Reinforcing the LINC Complex Connection to Actin Filaments: The

Reinforcing the LINC complex connection to actin ﬁlaments: the role of FHOD1 in TAN line formation and nuclear movement

Susumu Antoku1, Ruijun Zhu1, Stefan Kutscheidt2, Oliver T Fackler2, and Gregg G Gundersen1,* 1Department of Pathology & Cell Biology; Columbia University; New York, NY USA; 2Department of Infectious Diseases; Integrative Virology; University of Heidelberg; Heidelberg, Germany

ositioning the nucleus is critical for cytoskeleton and the nucleus established Pmany cellular processes including by specific LINC complexes.1,4,5 For the cell division, migration and differentia- actin-dependent nuclear movement in tion. The linker of nucleoskeleton and fibroblasts and myoblasts, the specific cytoskeleton (LINC) complex spans the LINC complex is composed of nesprin- inner and outer nuclear membranes and 2G, a ~800 kDa, actin-binding and spec- has emerged as a major factor in connect- trin repeat (SR)-containing outer nuclear ing the nucleus to the cytoskeleton for membrane protein and SUN2, an inner movement and positioning. Recently, we nuclear membrane protein that binds the discovered that the diaphanous formin KASH (Klarsicht, ANC-1, Syne homol- family member FHOD1 interacts with ogy) domain of nesprin-2G.3,6 The associ- the LINC complex component nesprin-2 ation of these proteins with dorsal actin giant (nesprin-2G) and that this interac- cables results in their assembly into linear tion plays essential roles in the formation arrays termed TAN lines (Fig. 1).3,6,7 of transmembrane actin-dependent TAN lines are anchored by association of nuclear (TAN) lines and nuclear move- SUN2 with A-type lamins.8 Two inner ment during cell polarization in fibro- nuclear membrane proteins, Samp1 and blasts. We found that FHOD1 emerin, are also found in TAN lines and 9,10 Keywords: actin filaments, Emery-Drei- strengthens the connection between may contribute to their anchoring. fuss muscular dystrophy, FHOD1, for- nesprin-2G and rearward moving dorsal Emerin additionally affects actin-depen- min, LINC Complex, nesprin, nuclear actin cables by providing a second site of dent nuclear movement by interacting movement, TAN lines interaction between nesprin-2G and the with myosin IIB and organizing the direc- actin cable. These results indicate that tionality of actin flow.10 Abbreviations: LINC, linker of nucleoske- the LINC complex connection to the TAN lines resemble other membrane leton and cytoskeleton; nesprin-2G, actin cytoskeleton can be enhanced by adhesions, such as focal adhesions and cad- nesprin-2 giant; TAN lines, transmem- cytoplasmic factors and suggest a new herin cell-cell adhesions, in that they form brane actin-dependent nuclear lines; SR, model for TAN line formation. We dis- by actin-dependent clustering of mem- spectrin repeat; KASH, Klarsicht; ANC-1, cuss how the nesprin-2G-FHOD1 inter- brane proteins and transmit force. The Syne homology; DRF, diaphanous related action may be regulated and its possible large number of proteins associated with formin; GBD, GTPase binding domain; functional significance for development focal and cell-cell adhesions suggests that DID, diaphanous inhibitory domain; FH, and disease. additional proteins are likely to be associ- formin homology; DAD, diaphanous ated with TAN lines. In a recent study,11 autoregulatory domain; ABS, actin bind- we identified the diaphanous related for- ing site; LPA, lysophosphatidic acid; Metazoans actively control the position min (DRF) FHOD1 as interaction partner GFP-mN2G, GFP-mini-nesprin-2G; of the nucleus in the cell to regulate cell of nesprin-2G and a new component of CH, calponin