ResearchArticle Nesprin-2 Interacts with Condensin...

Research ArticleNesprin-2 Interacts with Condensin Component SMC2

Xin Xing12 CarmenMroszlig12 Linlin Hao12 Martina Munck12

Alexandra Herzog12 Clara Mohr12 C P Unnikannan12 Pranav Kelkar12

Angelika A Noegel12 Ludwig Eichinger12 and Sascha Neumann12

1 Institute of Biochemistry I Medical Faculty University Hospital Cologne Joseph-Stelzmann-Str 52 50931 Cologne Germany2Center for Molecular Medicine Cologne (CMMC) and Cologne Cluster on Cellular Stress Responses inAging-Associated Diseases (CECAD) Medical Faculty University of Cologne Cologne Germany

Correspondence should be addressed to Angelika A Noegel noegeluni-koelnde andLudwig Eichinger ludwigeichingeruni-koelnde

Received 27 July 2017 Revised 17 November 2017 Accepted 7 December 2017 Published 27 December 2017

Academic Editor Arnoud Sonnenberg

Copyright copy 2017 Xin Xing et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The nuclear envelope proteins Nesprins have been primarily studied during interphase where they function inmaintaining nuclearshape size and positioning We analyze here the function of Nesprin-2 in chromatin interactions in interphase and dividing cellsWe characterize a region in the rod domain of Nesprin-2 that is predicted as SMC domain (aa 1436ndash1766) We show that thisdomain can interact with itself It furthermore has the capacity to bind to SMC2 and SMC4 the core subunits of condensin Theinteraction was observed during all phases of the cell cycle it was particularly strong during S phase and persisted also duringmitosisNesprin-2 knockdowndid not affect condensin distribution howeverwenoticed significantly higher numbers of chromatinbridges in Nesprin-2 knockdown cells in anaphase Thus Nesprin-2 may have an impact on chromosomes which might be due toits interaction with condensins or to indirect mechanisms provided by its interactions at the nuclear envelope

1 Introduction

The nucleus of a eukaryotic cell harbors the genetic materialthat is organized in long DNA polymers and is associatedwith numerous proteins to form chromatin Chromatin isseparated from the cytoplasm by the nuclear envelope (NE) acontinuous membrane system consisting of an inner (INM)and an outer nuclear membrane (ONM) enclosing the per-inuclear space (PNS) Both membranes are connected at thenuclear pore complexes the ONM continues into the endo-plasmic reticulum (ER) The NE is not a simple membranebarrier but is lined with and crossed by large protein assem-blies that provide it with various cellular functions Nesprins(nuclear envelope spectrin repeat proteins) together withSUN proteins are central components of the NE Currentlyfour Nesprins (Nesprins-1ndash4) are known in mammals Theyreside at the INM and ONM have different sizes and existin many isoforms [1] Nesprins are characterized by a varyingnumber of spectrin repeats followed by a C-terminal KASH(Klarsicht ANC-1 Syne Homology) domain which anchors

the proteins in the nuclear membrane and interacts with theSUN domain of SUN proteins in the perinuclear space [2]Nesprin-1 and Nesprin-2 harbor at their N-terminus pairedcalponin homology domains that mediate the binding to F-actin [3 4] The N-terminus of Nesprin-3 binds to plectin acytoskeletal crosslinker that establishes the connection to theintermediate filament system [5] Nesprin-4 interacts withkinesin-1 a motor protein that uses microtubules as cellularroutes [6]Microtubule interaction through kinesin-1 has alsobeen described for Nesprin-2 [7]

Based on the nucleo-cytoskeletal interactions Nesprinsintegrate the nucleus into the cytoskeleton of a cell and partic-ipate in the maintenance of nuclear shape and stability [8 9]The spectrin repeats (SRs) are platforms for protein-proteinor self-interactions [10] Furthermore the number of SRs andtherefore the length of the rod have been proposed to modu-late the size of the nucleus [11] In the central SR domain anadditional domain has been described in Nesprin-2 an SMC(structural maintenance of chromosomes) domain encom-passing amino acid residues 1464ndash1771 whichwas identified

HindawiInternational Journal of Cell BiologyVolume 2017 Article ID 8607532 15 pageshttpsdoiorg10115520178607532

2 International Journal of Cell Biology

by Dawe et al [12] as an interaction site for meckelin aprotein with functions in the formation of primary ciliaPrimary cilia are sensory organs that act as mechanorecep-tors in various signaling pathways or sensors of chemicalstimuli [13]

SMC proteins have core functions in regulating genomestability and the organization of the geneticmaterialThey arepresent from bacteria to man [14] Classical SMC proteins arecomposed of 1000ndash1300 amino acids They have two coiled-coil regions interrupted by a central hinge The coiled coilsfold back on themselves and form an extended structure Attheir ends the N- and C-termini of a molecule interact witheach other to form a globular ATP-binding domain [15] Thehinge regions are responsible for heterodimerization of SMCmolecules [16] Six SMCproteins have been described inmanSMC1ndash6 SMC13 form the core of the cohesin complexwhichmediates sister chromatid cohesion SMC24 are present inthe condensin complex that acts in chromosome assemblyand segregation They are present in two condensin com-plexes with distinct roles condensins I and II which containSMC2 and SMC4 in combination with different non-SMCsubunits Condensins I and II are associated sequentially withchromosomes during the cell cycle and have different rolesfor chromosome architecture Condensin I is not presentin the nucleus in interphase During mitosis condensin Iis required for removal of cohesin from chromosome armsand for chromosome shortening whereas condensin II playsa role in chromosome condensation during early prophase[17] Condensin I is however not completely excluded ininterphase from the nucleus since a small pool was foundin association with intergenic and intronic regions duringinterphase [18 19] By contrast condensin II is alwaysnuclear It is associated with DNA throughout interphaseand concentrates on chromosomes in prophase Based onits interphase distribution a role in nuclear architecture wasproposed [20 21] Cohesin and condensin complexes havealso roles in DNA repair and gene regulation throughout thecell cycle [20] Moreover condensin is involved in organizingthe chromatin allowing intrachromosomal associations ofgene loci as shown in fission yeast [22] SMC56 is mainlyimplicated in DNA damage repair and DNA recombinationand has specific roles in meiosis [23 24]

We have carried out a biochemical and functional charac-terization of the Nesprin-2-SMC domain hereafter referredto as Nesprin-2-SMC We show that it can self-assemble toform dimers trimers and higher order structures and caninteract with condensin proteins SMC2 and SMC4 Mono-clonal antibodies directed against the SMC domain showed adistribution of the Nesprin-2 isoforms containing the SMCdomain along the NE during interphase and a presence atthe chromosomes during mitosis We also uncovered animpact of Nesprin-2 on mitotic chromosomes that might bemediated by an interaction with the condensin core unitsSMC24

2 Materials and Methods

21 Cell Culture Transfection and Cell SynchronizationHaCaT (human keratinocyte cell line) COS7 (African green

monkey kidney fibroblasts) and HeLa (human cervical can-cer cells) cells were grown in a humidified atmosphere con-taining 5 CO

2at 37∘C in DMEM (high glucose Life Tech-

nologies) supplemented with 10 fetal bovine serum (FBS)2mM Glutamine (SIGMA) and 1 penicillinstreptomycinCells were transfected as described [11] To knock downNesprin-2 HaCaT cells were transfected twice at intervals of72 h using the Amaxa Nucleofector Kit V Solution (Lonza)The plasmids used for knockdown of Nesprin-2 targeting theN-terminus and the C-terminus (Nesprin-2 N-term shRNANe-2 N-term KD Nesprin-2 C-term shRNA Ne-2 C-termKD) as well as the control have been described previously[7] The newly generated plasmids are described belowFor cell cycle synchronization HaCaT cells were treatedwith thymidine (2mM) for 24 h and then with Nocodazole(100 ngml) for 12 h or alternatively first with 9120583M RO-3306(Santa Cruz Biotechnology sc-358700) for 20 to 22 h andthen approximately 3 h release (depending on the desiredmitotic phase) to obtain mitotic phases RO-3306 is a CDK1inhibitor and reversibly arrests proliferating cells at the G2Mphase of the cell cycle [26] FACS analysis of cell cycle stageswas performed with unsynchronized and synchronized cellsStaining was done with Nuclear-ID Red DNA (Enzo ENZ-52406)

Determination of cell proliferation was done by plating atone time point six wells each with the same number of cellsand then counting twowells after 24 h two after 48 h and twoafter 72 h

22 Cloning Strategies cDNAs from HaCaT cells encodingthe SMC domain in Nesprin-2 (AAN60443 aa 1436ndash1766and SRs 11ndash13) were used as PCR templates using theprimers for the following 51015840 GAATTCAATGAACTC-CTTAAAAATATTCAAGATGTG 31015840 rev 51015840 GAATTC-CTCGAGGGATTCAGTCATCCCGATCTGGGTCTTGG31015840 that contain EcoRI restriction sites for cloning intopGEX-4T1 (Amersham) yielding pGEX-4T1-Nesprin-2-SMCwhich encodes GST-Nesprin-2-SMC GST is located at theamino terminus of the protein Nesprin-2-SMC sequenceswere generated by PCR and cloned into pCMV-Myc (GEHealthcare) using pGEX-4T1 Nesprin-2-SMC as templateand primers with EcoRI or XhoI restriction sites SMC(SR11ndash13) for the following 51015840GAATTCTGAATGAACTCC-TTAAAAATATTCAAGATGTG 31015840 rev 51015840 CTCGAGCTA-GAGGGATTCAGTCATCCCGATCTGGGTCTT 31015840 SR11for 51015840 GAATTCTGAATGAACTCCTTAAAAATATTC-AAGATGTG 31015840 rev 51015840 CTCGAGCTATCTCCCACATTG-TTCAAGACATTCGGTGAC 31015840 SR12 for 51015840 CTCGAG-GTTTTGGAGCTCTTAAAACAATATCAGAAT 31015840 rev51015840 CTCGAGCTAACCAAGATTTTCATAGTAATCTTC-TGTCTT 31015840 SR13 for 51015840 GAATTCTGCGAGCTCTAGCTT-TGTGGGACAAACTTTTTA 31015840 rev 51015840 CTCGAGCTA-GAGGGATTCAGTCATCCCGATCTGGGTCTT 31015840 Myc-SR53ndash56 corresponding to residues 6146ndash6799 of Nesprin-2is described in Schneider et al [7]

A Nesprin-2 SMC domain specific shRNA (Ne-2SMC) was generated as described using the following

International Journal of Cell Biology 3

oligonucleotides sense 51015840-ATTCTCCTGTTAAGC-ACTTCTGTACATGGAAGCTTGCATGTATAGGAG-TGCTTAGCAGGAGAATCCATTTTTT-31015840 antisense51015840-GATCAAAAAATGGATTCTCCTGCTAAGCACTCC-TATACATGCAAGCTTCCATGTACAGAAGTGCTT-AACAGGAGAATCG-31015840 and a random control using sense51015840-CCTTTCAGATACGTCTTGTACAGGTATTGAAGC-TTGAATGCCTGTACAGGATGTATCTGAAAGGCG-ATTTTTT-31015840 and antisense 51015840 GATCAAAAAATCGCC-TTTCAGATACATCCTGTACAGGCATTCAAGCTT-CAATACCTGTACAAGACGTATCTGAAAGGCG-31015840oligonucleotides [27] The efficiency of the knockdown wasevaluated by immunofluorescence and western blot analysisKnockdown of SMC2 in COS7 cells was achieved withSMC2-specific siRNAs (E-006836-00-0005 Dharmacon GEHealthcare) For control corresponding scrambled shRNAwas used The cell line was recommended by the supplier incombination with the particular siRNAs Transfection wascarried out using Dharmafect transfection reagent accordingto the manufacturerrsquos protocol The cells were analyzed96 h after the transfection Successful knockdown wasassessed by immunofluorescence analysis using SMC2specific antibodies

23 Expression and Purification of GST Proteins and GSTPulldown Plasmids encoding GST fusion proteins weretransformed into E coli XL-1 blue and grown overnight anddiluted 1 50 into fresh LB mediaThe bacteria were grown toan OD

600of 06 to 08 when they were induced with 05mM

IPTG and the protein expression was continued overnightat 20∘C Bacteria were pelleted and washed with STE buffer(10mM Tris-HCl pH 80 50mM NaCl and 1mM EDTA)Lysis was achieved by the addition of 100 120583gml lysozyme andmechanical shearing in a Dounce homogenizer followed bycentrifugation Fusion proteins were bound to Glutathione-Sepharose 4B (GE Healthcare) The GST-Nesprin-2-SMCpolypeptide has a predicted molecular weight of 648 kDaIt was efficiently expressed in E coli XL-1 blue and purifiedas soluble proteins The protein was bound to Glutathione-Sepharose beads and Nesprin-2-SMC was released from theGST part by thrombin cleavage (Sigma-Aldrich) Alterna-tively GST-Nesprin-2-SMC was eluted from the beads withreduced glutathione (20mM) in 100mM Tris-HCl pH 80

