an initial screening assay for identifying possible differences in protein interaction
partners of SUN2Nt in control cells compared to patient cells, pull down experiments
with total control fibroblast cells lysates and total lysates from EDMD/CMT cells and
GST-SUN2Nt as bait were performed. Analysis was done by pre-separating the protein
complex by SDS-PAGE and followed by LC-MS (Liquid chromatography-mass
spectrometry) as described in 3.4.
Fig.31: Coomassie Blue stained PAA gel (12% acrylamide) showing proteins from pull down assays using
total control cell lysate and total EDMD/CMT cell lysate and GST-SUN2Nt as bait. Controls: beads
incubated with the indicated cell lysate. Arrows and grey brackets indicate areas of interest for protein
analysis. Black arrows: present in both samples; grey arrows: diminished in control cell/patient cell lysate;
red arrows: only found in control cell/patient cell lysate.
Table 2 represents a list of proteins which are categorized in proteins taking part in gene
regulatory events (A), in proteins present in RNA-processing complexes (B), in proteins
of architectural complexes (C) and proteins participating in signaling (D). The diagram in
figure 32 summarizes the proteins of each category and illustrates their distribution in
either total control cell lysates or EDMD/CMT cell lysates precipitated by GST-SUN2Nt
as bait. Proteins found in both lysates are illustrated in the intersection.
250 130
95
GST- SUN2Nt +EDMD/CMT- lysate
beads +EDMD/ CMT- lysate
beads +ctrl -lysate
GST- SUN2Nt +ctrl -lysate
72
55
Results
71
Tab. 2: Proteins detected in total control and patient fibroblast lysates precipitated by GST-SUN2Nt
categorized in gene regulatory (A), RNA processing (B), architectural complex (C) and signaling (D)
proteins. Identifier indicates the SwissProt accession number if not stated otherwise.
Identifier Name Category ctrl EDMD
/ CMT
Q9NVE4 (UniProtKB) CCDC87 A + +
P68104 (UniProtKB) EEF1A1 B - +
NP_991247.1 HNRNPH1 B + +
O00571 mRNA helicase 3X B + -
EEF2_HUMAN EEF2 B + -
P60842 (UniProtKB) EIF4A1 B + +
Q8N9N8 EEF1G B - +
Q9UHB6 LIMA1 C + -
Q8WZ42 titin C - +
O43707.2 α-actinin1 C + -
P12814.2 α-actinin4 C + -
P41351 tubulin α C + +
P05219 tubulin β C + +
Q9ULV4.1 coronin1C C + -
P02545.1 laminA/C C, D + +
P68032 actin C, D + -
P35579 myosin9 C, D + -
O00159 myosin1c C, D + -
Q8WXH0 nesprin2 C, D + -
P53618.3 coatomer β D + -
AAH20498.2 coatomer γ D + -
AAH20498.2 coatomer γ D - +
P22314 ubiquitin-like modifier-activating enzyme D - +
Only one protein taking part in DNA-processing events, the coiled coil domain
containing protein 87 (CCDC87), could be detected in lysates from control cells and in
lysates from EDMD/CMT affected patient cells. In both lysates equal amounts of
proteins participating in RNA-processing (B), like translation factors and HNRNPH1,
could be detected. In control cell lysates significantly more proteins contributing to the
cell architecture (C) and signaling (D) events could be detected which were absent from
the EDMD/CMT cell lysate. Missing proteins are mainly actin related proteins such as
Results
72
actin, myosin1C and myosin9. Notably, NESPRIN2 was only found in control cell
lysates. LMNA/C was identified in both lysates.
Fig. 32: Distribution of proteins in either total control fibroblasts or EDMD/CMT fibroblasts lysates
precipitated by GST-SUN2Nt as bait. Proteins found in both lysates are illustrated in the intersection.
tubulinα
tubulinβ LMNA/C
HNRNPH1 EIF4A1
coatomerγ
α-actinin1
α-actinin4 coronin1C
LIMA1 actin
myosin9 myosin1c
NESPRIN2
coatomerβ mRNA helicase 3X
EEF2 CCDC87
titin ubiquitin- like modifier-
activating enzyme EEF1A1 EEF1G
control
EDMD/CMT
Discussion
73
4 Discussion
4.1 Generation of a monoclonal antibody
In the present study mouse monoclonal antibodies directed against an N-terminal
peptide comprising amino acids 1-138 of human SUN2 protein were generated. Several
hybridoma clones showed a similar recognition pattern in western blot analysis.
Hybridoma subclone K80-207-11 detected a recombinant bacterially expressed protein,
the mammalian V5*6xHis-tagged fusion, and endogenous SUN2 protein in
immunoblotting and immunostaining and thus was selected for subsequent applications.
HeLa cells stained with emerin specific antibodies and K80-207-11 showed an
overlapping localization of the proteins at the nuclear envelope. In cell fractionation
studies, K80-207-11 recognized SUN2 exclusively in the nuclear fraction. These data
indicate that SUN2 is primarily located at the nuclear envelope. The cytoplasmic staining
that is observed in the immunofluorescence experiments may be derived from non-
specific interactions of mAb K80-207-11.
Westernblot analysis revealed the presence of 2 bands at ~75 and ~70 kDa in HeLa and
Pop10 cells. This could be due to differential splicing, although experimental evidence is
lacking so far (Hodzic et al, 2004). Alternatively, the faster migrating protein could be a
degradation product, or it might be a cross reactive protein.
4.2 Subcellular localization of endogenous SUN2 protein during the cell cycle
Immunostaining of HeLa cells revealed that SUN2 is largely segregated within the plane
of the NE in non mitotic cells. The nuclear rim staining by K80-207-11 persisted during
prophase. When the nuclear envelope is completely disassembled in metaphase, SUN2
associates with the condensed chromosomes. In anaphase SUN2 appears to
accumulate at distinct chromosome regions of the condensed chromatids which might
be telomere regions as described in meiotic cells for sad1 in S. pombe (Hagan et al.,
1995; Alsheimer et al., 2006).