homology; EDMD, division, differentiation, and migration.1 TAN lines. Like other DRFs, FHOD1 is Emery-Dreifuss muscular dystrophy In fibroblasts and myoblasts, actin retro- composed of an N-terminal GTPase bind- *Correspondence to: Gregg G Gundersen; Email: grade flow powers the rearward movement ing domain (GBD), followed by the diaph- [email protected] of the nucleus to orient the centrosome anous inhibitory domain (DID), the Submitted: 05/06/2015 and polarize cells for directed migration central formin homology (FH) domains 2,3 Accepted: 05/16/2015 (Fig. 1A). Many nuclear movements FH1 and the actin binding FH2, and the http://dx.doi.org/10.1080/15384101.2015.1053665 are mediated by connections between the C-terminal diaphanous autoregulatory

2200 Cell Cycle Volume 14 Issue 14 Figure 1. TAN line dependent nuclear movement. (A) Model for TAN line dependent nuclear movement in fibroblasts and myoblasts polarizing for migration. In serum-starved wounded monolayers of fibroblasts and myoblasts, LPA stimulation induces the retrograde flow of dorsal actin cables (blue). These actin cables engage nesprin-2G on the nuclear surface to form TAN lines (red) resulting in the rearward movement of the nucleus while the centrosome stays in the center of the cell. This establishes cell polarization by orienting the centrosome toward the wound edge. (B) Immunofluorescence images of TAN lines in NIH3T3 fibroblasts expressing the nesprin-2G chimera, GFP-mini-N2G. Cells were stimulated with LPA for one hr and the formation of TAN lines visualized by staining for GFP-mini-N2G and F-actin. The boxed region in the left panel is shown at higher magnification in the panels to the right. Arrows indicate TAN lines with colocalized GFP-mini-N2G and actin cables. Scale bar, 5 mm.

domain (DAD) (Fig. 2A). Despite a simi- autoinhibition occurs by phosphorylation if FHOD1 was required for these pro- lar domain organization, FHOD1 exhibits of the DAD (see below). cesses by knocking it down with siRNA features that distinguish it from other The FHOD1 N-terminus (residues oligonucleotides in NIH3T3 fibroblasts. DRFs. Importantly, It does not stimulate 1–339) is structurally different from other Similar to the phenotype of nesprin-2G actin polymerization in vitro, but instead DRFs13 and we used it as a bait in a yeast knockdown cells, FHOD1-depleted cells appears to cap actin filaments.12 Also 2-hybrid screen for FHOD1 interacting did not orient their centrosomes due to a unlike other DRFs, FHOD1 bundles actin proteins. We found that a fragment of failure to move their nuclei rearward after filaments both in vivo and in vitro.12-15 human nesprin-2G (residues 1340–1669) stimulation with lysophosphatidic acid This bundling activity requires dimeriza- interacted with the N-terminal fragment (LPA), a serum factor that stimulates these tion provided by the FH2 domain, but of FHOD1.11 By GST-pulldown and co- processes in starved NIH3T3 fibro- also a second F-actin binding domain in immunoprecipitation assays we confirmed blasts.16 Defects in centrosome orienta- the N-terminus between the DID and the the interaction of the proteins in mamma- tion and nuclear movement in FHOD1- FH1 domain that is unique to FHOD1 lian cells. The interaction of the full length depleted cells were rescued by re- among the 15 mammalian formins proteins was shown by co-immunoprecip- expression of wild-type FHOD1 but not (Fig. 2C). This second actin binding site itation of endogenous nesprin-2G with by re-expression of FHOD1 lacking resi- (ABS) also endows FHOD1 with the abil- over-expressed FHOD1. A directed yeast dues 1–339 that interact with nesprin-2G. ity to bind along the length of actin fila- 2-hybrid interaction screen with