GST pulldown assays were performed by lysingHaCaT orCOS7 cells in lysis buffer (50mM Tris-HCl pH 75 150mMNaCl 1Nonidet P-40 and 05 sodium deoxycholate) sup-plemented with protease inhibitor cocktail (Sigma-Aldrich)by pushing them through a 04mm needle followed bysonication and centrifugation Cell lysates were incubatedwith Glutathione-Sepharose beads overnight for binding tothe GST fusion proteins or GST and washed 5 times with PBSor lysis buffer supplemented with protease inhibitors Beadsbound protein complexes were analyzed by SDS-PAGE andwestern blot (WB)

24 Antibodies and Immunofluorescence (IF)Microscopy Thefollowing antibodies were used mouse monoclonal anti-Nesprin-2 mAb K20-478 raised against the actin bindingdomain (ABD) of Nesprin-2 (residues 1ndash285) [3] (IF 1 200

hybridoma supernatant WB 1 10) rabbit polyclonal anti-bodies pAbK1 raised against spectrin repeats in the C-terminal region of Nesprin-2 [28] (IF 1 100 WB 1 1000)Nesprin-1 specific mAb K43-322-2 raised against N-terminalspectrin repeats 10 and 11 of Nesprin-1 [29] (hybridomasupernatant undiluted) GFP-specific mAb K3-184-2 [30](hybridoma supernatant IF 1 2 WB 1 10) Myc-specificmAb 9E10 [31] (hybridoma supernatant IF undiluted WB1 10) pAb against GST [32] (WB 1 50000) mAb K84-913against GST (hybridoma supernatant WB 1 10) pAb LaminB1 (Abcam ab16048 IF 1 200 WB 1 4000) pAb SMC2(Novus Biologicals NB100-373 IF 1 100 WB 1 2000) WBmAb SMC4 (Abcam ab179803 1 2000) IF pAb SMC4(Abcam ab17958 1 500) pAb SMC1 (Abcam ab21583 WB1 1000) goat SMC3 (Santa Cruz Biotechnology sc-8135WB 1 50) rabbit CAP-H (Biomol-Bethyl A300-603A-TWB 1 1000) pAb CAP-H2 (Biomol-Bethyl A302-275A WB1 4000) mAb PDI (Abcam ab2792 1 100) pAb calreticulin(Thermo Fisher PA3-900 IF 1 50ndash200) and rat mAb YL12specific for 120572-tubulin (1 5) mAb K81-116-6 (hybridomasupernatant undiluted) directed against the SMC domainin Nesprin-2 was generated in this study The antibodieswere used for immunofluorescence and western blot analysisA polypeptide corresponding to Nesprin-2 aa 1436ndash1766(calculated molecular weight 3878 kDa) was produced asGST fusion polypeptide and bound toGlutathione-Sepharosebeads as described aboveThe SMCpolypeptide was liberatedby thrombin cleavage and used for production ofmonoclonalantibodies by immunization of mice as described [33] Alexa568 or 488 fluorescently labeled and highly cross absorbedand affinity purified secondary antibodieswere used (ThermoFisher) and 46-diamino-2-phenylindole (DAPI Sigma) wasused to visualize DNA For immunofluorescence cells grownon cover slips were fixed in 3 paraformaldehyde (PFA)in phosphate-buffered saline (PBS) for 15min followed by4min incubation with 05 Triton X-100PBS Alternativelycells were fixed by 10min incubation in ice cold methanolat minus20∘C Blocking was done with PBG (05 BSA 0045fish gelatine in PBS pH 74) at room temperature (RT) for30min Primary and secondary antibodies as well as DAPIwere diluted in PBG and applied to the cells for 1 h at RTor overnight at 4∘C Microscopy was performed by usingTCS-SP5 (Leica) or the Angstrom Opti Grid confocal micro-scope (Leica) For control cells were routinely labeled withsecondary antibodies only In no case was a signal obtained

To test the specificity of the newly established mAb K81-116-6 the antibodies were removed from the hybridomasupernatant (depletion) and the supernatant was then usedfor immunofluorescence analysis Depletion was performedin two ways For one the hybridoma supernatant wasincubated with Glutathione-Sepharose beads carrying GST-Nesprin-2-SMC polypeptides The beads were removed bycentrifugation (2000 rpm 2min) and the supernatant wasused for immunofluorescence analysis Alternatively GST-Nesprin-2-SMC was loaded onto a SDS-polyacrylamide gelthe protein was then transferred to a nitrocellulose mem-brane detected by Ponceau S staining and the part of themembrane carrying GST-Nesprin-2-SMC protein was cutout and incubated with mAb K81-116-6 After overnight


incubation (4∘C) the solution was removed from the mem-brane and applied for IF For both approaches an aliquot ofthe antibody solution before depletion was kept for control

25 Immunoprecipitation For immunoprecipitation (IP)HaCaT cells were harvested and lysed in lysis buffer (50mMTris-HCl pH 75 150mM NaCl 1 Nonidet P-40 05sodium deoxycholate and protease inhibitor cocktail) Cellswere lysed by pushing and pulling through a 04mm needleand centrifuged (12000 rpm 20min) Supernatants wereincubated for 1 h with protein A Sepharose CL-4B beads(GE Healthcare) for preclearing Subsequently beads wereremoved by centrifugation (2000 rpm 2min) and cell lysatesincubated with 5ndash8120583g of the antibody of interest for 2 h atRT Protein A Sepharose CL-4B beads equilibrated with lysisbuffer were then added to the cell lysates and incubationwas continued overnight at 4∘C The beads were collectedby centrifugation and washed five times with PBS and thebound proteins released from the beads by addition of SDSsample buffer and heating to 95∘C for 5min and analyzed bySDS-PAGE (3ndash12 acrylamide for gradient gels 10 and 12acrylamide as appropriate) and western blotting Transferof high molecular weight Nesprin-2 giant to nitrocellulosemembranes (022120583m pore size) was done by wet blottingtechnique for two to three days

26 Gel Filtration and Chemical Cross-Linking To assessthe oligomeric state of the native protein the samplewas applied to a gel filtration column (Sephadex G-200 GE Healthcare) as described [34] For molecularweight determination molecular weight standards (GEHealthcare) were separated under identical conditionsChemical cross-linking of Nesprin-2-SMC (1mgml) wasperformed with the zero-length cross-linking reagent EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydro-chloride) (Thermo Fisher) together with sNHS (Sulfo-N-hydroxysuccinimide) in 01M MES buffer (pH 65)[35]

3 Results

31 Nesprin-2 Contains an SMC Domain in Its Rod DomainWe investigate here a region in the SR containing rod domainof Nesprin-2 with homology to the SMC (Structural Mainte-nance of Chromosomes) domain (119864 value 934119890 minus 03) Thisdomain encompasses amino acids 1436ndash1766 and extendsover SR11ndash13 designatedNesprin-2-SMC (Figure 1(a)) [36] Ina comparison withmammalian SMC proteins we found highdegrees of homology with the coiled-coil regions of SMC2and SMC4 (197 identity 529 similarity and 215 iden-tity 539 similarity resp) (Figure 1(b)) To assess whetherNesprin-2-SMC can undergo self-interactions we expressedit as GST fusion protein and analyzed the elution behaviorof the 39 kDa polypeptide which had been released fromGSTby thrombin cleavage by size exclusion chromatographyThe protein eluted in two peaks one eluting at sim50 kDa andcorresponding to the monomer and a broader and larger oneeluting between 75 kDa and 158 kDa indicative of oligomers(Figure 1(c))The proteins used for calibrating the column are

globular proteins whereasNesprin-2-SMC is expected to be arod shaped molecule presumably affecting the elution behav-ior The elution pattern was also confirmed by SDS-PAGEand staining with Coomassie Blue which showed that theprotein eluted in fractions in front of ovalbumin indicatingan oligomeric state (Figure S1(a)) Cross-linking experimentsusing varying concentrations of the zero-length cross-linkingreagent EDC showed the presence of monomers dimerstrimers and even higher molecular weight complexes Withdecreasing EDC concentration the amount of higher molec-ular weight forms decreased whereas the monomeric formincreased (Figure 1(d)) The oligomerization property ofNesprin-2-SMCwas supported by data frompulldown exper-iments in which GST-Nesprin-2-SMC precipitated Nesprin-2 giant from HaCaT cell lysates (see Materials and Methodsfor experimental details) Human Nesprin-2 giant is a 6885-amino-acid protein with a predicted molecular weight of796 kDaMass spectrometric analysis identified peptides cov-ering the entire Nesprin-2 giant molecule in the precipitate(Figure S1(b)) The high coverage of the sequence locatedbetween residues 1436 and 1766 was due to the polypeptideused for the pulldown GST did not precipitate Nesprin-2

We further expressedMyc-tagged Nesprin-2-SMC (Myc-Nesprin-2-SMC) corresponding to the full length SMCdomain of Nesprin-2 and Myc-tagged polypeptides corre-sponding to its individual SR domains in COS7 cells andused the cell lysates for pulldown experiments with GST-Nesprin-2-SMC (Figure 1(e)) GST-Nesprin-2-SMC precipi-tatedMyc-Nesprin-2-SMC and its individual SRs fromCOS7cell lysates as shown in the immunoblot using Myc-specificantibody mAb 9E10 (Figure 1(f)) Taken together the resultssuggest that the Nesprin-2-SMC domain has the potentialto oligomerize We then asked whether this interaction isspecific to this Nesprin-2 domain and tested whether GST-Nesprin-2-SMC could interact with other spectrin repeats ofNesprin-2 We therefore expressed Myc-SR53ndash56 composedof the last four spectrin repeats of Nesprin-2 (SR53ndashSR56 aa6116ndash6799 Figure 1(a)) in COS7 cells and carried out pull-down assays with GST for control and GST-Nesprin-2-SMC[7] GST-Nesprin-2-SMC did not precipitate Myc-SR53ndash56underlining the specificity of the interaction (Figure 1(g))

32 Monoclonal Nesprin-2-SMC Domain Specific Antibod-ies Detect a High Molecular Weight Protein and Stain theNuclear Envelope To study Nesprin-2 isoforms harboringthe SMC domain we generated monoclonal antibodies byimmunizing mice with Nesprin-2-SMC polypeptide that hadbeen released from the GST part by thrombin cleavage Inwestern blots of HaCaT cell homogenates that had beenseparated in gradient gels (3ndash12 acrylamide) mAb K81-116-6 recognized primarily a high molecular weight proteinwhich we presume corresponds to the sim800 kDa Nesprin-2giant [3] Faint bands below could be degradation productsor N-terminal isoforms [1] (Figure 2(a)) In independentexperiments in which we immunoprecipitated Nesprin-2from HaCaT cells and probed the precipitate with SMC2and SMC4 antibodies we excluded that any of the lowermolecular weight bands corresponded to SMC proteins dueto cross reactivity of the antibodies (data not shown) In


ABD 1

1 285

11 12 13 KASH

6885

Nesprin-2 giant53 54 55 56

mAb K20-478 mAb K81-116-6 pAbK1

1431 1766 6146 6799

(a)

1470 1510150014901480KKSLIRLDKVLDEYEEEKRHLQEMANSLPHF--KDGREKTVNQQCQNT

EKNMVEDSKTLAAKEKEVKKITDGLHALQEASNKDAEALAAAQQHFNA

340 380370360350

1520 15701560155015401530VVLWENTKALVTECLEQCGRVLELLKQYQNFKSILTTLIQKEESVISLQASYMGKENLKK

V-----SAGLSSN---EDGAEATLAGQMMACKNDISK-AQTEAKQAQMKLKHAQQE-LKN

390 430420410400

1630 16801670166016501640INEKTEDYYENLGRALALWDKLFNLKNVIDEWTEKALQKMELHQLTEEDRERLKE-ELQV

IGRLKETYEALLARFPNL---RFAYKDPEKNWNRNCVKGLVASLISVKDTSATTALELVA

490 540530520510500

1580 1620161016001590RIAEIEIVKEEFNEHLEVVDKINQVCKNLQFYLNKMKTFEEPPFEKEANIIVDRWL---D

KQAEVKKMDSGYRKDQEALEAVKRLKEKLEAEMKKLN-YEE---NKEESLLEKRRQLSRD

440 4804704604501690 17401730172017101700

HEQKTSEFSRRVAEIQFLLQSSE-------IPLELQVMESSILNKMEHVQKCLTGESNCH

GERLYNVVVDTEVTGKKLLERGELKRRYTIIPLN-KISARCIAPETLRVAQNLVGPDNVH

550 600590580570560

Ne-2

SMC2

1470 15201510150014901480KKSLIRLDKVLDEYEEEKRHLQEMANSLPHFKDGREKTVNQQCQN----TVVLWENTKAL

QKRIAEMETQKEKIHEDTKEINEKSNILSNEMKAKNKDVKDTEKKLNKITKFIEENKEKF

340 390380370360350

1530 1570156015501540VTECLEQCGRVLELLKQYQNFKSILTTLIQKEESVISLQASYMGKEN-----LKKRIAEI

TQLDLEDV-QVREKLKHATSKAKKLEKQLQKDKEKVEEFKSIPAKSNNIINETTTRNNAL

400 4404304204101580 16301620161016001590EIVKEEFNEHL-EVVDKINQVCKNLQFYLNKMKTFEEPPFEKEANIIVDRWLDINEKTED

EKEKEKEEKKLKEVMDSLKQETQGLQ-KEKESREKELMGFSKSVNEARSK-MDVAQSELD

450 500490480470460

1640 1680167016601650YYENLGRALALWDKLFNLKNVIDEWTEK------ALQKME--LHQLTEEDRERLKEELQV

IY--LSRHNTAVSQLTKAKEALIAASETLKERKAAIRDIEGKLPQTEQELKEKEKE-LQK

510 5605505405305201690 1730172017101700

HEQKTSEFSRRVAEIQFLLQSSEIPLELQVMESSILNKMEHVQK

LTQEETNFKSLVHDLFQKVEEAKSSLAMNRSRGKVLDAIIQEKK

570 600590580

Ne-2

SMC4

(b)