In metaphase, SUN2 can be detected in two dot-like structures which colocalize with
pericentrin and therefore indicate a centrosomal localization of SUN2. Similar findings
have been reported by Wang et al. (2006). In C. elegans the KASH protein zyg1 and
sun1 are essential for tethering the centrosome to the NE and therefore for pronuclear
migration (Gönczy et al., 2004; Malone et al., 2003). In Dictyostelium, Sun1 is required
for centrosome attachment (Xiong et al. 2008), and mouse Nesprin1/2 double knockout
cells have a centrosome detachment phenotype (Zhang et al. 2009). The role of KASH-
Discussion
74
SUN bridges in centrosome attachment to the nucleus is less clear in other tissues and
systems. Since SUN2 colocalizes with pericentrin in mitotic HeLa cells, there is
evidence that SUN2 proteins associate with the centrosomes and provide a molecular
linkage between the NE and the centrosome. If this linkage is maintained via nesprins
and the microtubule cytoskeleton or if novel interaction partners are involved has to be
further elucidated.
Upon nuclear envelope reassembly during late anaphase and telophase, K80-207-11
detects the SUN2 protein in a rim like pattern. SUN2 is enriched at a distinct core region
of each newly separated chromatin mass. At late anaphase to early telophase,
reforming nuclei exhibit a distinct distribution of nuclear pore complexes (NPC) and NE
components and the core region is typically deficient in NPC reformation while NPC
assembly is initiated on the lateral margins of the chromatid masses (Liu et al., 2001).
Pore-free islands are directly connected with gene silent heterochromatin regions
beneath them (Casolari et al., 2004). Pore-rich and pore-poor subdomains on the
nuclear envelope might reflect gene activation and silencing states of the corresponding
nuclear surface of certain chromosome territories. So far LMNA/C has been shown to
play an essential structural and regulatory role in the formation of pore-free islands
(Maeshima et al., 2006). Higher proliferation ability and aggressiveness in human
malignancies including leukemias and lymphomas, small-cell lung carcinoma and skin
carcinomas is correlated with the absence or downregulation of LMNA/C (Stadelmann et
al., 1990; Broers and Ramaekers, 1993; Venables et al., 2001). These reports support
the hypothesis that LMNA/C has a negative influence on cell proliferation and that the
suppression of LMNA/C might contribute to tumorigenesis through increasing pore
density. SUN2 as a direct LMNA/C interactor might therefore be involved in the
regulation of cell cycle depending dynamics and nuclear envelope subdomain
organization by controlling the reorganization of inner nuclear structures.
4.3 Protein networks formed by SUN2
Since distribution and localization of SUN2 changes during the cell cycle, a change in
interaction partners during nuclear envelope breakdown is conceivable. In an initial
approach, proteins which interacted with the N-terminus of SUN2 from total cell lysates
of normally grown HaCaT cells (HaCaT cell lysate) and HaCaT cells arrested in
prometaphase (mHaCaT cell lysate) were analyzed by LC-MS and compared to each
other. The proteins identified in these experiments can be categorized in proteins
Discussion
75
participating in DNA-replication and chromatin remodeling, components of RNA
processing complexes, proteins of the architectural complex and proteins participating in
signaling.
Proteins contributing to DNA replication and chromatin remodeling are found in almost
equal amounts in the SUN2 precipitates from HaCaT and mHaCaT cell lysates. This
complex includes components of the nucleosome like H4 and H3-like and proteins that
mediate gene silencing via histone modifications like RNA-Pol II associated factor 1
(PAF1), Histone deacetylase (HDAC) and RUVB-like1/2 which are components of the
histone acetyltransferase (HAT)-complex. Also involved in histone modification and
transcription repression are C-terminus-binding protein 1 (CTB1), YLB-motif containing
protein 1 (YLB1) and metastasis associated protein 2 (MTA2). Both lysates contained
these proteins that predominantly participate in gene repression events in approximately
same amounts (Protein information obtained from www.ncbi.nlm.nih.gov).
Gene activation and repression are mostly regulated through DNA methylation,
chromatin remodeling and histone modifications. A variety of proteins are involved in
chromatin regulation including DNA methyltransferases, chromatin-remodeling
complexes, DNA transcription factors and chromatin-modifying complexes. Chromatin-
remodeling complexes modify nucleosome structure and modulate the accessibility of
DNA for transcription factors. Human Sin3-HDAC complex includes HDAC1, HDAC2
and the histone-binding proteins RbAp46 and RbAp48 (Perissi et al., 2010). RbAp48 is
also termed RBBP4 and was found in pull down experiments using the N-terminus of
SUN2 in both HaCaT cell lysates together with HDAC1. HDAC1 is also involved in the
CoREST and NURD complexes which are important transcription silencing machines
(Huang et al., 2011). CTB, a part of the CoREST complex, was also found in the lysates
while MTA2, a component of the NURD complex, was only detected in mHaCaT lysates.
A recent study reported the nuclear actin binding protein coronin2A as part of the NCoR
(nuclear receptor co-repressor) and SMRT (Silencing Mediator of Retinoid acid and
Thyroid hormone receptor) complex (Perissi et al., 2010). Here, a related coronin,
coronin1C, was found in pulldown experiments.
Links between NCoR and SMRT to several types of leukaemias including acute
promyelocytic leukaemia, acute myeloid leukaemia and paediatric b-cell acute
leukaemia have been reported (Karagianni and Wong, 2007). A correlation between
NCoR expression and the most common and aggressive type of primary brain tumour,
astrocyte-derived cancer glioblastoma multiforme (GbM), has been observed. In severe
Discussion
76
grades of astrocytomas NCoR is dramatically increased and correlates with progress
from WHO (World Health Organization) grade II to grade IV glioma (Lubensky et al.,
2006; Park et al., 2007). Likewise, coronin1C expression is significantly altered in
gliomas. In highgrade anaplastic astrocytomas, anaplastic oligodendrogliomas,
anaplastic oligoastrocytomas and glioblastomas high numbers of coronin1C-positive
tumor cells have been found, which is suggestive for a contribution of the protein in the
malignant progression of diffuse gliomas (Roadcap et al., 2008; Xavier et al., 2009). In
addition, SUN2 staining was significantly increased in the human glioblastoma-
astrocytoma cell line U373. Whether the observed interaction is direct or indirect is not
yet known. However a participation of the LINC complex protein SUN2 in the
development of tumorigenesis is intriguing. It is conceivable that increased SUN2
localization at the NE recruits increasing levels of coronin1C contributing to tumor
malignancy.