nesprin- Consistent with the importance of the ments, unlike other DRFs, which interact 2G fragments covering the length of interaction between FHOD1 and nesprin- transiently with actin filament barbed nesprin-2G and additional GST-pull- 2G, overexpression of the interacting frag- ends. FHOD1 is regulated by autoinhibi- down assays revealed that the only site of ments of nesprin-2G and FHOD1 inhib- tion which is accomplished by binding of FHOD1 interaction on nesprin-2G was ited centrosome orientation and nuclear the DAD to the DID. Although for most with SRs 11 and 12. movement. DRFs release of autoinhibition is mediated Because of the importance of nesprin- Rearward nuclear movement requires by binding of specific Rho GTPase(s) to 2G for centrosome orientation and the formation and retrograde flow of dor- the GBD, for FHOD1 release of nuclear movement in fibroblasts, we tested sal actin cables that engage the nucleus

www.tandfonline.com Cell Cycle 2201 Figure 2. Multi-site attachment model for TAN lines. (A) Schematic of the autoinhibited form of FHOD1 (shown as a monomer to emphasize domains). Individual domains are described in the text. (B) Model for the multi-site attachment of nesprin-2G (N2G) and FHOD1 to actin cables in TAN lines. The interaction of nesprin-2G with FHOD1 forms a branched connection between nesprin-2G and the actin cable with one connection provided by nesprin- 2G’s CH domains and the other by FHOD1s unique ABS. This branched connection is proposed to strengthen the association between the nesprin and the actin cable. In the perinuclear space between the inner (INM) and outer nuclear membrane (ONM), KASH domains of nesprin-2G interact with the SUN2 trimer. In the nucleoplasm, SUN2 is anchored by interaction with lamin A/C of the lamina and with the INM proteins Samp1 and emerin. (C)A detailed view showing FHOD1 interacting with SRs 11–12 of nesprin-2G through its N-terminal GBD and DID and to the actin cable through its ABS. The dimeric nature of FHOD1 may bring multiple nesprin-2Gs together. through TAN lines.6,10 Despite its pro- expressing various forms of FHOD1 in chimera rescued the centrosome orienta- posed role in regulating the actin cytoskel- FHOD1 depleted cells. We found that a tion and nuclear movement defects in eton, FHOD1 knockdown cells did not FHOD1 construct comprising the entire FHOD1 knockdown cells as well as have significant differences in total levels N-terminus (residues 1–569) and lacking FHOD1 1–569, implying that the of F-actin, the number of dorsal actin the canonical formin FH1 and FH2 FHOD1-nesprin-2G interaction provides cables above the nucleus, or the velocity of domains localized in TAN lines, rescued a second linkage to actin cables in addition actin retrograde flow after LPA stimula- centrosome orientation and partially res- to that provided by the CH domains of tion compared to control cells. However, cued nuclear movement in FHOD1 defi- nesprin-2G. TAN line formation was severely impaired cient cells. In addition to a nesprin-2G These findings support a new model in FHOD1 knockdown cells. Impor- binding site, FHOD1 1–569 contains the for TAN line engagement of actin cables tantly, expressed RFP-FHOD1 localized unique ABS of FHOD1. This consider- (Fig. 2B). In this model, nesprin-2G to TAN lines, which were visualized by ation raised the possibility that FHOD1 attaches to actin cables by 2 sites of inter- staining of either expressed GFP-mini- bound to nesprin-2G contributes to TAN action: its own CH domains and the nesprin-2G (GFP-mN2G, a minimal line formation by providing a second F- unique ABS in the N-terminus of functional nesprin-2G containing the N- actin binding site in addition to the