11 12 13

1500 2000 2500

(ml)

minus0007

minus0002

minus0003

0008

0013

OD

280

nm

158 kDa 75 kDa 43 kDa

(c)

Highersim120

sim80

sim40

Decreasing EDC concentration

(kD

a)

(d)

Myc-SR11 1436

Myc-SR12 1532

Myc-SR13 1642

Myc-

Myc-

Myc-

Myc-

11 12 13 1766

1531

1641

1766

1436

11

12

13

Myc-Nesprin-2-SMC

(e)

Figure 1 Continued


Input Pulldown

kDa

35

27

15

lowast

COS7

Myc

-SR1

1

Myc

-SR1

2

Myc

-SR1

3

Myc

-Nes

prin

-2-S

MC

COS7

Myc

-SR1

1

Myc

-SR1

2

Myc

-SR1

3

Myc

-Nes

prin

-2-S

MC

(f)

Ponceau S WB Myc

COS7

COS7

COS7

Myc

-SR5

3ndash56

COS7

Myc

-SR5

3ndash56

GST

GST

GST

-Nes

prin

-2-S

MC

MW

GST

-Ne-

2-SM

C

(g)

Figure 1 Characterization of the SMC domain of Nesprin-2 (a) Schematic of Nesprin-2 (not drawn to scale) The location of the SMCdomain (spectrin repeats 11ndash13) and the C-terminal spectrin repeats (53ndash56) is shown Epitopes of antibodies used are indicated above theschematic ABD actin binding domain ovals spectrin repeats The spectrin repeat domain starts at position 308 (b) Sequence comparisonof the Nesprin-2-SMC domain with coiled-coil regions of SMC2 and SMC4 The sequence comparison was performed using LALIGN thePairwise Sequence Alignment tool from EMBL-EBI (httpswwwebiacukToolspsalalign) Nesprin-2 (NCBI GenBank accession numberAF4350111) SMC2 (NCBI GenBank accession number O953472) and SMC4 (NCBI GenBank accession number Q8WXH03) were used identical amino acid conservative substitution (c) Analysis of Nesprin-2-SMC by gel filtration chromatography UV traces of the elutionprofile are shown Nesprin-2 SMC (calculated molecular weight 39 kDa) Molecular weight markers were ovalbumin (43 kDa) conalbumin(75 kDa) and aldolase (158 kDa) (d) Analysis of chemically crosslinked Nesprin-2-SMC Zero-length cross-linking reagent EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) was used at decreasing concentrations The proteins were separated by SDS-PAGE(10 acrylamide) and stained with Coomassie Blue (e) Schematic representation of Myc-tagged Nesprin-2-SMC polypeptides Amino acidpositions refer to humanNesprin-2 giant (accession number AF4350111) (f) Interaction of GST-Nesprin-2-SMCwith individualMyc-taggedspectrin repeats derived from Nesprin-2-SMC and expressed in COS7 cells GST-Nesprin-2-SMC was used for pulldown (right panel)Western blots were probed with mAb 9E10 specific forMyc Asterisk endogenousMyc [25] (g) Specificity of the Nesprin-2-SMC interactionMyc-SR53ndash56 expressed inCOS7 cells was used for pulldownswithGST for control andGST-Nesprin-2-SMCCOS7 andCOS7Myc-SR53ndash56represent whole cell lysatesThe Ponceau S stained blot and the corresponding blot probed with mAb 9E10 are shown MWmolecular weightmarker (from top to bottom 200 130 100 70 55 35 and 25 kDa)

immunofluorescence analysis mAb K81-116-6 labeled the NEinHaCaT andHeLa cells overlappingwith the pAbK1 staining(Figure 2(b))The previously characterized pAbK1 polyclonalantibodies had been generated against the four C-terminalspectrin repeats of Nesprin-2 and are specific for Nesprin-2 (Figure 1(a)) [28] In addition mAb K81-116-6 stainedstructures in the cytoplasm in the vicinity of the nucleuswhich are possibly membranes of the endoplasmic reticulum(ER) as we observed colocalization with calreticulin an ERprotein (Figure 2(b) lower panel) The cytoplasmic stainingwas comparatively faint in HaCaT cells whereas inHeLa cellsit wasmore pronounced pAbK1 also stained these structureshowever the staining was less intense which might be due todifferent accessibility of the epitopes (Figure 2(b)) Nesprin-2 is a tail-anchored protein and its mRNA has been foundanchored to the ER where it is translated This might explainthe observed localization [37]

To prove the specificity of mAb K81-116-6 we carriedout antibody depletion studies We found that the stainingof the NE as well as the cytoplasmic staining was com-pletely abrogated after depletion of mAb K81-116-6 from

the hybridoma supernatant by incubating the supernatantwith nitrocellulose membrane strips carrying GST-Nesprin-2-SMC or with Glutathione-Sepharose 4B beads carryingGST-Nesprin-2-SMC By contrast the NE was still labeledby pAbK1 (Figure 2(c)) Furthermore the protein was nolonger detected in cell lysates after knocking down Nesprin-2using shRNAdirected against the SMCdomain (Figure S2(a))and no signals were detected when cells were analyzed byimmunofluorescence (see below Figures 4(b) and 4(c))

33 SMC2 Is a Nesprin-2 Binding Partner To identify bindingpartners for Nesprin-2 we performed immunoprecipitationexperiments using mAb K20-478 directed against the N-terminus of Nesprin-2 and pAbK1 (Figure 1(a)) The proteinswere separated by SDS-PAGE and stained with CoomassieBlue bands were cut out and the proteins were identified bymass spectrometry For control GFP-specific antibody mAbK3-184-2 was used Among the precipitated proteins werehistones SUN1 Lamin AC and SMC2 which were foundin the immunoprecipitate of mAb K20-478 The SUN1 andLamin AC interactions have been previously described and


kDa

250

130

100

800

(a)

mAb K81-116-6 pAbK1 Merge

HaC

aTH

eLa

mAb K81-116-6 Merge

HaC

aT

Calreticulin

(b)

mAb K81-116-6DAPI pAbK1 Merge

mAb

K81

-116

-6

Ant

ibod

y so

lutio

n aft

erde

plet

ion

on G

ST-

Nes

prin

-2-S

MC

load

edm

embr

anes

Ant

ibod

y so

lutio

n aft

erde

plet

ion

on G

ST-

Nes

prin

-2-S

MC

load

edSe

phar

ose b

eads

(c)

Figure 2 Characterization of monoclonal antibodies directed against the SMC domain (a) Detection of Nesprin-2 with mAb K81-116-6in HaCaT cell lysates Proteins were separated by SDS-PAGE (3ndash12 acrylamide) (b) mAb K81-116-6 staining of HaCaT and HeLa cellspAbK1 was used as bona fide Nesprin-2 antibody DAPI stains the DNA (in Merge) Bar 10 120583m Lower panel colocalization of Nesprin-2 detected by mAb K81-116-6 with ER marker calreticulin in HaCaT cells Bar 5120583m (c) Analysis of the specificity of mAb K81-116-6Antibodies were depleted from the hybridoma supernatant by the indicated procedures Antibody depleted supernatants were then usedfor immunofluorescence analysis Bar 10120583m

are well characterized the histone and SMC2 interactions arenovel findings [2 28 38] Here we followed up the SMC2interaction Because of the SMC homology in Nesprin-2 wespeculated that this domain could interact with SMC2 andcarried out pulldown assays with Glutathione-Sepharose 4Bbeads loaded with GST-Nesprin-2-SMC using HaCaT cell

lysates as described in Materials and Methods and probedthe pulldown for the presence of SMC2 GST loaded beadsserved as control We could indeed detect SMC2 in the GST-Nesprin-2-SMC precipitate by SMC2 specific antibodiesSMC4 which forms a complex with SMC2 in condensinwas also pulled down by GST-Nesprin-2-SMC GST did not


Coo

mas

sie

WB anti-SMC2

WB anti-SMC4

kDa70

45

30

SPN

SPN

PD

PD

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(a)

800

155

160

kDa

WB anti-SMC4

WB anti-SMC2

WB mAb K20-478

IP

mAb

K20

-478

GFP

SMC2

Inpu

t

(b)

WB mAb CAP-H270kDa

WB mAb CAP-H100

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(c)

WB anti-SMC2155

GST GST-Nesprin-2-SMC

kDa

G0G1 S M Untreated

MS

G0G1

G0

G1

G0

G1

S ph

ase

S ph

ase

Mito

sis

Mito

sis

Unt

reat

ed

Unt

reat

ed

0102030405060708090

100

Perc

enta

ge o

f cel

ls

(d)DAPI mAb K81-116-6 Merge

Prop

hase

Met

apha

seTe

loph

ase

Ana

phas

eIn

terp

hase

Met

apha

se

(e)

Figure 3 Continued


DAPI mAb K20-478 pAbK1 Merge

Prop

hase

Telo

phas

eIn

terp

hase

Met

apha

seA

naph

ase

(f)

DAPI mAb K20-478 Merge

(g)

Figure 3 Interaction of Nesprin-2-SMC and Nesprin-2 with SMC2 and SMC4 (a) Precipitation of SMC2 and SMC4 with GST-Nesprin-2-SMC from HaCaT cell lysates Precipitates were resolved on SDS-polyacrylamide gels (10 acrylamide) and probed with SMC2 and SMC4specific antibodies SPN supernatant after pulldown PD pulldown The lower molecular weight band in the SMC2 pulldown is presumablya breakdown product (b) Immunoprecipitation of SMC2 from HaCaT cell lysates with Nesprin-2 specific mAbK20-478 and of Nesprin-2with SMC2 specific antibodies GFP-specific monoclonal antibodies were used for control The antibodies used for immunoprecipitation areindicated above the panels (IP) The blots were probed with the antibodies listed on the right (WB) Immunoprecipitates were resolved ongradient gels (3ndash12 acrylamide) and 10 acrylamide gels as appropriate The data are from one blot however the input was not directlyadjacent to the SMC2 IP (c) Interaction ofCAP-H2 (condensin II) andCAP-H (condensin I)withNesprin-2-SMC Pulldownswere performedwith HaCaT cell lysates and GST for control and GST-Nesprin-2-SMC as indicated Unsynchronized cells were used for the experimentsshown in (a)ndash(c) (d) Analysis of the Nesprin-2-SMC interaction with SMC2 during the cell cycle HaCaT cells were synchronized with RO-3306 or other reagents as described inMaterials andMethods in order to obtain the relevant cell cycle phases Cell cycle phases were assessedby FACS analysis the results are depicted in the accompanying diagram Pulldown was carried out with GST-Nesprin-2-SMC bound to GST-Sepharose GST was used for controlThe blot was probed with SMC2 specific antibodies (e) Localization of Nesprin-2 as detected with mAbK81-116-6 (green) duringmitosis in HaCaT cells DNAwas stained with DAPI Arrow points to filamentous staining across the chromosomes(f) Nesprin-2 distribution in HaCaT cells during mitosis as detected with mAb K20-478 (green) and pAbK1 (red) DNA was detected withDAPI Bar 10 120583m (g) Nesprin-2 presence on chromosomes Different Z-stacks (from top to bottom 0 120583m 021 120583m 042 120583m and 084 120583m)from a COS7 cell in anaphase stained with mAb K20-478 DNA was stained with DAPI Bar 5 120583m

precipitate SMC2 or SMC4 (Figure 3(a)) Further proof foran interaction came from immunoprecipitation experimentsfrom HaCaT cells with mAb K20-478 to precipitate Nesprin-2 In the Nesprin-2 pulldown we detected SMC2 and SMC4In the reverse experiment using SMC2 specific antibodiesNesprin-2 was detected in the precipitate withmAbK20-478GFP antibodies used for control did not bring down any of theproteins tested (Figure 3(b))