Altered mRNA synthesis and processing has been reported to be involved in a broad
spectrum of human diseases, including cancer, spinal muscular atrophy and
Hutchinson-Gilford Progeria syndrome (Morares, 2009). In the present study, putative
interactions with proteins of the RNA processing complex suggest a novel role for SUN2
in RNA processing and splicing. In the proteins categorized as RNA processing
components an equal number of proteins could be detected in both cell lysates. While
mainly heterogeneous nuclear ribonucleoproteins (hnRNPs) and splicing factors were
detected in lysates of asynchronously growing HaCaT cells, lysates of mHaCaT cells
contained three translation factors absent from the HaCaT cell lysates. Eukaryotic inition
factor 4A is a nuclear protein that specifically functions during nonsense-mediated
decay (NMD), a RNA surveillance mechanism that degrades mRNA bearing a
premature termination codon (PTC) (Wagner and Andersen, 2001; Ferraiuolo et al.,
2004). About 30% of inherited disorders including β-thalassemia, myotonia congenita
and retinal degeneration are the consequences of mutations that create a PTC
(Frischmeyer et al., 1999). The presence of eukaryotic translation initiation factors found
in the lysates is irritating at first glance. However reports exist describing that some
nuclear translation closely coupled to transcription takes place (Iborra et al., 2004). The
presence of translation initiation factors in the nucleus and how they act in the nucleus
has to be further elucidated.
More than twice the number of proteins contributing to cell architecture was detected in
mHaCaT cell lysates in comparison to HaCaT cell lysates. Proteins found only in the
Discussion
77
mHaCaT cell lysates participate mainly in the actin cytoskeleton. Many proteins with
known structural roles in the cytoskeleton are either localized in the nucleus or shuttle in
and out of the nucleus, supporting the existence of both cytoplasmic and nuclear
isoforms of key structural proteins. For actin it is reported that it shuttles between the
nucleus and the cytoplasm and assembles to nuclear-specific short polymers (Pederson
and Aebi, 2002; Pederson and Aebi 2005; McDonald et al., 2006). Diverse roles are
reported for nuclear actin including chromatin remodeling and transcription, processing
and export of mRNA (Bettinger et al., 2004, Olave et al., 2004). Included in the actin
network is α-actinin, Ras-GTPase activating like protein and myosin. Myoferlin is
required for myotube formation (Doherty et al., 2005). It has also been reported that
actin binds directly to LMNA/C at two actin binding sites in the tail region of LMNA and
at one region in LMNC (Simon et al., 2010). Therefore, actin and actin-related proteins
might be present in the detected protein pool due to interactions with lamin contained in
both lysates, and which interacts with SUN2.
In most cells tubulin resides only in the cytosol and not in the nucleus. The βII- isotype
of tubulin was recently found in the nuclei of several tumor cells but could not be
detected in biopsy samples of normal human tissues suggesting that the presence of
nuclear βII-tubulin may be correlated with the cancerous state of cells (Xu et al., 2002,
Yeh et al., 2004). However, the function of βII-tubulin in the nucleus is still unknown.
Microtubule polymers formed by α- and β-tubulin have been detected in both lysates.
Since only the N-terminus of SUN2 was used as bait, no indirect interactions with
microtubule through ONM-located nesprins can be responsible for the presence of
tubulin. Also, with regard to the reported findings of tubulin isoforms in tumor cell nuclei
HaCaT cells and not HeLa (human cervical cancer) cells have been used for pull down
assays. Therefore, the presence of tubulin might be due to microtubules that have
invaded the nuclear space upon nuclear envelope disassembly in prometaphase and
suggest a role for SUN2 in coupling the mitotic spindle microtubules to kinetochores or
to opposing microtubules (Loubery et al., 2008). Tubulin pools can be found in both
lysates because the normally grown HaCaT cells are not synchronized and therefore
some of the cells underwent mitosis when harvested for the assay.
Several subunits of coat protein complex I (COPI), namely COPα, β, γ, δ, could be
pulled down by the N-terminus of SUN2. Trafficking of many membrane proteins within
the Golgi and between Golgi and ER relies on the recognition of ER localization signals
by COPI. As described in 3.1 and shown in figure 4 and 5, four arginine residues at
Discussion
78
amino acid position 102-105 conserved in mammalian SUN2 but not in SUN1, provide a
ER localization signal as previously described for membrane proteins (Michelsen et al.,
2005). This 4R motif of mammalian SUN2 was recently shown to interact with COPI,
and mutations in this motif resulted in loss of the association (Turgay et al, 2010).
These observations underline not only the structural importance of SUN2 during the cell
cycle, but also point to SUN2 as a protein that scaffolds a variety of multi-protein
complexes at the inner nuclear membrane. The identity of the putative interactors
suggests that SUN2 might exhibit a network function at the inner nuclear membrane by
interlinking proteins with diverse functions. Also, the presence of several RNA-
processing proteins may point to a novel role for SUN2 in the processing of RNA, and
could extend the mode of operation for SUN2 in cellular processes. Therefore, SUN2
may not only be essential in the transmission of signals via the LINC complex, but may
also influence and mediate gene regulation at various levels and functions. Such
proposals are however still hypothetical as the interactions have not been verified by
different methods yet.
4.4 Direct interactions of LMNA/C with the N-terminus of SUN2 in vitro
A previous study (Crisp et al, 2006) reported the in vitro interaction of a GST-fusion
protein containing the first 165 amino acids of SUN2 with four lamin proteins: LMNB1,
LMNC, full-length LMNA, and mature LMNA, although the interaction with LMNB1 and C
appeared barely more than the background observed with GST as control alone. Since
LMNA/C was detected in the pull down experiments from HaCaT cell lysates, the
respective interacting domains of LMNA/C with SUN2 was mapped. Five GST-LMNC
fragments have been tested for interaction with the N-terminus of recombinant SUN2 by
GST pulldown assays: GST-LMNC N-term/LMNA (aa 1-127), GST-LMNC coil1B-∆ (aa
128-218), GST-LMNC coil2 (aa 243- 387), GST-LMNC tail (aa 384-566), GST-∆LMNC
(aa 128-572). The GST-fusion constructs LMNNt and coil1B∆ which are contained
within ∆LMNC could precipitate high amounts of SUN2Nt whereas decreased amounts
of SUN2Nt have been precipitated by ∆LMNC. These differences in the binding affinity
for ∆LMNC compared with LMNNt might be due to varying folding and dimerization
processes in the SUN2Nt protein as well as in the lamin-constructs. Both lamin
polypeptides (A- and B-types) harbor α-helical regions and form parallel coiled–coil
homodimers, which can in turn assemble into higher-order filamentous structures and
Discussion
79
therefore compete for binding with SUN2Nt. The data point to an extended interaction
area for SUN2 and LMNA/C. Multiple or extended interaction zones have also been
reported for c-Fos and LMNA/C (Maraldi et al., 2010).