actin FHOD1. We hypothesize that this terminal calponin homology (CH) binding CH domains of nesprin-2G. To branched connection reinforces the domains and the C-terminal KASH test this possibility, we fused the nesprin- nesprin-actin interaction and allows it to domain6) or endogenous nesprin-2G. 2G interacting site of FHOD1 (residues resist the high forces necessary to move We further dissected the role of 1–339) with the F-actin binding CH such a large organelle as the nucleus. The FHOD1 in TAN line formation by domains of a-actinin. Expression of this model also implies that the orientation of

2202 Cell Cycle Volume 14 Issue 14 nesprin-2G relative to the actin cable is inhibition through an intra-molecular domains at the N-terminus of nesprin- likely to be nearly parallel (at least for the interaction between its N-terminal DID 2G, SRs 49–53 are at the other end of the region between the CH domains and the and C-terminal DAD.20 By co-immuno- molecule near the transmembrane SR11–12) rather than the end-on orienta- precipitation of FHOD1 transfected domain.24 A number of proteins bind to tion that was originally envisioned.6,7 293T cells, in preliminary experiments we this region29 raising the possibility that This conclusion is based on the extended have observed that a nesprin-2G fragment additional bridging proteins will be structure of SR proteins and average 6 nm containing SR11–12 co-immunoprecipi- important for TAN line formation and length of individual SRs, yielding a dis- tated the constitutively active FHOD1 nuclear movement. tance of at least 60 nm between the CH DDAD more efficiently than it did wild- Despite the importance of the domains and SRs11–12 of nesprin- type FHOD1. This implies that the intra- FHOD1-nesprin-2G interaction for 2G.17,18 Coupled with the predicted molecular interaction between FHOD1’s nuclear movement and polarity regulation length of the FHOD1 N-terminal frag- DID and DAD reduces the interaction of cells in culture, it remains to be tested ment that interacts with both nesprin-2G between FHOD1 and nesprin-2G and whether the FHOD1-nesprin-2G interac- and the actin cable (<10 nm),13 the only that the release of FHOD1’s autoinhibition is involved in animal development, way that both proteins can interact with tion may be a pre-requisite for efficient differentiation or pathogenesis. Nesprin-2 the actin cable is if nesprin-2G lies along FHOD1-nesprin-2G interaction. has numerous splice isoforms from the the axis of the actin cable. Lastly, FHOD1 There have been several mechanistic giant nesprin-2G to shorter isoforms that is hypothesized to form a dimer through models proposed for how the autoinhibi- lack actin interacting CH domains or even its FH2 domains.14 Thus, FHOD1 has tion of FHOD1 is released. The GBD of the transmembrane KASH domain.30 the potential to bring two nesprin-2Gs FHOD1 interacts with Rac1-GTP, but When the exon of Syne2 (the gene encod- together. In this way, FHOD1 may addi- this does not seem to activate ing nesprin-2) specifying the KASH tionally contribute to the assembly of FHOD1.13,20,21 Phosphorylation of domain is deleted, mice exhibit defects in TAN lines by enhancing the avidity of FHOD1 at three serine/threonine residues laminar formation in the cerebral cortex nesprin-2G for the actin cables. Perhaps, by ROCK1 partially releases autoinhib- and hippocampus during brain develop- this explains why monomeric constructs tion.14,22 Additionally, phosphorylation ment resulting in poor learning skills.31 based on the N-terminus of FHOD1 did of FHOD1 at Y99 by Src was shown to be Nesprin-1G is structurally similar to not fully rescue nuclear movement. a prerequisite for phosphorylation of the nesprin-2G, but lacks the set of nesprin- In muscle cells there are 2 splice var- DAD by ROCK1.23 Phosphorylation of 2G conserved SRs that interact with iants of FHOD1 in addition to the widely FHOD1 by Src and ROCK1 to release FHOD1.24,25 Unlike the KASH null expressed version in fibroblasts: one vari- autoinhibition might be relevant to acti- nesprin-2 mouse, the KASH null nesprin- ant contains a 28 aa insert in the ABS, the vate FHOD1 for TAN line formation. 