As condensin exists in two complexes condensin I andcondensin II [18] we used CAP-H (kleisin 120574 non-SMCcondensin I complex subunit H) and CAP-H2 (kleisin 120573non-SMC condensin II complex subunit H2) antibodiesto probe the GST-Nesprin-2-SMC pulldown and identifiedCAP-H and CAP-H2 in the precipitate (Figure 3(c)) We alsoprobedwhether other SMCproteins interactedwithNesprin-2 However the cohesin components SMC1 and SMC3 werenot seen in the precipitate after carrying out a pulldown withGST-Nesprin-2-SMC (Figure S2(b)) These results make theinteraction a specific one between condensin and Nesprin-2 Although SMC proteins are present in all phases of thecell cycle they have specific roles in specific phases [17] Tofind out whether the interaction is confined to a particularstage of the cell cycle we used lysates from HaCaT cells

that had been treated with various reagents as described inMaterials and Methods This led to the enrichment of cellsin particular cell cycle stages Pulldown assays were carriedout with GST-Nesprin-2-SMC and GST loaded Glutathione-Sepharose beads and the precipitates probed for the presenceof SMC2 SMC2 was present in the precipitates obtainedfrom lysates of untreated cells cells in G0G1 and from cellsamples enriched for S and M phase The signal was mostprominent in lysates from S phase enriched cells followed byM phase cells The GST-control did not bring down SMC2(Figure 3(d)) The cell cycle stages were controlled by FACSanalysis (Figure 3(d) bar graph)

A colocalization of SMC2 and SMC4 with Nesprin-2was difficult to visualize at the immunofluorescence levelbecause of the very strong signals for SMC2 and SMC4However some overlap indicating a colocalization could beseen particularly in telophase (see below Figures 5(a) and5(b) upper panels see telophases of control cells for overlap)

34 Nesprin-2 Localization during Mitosis For studyingNesprin-2 localization during mitosis we performedimmunofluorescence analysis using mAb K81-116-6 mAbK20-478 and pAbK1 (Figures 3(e) 3(f) and 3(g)) All


Nesprin-2

Lamin B1

kDa

800

68

ctrl

Ne-

2 C-

term

KD

Ne-

2 N

-term

KD

ctrl

Ne-

2 SM

C KD

(a)

K20-478 pAbK1 Merge

ctrl

Ne-

2C-

term

KD

Ne-

2N

-term

KD

Ne-

2SM

C KD

(b)

DAPI K81-116-6

Ne-

2SM

C KD

(c)

Figure 4 Knockdown of Nesprin-2 using shRNA directed against C-terminal N-terminal and SMC domain sequences (a) Western blotsshowing the efficiency of the shRNA treatment at the protein level HaCaT cells were transfected with shRNAs targeting the various regionsand for control (ctrl) with the corresponding scrambled shRNAs Nesprin-2 at sim800 kDa was detected by mAb K20-478 Lamin B1 was usedfor loading control (b) Immunofluorescence analysis of HaCaT cells treated with shRNAs targeting the C-terminus (Ne-2 C-term KD) theN-terminus (Ne-2 N-term KD) or the SMC domain (Ne-2 SMC KD) Cells were stained with antibodies directed against the N-terminus(mAb K20-478 green) and the C-terminus (pAbK1 red) of Nesprin-2 DAPI was used to visualize DNA Arrowhead indicates cells withsuccessful knockdown asterisk indicates cells which still express Nesprin-2 Bar 10 120583m (c) Immunolabelling of Ne-2 SMC KD cells withmAb K81-116-6 Nuclei were labeled with DAPI Asterisk indicates a cell which still expresses Nesprin-2 Bar 10 120583M

antibodies showed that Nesprin-2 relocated to the cytoplasmupon nuclear envelope breakdown where it colocalized withthe ER as revealed by costaining with an antibody specificfor the ER marker PDI (protein disulfide isomerase) (FigureS3) It also still surrounded the condensed chromosomesand Nesprin-2 positive structures extended across thechromosomes in all mitotic phases (Figures 3(e) 3(f)3(g) and S4) Serial sections through the chromosomesof a mitotic cell confirmed the distribution of Nesprin-2 (Figure 3(g)) At the beginning of anaphase untiltelophase we found signals at opposing ends of the dividingchromosome material presumably showing the reformationof the NE (Figure 3(f)) This localization was specific forNesprin-2 as staining for Nesprin-1 with mAb K43-322-2 didnot reveal an association with the chromosomes (Figure S5)

35 Nesprin-2 Knockdown Does Not Affect Condensin Dis-tribution To specifically explore the role of SMC domaincontaining Nesprin-2 isoforms HaCaT cells were treated

withNesprin-2-SMCshRNAs (Ne-2 SMCKD) and comparedto cells treated with shRNAs targeting the Nesprin-2 N-terminus or the Nesprin-2 C-terminus (Ne-2 N-term KDNe-2 C-term KD) [7] The sequences for the generation ofthe SMC-specific shRNAs were carefully chosen in order toexclude off-target effects due to homology to SMC sequencesIn western blots labeling withmAbK20-478 revealed a strongreduction of Nesprin-2 giant at sim800 kDa in lysates fromcells treated with Ne-2 C-term and Ne-2 SMC shRNAs (Fig-ure 4(a)) Similar results were obtained with mAb K81-116-6(see above and Figure S2(a))The knockdown was confirmedat the immunofluorescence level with mAb K20-478 pAbK1andmAbK81-116-6 (Figures 4(b) and 4(c)) Cell proliferationwas not altered in the knockdown cells as compared toHaCaT control cells (two independent experiments FigureS6(a)) Similarly FACS analysis did not reveal changes inthe progression through the cell cycle (three independentexperiments Figure S6(b)) Nesprin-2 depletion using Ne-2 SMC shRNA did not have an obvious effect on SMC24


Prop

hase

Met

apha

seA

naph

ase

Telo

phas

eTe

loph

ase

Ana

phas

eM

etap

hase

Prop

hase

ctrl

Ne-

2 SM

C KD

DAPI mAB K20-478 SMC2 Merge

(a)

Telo

phas

eTe

loph

ase

Ana

phas

eA

naph

ase

Met

apha

seM

etap

hase

Prop

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(b)

DAPI mAb K20-478 SMC2

ctrl

SMC2

KD

(c)ctrl SMC2 KD

0102030405060708090

100

Fluo

resc

ence

inte

nsity

(au

)

lowastlowastlowast

(d)

Figure 5 SMC2 (a) and SMC4 (b) in HaCaT keratinocytes treated with control shRNA (upper panels) and treated with Nesprin-2-SMCdomain specific shRNA (lower panels) Nesprin-2 was detected with mAb K20-478 Bar 10 120583m (c) Localization of Nesprin-2 after siRNAmediated knockdown of SMC2 in COS7 cells Staining was with SMC2 specific antibodies and mAb K20-478 for Nesprin-2 Bar 5 120583m (d)Evaluation of the SMC2 knockdown SMC2 fluorescence intensity wasmeasured in the center of mitotic chromosomes 10 siRNA treated cellsand 12 control cells (control treatment) were analyzed (lowastlowastlowast119875 value = 00001)


location as the staining in immunofluorescence analysiswas comparable to control cells Also SMC24 distributionduring mitosis was not affected and the proteins had anapparently unaltered association with mitotic chromosomesat the level of analysis (Figures 5(a) and 5(b)) Furthermorethe protein levels appeared unaltered (Figure S2(c))

We also performed the converse experiment by downreg-ulating SMC2 in COS7 cells by transfection with a siRNApool targeting SMC2 Since the knockdown was not com-plete we searched for mitotic cells with reduced SMC2 stain-ing and analyzed the Nesprin-2 distribution We found thatNesprin-2 still surrounded the chromosomal mass indicatingthat Nesprin-2 localization is not strictly dependent on SMC2(Figures 5(c) and 5(d))

However the analyses of the Nesprin-2 depleted cellsrevealed the presence of chromatin bridges during ana- andtelophase When we determined the chromatin bridges incells transfected with SMC control and Ne-2 SMC shRNAat ana- and telophase we observed that 44 (mean value)of control cells harbored chromatin bridges In the Nesprin-2 knockdown cells this number was increased to 103 (119875value 001 440 and 544 ana- and telophases evaluated resp)This is a Nesprin-2 specific result as the Ne-2 N-termKD alsoled to enhanced chromatin bridge formation (1525 445ana- and telophases evaluated) Increased number of chro-matin bridges in anaphase has been described for condensinII knockout cells as well as condensins I and II depleted cells[39 40]

4 Discussion

Research on the Nesprins primarily focuses on the interphasenucleus and their role in nuclear positioning maintainingmechanical and structural properties of the nucleus and theperinuclear cytoskeleton and their role in signal transduction[1 41 42] We found that during mitosis Nesprin-2 waspresent along mitotic condensed DNA In previous studieswe reported that Nesprin-2 interacts with chromatin inparticular centromeric and other heterochromatic reads wereenriched in the ChIP-seq data [9] However the nature ofthis interaction is unclear and it might well be an indirectone since Nesprin-2 interacts with proteins present in thechromatin such as histones or SMCproteinsWe focused herespecifically on the interaction with SMC proteins In openmitosis the NE breakdown (NEBD) starts during prophaseresulting in a removal of the NE from chromatin We foundthat Nesprin-2 was still associated withmitotic chromosomesandNesprin-2 knockdown cells harbored increased numbersof chromatin bridges in anaphase cells

In vertebrates condensins I and II are both composedof the SMC24 heterodimer together with distinct additionalnon-SMC subunits CAP-GG2 CAP-D2D3 and CAP-HH2 [18] A depletion of condensin I or II or a combi-nation of both in HeLa cells led to delayed chromosomecondensation and caused segregation problems resulting incells with bridged or lagging chromosomes [17 41] In mouseembryonic stem cells RNA interference studies revealed thatcondensins I and II are required for ES cell proliferationand that their loss leads to delayed initiation of anaphase

and formation of enlarged and misshapen interphase nuclei[43] Altered nuclear architecture and size after condensin IIknockdown were also described more recently [44]

Since we propose a role for Nesprin-2 on chromosomesand also on mitotic chromosomes we searched publicationsreporting chromatin proteomes for the presence of Nesprin-2 Nesprin-2 was present in interphase chromatin [45]where it was listed in the category ldquonon-expected chromatinfunctionrdquo and Nesprin-2 peptides were also identified in areport on nascent chromatin capture proteomics [46] Bycontrast in a publication describing the mitotic proteomeonly Nesprin-1 was listed [47] Taken together data fromindependent proteomic approaches support our findings onthe presence of Nesprin-2 on chromatin

Based on the well-known structure and assembly ofSMC monomers into pentameric ring complexes it appearsunlikely that the predicted SMC domain in Nesprin-2 fulfillsthe role of a classical SMC protein SMC proteins formheterodimers and each dimer consists of a single polypep-tide that follows a V-shaped topology SMC monomers areconnected along the hinge region and the terminal endsform catalytically active ATPases [16] Currently no Nesprin-2 isoform has been described that might exist as a separateisoform composed of the SMC domain only [48] It mightrather be that the SMC domain in Nesprin-2 interacts withSMC24 along their coiled coils Alternatively the interac-tion between condensin and Nesprin-2 is an indirect oneInterestingly Nesprin-2 knockdown does not have an effecton mitotic progression but preliminary data indicate that thechromosomes inmetaphase cells have a fuzzy appearance anda larger volume [49 50] Similar observationsweremade afterSMC knockdown and this observation could place Nesprin-2 in this pathway [51] In this context Nesprin-2 mightadopt a role similar to the one previously suggested for NEproteins in transcriptional regulation where they are thoughtto regulate the spatiotemporal accessibility of transcriptionalregulators to their nuclear targets instead of directly actingas transcriptional regulators in the proximity of genes [5253] Nesprin-2 might act on SMC24 in a similar way Ourdata indicate that a loss of Nesprin did not prevent SMC24proteins to assemble along mitotic chromosomes but anincreased number of chromatin bridges were observed whichhints at changes in the process of chromosome separationIt could therefore well be that Nesprin-2 affects directly orindirectly the spatiotemporal assembly or the function ofSMC proteins along chromosomes

In our analysis we observed that the condensin Nesprin-2 interaction occurred throughout the cell cycle Interest-ingly condensins have roles not only during mitosis butalso in interphase where they are important particularly ingene regulation For instance a function in transcriptionalregulation has been reported for condensins I and II byLi et al [19] who found them on enhancers that hadthe estrogen receptor 120572 bound This led to full enhanceractivation and efficient transcription of the respective genes[19] Furthermore Zhang et al [54] reported that condensin Idownregulation in chickenDT40 cells caused amisregulationof gene expression underlining its role in transcriptionalregulation during interphase Related findings were reported


earlier for C elegans where condensins were found at tRNAgenes promoters and enhancers in interphase and con-densin II binding was associated with a repressive effect ontranscription [55] By contrast in mouse embryonic stemcells condensin II and cohesinwere present at transcriptionalelements of active genes during interphase and affected geneactivity in a positive way [56]