In experiments in HeLa cells in which A-type lamins had been eliminated by RNA
interference, the NE localization of SUN2 was barely affected (Crisp et al, 2006).
Evidently, although A-type lamins can contribute to SUN2 localization, they are not the
only determinants. As recently published, three features are thought to jointly contribute
to the NE-localization of SUN2: 1) The N- terminal domain (aa38-52) recognized by
Importin α/β; 2) The 4R-motif serves as binding platform for coatomer I complex, and 3)
The C-terminal SUN domain establishing SUN-KASH interactions to stabilize the NE
localization (Turgay et al, 2010). Therefore, the direct interaction of SUN2 and LMNA/C
might be affected by mutations in LMNA and result in a defective interaction despite the
regular presence of both components.
Discussion
80
4.5 Characterization of fibroblast from Stiff skin syndrome (SSS), Duchenne
muscular dystrophy (DMD) and Emery-Dreifuss muscular dystrophy / Charcot-
Marie-Tooth syndrome (EDMD/CMT) patients
4.5.1 Stiff skin syndrome (SSS) patients
Stiff skin syndrome is characterized by hard, thick skin that limits joint mobility and
causes flexion contractures. Hypertrichosis, postural and thoracic wall abnormalities are
associated, and occasional findings include focal lipodystrophy and muscle weakness
(Liu et al., 2008). Although about forty cases have been described in literature, definitive
assignment of the inheritance pattern is precluded due to the lack of large multiplex
families. Thus, prior work concerning the pathogenesis of SSS has been mainly
observational with few mechanistic insights (Esterly et al., 1971; Amoric et al., 1991;
Bodemer et al., 1991; Fidzianska et al., 2000; Jablonska et al., 2004; Ferrari et al.,
2005; Geng et al., 2006; Pages et al, 2007). Findings include increased collagen
production with sclerotic collagen bundles in the deep reticular dermis and/or
subcutaneous septa. Also, increased levels of cytokines including TNFα, IL-6 and
TGFβ2 have been described (Jablonska et al., 2000; Loeys et al., 2010).
In this study, dermal fibroblast from a SSS patient are described which exhibit nuclear
alterations similar to laminopathy patient cells. Other findings revealed restricted growth,
decreased SUN2 transcript levels in senescent cells but overexpression of SUN2 in low
passages. Also, in comparison to control cells, cell adhesion and spreading was
remarkably decreased.
Ordered polymers of fibrillin1 (termed microfibrils) encoded by FBN1 initiate elastic fiber
assembly and bind to and regulate the activation of the profibrotic cytokine transforming
growth factor β (TGFβ) (Isogai et al., 2003). Excessive microfibrillar deposition is
accompanied by increased TGFβ concentration in stiff skin patients as described in a
recent report (Loeys et al., 2010). The study describes mutations in the only Arg-Gly-
Asp (RGD) sequence–encoding domain of fibrillin1 that mediates integrin binding as
causative for Stiff skin syndrome. Integrins that bind via this RGD-motif are integrin
αvβ3, αvβ6, and α5β1 which are also known to activate TGFβ (Neil et al., 2006; Galliher
et al., 2006).
Several studies have implicated aberrant integrin expression or function in fibrotic
phenotypes (Asano et al., 2005; Yang et al., 2007; Wipff et al., 2008). Integrins activate
a variety of adhesion-dependent signal cascades including FAK, MAPK and PI3K/PKB
Discussion
81
which regulate cell proliferation. Integrin β1 overexpression has been reported to inhibit
cell adhesion and accordingly, reduces PI3K/PKB pathway activity, subsequently
resulting in reduced cell proliferation through upregulation of the cyclin-dependent
kinase (CDC) inhibitor p21Kip1 (Fu et al, 2007).
Contrary, no disease relevant mutations could be found in the FBN1 gene for the patient
fibroblasts described in the present study. This is compatible with findings in further SSS
patients in which the FBN1 gene was also unaltered (personal communication Dr. P.
Robinson, Charité, Berlin). Thus, diminished growth and a remarkable decrease in cell
adhesion and spreading on the substratum observed in SSS fibroblasts described in this
study is most probably due to so far unknown mechanisms independent from FBN1
mutations. These results underline the hypothesis that microfibrills present in SSS
patients without mutations in FBN1 bind to and regulate the activation of TGFβ in a
concentration depending manner (Loeys et al., 2010). As a consequence of enhanced
nuclear signaling fibroblasts produce more collagen. Increased TGFβ concentration thus
results in increased signaling in the dermis, contributing to the disease phenotype.
Overexpression of microfibrillar bundles might also contribute to altered SUN2 transcript
levels observed in our SSS patient cells. Increased SUN2 protein levels at the NE might
strengthen links both to the nuclear lamina and, via the LINC complex, to the
cytoskeleton and therefore contribute to the cell’s stiffness. Consistent with this is the
observation of broadened rim staining for SUN2 indicating a thickened NE with
increased SUN2 accumulation. Similar observations were made for LMNA/C, LMNB1
and NESPRIN1. Therefore, presently unknown mutations might affect LINC complex
proteins resulting in a disruption of the LINC complexes potentially leading to an
enlargement of the perinuclear space between the ONM and the INM. In fact, previous
studies reported significant enlargement of the perinuclear space due to disruption of
LINC complexes (Crisp et al., 2006), supporting the involvement of LINC complex
proteins in the etiology of Stiff skin syndrome.