1 mouse does not have brain developmen- other variant is a short N-terminal form However, expression of dominant negative tal defects.30 This difference in phenotype that includes a unique 7 aa insert and ter- RhoA does not inhibit centrosome orien- may reflect FHOD1’s specific interaction minates within the ABS.19 Neither of tation, suggesting that activation of with nesprin-2G and raises the possibility these splice variants have been functionally FHOD1 by ROCK is not required for that the nesprin-2G-FHOD1 interaction characterized, but it is interesting to note rearward nuclear movement.16 Perhaps is important for brain development. that the short variant is similar to the con- for FHOD1’s function in linking nesprin- There is an intriguing correlation struct that we used to rescue the nuclear 2G to actin cables in TAN lines, only the between proteins contributing to TAN movement defect in FHOD1 depleted phosphorylation of the N-terminus by Src line- dependent nuclear movement in cells. Additional studies to refine the loca- is required. It will be interesting to test fibroblasts and myoblasts and variants in tion of the actin binding site may reveal whether Src is required for TAN line for- their genes causing Emery-Dreifuss mus- whether this short variant retains the mation and nuclear movement. cular dystrophy (EDMD). Mutations in capacity to bind actin filaments. If so, it SRs11–12 are in one of 2 clusters of genes encoding emerin32-34 and A-type has the potential to reinforce the nesprin- SRs in nesprin-2G (SRs11–13 and SRs lamins35 were the first to be associated 2G-actin cable interaction; if not, it may 49–53) that are phylogenetically con- with EDMD, but more recent studies act as an endogenous inhibitor of the served and are predicted to interact with have identified variants in nesprin-236 and interaction. proteins based on their probability of sur- SUN2.37 In addition to skeletal dystro- The N-terminal site on FHOD1 that face exposure.24,25 Interestingly, an addi- phy, EDMD frequently leads to dilated interacts with nesprin-2G is in the same tional protein meckelin, which is localized cardiomyopathy. Furthermore, mice with region that is involved regulating its activ- in plasma membrane and ER, was found KASH null nesprin-1 and 2 genes in car- ity. This raises interesting questions con- to bind to the SRs 11–13.26-28 Taking diomyocytes develop cardiomyopathy, cerning how FHOD1 interaction with this into account, FHOD1 and meckelin and various mutations of nesprin-2 have nesprin-2G may be regulated and whether binding to the SRs could work indepen- been found in cardiomyopathy patients.38 nesprin-2G interaction with FHOD1 may dently, cooperatively, or competitively to Expression of EDMD variants of lamin A maintain it in an active state. Like other regulate the formation of TAN lines. and SUN2 or depletion of emerin, as DRFs, FHOD1 is regulated by auto- While SRs 11–13 are close to the CH occurs with many EDMD emerin