In summary we report a novel interaction partner ofNesprin-2 giant and show that the Nesprin-2 condensininteraction has an impact onmitotic chromosomesThe tightpackaging of chromosomes during mitosis to which theNesprin-2 interactionmight contribute ensures their faithfulsegregation and allows them to withstand forces duringsegregation Malfunctions in this process can cause DNAbridges which result in chromosome segregation errors andlead tomicronucleus formation and canmake chromosomesmore prone to DNA damage It could well be that Nesprinsand further NE proteins contribute to this chromosomephenotype Therefore mutations in these proteins have thepotential to contribute to the formation of distinct clinicalmanifestations associated with condensin linked diseases[57] Furthermore since theNesprin-2 condensin interactionalso takes place during other phases of the cell cycle andsince condensins have additional functions in interphasethe Nesprin-2 condensin complex could also affect theseprocesses

Disclosure

The present address of Linlin Hao is Animal BiotechnologyDepartment Jilin University Changchun 130062 China Partof this work was carried out as PhD thesis (Xin Xing andCarmen Mroszlig)

Conflicts of Interest

Theauthors declare that they have no conflicts of interest withthe contents of this article

Authorsrsquo Contributions

Xin Xing and CarmenMroszlig designed and carried out exper-iments analyzed the data prepared the figures and wrote themanuscript Linlin Hao Martina Munck Alexandra HerzogClaraMohr C P Unnikannan and Pranav Kelkar performedadditional experiments and analyzed data Sascha NeumannLudwig Eichinger and Angelika A Noegel conceived thestudy reviewed all data and prepared the final versions of themanuscript text and figures Xin Xing and Carmen Mroszlighave equal contribution to this work

Acknowledgments

Xin Xing was supported by a fellowship from the ChinaScholarship Council (CSC) Linlin Hao was supported by afellowship from the Deutsche Akademische Austauschdienst(DAAD) andCarmenMroszlig is amember of the InternationalGraduate School in Development Health and Disease (IGS-DHD) The work was supported by the CMMC (C6) and

CECAD (TPC05) and by a grant to Sascha Neumann fromthe Marga und Walter Boll-Stiftung The authors thank DrM Schleicher for help with chemical cross-linking BertholdGaszligen for help with the generation of monoclonal Nesprin-2-SMC antibodies Maria Stumpf for help with microscopyand Rolf Muller for cloning protein analysis and invaluablehelp with figures They thank Dr Astrid Schauszlig and NikolayKladt from the CECAD imaging facility and Drs S Mullerand G Rappl for mass spectrometry and FACS cell sortinganalysis respectively at the central facilities of the CMMC

Supplementary Materials

Supplementary 1 Figure S1 (a) analysis of Nesprin-2 SMCby gel filtration chromatography followed by SDS-PAGEThe elution profile of the Nesprin-2-SMC polypeptide andovalbumin is shown (b) GST-Nesprin-2-SMC pulls downNesprin-2 giant from HaCaT whole cell lysates SeveralNesprin-2 polypeptides derived from the 6885 amino acidsprotein were identified by mass spectrometry Amino acidpositions are given at the beginning and end of the identifiedsequences and refer to human Nesprin-2 giant (NCBI acces-sion number AF4350111)Supplementary 2 Figure S2 (a) Nesprin-2 giant is no longerdetected by mAb K81-116-6 in lysates from HaCaT cellstreatedwith shRNAdirected against the SMCdomain and theN-terminus ofNesprin-2Whole cell lysates fromcells treatedwith the indicated knockdown plasmids were separated ina gradient gel (3 to 12 acrylamide) and probed with mAbK81-116-6 Ne-2 ctrl KD corresponds to a scrambled SMColigonucleotide (b) SMC1 and SMC3 do not interact withGST-Nesprin-2-SMC HaCaT cell lysates (input) were usedfor precipitation experiments employing GST GST-Nesprin-2-SMC and Glutathione-Sepharose beads respectively asindicated above the panels Proteins were separated by SDS-PAGE (10 acrylamide) and the resulting western blotswere probed with the antibodies indicated on the right (c)SMC2 and SMC4 protein levels are not affected in Nesprin-2 knockdown cells Whole cell lysates from cells treated withthe indicated knockdown plasmids were separated by SDS-PAGE (10 acrylamide) and probed for SMC2 and SMC4Lamin B1 served as controlSupplementary 3 Figure S3 colocalization of Nesprin-2 andan ER marker in mitotic cells HaCaT cells were stained withpAbK1 for Nesprin-2 and with protein disulfide isomerase(PDI) specific monoclonal antibodies as ER marker DNAwas stained with DAPISupplementary 4 Figure S4 Nesprin-2 distribution duringmitosis HaCaT cells were labeled with pAbK1 mAb YL12specific for 120572-tubulin and DAPI for DNA Bar 5 120583mSupplementary 5 Figure S5 specificity of Nesprin-2 associa-tion with chromosomes in mitosis HaCaT cells were stainedwith pAbK1 for Nesprin-2 andmAbK43-322-2 for Nesprin-1Bar 5 120583mSupplementary 6 Figure S6 (a) proliferation of Nesprin-2-SMC knockdownHaCaT cellsThemean of two independentexperiments is shown (b) Cell cycle progression is unaffected


by the loss of Nesprin-2 The experiment was carried outfor HaCaT control cells Nesprin-2-SMC knockdown (Ne-2-SMCKD) and cells treatedwith a control plasmid containingscrambled sequences The data show the mean of threeindependent experiments No significant differences werenoted M mitosis S S phase G0G1 G0 G1 phase

References

[1] D Rajgor and C M Shanahan ldquoNesprins from the nuclearenvelope and beyondrdquo Expert Reviews in Molecular Medicinevol 15 no e5 2013

[2] V C Padmakumar T Libotte W Lu et al ldquoThe inner nuclearmembrane protein Sun1mediates the anchorage ofNesprin-2 tothe nuclear enveloperdquo Journal of Cell Science vol 118 no 15 pp3419ndash3430 2005

[3] Y-Y Zhen T Libotte M Munck A A Noegel and EKorenbaum ldquoNUANCE a giant protein connecting the nucleusand actin cytoskeletonrdquo Journal of Cell Science vol 115 no 15pp 3207ndash3222 2002

[4] V C Padmakumar S Abraham S Braune et al ldquoEnaptin agiant actin-binding protein is an element of the nuclear mem-brane and the actin cytoskeletonrdquo Experimental Cell Researchvol 295 no 2 pp 330ndash339 2004

[5] K Wilhelmsen S H M Litjens I Kuikman et al ldquoNesprin-3 a novel outer nuclear membrane protein associates with thecytoskeletal linker protein plectinrdquo The Journal of Cell Biologyvol 171 no 5 pp 799ndash810 2005

[6] K J Roux M L Crisp Q Liu et al ldquoNesprin 4 is an outernuclear membrane protein that can induce kinesin-mediatedcell polarizationrdquo Proceedings of the National Acadamy ofSciences of the United States of America vol 106 no 7 pp 2194ndash2199 2009

[7] M SchneiderW Lu SNeumann et al ldquoMolecularmechanismsof centrosome and cytoskeleton anchorage at the nuclearenveloperdquo Cellular and Molecular Life Sciences vol 68 no 9pp 1593ndash1610 2011

[8] M L Lombardi D E Jaalouk C M Shanahan B Burke KJ Roux and J Lammerding ldquoThe interaction between nesprinsand sun proteins at the nuclear envelope is critical for forcetransmission between the nucleus and cytoskeletonrdquo The Jour-nal of Biological Chemistry vol 286 no 30 pp 26743ndash267532011

[9] R N Rashmi B Eckes G Glockner et al ldquoThe nuclearenvelope protein Nesprin-2 has roles in cell proliferation anddifferentiation during wound healingrdquo Nucleus (Austin Tex)vol 3 no 2 pp 172ndash186 2012

[10] K Djinovic-Carugo M Gautel J Ylanne and P Young ldquoThespectrin repeat a structural platform for cytoskeletal proteinassembliesrdquo FEBS Letters vol 513 no 1 pp 119ndash123 2002

[11] W Lu M Schneider S Neumann et al ldquoNesprin interchainassociations control nuclear sizerdquo Cellular and Molecular LifeSciences vol 69 no 20 pp 3493ndash3509 2012

[12] H R Dawe M Adams G Wheway et al ldquoNesprin-2 interactswith meckelin and mediates ciliogenesis via remodelling of theactin cytoskeletonrdquo Journal of Cell Science vol 122 no 15 pp2716ndash2726 2009

[13] P Satir L B Pedersen and S T Christensen ldquoThe primarycilium at a glancerdquo Journal of Cell Science vol 123 no 4 pp499ndash503 2010

[14] T Hirano ldquoSMC proteins and chromosome mechanics frombacteria to humansrdquo Philosophical Transactions of the RoyalSociety B Biological Sciences vol 360 no 1455 pp 507ndash5142005

[15] T Hirano ldquoAt the heart of the chromosome SMC proteins inactionrdquo Nature Reviews Molecular Cell Biology vol 7 no 5 pp311ndash322 2006

[16] C H Haering J Lowe A Hochwagen and K NasmythldquoMolecular architecture of SMC proteins and the yeast cohesincomplexrdquoMolecular Cell vol 9 no 4 pp 773ndash788 2002

[17] T Hirota D Gerlich B Koch J Ellenberg and J-M PetersldquoDistinct functions of condensin I and II in mitotic chromo-some assemblyrdquo Journal of Cell Science vol 117 no 26 pp 6435ndash6445 2004

[18] I Piazza C H Haering and A Rutkowska ldquoCondensin craft-ing the chromosome landscaperdquo Chromosoma vol 122 no 3pp 175ndash190 2013

[19] W Li Y Hu S Oh et al ldquoCondensin I and II complexeslicense full estrogen receptor120572-dependent enhancer activationrdquoMolecular Cell vol 59 no 2 pp 188ndash202 2015

[20] A J Wood A F Severson and B J Meyer ldquoCondensin andcohesin complexity the expanding repertoire of functionsrdquoNature Reviews Genetics vol 11 no 6 pp 391ndash404 2010

[21] C R Bauer T A Hartl and G Bosco ldquoCondensin II promotesthe formation of chromosome territories by inducing axial com-paction of polyploid interphase chromosomesrdquo PLoS Geneticsvol 8 no 8 Article ID e1002873 2012

[22] O Iwasaki C J Corcoran and K-I Noma ldquoInvolvement ofcondensin-directed gene associations in the organization andregulation of chromosome territories during the cell cyclerdquoNucleic Acids Research vol 44 no 8 pp 3618ndash3628 2016

[23] E Ampatzidou A Irmisch M J OrsquoConnell and J M MurrayldquoSmc56 is required for repair at collapsed replication forksrdquoMolecular and Cellular Biology vol 26 no 24 pp 9387ndash94012006

[24] D E Verver GHHwang PW Jordan andGHamer ldquoResolv-ing complex chromosome structures during meiosis versatiledeployment of Smc56rdquo Chromosoma vol 125 no 1 pp15ndash27 2016

[25] G D Spotts S V Patel Q Xiao and S R Hann ldquoIdentificationof downstream-initiated c-Myc proteins which are dominant-negative inhibitors of transactivation by full-length c-MycproteinsrdquoMolecular and Cellular Biology vol 17 no 3 pp 1459ndash1468 1997

[26] L T Vassilev C Tovar S Chen et al ldquoSelective small-moleculeinhibitor reveals critical mitotic functions of human CDK1rdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 103 no 28 pp 10660ndash10665 2006

[27] P J Paddison A A Caudy E Bernstein G J Hannon and DS Conklin ldquoShort hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cellsrdquo Genes amp Developmentvol 16 no 8 pp 948ndash958 2002

[28] T Libotte H Zaim S Abraham et al ldquoLamin AC-dependentlocalization of Nesprin-2 a giant scaffolder at the nuclearenveloperdquo Molecular Biology of the Cell (MBoC) vol 16 no 7pp 3411ndash3424 2005

[29] S Taranum I Sur R Muller et al ldquoCytoskeletal interactionsat the nuclear envelope mediated by Nesprinsrdquo InternationalJournal of Cell Biology vol 2012 Article ID 736524 11 pages2012


[30] A A Noegel R Blau-Wasser H Sultana et al ldquoThe Cyclase-associated protein CAP as regulator of cell polarity andcAMP signaling in dictyosteliumrdquoMolecular Biology of the Cell(MBoC) vol 15 no 2 pp 934ndash945 2004