Discussion
82
4.5.2 Duchenne muscular dystrophy (DMD) and Emery-Dreifuss muscular
dystrophy / Charcot-Marie-Tooth syndrome (EDMD/CMT)
It has been reported that alterations in nuclear morphology correlate with chromatin
arrangement possibly involved in the control of gene expression. Defects in nuclear
architecture are associated with X-EDMD, including aberrant heterochromatin
distribution and leakage of amino acids into the cytoplasm, conceivably due to NE
fragility (Fidzianska et al, 2003; Muchir et al., 2004).
These changes include altered subnuclear targeting of transcription factors and/or
nuclear domains. Principal components of chromatin remodeling complexes include
actin and actin regulatory proteins. Between nuclear actin, LMNA/C and emerin a
molecular link has been suspected to exist (Fairly et al., 1999; Clements et al., 2000;
Vaughan et al., 2001). Previously reported and further elucidated in the study by Crisp
et al (2006), is a direct interaction of LMNA/C and SUN2. Loss or mutations that inhibit
the interaction between one of those proteins could result in an altered relationship
between the NE and chromatin. Subsequently, this is also true for any protein taking
part in the extended LINC-complex. Strikingly, putative interaction partners of SUN2
differ dramatically as far as architectural proteins are concerned in EDMD/CMT cell
lysates in comparison to control cell lysates. Actin and actin related proteins like α-
actinin, myosin and nesprin were only detected in control lysates. Since LMNA/C was
still detected in EDMD/CMT cell lysates, it is conceivable that the LMNA/C-SUN2
interaction is maintained in EDMD/CMT cells, and the interruption might be therefore up-
or downstream of the LMNA/C-SUN2 connection. The absence of the actin complex in
this patient cell lysate points to a possibly altered interaction with these proteins. The
NESPRIN2 peptide identified in control cells matches to a sequence in the last spectrin
repeat in all NESPRIN2 isoforms, namely NESPRIN2 Giant/NUANCE, NESPRIN2α2
and NESPRIN2α1. Spectrins are known to associate with actin (Nowak et al., 2009). In
a hypothetical scenario, mutated NESPRIN1α in EDMD/CMT patient might weaken the
actin complex interaction and by doing so, contribute to or be the crucial factor leading
to the disease phenotype. It is therefore conceivable that altered expression of LINC-
complex proteins and/or associated proteins which interact with a nuclear actin scaffold
may affect gene expression in repair and/or maintenance of muscle cells in laminopathy
patients. Further experiments assessing the interactions between LINC complex
proteins and actin complex proteins will shed light on these important questions.
Discussion
83
As already mentioned in 4.3, RNA processing is linked to several severe human
diseases. However, the putative proteome of SUN2NT in EDMD/CMT patient cell
lysates revealed no significant difference in RNA processing proteins compared to
control cell lysates. Still, further studies beyond pulldown assays are required to prove
SUN2 interactions with the RNA processing complex.
The diminished SUN2 transcript levels in senescent patient cells observed in this study
might be due to presently unknown mutations in LINC complex proteins abolishing or
weakening the interaction with SUN2 or through perturped mechanotransduction and
subsequent aberrant signaling pathways which can in turn adjust cellular and
extracellular structure. Since it is reported that protein-protein interactions stabilize the
NE localization of SUN2 (Turgay et al, 2010), it is conceivable that mutations in SUN2
interacting partners affect the efficiency of their recruitment to the NE, leading to an
enhanced degradation. Findings in this study underline the importance of SUN2 and its
interacting partners as part of the LINC complex in disease development. Interestingly,
the N-terminus of SUN2 is a serine rich region, which in general is suspected to be a
precondition for alternate protein interactions in a tissue specific way. The analysis of
phosphorylation sites and of tissue specific SUN2 interacting partners will give more
insights into the tissue specific manifestation and variable penetrance seen in the
diverse laminopathy phenotypes.
In muscular dystrophy disorders, there is a constant need for regeneration in recurrent
myofiber damage in the presence of even mild stress. Satellite cells play a major role in
postnatal muscle growth and repair. Clinical manifestations of severe muscle wasting
caused by fibrosis, calcium deposits and adipose accumulation supplant muscle tissue,
is postulated due to impaired function of muscle stem cells (Wilson et al., 2000; Sacco
et al., 2008; Sacco et al., 2010). Damaged muscle fibers require gene activation that
might be affected by chromatin arrangements occurring in laminopathy conditions. In
fact, in X-EDMD patients a certain percentage of muscle fibre nuclei show nuclear
lamina and chromatin alterations (Ognibene et al., 1999).
The mutation in the dystrophin gene found in patient 1 converts glutamic acid encoded
by GAA/GAG into a premature termination codon resulting subsequently in a truncated
protein. This loss of protein function disconnects the extra-cellular matrix from the
cytoskeleton. The dystrophin gene is highly complex, containing at least eight
independent, tissue-specific promoters and two polyA-addition sites. Dystrophin RNA is
Discussion
84
differentially spliced, producing a range of different transcripts encoding a large set of
protein isoforms. The dystrophin protein as encoded by the Dp427 transcript is a large,
rod-like cytoskeletal protein found at the inner surface of muscle fibers. Dystrophin is
part of the dystrophin-glycoprotein complex (DGC), which connects the extra-cellular
matrix with the inner cytoskeleton (F-actin) (NCBI RefSeq database; Soltanzadeh et al.,
2010). Although loss of the structural protein dystrophin is the primary defect in
Duchenne muscular dystrophy, the secondary molecular machinery based on the
hypothesis that mitochondrial dysfunction caused by alterations of the calcium signaling
system resulting in a deleterious amplification of stress-induced cytosolic calcium
signals and in an amplification of stress-induced ROS production, is not fully understood
(Shkryl et al, 2009).