www.tandfonline.com Cell Cycle 2203 variants, leads to defective nuclear move- 5. Starr DA. A nuclear-envelope bridge positions nuclei responsive aminopeptidase and profilin IIa. Mol Endo- ment and centrosome orientation in fibro- and moves chromosomes. J Cell Sci 2009; 122:577-86; crinol 2003; 17:1216-29; PMID:12677009; http://dx. 8,10,37 PMID:19225124; http://dx.doi.org/10.1242/jcs.037622 doi.org/10.1210/me.2003-0056 blasts. Expression of FHOD1 is 6. Luxton GW, Gomes ER, Folker ES, Vintinner E, Gun- 20. Westendorf JJ. The formin/diaphanous-related protein, found in many tissues and like nesprin- dersen GG. Linear arrays of nuclear envelope proteins FHOS, interacts with Rac1 and activates transcription harness retrograde actin flow for nuclear movement. from the serum response element. J Biol Chem 2001; 2G it is particularly high in heart, skeletal Science 2010; 329:956-9; PMID:20724637; http://dx. 276:46453-9; PMID:11590143 19,30 muscle, and spleen. The coincidence doi.org/10.1126/science.1189072 21. Gasteier JE, Madrid R, Krautkramer E, Schroder S, of expression patterns of FHOD1 and 7. Luxton GW, Gomes ER, Folker ES, Worman HJ, Muranyi W, Benichou S, Fackler OT. Activation of the Gundersen GG. TAN lines: a novel nuclear envelope Rac-binding partner FHOD1 induces actin stress fibers nesprin-2G and their interaction suggest structure involved in nuclear positioning. Nucleus via a ROCK-dependent mechanism. J Biol Chem that FHOD1 like nesprin-2 plays a pivotal 2011; 2:173-81; PMID:21818410; http://dx.doi.org/ 2003; 278:38902-12; PMID:12857739 10.4161/nucl.2.3.16243 22. Hannemann S, Madrid R, Stastna J, Kitzing T, Gasteier role in the maintenance of striated muscle, 8. Folker ES, Ostlund C, Luxton GW, Worman HJ, Gun- J, Schonichen A, Bouchet J, Jimenez A, Geyer M, and the disruption of the interaction dersen GG. Lamin A variants that cause striated muscle Grosse R, et al. The Diaphanous-related Formin disease are defective in anchoring transmembrane actin- FHOD1 associates with ROCK1 and promotes Src- between nesprin-2G and FHOD1 may associated nuclear lines for nuclear movement. Proc Natl dependent plasma membrane blebbing. J Biol Chem lead to muscular dystrophies. Although Acad Sci U S A 2011; 108:131-6; PMID:21173262; 2008; 283:27891-903; PMID:18694941 variants of FHOD1 have not been associ- http://dx.doi.org/10.1073/pnas.1000824108 23. Iskratsch T, Yu CH, Mathur A, Liu S, Stevenin V, 9. Borrego-Pinto J, Jegou T, Osorio DS, Aurade F, Gorja- Dwyer J, Hone J, Ehler E, Sheetz M. FHOD1 is needed ated with disease, FHOD1’s closet rela- nacz M, Koch B, Mattaj IW, Gomes ER. Samp1 is a com- for directed forces and adhesion maturation during cell tive, FHOD3 is expressed in the heart and ponent of TAN lines and is required for nuclear spreading and migration. Dev Cell 2013; 27:545-59; movement. J Cell Sci 2012; 125:1099-105; PMID:24331927; http://dx.doi.org/10.1016/j.devcel. a missense variant of FHOD3 has been PMID:22349700; http://dx.doi.org/10.1242/jcs.087049 2013.11.003 found in a patient case with adult-onset 10. Chang W, Folker ES, Worman HJ, Gundersen GG. 24. Autore F, Pfuhl M, Quan X, Williams A, Roberts RG, familial dilated cardiomyopathy.39 Inter- Emerin organizes actin flow for nuclear movement and Shanahan CM, Fraternali F. Large-scale modelling of centrosome orientation in migrating fibroblasts. Mol the divergent spectrin repeats in nesprins: giant modular estingly, FHOD1 localizes in intercalated Biol Cell 2013; 24:3869-80; PMID:24152738; http:// proteins. PloS One 2013; 8:e63633; PMID:23671687; discs and costameres in cardiomyocytes, dx.doi.org/10.1091/mbc.E13-06-0307 http://dx.doi.org/10.1371/journal.pone.0063633 11. Kutscheidt S, Zhu R, Antoku S, Luxton GW, Stagljar I, 25. Simpson JG, Roberts RG. Patterns of evolutionary conser- unlike FHOD3’s cross-striated pat- Fackler OT, Gundersen GG. FHOD1 interaction with vation in the nesprin genes highlight probable functionally 40,41 tern, suggesting a distinct role