[31] G I Evan G K Lewis G Ramsay and J M Bishop ldquoIsolationof monoclonal antibodies specific for human c-myc proto-oncogene productrdquo Molecular and Cellular Biology vol 5 no12 pp 3610ndash3616 1985

[32] H Xiong F Rivero U Euteneuer et al ldquoDictyostelium Sun-1connects the centrosome to chromatin and ensures genomestabilityrdquo Traffic vol 9 no 5 pp 708ndash724 2008

[33] M Schleicher G Gerisch and G Isenberg ldquoNew actin-bindingproteins fromDictyostelium discoideumrdquo EMBO Journal vol 3no 9 pp 2095ndash2100 1984

[34] P Fucini B Koppel M Schleicher et al ldquoMolecular architec-ture of the rod domain of the Dictyostelium gelation factor(ABP120)rdquo Journal ofMolecular Biology vol 291 no 5 pp 1017ndash1023 1999

[35] Z Grabarek and J Gergely ldquoZero-length crosslinking proce-dure with the use of active estersrdquo Analytical Biochemistry vol185 no 1 pp 131ndash135 1990

[36] J G Simpson and R G Roberts ldquoPatterns of evolutionary con-servation in the nesprin genes highlight probable functionallyimportant protein domains and isoformsrdquo Biochemical SocietyTransactions vol 36 no 6 pp 1359ndash1367 2008

[37] X A Cui H Zhang L Ilan A X Liu I Kharchuk and A FPalazzo ldquomRNA encoding Sec61120573 a tail-anchored protein islocalized on the endoplasmic reticulumrdquo Journal of Cell Sciencevol 128 no 18 pp 3398ndash3410 2015

[38] L Yang M Munck K Swaminathan L E Kapinos A ANoegel and S Neumann ldquoMutations in LMNA modulatethe lamin AmdashNesprin-2 interaction and cause LINC complexalterationsrdquo PLoS ONE vol 8 no 8 Article ID e71850 2013

[39] L C Green P Kalitsis T M Chang et al ldquoContrastingroles of condensin I and condensin II in mitotic chromosomeformationrdquo Journal of Cell Science vol 125 no 6 pp 1591ndash16042012

[40] D Gerlich T Hirota B Koch J-M Peters and J EllenbergldquoCondensin I stabilizes chromosomes mechanically through adynamic interaction in live cellsrdquo Current Biology vol 16 no 4pp 333ndash344 2006

[41] D T Warren T Tajsic J A Mellad R Searles Q Zhangand C M Shanahan ldquoNovel nuclear nesprin-2 variants tetheractive extracellular signal-regulated MAPK1 and MAPK2 atpromyelocytic leukemia protein nuclear bodies and act to regu-late smooth muscle cell proliferationrdquoThe Journal of BiologicalChemistry vol 285 no 2 pp 1311ndash1320 2010

[42] J T Morgan E R Pfeiffer T L Thirkill et al ldquoNesprin-3regulates endothelial cell morphology perinuclear cytoskeletalarchitecture and flow-induced polarizationrdquoMolecular Biologyof the Cell (MBoC) vol 22 no 22 pp 4324ndash4334 2011

[43] T G Fazzio and B Panning ldquoCondensin complexes regulatemitotic progression and interphase chromatin structure inembryonic stem cellsrdquo The Journal of Cell Biology vol 188 no4 pp 491ndash503 2010

[44] C George J Bozler H Nguyen and G Bosco ldquoCondensins arerequired for maintenance of nuclear architecturerdquo Cells vol 3no 3 pp 865ndash882 2014

[45] G Kustatscher N Hegarat K L HWills et al ldquoProteomics of afuzzy organelle interphase chromatinrdquo EMBO Journal vol 33no 6 pp 648ndash664 2014

[46] C Alabert J-C Bukowski-Wills S-B Lee et al ldquoNascentchromatin capture proteomics determines chromatin dynamicsduring DNA replication and identifies unknown fork compo-nentsrdquo Nature Cell Biology vol 16 no 3 pp 281ndash291 2014

[47] S Ohta J-C Bukowski-Wills L Sanchez-Pulido et al ldquoTheprotein composition ofmitotic chromosomes determined usingmulticlassifier combinatorial proteomicsrdquo Cell vol 142 no 5pp 810ndash821 2010

[48] D Rajgor J A Mellad F Autore Q Zhang and C MShanahan ldquoMultiple novel nesprin-1 and nesprin-2 variants actas versatile tissue-specific intracellular scaffoldsrdquo PLoS ONEvol 7 no 7 Article ID e40098 2012

[49] X Xing Functional Characterization of The Predicted SMCDomain in Nesprin-2 [PhD thesis] Math-Nat Fac Universityof Cologne Germany 2013

[50] C Mroszlig Novel Functions of Nesprin-2 and Analysis of Its InVivo Role [PhD thesis] Math-Nat Fac University of CologneGermany 2017

[51] T Ono A Losada M Hirano M P Myers A F Neuwaldand T Hirano ldquoDifferential contributions of condensin I andcondensin II to mitotic chromosome architecture in vertebratecellsrdquo Cell vol 115 no 1 pp 109ndash121 2003

[52] S Heessen and M Fornerod ldquoThe inner nuclear envelope as atranscription factor resting placerdquo EMBO Reports vol 8 no 10pp 914ndash919 2007

[53] C Ivorra M Kubicek J M Gonzalez et al ldquoA mechanism ofAP-1 suppression through interaction of c-Fos with lamin ACrdquoGenes amp Development vol 20 no 3 pp 307ndash320 2006

[54] T Zhang J R PaulsonM Bakhrebah et al ldquoCondensin I and IIbehaviour in interphase nuclei and cells undergoing prematurechromosome condensationrdquo Chromosome Research vol 24 no2 pp 243ndash269 2016

[55] A-L Kranz C-Y Jiao L H Winterkorn S E Albritton MKramer and S Ercan ldquoGenome-wide analysis of condensinbinding in Caenorhabditis elegansrdquoGenome Biology vol 14 no10 article no R112 2013

[56] J M Dowen S Bilodeau D A Orlando et al ldquoMultiple struc-tural maintenance of chromosome complexes at transcriptionalregulatory elementsrdquo StemCell Reports vol 1 no 5 pp 371ndash3782013

[57] C-A Martin J E Murray P Carroll et al ldquoMutations in genesencoding condensin complex proteins cause microcephalythrough decatenation failure at mitosisrdquo Genes amp Developmentvol 30 no 19 pp 2158ndash2172 2016

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


by Dawe et al [12] as an interaction site for meckelin aprotein with functions in the formation of primary ciliaPrimary cilia are sensory organs that act as mechanorecep-tors in various signaling pathways or sensors of chemicalstimuli [13]

SMC proteins have core functions in regulating genomestability and the organization of the geneticmaterialThey arepresent from bacteria to man [14] Classical SMC proteins arecomposed of 1000ndash1300 amino acids They have two coiled-coil regions interrupted by a central hinge The coiled coilsfold back on themselves and form an extended structure Attheir ends the N- and C-termini of a molecule interact witheach other to form a globular ATP-binding domain [15] Thehinge regions are responsible for heterodimerization of SMCmolecules [16] Six SMCproteins have been described inmanSMC1ndash6 SMC13 form the core of the cohesin complexwhichmediates sister chromatid cohesion SMC24 are present inthe condensin complex that acts in chromosome assemblyand segregation They are present in two condensin com-plexes with distinct roles condensins I and II which containSMC2 and SMC4 in combination with different non-SMCsubunits Condensins I and II are associated sequentially withchromosomes during the cell cycle and have different rolesfor chromosome architecture Condensin I is not presentin the nucleus in interphase During mitosis condensin Iis required for removal of cohesin from chromosome armsand for chromosome shortening whereas condensin II playsa role in chromosome condensation during early prophase[17] Condensin I is however not completely excluded ininterphase from the nucleus since a small pool was foundin association with intergenic and intronic regions duringinterphase [18 19] By contrast condensin II is alwaysnuclear It is associated with DNA throughout interphaseand concentrates on chromosomes in prophase Based onits interphase distribution a role in nuclear architecture wasproposed [20 21] Cohesin and condensin complexes havealso roles in DNA repair and gene regulation throughout thecell cycle [20] Moreover condensin is involved in organizingthe chromatin allowing intrachromosomal associations ofgene loci as shown in fission yeast [22] SMC56 is mainlyimplicated in DNA damage repair and DNA recombinationand has specific roles in meiosis [23 24]

We have carried out a biochemical and functional charac-terization of the Nesprin-2-SMC domain hereafter referredto as Nesprin-2-SMC We show that it can self-assemble toform dimers trimers and higher order structures and caninteract with condensin proteins SMC2 and SMC4 Mono-clonal antibodies directed against the SMC domain showed adistribution of the Nesprin-2 isoforms containing the SMCdomain along the NE during interphase and a presence atthe chromosomes during mitosis We also uncovered animpact of Nesprin-2 on mitotic chromosomes that might bemediated by an interaction with the condensin core unitsSMC24

2 Materials and Methods

21 Cell Culture Transfection and Cell SynchronizationHaCaT (human keratinocyte cell line) COS7 (African green

monkey kidney fibroblasts) and HeLa (human cervical can-cer cells) cells were grown in a humidified atmosphere con-taining 5 CO

2at 37∘C in DMEM (high glucose Life Tech-

nologies) supplemented with 10 fetal bovine serum (FBS)2mM Glutamine (SIGMA) and 1 penicillinstreptomycinCells were transfected as described [11] To knock downNesprin-2 HaCaT cells were transfected twice at intervals of72 h using the Amaxa Nucleofector Kit V Solution (Lonza)The plasmids used for knockdown of Nesprin-2 targeting theN-terminus and the C-terminus (Nesprin-2 N-term shRNANe-2 N-term KD Nesprin-2 C-term shRNA Ne-2 C-termKD) as well as the control have been described previously[7] The newly generated plasmids are described belowFor cell cycle synchronization HaCaT cells were treatedwith thymidine (2mM) for 24 h and then with Nocodazole(100 ngml) for 12 h or alternatively first with 9120583M RO-3306(Santa Cruz Biotechnology sc-358700) for 20 to 22 h andthen approximately 3 h release (depending on the desiredmitotic phase) to obtain mitotic phases RO-3306 is a CDK1inhibitor and reversibly arrests proliferating cells at the G2Mphase of the cell cycle [26] FACS analysis of cell cycle stageswas performed with unsynchronized and synchronized cellsStaining was done with Nuclear-ID Red DNA (Enzo ENZ-52406)

Determination of cell proliferation was done by plating atone time point six wells each with the same number of cellsand then counting twowells after 24 h two after 48 h and twoafter 72 h

22 Cloning Strategies cDNAs from HaCaT cells encodingthe SMC domain in Nesprin-2 (AAN60443 aa 1436ndash1766and SRs 11ndash13) were used as PCR templates using theprimers for the following 51015840 GAATTCAATGAACTC-CTTAAAAATATTCAAGATGTG 31015840 rev 51015840 GAATTC-CTCGAGGGATTCAGTCATCCCGATCTGGGTCTTGG31015840 that contain EcoRI restriction sites for cloning intopGEX-4T1 (Amersham) yielding pGEX-4T1-Nesprin-2-SMCwhich encodes GST-Nesprin-2-SMC GST is located at theamino terminus of the protein Nesprin-2-SMC sequenceswere generated by PCR and cloned into pCMV-Myc (GEHealthcare) using pGEX-4T1 Nesprin-2-SMC as templateand primers with EcoRI or XhoI restriction sites SMC(SR11ndash13) for the following 51015840GAATTCTGAATGAACTCC-TTAAAAATATTCAAGATGTG 31015840 rev 51015840 CTCGAGCTA-GAGGGATTCAGTCATCCCGATCTGGGTCTT 31015840 SR11for 51015840 GAATTCTGAATGAACTCCTTAAAAATATTC-AAGATGTG 31015840 rev 51015840 CTCGAGCTATCTCCCACATTG-TTCAAGACATTCGGTGAC 31015840 SR12 for 51015840 CTCGAG-GTTTTGGAGCTCTTAAAACAATATCAGAAT 31015840 rev51015840 CTCGAGCTAACCAAGATTTTCATAGTAATCTTC-TGTCTT 31015840 SR13 for 51015840 GAATTCTGCGAGCTCTAGCTT-TGTGGGACAAACTTTTTA 31015840 rev 51015840 CTCGAGCTA-GAGGGATTCAGTCATCCCGATCTGGGTCTT 31015840 Myc-SR53ndash56 corresponding to residues 6146ndash6799 of Nesprin-2is described in Schneider et al [7]

A Nesprin-2 SMC domain specific shRNA (Ne-2SMC) was generated as described using the following














3 Results






ABD 1

1 285

11 12 13 KASH

6885


mAb K20-478 mAb K81-116-6 pAbK1

1431 1766 6146 6799

(a)