Defects in dystrophin have been associated with reduced nitric oxide (NO) production,
chronic inflammation and tissue degeneration resulting in altered gene expression
profiles and deficient regeneration (Gucuyener et al, 2000; Kasai et al, 2004; Judge et
al, 2006). There is evidence that during the mechanical stretching or regeneration of
dystrophic muscles signal transduction pathways, including PI3K-AKT, are altered. AKT
is also involved in the regulation of the intracellular NO synthesis (Dimmeler et al, 1999;
Dogra et al, 2006; Peter et al., 2006). Protein phosphatase 2A activity is NO dependent,
and its reduced activity results in altered regulation of class IIa histone deacetylases
(HDACs) 4 and 5 which have been found altered in DMD patient muscle cells (Illi et al,
2008). HDAC activity is involved in the regulation of genes, including c-Fos
downregulation which is necessary for satellite cell conversion to myoblasts. In
comparison to healthy individuals, altered pattern of global histone modifications have
been found in DMD patient muscle cells (Cohen et al, 2007). Therefore, the connection
between NO signaling and the altered epigenetic profile described in muscle cells
deficient for dystrophin indicates a link between the dystrophin-activated NO signaling
and the remodeling of chromatin and therefore an epigenetic contribution to the
pathogenesis and progression of DMD (Colussi et al. 2009).
Through mechanotransduction mechanical forces are translated into biochemical
signals and activate diverse signaling pathways which can in turn adjust cellular and
extracellular structure. By this mechanosensitive feedback, cellular functions like
migration, proliferation, differentiation and apoptosis are modulatet. Multiple and
overlapping cellular signaling pathways are activated by mechanosensors that can be
activated by stretch even in the absence of ligands, including extracellular signal-
Discussion
85
regulated kinase 1/2 (ERK1/2) and the mitogen-activated protein kinase (MAPK)
signaling (Jaalouk and Lammerding, 2009). It is reported, that dystrophin deficiency
causes an aberrant mechanotransduction in muscle fibers and leads to deregulation of
only ERK1/2 among the MAP kinase signaling pathways (Kumar et al, 2004).
Additionally, the mutation found in NESPRIN1α2 in patient 1 and in NESPRIN1α1 in
patient 2 being without consequences in healthy individuals, might in these cases
contribute to the perturbed mechanotransduction and weaken LINC complex protein
interactions. In immunofluorescence analysis, diminished staining for NESPRIN1
detected in EDMD/CMT cells and cytoplasmic localization of NESPRIN1 in DMD
fibroblasts further support this hypothesis. The nucleus-centrosome distance maintained
by SUN-NESPRIN1/2 interactions might also be affected by these nesprin mutations.
However, a previous report identified emerin as a novel microtubule interacting protein
anchoring the centrosome to the nucleus (Salpingidou et al, 2007). These data are
further confirmed by findings that the centrosome-nucleus distance is increased in
emerin-null human dermal fibroblasts. Fibroblasts with similar nuclear morphological
defects but normal for emerin from a Greenberg dysplasia patient did not exhibit altered
nucleus-centrosome distance, suggesting that centrosome mislocalization is specific to
the loss of emerin from the NE (Hale et al., 2008; Hutchison et al., 2007). Therefore, the
so far unknown underlying mutations in the fibroblasts of the three patients described in
this study are conceivable to affect proteins that interact either directly or indirectly with
emerin. Since SUN2 binds directly to emerin and is diminished in senescence patient
cells, SUN2 might play an essential role. Also, presently unknown interactions of NE
proteins with microtubules are possible. Tubulinα and tubulinβ were pulled down by
SUN2Nt from control and EDMD/CMT patient cell lysates, thus SUN2 might be a
promising candidate for microtubule interactions.
LINC complex proteins and associated proteins are ubiquitously expressed; yet
mutations in one of these proteins affect predominantly cardiac and skeletal muscle
tissue (Cohen et al., 2001). The hypothesis that nuclear fragility, although present in all
cell types, is critical only for certain tissues does not explain arrhythmia heart defects
involving pace-making cells that mediate the conduction pathway, and is also not
sufficient to explain the pathogenesis of lipodystrophy since adipose tissue is not
subjected to critical mechanical stress levels (information obtained from Muscular
Dystrophy Association, http://www.mdausa.org). Tissue specific signaling pathways
mediated by tissue specific composition of LINC complex proteins and associated
Discussion
86
proteins might be an alternative model. In such a model, nuclear envelopathies might be
caused by defects in gene expression due to loss or mutations in proteins involved
either directly or indirectly in the maintenance of proper chromatin arrangement which is
crucial for tissue specific regulation of transcription. In fact, perturbed ERK and JNK
branches of the MAPK signaling cascade was found in hearts of a mouse model for
autosomal EDMD and X-linked EDMD (Muchir et al., 2007; Muchir and Wormann,
2007). Chronically increased ERK and JNK activation is deleterious for hearts and
treatment of EDMD mice with PD98059, an inhibitor of ERK signaling, prevents
development of cardiomyopathy (Muchir et al., 2009). These findings were consistent
with known alterations in MAPK signaling in cardiomypathy (Molkentin et al., 2004). This
model also includes that no single mechanism will account for the varying phenotypes
among laminopathies.
Also, the complexity of polynucleated skeletal muscle maintenance and recurrent repair
and differentiation processes in muscular dystrophies suggest that a large number of
molecular mechanisms might be involved in the pathogenic development. One complex
mechanism is proper nuclear positioning relative to the cell body which is important for
many cellular processes during mammalian development (Zhang et al., 2008,
Gundersen et al., 2011). In the syncytial skeletal muscle cells, more than 100 nuclei are
evenly distributed at the periphery of each cell, with 3–8 nuclei anchored beneath the
neuromuscular junction. These postsynaptic nuclei cluster together under the plasma
membrane at sites of neuronal contacts forming the neuromuscular junction and
synthesize the components of the neuromuscular junction that specify the overlying
membrane as the target site for innervation. Therefore, in another model, failure in
nuclear positioning due to disruption of the extended LINC complex leads to a failure of
correct anchoring and correct positioning of the nucleus within the cell, and
subsequently to failure in innervation and to the disease phenotype of muscular
dystrophy (Bruusgaard, 2003; Starr et al, 2005; Zhang et al, 2008). Taken together,
comparative studies on the three different laminopathy affected patient cells revealed
similar cellular phenotypes with regard to the experiments carried out in this study. This
emphasizes the hypothesis that interruption of mechanotransduction and subsequent
signaling pathways caused at multiple levels, leading to disease; schematical displayed
in figure 33. Identifying the proteins taking part in the extended linker complex and their
function in biochemical pathways will provide new possibilities of therapeutic
approaches for these diseases.