for nesprin-2G mediates TAN line formation and nuclear important protein domains and isoforms. Biochem Soc FHOD1 in cardiomyocyte function. It movement. Nat Cell Biol 2014; 16:708-15; Transact 2008; 36:1359-67; PMID:19021556; http://dx. PMID:24880667; http://dx.doi.org/10.1038/ncb2981 doi.org/10.1042/BST0361359 will be interesting to see if FHOD1 muta- 12. Schonichen A, Mannherz HG, Behrmann E, Mazur AJ, 26. Dawe HR, Adams M, Wheway G, Szymanska K, Logan tions are associated with skeletal or cardiac Kuhn S, Silvan U, Schoenenberger CA, Fackler OT, CV, Noegel AA, Gull K, Johnson CA. Nesprin-2 inter- Raunser S, Dehmelt L, et al. FHOD1 is a combined acts with meckelin and mediates ciliogenesis via remod- muscle diseases. actin filament capping and bundling factor that selec- elling of the actin cytoskeleton. J Cell Sci 2009; tively associates with actin arcs and stress fibers. J Cell 122:2716-26; PMID:19596800; http://dx.doi.org/ Sci 2013; 126:1891-901; PMID:23444374; http://dx. 10.1242/jcs.043794 doi.org/10.1242/jcs.126706 27. Dawe HR, Smith UM, Cullinane AR, Gerrelli D, Cox Disclosure of Potential Conflicts of Interest 13. Schulte A, Stolp B, Schonichen A, Pylypenko O, Rak A, P, Badano JL, Blair-Reid S, Sriram N, Katsanis N, Fackler OT, Geyer M. The human formin FHOD1 Attie-Bitach T, et al. The Meckel-Gruber Syndrome No potential conflicts of interest were contains a bipartite structure of FH3 and GTPase-bind- proteins MKS1 and meckelin interact and are required disclosed. ing domains required for activation. Structure 2008; for primary cilium formation. Hum Mol Genet 2007; 16:1313-23; PMID:18786395; http://dx.doi.org/ 16:173-86; PMID:17185389; http://dx.doi.org/ 10.1016/j.str.2008.06.008 10.1093/hmg/ddl459 14. Takeya R, Taniguchi K, Narumiya S, Sumimoto H. 28. Wang M, Bridges JP, Na CL, Xu Y, Weaver TE. Funding The mammalian formin FHOD1 is activated through Meckel-Gruber syndrome protein MKS3 is required phosphorylation by ROCK and mediates thrombin- for endoplasmic reticulum-associated degradation of Our work on nuclear movement is sup- induced stress fibre formation in endothelial cells. surfactant protein C. J Biol Chem 2009; 284:33377- ported by NIH (grant GM 099481 and EMBO J 2008; 27:618-28; PMID:18239683; http:// 83; PMID:19815549 dx.doi.org/10.1038/emboj.2008.7 29. Chang W, Worman HJ, Gundersen GG. Accessorizing NS059352 to GGG) and the Deutsche 15. Koka S, Neudauer CL, Li X, Lewis RE, McCarthy JB, and anchoring the LINC complex for multifunctional- Forschungsgemeinschaft (grant FA 378/ Westendorf JJ. The formin-homology-domain-contain- ity. J Cell Biol 2015; 208:11-22; PMID:25559183; 15–1 to OTF). ing protein FHOD1 enhances cell migration. J Cell Sci http://dx.doi.org/10.1083/jcb.201409047 2003; 116:1745-55; PMID:12665555; http://dx.doi. 30. Zhang Q, Ragnauth CD, Skepper JN, Worth NF, War- org/10.1242/jcs.00386 ren DT, Roberts RG, Weissberg PL, Ellis JA, Shanahan 16. Palazzo AF, Joseph HL, Chen YJ, Dujardin DL, Alberts CM. Nesprin-2 is a multi-isomeric protein that binds References AS, Pfister KK, Vallee RB, Gundersen GG. Cdc42, lamin and emerin at the nuclear envelope and forms a dynein, and dynactin regulate MTOC reorientation subcellular network in skeletal muscle. J Cell Sci 2005; 1. Gundersen GG, Worman HJ. Nuclear positioning. independent of Rho-regulated microtubule stabiliza- 118:673-87; PMID:15671068; http://dx.doi.org/ Cell 2013; 152:1376-89; PMID:23498944; http://dx. tion. Curr Biol 2001; 11:1536-41; PMID:11591323; 10.1242/jcs.01642 doi.org/10.1016/j.cell.2013.02.031 http://dx.doi.org/10.1016/S0960-9822(01)00475-4 31. Zhang X, Lei K, Yuan X, Wu X, Zhuang Y, Xu T, Xu 2. Gomes ER, Jani S, Gundersen GG. Nuclear movement 17. Muthu