340 380370360350



390 430420410400



490 540530520510500



440 4804704604501690 17401730172017101700



550 600590580570560

Ne-2

SMC2



340 390380370360350





450 500490480470460



510 5605505405305201690 1730172017101700



570 600590580

Ne-2

SMC4

(b)

11 12 13

1500 2000 2500

(ml)

minus0007

minus0002

minus0003

0008

0013

OD

280

nm


(c)

Highersim120

sim80

sim40


(kD

a)

(d)

Myc-SR11 1436

Myc-SR12 1532

Myc-SR13 1642

Myc-

Myc-

Myc-

Myc-

11 12 13 1766

1531

1641

1766

1436

11

12

13

Myc-Nesprin-2-SMC

(e)

Figure 1 Continued


Input Pulldown

kDa

35

27

15

lowast

COS7

Myc

-SR1

1

Myc

-SR1

2

Myc

-SR1

3

Myc

-Nes

prin

-2-S

MC

COS7

Myc

-SR1

1

Myc

-SR1

2

Myc

-SR1

3

Myc

-Nes

prin

-2-S

MC

(f)

Ponceau S WB Myc

COS7

COS7

COS7

Myc

-SR5

3ndash56

COS7

Myc

-SR5

3ndash56

GST

GST

GST

-Nes

prin

-2-S

MC

MW

GST

-Ne-

2-SM

C

(g)







kDa

250

130

100

800

(a)


HaC

aTH

eLa

mAb K81-116-6 Merge

HaC

aT

Calreticulin

(b)


mAb

K81

-116

-6

Ant

ibod

y so

lutio

n aft

erde

plet

ion

on G

ST-

Nes

prin

-2-S

MC

load

edm

embr

anes

Ant

ibod

y so

lutio

n aft

erde

plet

ion

on G

ST-

Nes

prin

-2-S

MC

load

edSe

phar

ose b

eads

(c)





Coo

mas

sie

WB anti-SMC2

WB anti-SMC4

kDa70

45

30

SPN

SPN

PD

PD

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(a)

800

155

160

kDa

WB anti-SMC4

WB anti-SMC2

WB mAb K20-478

IP

mAb

K20

-478

GFP

SMC2

Inpu

t

(b)

WB mAb CAP-H270kDa

WB mAb CAP-H100

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(c)

WB anti-SMC2155


kDa

G0G1 S M Untreated

MS

G0G1

G0

G1

G0

G1

S ph

ase

S ph

ase

Mito

sis

Mito

sis

Unt

reat

ed

Unt

reat

ed

0102030405060708090

100

Perc

enta

ge o

f cel

ls


Prop

hase

Met

apha

seTe

loph

ase

Ana

phas

eIn

terp

hase

Met

apha

se

(e)

Figure 3 Continued



Prop

hase

Telo

phas

eIn

terp

hase

Met

apha

seA

naph

ase

(f)


(g)








Nesprin-2

Lamin B1

kDa

800

68

ctrl

Ne-

2 C-

term

KD

Ne-

2 N

-term

KD

ctrl

Ne-

2 SM

C KD

(a)

K20-478 pAbK1 Merge

ctrl

Ne-

2C-

term

KD

Ne-

2N

-term

KD

Ne-

2SM

C KD

(b)

DAPI K81-116-6

Ne-

2SM

C KD

(c)






Prop

hase

Met

apha

seA

naph

ase

Telo

phas

eTe

loph

ase

Ana

phas

eM

etap

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(a)

Telo

phas

eTe

loph

ase

Ana

phas

eA

naph

ase

Met

apha

seM

etap

hase

Prop

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(b)


ctrl

SMC2

KD

(c)ctrl SMC2 KD

0102030405060708090

100

Fluo

resc

ence

inte

nsity

(au

)

lowastlowastlowast

(d)






4 Discussion










Disclosure






Acknowledgments







References


































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














3 Results






ABD 1

1 285

11 12 13 KASH

6885


mAb K20-478 mAb K81-116-6 pAbK1

1431 1766 6146 6799

(a)



340 380370360350



390 430420410400



490 540530520510500



440 4804704604501690 17401730172017101700



550 600590580570560

Ne-2

SMC2



340 390380370360350





450 500490480470460



510 5605505405305201690 1730172017101700



570 600590580

Ne-2

SMC4

(b)

11 12 13

1500 2000 2500

(ml)

minus0007

minus0002

minus0003

0008

0013

OD

280

nm


(c)

Highersim120

sim80

sim40


(kD

a)

(d)

Myc-SR11 1436

Myc-SR12 1532

Myc-SR13 1642

Myc-

Myc-

Myc-

Myc-

11 12 13 1766

1531

1641

1766

1436

11

12

13

Myc-Nesprin-2-SMC

(e)

Figure 1 Continued


Input Pulldown

kDa

35

27

15

lowast

COS7

Myc

-SR1

1

Myc

-SR1

2

Myc

-SR1

3

Myc

-Nes

prin

-2-S

MC

COS7

Myc

-SR1

1

Myc

-SR1

2

Myc

-SR1

3

Myc

-Nes

prin

-2-S

MC

(f)

Ponceau S WB Myc

COS7

COS7

COS7

Myc

-SR5

3ndash56

COS7

Myc

-SR5

3ndash56

GST

GST

GST

-Nes

prin

-2-S

MC

MW

GST

-Ne-

2-SM

C

(g)







kDa

250

130

100

800

(a)


HaC

aTH

eLa

mAb K81-116-6 Merge

HaC

aT

Calreticulin

(b)


mAb

K81

-116

-6

Ant

ibod

y so

lutio

n aft

erde

plet

ion

on G

ST-

Nes

prin

-2-S

MC

load

edm

embr

anes

Ant

ibod

y so

lutio

n aft

erde

plet

ion

on G

ST-

Nes

prin

-2-S

MC

load

edSe

phar

ose b

eads

(c)





Coo

mas

sie

WB anti-SMC2

WB anti-SMC4

kDa70

45

30

SPN

SPN

PD

PD

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(a)

800

155

160

kDa

WB anti-SMC4

WB anti-SMC2

WB mAb K20-478

IP

mAb

K20

-478

GFP

SMC2

Inpu

t

(b)

WB mAb CAP-H270kDa

WB mAb CAP-H100

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(c)

WB anti-SMC2155


kDa

G0G1 S M Untreated

MS

G0G1

G0

G1

G0

G1

S ph

ase

S ph

ase

Mito

sis

Mito

sis

Unt

reat

ed

Unt

reat

ed

0102030405060708090

100

Perc

enta

ge o

f cel

ls


Prop

hase

Met

apha

seTe

loph

ase

Ana

phas

eIn

terp

hase

Met

apha

se

(e)

Figure 3 Continued



Prop

hase

Telo

phas

eIn

terp

hase

Met

apha

seA

naph

ase

(f)


(g)








Nesprin-2

Lamin B1

kDa

800

68

ctrl

Ne-

2 C-

term

KD

Ne-

2 N

-term

KD

ctrl

Ne-

2 SM

C KD

(a)

K20-478 pAbK1 Merge

ctrl

Ne-

2C-

term

KD

Ne-

2N

-term

KD

Ne-

2SM

C KD

(b)

DAPI K81-116-6

Ne-

2SM

C KD

(c)






Prop

hase

Met

apha

seA

naph

ase

Telo

phas

eTe

loph

ase

Ana

phas

eM

etap

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(a)

Telo

phas

eTe

loph

ase

Ana

phas

eA

naph

ase

Met

apha

seM

etap

hase

Prop

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(b)


ctrl

SMC2

KD

(c)ctrl SMC2 KD

0102030405060708090

100

Fluo

resc

ence

inte

nsity

(au

)

lowastlowastlowast

(d)






4 Discussion










Disclosure






Acknowledgments







References


































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results






ABD 1

1 285

11 12 13 KASH

6885


mAb K20-478 mAb K81-116-6 pAbK1

1431 1766 6146 6799

(a)



340 380370360350



390 430420410400



490 540530520510500



440 4804704604501690 17401730172017101700



550 600590580570560

Ne-2

SMC2



340 390380370360350





450 500490480470460



510 5605505405305201690 1730172017101700



570 600590580

Ne-2

SMC4

(b)

11 12 13

1500 2000 2500

(ml)

minus0007

minus0002

minus0003

0008

0013

OD

280

nm


(c)

Highersim120

sim80

sim40


(kD

a)

(d)

Myc-SR11 1436

Myc-SR12 1532

Myc-SR13 1642

Myc-

Myc-

Myc-

Myc-

11 12 13 1766

1531

1641

1766

1436

11

12

13

Myc-Nesprin-2-SMC

(e)

Figure 1 Continued


Input Pulldown

kDa

35

27

15

lowast

COS7

Myc

-SR1

1

Myc

-SR1

2

Myc

-SR1

3

Myc

-Nes

prin

-2-S

MC

COS7

Myc

-SR1

1

Myc

-SR1

2

Myc

-SR1

3

Myc

-Nes

prin

-2-S

MC

(f)

Ponceau S WB Myc

COS7

COS7

COS7

Myc

-SR5

3ndash56

COS7

Myc

-SR5

3ndash56

GST

GST

GST

-Nes

prin

-2-S

MC

MW

GST

-Ne-

2-SM

C

(g)







kDa

250

130

100

800

(a)


HaC

aTH

eLa

mAb K81-116-6 Merge

HaC

aT

Calreticulin

(b)


mAb

K81

-116

-6

Ant

ibod

y so

lutio

n aft

erde

plet

ion

on G

ST-

Nes

prin

-2-S

MC

load

edm

embr

anes

Ant

ibod

y so

lutio

n aft

erde

plet

ion

on G

ST-

Nes

prin

-2-S

MC

load

edSe

phar

ose b

eads

(c)





Coo

mas

sie

WB anti-SMC2

WB anti-SMC4

kDa70

45

30

SPN

SPN

PD

PD

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(a)

800

155

160

kDa

WB anti-SMC4

WB anti-SMC2

WB mAb K20-478

IP

mAb

K20

-478

GFP

SMC2

Inpu

t

(b)

WB mAb CAP-H270kDa

WB mAb CAP-H100

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(c)

WB anti-SMC2155


kDa

G0G1 S M Untreated

MS

G0G1

G0

G1

G0

G1

S ph

ase

S ph

ase

Mito

sis

Mito

sis

Unt

reat

ed

Unt

reat

ed

0102030405060708090

100

Perc

enta

ge o

f cel

ls


Prop

hase

Met

apha

seTe

loph

ase

Ana

phas

eIn

terp

hase

Met

apha

se

(e)

Figure 3 Continued



Prop

hase

Telo

phas

eIn

terp

hase

Met

apha

seA

naph

ase

(f)


(g)








Nesprin-2

Lamin B1

kDa

800

68

ctrl

Ne-

2 C-

term

KD

Ne-

2 N

-term

KD

ctrl

Ne-

2 SM

C KD

(a)

K20-478 pAbK1 Merge

ctrl

Ne-

2C-

term

KD

Ne-

2N

-term

KD

Ne-

2SM

C KD

(b)

DAPI K81-116-6

Ne-

2SM

C KD

(c)






Prop

hase

Met

apha

seA

naph

ase

Telo

phas

eTe

loph

ase

Ana

phas

eM

etap

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(a)

Telo

phas

eTe

loph

ase

Ana

phas

eA

naph

ase

Met

apha

seM

etap

hase

Prop

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(b)


ctrl

SMC2

KD

(c)ctrl SMC2 KD

0102030405060708090

100

Fluo

resc

ence

inte

nsity

(au

)

lowastlowastlowast

(d)






4 Discussion










Disclosure






Acknowledgments







References


































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


ABD 1

1 285

11 12 13 KASH

6885


mAb K20-478 mAb K81-116-6 pAbK1

1431 1766 6146 6799

(a)



340 380370360350



390 430420410400



490 540530520510500



440 4804704604501690 17401730172017101700



550 600590580570560

Ne-2

SMC2



340 390380370360350





450 500490480470460



510 5605505405305201690 1730172017101700



570 600590580

Ne-2

SMC4

(b)

11 12 13

1500 2000 2500

(ml)

minus0007

minus0002

minus0003

0008

0013

OD

280

nm


(c)

Highersim120

sim80

sim40


(kD

a)

(d)

Myc-SR11 1436

Myc-SR12 1532

Myc-SR13 1642

Myc-

Myc-

Myc-

Myc-

11 12 13 1766

1531

1641

1766

1436

11

12

13

Myc-Nesprin-2-SMC

(e)

Figure 1 Continued


Input Pulldown

kDa

35

27

15

lowast

COS7

Myc

-SR1

1

Myc

-SR1

2

Myc

-SR1

3

Myc

-Nes

prin

-2-S

MC

COS7

Myc

-SR1

1

Myc

-SR1

2

Myc

-SR1

3

Myc

-Nes

prin

-2-S

MC

(f)