Discussion
87
Fig. 33: Scheme of the LINC complex and associated proteins involved in chromatin association
maintenance and transduction of cytoplasmic forces. Question marks indicate suggested but not proven
interactions. Black arrows show signal transduction from the cytoplasm or the cytoplasmic membrane into
the nucleus via the LINC complex. Gray dashed arrows crossed by red line illustrate interrupted signal
transduction from the cytoplasm or the cytoplasmic membrane into the nucleus via the LINC complex.
Gray brackets with red arrows point to potential mutations/interruptions of cytoskeletal/LINC complex
proteins leading ultimately to altert signaling and disturbed gene transcription.
assembled cytosceletal/LINC complex
gene silencing
disassembled cytosceletal/LINC complex
disturbed gene transcription:disease phenotype
nucleus
cytoplasm
MTOC
? during mitosis
ONM
INM
mechano transduction/
signaling
collagen/fibronectin
plasma membrane
actin-filaments/myosin emerin heterochromatin LMNA/C microtubules NESPRIN1/2 intermediate filaments repression complex SUN2 transcription complex
laminin
dystroglycan complex
dystrohin
integrin receptors
talin vinculin
Summary
88
Summary
LINC complexes serve in several cellular processes providing a connection between
cellular components and organelles. Here I have generated monoclonal antibodies
directed against the N-terminus of human SUN2 and used it for immunofluorescence
studies. I observed that during the cell cycle SUN2 is evenly distributed in a rim like
pattern localized to the inner nuclear membrane in inter- and prophase, and
associates with condensed chromatin during metaphase. During nuclear envelope
reassembly in anaphase, SUN2 again shows a rim like pattern and is enriched in
NPC-poor regions further supporting the proposed role of SUN2 in heterochromatin
maintenance. SUN2 colocalizes also with pericentrin at the centrosome.
An increased SUN2 presence at the NE was detected in human glioblastoma cells,
pointing to a putative role in tumorigenesis possibly due to aberrant recruitment of
interaction partners that are involved in the development of malignancy.
A first proteomic study of the N-terminus of SUN2 revealed interactions with proteins
of the chromatin remodeling complex, suggesting SUN2 might be involved in
maintaining gene silencing during the cell cycle by recruiting corepressor complexes
to the nuclear periphery. Also, the analysis of the putative proteome revealed the first
described interaction with the RNA processing complex, suggesting a novel role for
SUN2 in RNA processing and splicing.
Characterization of Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular
dystrophy/Charcot-Marie-Tooth syndrome (EDMD/CMT) and Stiff skin syndrome
(SSS) patient fibroblasts revealed various nuclear deformations, diminished cell
adhesion and cell spreading on the substratum. Furthermore, the nucleus-
centrosome distance was increased in all three patient cells.
In precipitation assays using the N-terminus of SUN2 as GST-fusion and carried out
with EDMD/CMT patient cell lysates several actin related proteins were not present
which have been detected in control cell lysates. This supports the hypothesis that
protein interactions that involve the extended LINC complex are weakened or
interrupted.
Taken together, the findings in this study are consistent with proposed disease
mechanisms involving altered cell stability and/or altered gene transcription and
underline the importance of SUN2 and its interactions as part of the LINC complex.
Furthermore, the analysis of tissue specific SUN2 interactions will give more insights
Summary
89
into the tissue specific manifestation and variable penetrance of the various
laminopathy phenotypes.
Zusammenfassung
90
Zusammenfassung
Der LINC-Komplex stellt eine Verbindung zwischen Zellkern und Zytoplasma dar und ist
an vielen zellulären Prozessen beteiligt. Ein in dieser Arbeit neu generierter
monoklonaler Antikörper gegen die N-terminale Domäne des humanen SUN2 Proteins
zeigte eine regelmäßige ringförmige Verteilung von SUN2 an der Kernmembran von
Inter- und Prophase-Kernen in HeLa Zellen. Während der Metaphase ko-lokalisiert
SUN2 mit den kondensierten Chromosomen. Außerdem konnte eine Kolokalisation mit
Pericentrin am Zentrosom gezeigt werden. Bei der Reassemblierung der Kernhülle in
der Anaphase ist SUN2 wieder im Bereich der Kernhülle detektierbar und ist besonders
in NPC-armen Bereichen angereichert. Dies ist ein weiterer Hinweis darauf, dass SUN2
an der Aufrechterhaltung von Heterochromatin beteiligt ist.
Die in Immunofluoreszenzanalysen beobachteten signifikant erhöhten SUN2 Mengen in
humanen Glioblastomzellen weisen auch auf eine mögliche Rolle von SUN2 bei der
Tumorentstehung hin. Dies könnte durch eine übermäßige Rekrutierung von
Interaktionspartnern erfolgen, die ihrerseits an der Zellentartung beteiligt sind.
In Präzipitationsversuchen mit dem N-Terminus von SUN2 und Lysaten von humanen
Keratinozytenzellen wurden putative SUN2 Interaktionspartner identifiziert. Dabei
konnte gezeigt werden, dass der nukleoplasmatisch lokalisierte N-Terminus nicht nur
direkt mit LaminA/C sondern auch mit einer Vielzahl von Komponenten des Chromatin-
Remodelierungskomplexes direkt oder indirekt interagiert. Dies lässt vermuten, dass
SUN2 durch die Rekrutierung von Ko-Repressoren an die Kernperipherie an der
Aufrechterhaltung von Heterochromatin beteiligt ist. Auch konnte durch die Analyse des
putativen Proteoms eine bisher nicht beschriebene Interaktion von SUN2 mit dem RNA-
prozessierendem Proteinkomplex gezeigt werden.
Bei der Charakterisierung von Duchenne Muskeldystrophie (DMD), Emery-Dreifuss
Muskeldystrophie /Charcot-Marie-Tooth Syndrom (EDMD/CMT) und Stiff skin Syndrom
(SSS) Patientenzellen konnten verschiedene Zellkerndeformationen und eine
wesentlich verminderte Zellsubstratanheftung und Zellausbreitung festgestellt werden.
Des Weiteren war das Expressionsprofil für SUN2 verändert und der Zentrosomen-
Nukleus Abstand vergrössert. Ein putatives SUN2-Proteom aus Lysaten von EMD/CMT
Patientenzellen gibt Hinweise auf eine mögliche Interaktionsstörung des LINC-Komplex
Proteins mit dem nukleären Aktin-Komplex.