Ponceau S WB Myc

COS7

COS7

COS7

Myc

-SR5

3ndash56

COS7

Myc

-SR5

3ndash56

GST

GST

GST

-Nes

prin

-2-S

MC

MW

GST

-Ne-

2-SM

C

(g)







kDa

250

130

100

800

(a)


HaC

aTH

eLa

mAb K81-116-6 Merge

HaC

aT

Calreticulin

(b)


mAb

K81

-116

-6

Ant

ibod

y so

lutio

n aft

erde

plet

ion

on G

ST-

Nes

prin

-2-S

MC

load

edm

embr

anes

Ant

ibod

y so

lutio

n aft

erde

plet

ion

on G

ST-

Nes

prin

-2-S

MC

load

edSe

phar

ose b

eads

(c)





Coo

mas

sie

WB anti-SMC2

WB anti-SMC4

kDa70

45

30

SPN

SPN

PD

PD

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(a)

800

155

160

kDa

WB anti-SMC4

WB anti-SMC2

WB mAb K20-478

IP

mAb

K20

-478

GFP

SMC2

Inpu

t

(b)

WB mAb CAP-H270kDa

WB mAb CAP-H100

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(c)

WB anti-SMC2155


kDa

G0G1 S M Untreated

MS

G0G1

G0

G1

G0

G1

S ph

ase

S ph

ase

Mito

sis

Mito

sis

Unt

reat

ed

Unt

reat

ed

0102030405060708090

100

Perc

enta

ge o

f cel

ls


Prop

hase

Met

apha

seTe

loph

ase

Ana

phas

eIn

terp

hase

Met

apha

se

(e)

Figure 3 Continued



Prop

hase

Telo

phas

eIn

terp

hase

Met

apha

seA

naph

ase

(f)


(g)








Nesprin-2

Lamin B1

kDa

800

68

ctrl

Ne-

2 C-

term

KD

Ne-

2 N

-term

KD

ctrl

Ne-

2 SM

C KD

(a)

K20-478 pAbK1 Merge

ctrl

Ne-

2C-

term

KD

Ne-

2N

-term

KD

Ne-

2SM

C KD

(b)

DAPI K81-116-6

Ne-

2SM

C KD

(c)






Prop

hase

Met

apha

seA

naph

ase

Telo

phas

eTe

loph

ase

Ana

phas

eM

etap

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(a)

Telo

phas

eTe

loph

ase

Ana

phas

eA

naph

ase

Met

apha

seM

etap

hase

Prop

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(b)


ctrl

SMC2

KD

(c)ctrl SMC2 KD

0102030405060708090

100

Fluo

resc

ence

inte

nsity

(au

)

lowastlowastlowast

(d)






4 Discussion










Disclosure






Acknowledgments







References


































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Input Pulldown

kDa

35

27

15

lowast

COS7

Myc

-SR1

1

Myc

-SR1

2

Myc

-SR1

3

Myc

-Nes

prin

-2-S

MC

COS7

Myc

-SR1

1

Myc

-SR1

2

Myc

-SR1

3

Myc

-Nes

prin

-2-S

MC

(f)

Ponceau S WB Myc

COS7

COS7

COS7

Myc

-SR5

3ndash56

COS7

Myc

-SR5

3ndash56

GST

GST

GST

-Nes

prin

-2-S

MC

MW

GST

-Ne-

2-SM

C

(g)







kDa

250

130

100

800

(a)


HaC

aTH

eLa

mAb K81-116-6 Merge

HaC

aT

Calreticulin

(b)


mAb

K81

-116

-6

Ant

ibod

y so

lutio

n aft

erde

plet

ion

on G

ST-

Nes

prin

-2-S

MC

load

edm

embr

anes

Ant

ibod

y so

lutio

n aft

erde

plet

ion

on G

ST-

Nes

prin

-2-S

MC

load

edSe

phar

ose b

eads

(c)





Coo

mas

sie

WB anti-SMC2

WB anti-SMC4

kDa70

45

30

SPN

SPN

PD

PD

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(a)

800

155

160

kDa

WB anti-SMC4

WB anti-SMC2

WB mAb K20-478

IP

mAb

K20

-478

GFP

SMC2

Inpu

t

(b)

WB mAb CAP-H270kDa

WB mAb CAP-H100

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(c)

WB anti-SMC2155


kDa

G0G1 S M Untreated

MS

G0G1

G0

G1

G0

G1

S ph

ase

S ph

ase

Mito

sis

Mito

sis

Unt

reat

ed

Unt

reat

ed

0102030405060708090

100

Perc

enta

ge o

f cel

ls


Prop

hase

Met

apha

seTe

loph

ase

Ana

phas

eIn

terp

hase

Met

apha

se

(e)

Figure 3 Continued



Prop

hase

Telo

phas

eIn

terp

hase

Met

apha

seA

naph

ase

(f)


(g)








Nesprin-2

Lamin B1

kDa

800

68

ctrl

Ne-

2 C-

term

KD

Ne-

2 N

-term

KD

ctrl

Ne-

2 SM

C KD

(a)

K20-478 pAbK1 Merge

ctrl

Ne-

2C-

term

KD

Ne-

2N

-term

KD

Ne-

2SM

C KD

(b)

DAPI K81-116-6

Ne-

2SM

C KD

(c)






Prop

hase

Met

apha

seA

naph

ase

Telo

phas

eTe

loph

ase

Ana

phas

eM

etap

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(a)

Telo

phas

eTe

loph

ase

Ana

phas

eA

naph

ase

Met

apha

seM

etap

hase

Prop

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(b)


ctrl

SMC2

KD

(c)ctrl SMC2 KD

0102030405060708090

100

Fluo

resc

ence

inte

nsity

(au

)

lowastlowastlowast

(d)






4 Discussion










Disclosure






Acknowledgments







References


































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


kDa

250

130

100

800

(a)


HaC

aTH

eLa

mAb K81-116-6 Merge

HaC

aT

Calreticulin

(b)


mAb

K81

-116

-6

Ant

ibod

y so

lutio

n aft

erde

plet

ion

on G

ST-

Nes

prin

-2-S

MC

load

edm

embr

anes

Ant

ibod

y so

lutio

n aft

erde

plet

ion

on G

ST-

Nes

prin

-2-S

MC

load

edSe

phar

ose b

eads

(c)





Coo

mas

sie

WB anti-SMC2

WB anti-SMC4

kDa70

45

30

SPN

SPN

PD

PD

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(a)

800

155

160

kDa

WB anti-SMC4

WB anti-SMC2

WB mAb K20-478

IP

mAb

K20

-478

GFP

SMC2

Inpu

t

(b)

WB mAb CAP-H270kDa

WB mAb CAP-H100

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(c)

WB anti-SMC2155


kDa

G0G1 S M Untreated

MS

G0G1

G0

G1

G0

G1

S ph

ase

S ph

ase

Mito

sis

Mito

sis

Unt

reat

ed

Unt

reat

ed

0102030405060708090

100

Perc

enta

ge o

f cel

ls


Prop

hase

Met

apha

seTe

loph

ase

Ana

phas

eIn

terp

hase

Met

apha

se

(e)

Figure 3 Continued



Prop

hase

Telo

phas

eIn

terp

hase

Met

apha

seA

naph

ase

(f)


(g)








Nesprin-2

Lamin B1

kDa

800

68

ctrl

Ne-

2 C-

term

KD

Ne-

2 N

-term

KD

ctrl

Ne-

2 SM

C KD

(a)

K20-478 pAbK1 Merge

ctrl

Ne-

2C-

term

KD

Ne-

2N

-term

KD

Ne-

2SM

C KD

(b)

DAPI K81-116-6

Ne-

2SM

C KD

(c)






Prop

hase

Met

apha

seA

naph

ase

Telo

phas

eTe

loph

ase

Ana

phas

eM

etap

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(a)

Telo

phas

eTe

loph

ase

Ana

phas

eA

naph

ase

Met

apha

seM

etap

hase

Prop

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(b)


ctrl

SMC2

KD

(c)ctrl SMC2 KD

0102030405060708090

100

Fluo

resc

ence

inte

nsity

(au

)

lowastlowastlowast

(d)






4 Discussion










Disclosure






Acknowledgments







References


































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Coo

mas

sie

WB anti-SMC2

WB anti-SMC4

kDa70

45

30

SPN

SPN

PD

PD

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(a)

800

155

160

kDa

WB anti-SMC4

WB anti-SMC2

WB mAb K20-478

IP

mAb

K20

-478

GFP

SMC2

Inpu

t

(b)

WB mAb CAP-H270kDa

WB mAb CAP-H100

Inpu

t

GST

GST

-Nes

prin

-2-S

MC

(c)

WB anti-SMC2155


kDa

G0G1 S M Untreated

MS

G0G1

G0

G1

G0

G1

S ph

ase

S ph

ase

Mito

sis

Mito

sis

Unt

reat

ed

Unt

reat

ed

0102030405060708090

100

Perc

enta

ge o

f cel

ls


Prop

hase

Met

apha

seTe

loph

ase

Ana

phas

eIn

terp

hase

Met

apha

se

(e)

Figure 3 Continued



Prop

hase

Telo

phas

eIn

terp

hase

Met

apha

seA

naph

ase

(f)


(g)








Nesprin-2

Lamin B1

kDa

800

68

ctrl

Ne-

2 C-

term

KD

Ne-

2 N

-term

KD

ctrl

Ne-

2 SM

C KD

(a)

K20-478 pAbK1 Merge

ctrl

Ne-

2C-

term

KD

Ne-

2N

-term

KD

Ne-

2SM

C KD

(b)

DAPI K81-116-6

Ne-

2SM

C KD

(c)






Prop

hase

Met

apha

seA

naph

ase

Telo

phas

eTe

loph

ase

Ana

phas

eM

etap

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(a)

Telo

phas

eTe

loph

ase

Ana

phas

eA

naph

ase

Met

apha

seM

etap

hase

Prop

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(b)


ctrl

SMC2

KD

(c)ctrl SMC2 KD

0102030405060708090

100

Fluo

resc

ence

inte

nsity

(au

)

lowastlowastlowast

(d)






4 Discussion










Disclosure






Acknowledgments







References


































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Prop

hase

Telo

phas

eIn

terp

hase

Met

apha

seA

naph

ase

(f)


(g)








Nesprin-2

Lamin B1

kDa

800

68

ctrl

Ne-

2 C-

term

KD

Ne-

2 N

-term

KD

ctrl

Ne-

2 SM

C KD

(a)

K20-478 pAbK1 Merge

ctrl

Ne-

2C-

term

KD

Ne-

2N

-term

KD

Ne-

2SM

C KD

(b)

DAPI K81-116-6

Ne-

2SM

C KD

(c)






Prop

hase

Met

apha

seA

naph

ase

Telo

phas

eTe

loph

ase

Ana

phas

eM

etap

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(a)

Telo

phas

eTe

loph

ase

Ana

phas

eA

naph

ase

Met

apha

seM

etap

hase

Prop

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(b)


ctrl

SMC2

KD

(c)ctrl SMC2 KD

0102030405060708090

100

Fluo

resc

ence

inte

nsity

(au

)

lowastlowastlowast

(d)






4 Discussion










Disclosure






Acknowledgments







References


































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Nesprin-2

Lamin B1

kDa

800

68

ctrl

Ne-

2 C-

term

KD

Ne-

2 N

-term

KD

ctrl

Ne-

2 SM

C KD

(a)

K20-478 pAbK1 Merge

ctrl

Ne-

2C-

term

KD

Ne-

2N

-term

KD

Ne-

2SM

C KD

(b)

DAPI K81-116-6

Ne-

2SM

C KD

(c)






Prop

hase

Met

apha

seA

naph

ase

Telo

phas

eTe

loph

ase

Ana

phas

eM

etap

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(a)

Telo

phas

eTe

loph

ase

Ana

phas

eA

naph

ase

Met

apha

seM

etap

hase

Prop

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(b)


ctrl

SMC2

KD

(c)ctrl SMC2 KD

0102030405060708090

100

Fluo

resc

ence

inte

nsity

(au

)

lowastlowastlowast

(d)






4 Discussion










Disclosure






Acknowledgments







References


































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Prop

hase

Met

apha

seA

naph

ase

Telo

phas

eTe

loph

ase

Ana

phas

eM

etap

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(a)

Telo

phas

eTe

loph

ase

Ana

phas

eA

naph

ase

Met

apha

seM

etap

hase

Prop

hase

Prop

hase

ctrl

Ne-

2 SM

C KD


(b)


ctrl

SMC2

KD

(c)ctrl SMC2 KD

0102030405060708090

100

Fluo

resc

ence

inte

nsity

(au

)

lowastlowastlowast

(d)






4 Discussion










Disclosure






Acknowledgments







References


































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





4 Discussion










Disclosure






Acknowledgments







References


































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Disclosure






Acknowledgments







References


































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



References


































































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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