Zusammenfassend unterstützen die in dieser Arbeit gewonnen Ergebnisse in der
Literatur beschriebene Pathomechanismen, die sowohl eine erhöhte
Zusammenfassung
91
Mechanosensitivität der Zellen als auch eine veränderte Transkriptionsregulation
fordern, und stellen die Bedeutung von Proteinen des LINC-Komplex und damit
assoziierter Proteinkomplexe heraus. Detaillierte Bindungsstudien werden einen
weiteren Aufschluss über die Pathomechanismen geben, die zu den phänotypisch
vielfältigen und gewebespezifischen Ausprägungen der Laminopathien führen.
References
92
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Preliminary Puplications
103
Preliminary Puplications:
• Poster presentation, Annual Conference of German Society of Cell Biology
(Deutsche Gesellschaft für Zellbiologie, DGZ) 2011: Interactions and subcellular
distribution of human SUN2
Puplications (submitted soon):
• Vaylann, E., 1, 2 Noegel, A.A. 1, 2 (2011): Subcellular distribution and interactions
of human SUN2.
1Institute for Biochemistry I, Medical Faculty, University of Cologne, Cologne,
Germany 2Center for Molecular Medicine Cologne (CMMC) and Cologne
Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases
(CECAD), Medical Faculty, University of Cologne, Cologne, Germany
• Taranum, S.,1,2#, Vaylann, E., 1,2# Abraham, S., 1,2 Karakesisoglou, I., 3, Wehnert,
M., 5, Noegel, A.A., 1,2 (2011): Characterization of primary fibroblast of Emery-
Dreifuss muscular dystrophy (DMD) and Emery-Dreifuss muscular dystrophy/
Charcot-Marie-Tooth syndrome (EDMD/CMT) patients.
1Institute for Biochemistry I, Medical Faculty, University of Cologne, Cologne,
Germany, 2Center for Molecular Medicine Cologne (CMMC) and Cologne
Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases
(CECAD), Medical Faculty, University of Cologne, Cologne, Germany,
3Department of Biological Sciences, The School of Biological and Biomedical
Sciences, The University of Durham, Durham, UK, 5Ernst-Moritz-Arndt-University,
Institute of Human Genetics, Greifswald, Germany
# both authors contributed equally to this work
104
Curriculum vitae
Personal details
Name: Eva Mawina Vaylann, nee Stürz
Date of Birth: 25.07.1978
Nationality: german
Doctoral studies, Professional experience, University studies
April 2009 – March 2011 February 2009 - April 2009 April 2008 –February 2009 September 2007 - March 2008 April 2007 - September 2007 November 2003 - April 2007 2003 - 2007
Doctoral studies, Institute for Biochemistry I, Medical Faculty, University of Cologne, Germany, Supervisor: Prof. Dr. Angelika A. Noegel, Title: Interactions and subcellular distribution of human SUN2 Research assistant, Kagando Hospital and Rural Development Center, Uganda, Africa Research assistant, Internal Medicine I, University Hospital/ Institute for Biochemistry I, Medical Faculty, University of Cologne temporary employment abroad in Africa (South Africa, Zimbabwe, Zambia and Mozambique), founding and supervision of a children’s care project „Children-in-need-africa“ in South Africa and Mozambique, Africa Research assistant, Institute for Biochemistry, Faculty of Mathematics and Natural Science, University of Cologne, Germany (Group Prof. Dr. Krämer, Dr. Marin) Student research assistant, Institute for Zoology (Prof. Dr. Plickert); Institute for Biochemistry (Group Prof. Dr. Krämer, PD. Dr. Niefind) Faculty of Mathematics and Natural Science, University of Cologne, Germany Diploma studies, major subjects: Biochemistry, Genetics, Pharmacology, Diploma thesis in Biochemistry, Institute for Biochemistry, Faculty of Mathematics and Natural Science, University of Cologne, Germany, Supervisor: Prof. Dr. Schomburg, Title: „Metabolom analysis during the diauxic shift in Saccharomyces cerevisiae“
105
Lebenslauf
Persönliche Daten:
Name, Vorname: Eva Mawina Vaylann, geb. Stürz
Geburtsdatum: 25.07.1978
Staatsangehörigkeit: deutsch
Promotion, Berufspraxis, Hochschulstudium April 2009 – März 2011 Februar 2009 – April 2009 April 2008 –Februar 2009 September 2007 - März 2008 April 2007 - Sebtember 2007 November 2003 - April 2007 2003 - 2007
Promotion am Institut für Biochemie I der Medizinischen Fakultät der Universität zu Köln, Universität zu Köln, Betreuerin: Prof. Dr. Angelika A. Noegel, Thema: Interaktionen und subzelluläre Verteilung von humanem SUN2 Wissenschaftliche Mitarbeiterin, Kagando Hospital und Entwicklungszenter, Kagando, Uganda, Afrika Tätigkeit als Wissenschaftliche Mitarbeiterin, Inneren Medizin I, Universitätsklinik/ Institut für Biochemie I, Medizinische Fakultät der Universität zu Köln Auslandsaufenthalt in Afrika ( Südafrika, Zimbabwe, Sambia und Mosambik), Gründung und Betreuung eines „Children-in- need-africa“ Hilfsprojektes in Südafrika und Mosambik, Afrika Tätigkeit als Wissenschaftliche Hilfskraft am Institut für Biochemie der Universität zu Köln, Mathematisch-Naturwissenschaftliche Fakultät (AG Prof. Dr. Krämer, Dr. Marin) Tätigkeit als Studentische Hilfskraft am Institut für Zoologie der Universität zu Köln, Molekulare Grundlagen von Entwicklungs- prozessen (Prof. Dr. Plickert); Institut für Biochemie der Universität zu Köln, Mathematisch-Naturwissenschaftliche Fakultät (AG Prof. Dr. Krämer, PD. Dr. Niefind) Biologiestudium an der Universität zu Köln, Mathematisch-Naturwissenschaftliche Fakultät, Hauptstudium: Biochemie, Genetik, Pharmakologie, Diplomarbeit am Institut für Biochemie, AG Prof. Dr. Schomburg: „Meta-bolomanalyse des diauxischen Wechsels bei Glucoselimitation in S. cerevisiae“
106
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