M, Richardson KA, Sutherland-Smith AJ. The R, Han M. SUN1/2 and Syne/Nesprin-1/2 complexes regulated by Cdc42, MRCK, myosin, and actin flow crystal structures of dystrophin and utrophin spectrin connect centrosome to the nucleus during neurogenesis establishes MTOC polarization in migrating cells. Cell repeats: implications for domain boundaries. PloS One and neuronal migration in mice. Neuron 2009; 2005; 121:451-63; PMID:15882626; http://dx.doi. 2012; 7:e40066; PMID:22911693; http://dx.doi.org/ 64:173-87; http://dx.doi.org/10.1016/j.neuron.2009. org/10.1016/j.cell.2005.02.022 10.1371/journal.pone.0040066 08.018 3. Chang W, Antoku S, Ostlund C, Worman HJ, Gun- 18. Ylanne J, Scheffzek K, Young P, Saraste M. Crystal 32. Bione S, Maestrini E, Rivella S, Mancini M, Regis S, dersen GG. Linker of nucleoskeleton and cytoskeleton structure of the alpha-actinin rod reveals an extensive Romeo G, Toniolo D. Identification of a novel (LINC) complex-mediated actin-dependent nuclear torsional twist. Structure 2001; 9:597-604; X-linked gene responsible for Emery-Dreifuss muscular positioning orients centrosomes in migrating myoblasts. PMID:11470434; http://dx.doi.org/10.1016/S0969- dystrophy. Nat Genet 1994; 8:323-7; PMID:7894480 Nucleus 2015; 6:77-88; PMID:25587885 2126(01)00619-0 33. Manilal S, Nguyen TM, Sewry CA, Morris GE. The 4. Razafsky D, Zang S, Hodzic D. UnLINCing the 19. Tojo H, Kaieda I, Hattori H, Katayama N, Yoshimura Emery-Dreifuss muscular dystrophy protein, emerin, is nuclear envelope: towards an understanding of the K, Kakimoto S, Fujisawa Y, Presman E, Brooks CC, a nuclear membrane protein. Hum Mol Genet 1996; physiological significance of nuclear positioning. Bio- Pilch PF. The Formin family protein, formin homolog 5:801-8; PMID:8776595; http://dx.doi.org/10.1093/ chem Soc Trans 2011; 39:1790-4; PMID:22103527; overexpressed in spleen, interacts with the insulin- hmg/5.6.801 http://dx.doi.org/10.1042/BST20110660

2204 Cell Cycle Volume 14 Issue 14 34. Nagano A, Koga R, Ogawa M, Kurano Y, Kawada J, 2007; 16:2816-33; PMID:17761684; http://dx.doi. 39. Arimura T, Takeya R, Ishikawa T, Yamano T, Matsuo Okada R, Hayashi YK, Tsukahara T, Arahata K. org/10.1093/hmg/ddm238 A, Tatsumi T, Nomura T, Sumimoto H, Kimura A. Emerin deﬁciency at the nuclear membrane in patients 37. Meinke P, Mattioli E, Haque F, Antoku S, Columbaro Dilated cardiomyopathy-associated FHOD3 variant with Emery-Dreifuss muscular dystrophy. Nat Genet M, Straatman KR, Worman HJ, Gundersen GG, Lat- impairs the ability to induce activation of transcription 1996; 12:254-9; PMID:8589715 tanzi G, Wehnert M, et al. Muscular dystrophy-associ- factor serum response factor. Circul J 2013; 77:2990-6; 35. Bonne G, Di Barletta MR, Varnous S, Becane HM, ated SUN1 and SUN2 variants disrupt nuclear- PMID:24088304; http://dx.doi.org/10.1253/circj.CJ- Hammouda EH, Merlini L, Muntoni F, Greenberg cytoskeletal connections and myonuclear organization. 13-0255 CR, Gary F, Urtizberea JA, et al. Mutations in the gene PLoS Genet 2014; 10:e1004605; PMID:25210889; 40. Al Haj A, Mazur AJ, Radaszkiewicz K, Radaszkiewicz encoding lamin A/C cause autosomal dominant Emery- http://dx.doi.org/10.1371/journal.pgen.1004605 T, Makowiecka A, Stopschinski BE, Schonichen A, Dreifuss muscular dystrophy. Nat Genet 1999; 21:285- 38. Banerjee I, Zhang J, Moore-Morris T, Pfeiffer E, Buch- Geyer M, Mannherz HG. Distribution of formins in 8; PMID:10080180 holz KS, Liu A, Ouyang K, Stroud MJ, Gerace L, Evans cardiac muscle: FHOD1 is a component of intercalated 36. Zhang Q, Bethmann C, Worth NF, Davies JD, Wasner SM, et al. Targeted ablation of nesprin 1 and nesprin 2 discs and costameres. Eur J Cell Biol 2015; 94:101-13; C, Feuer A, Ragnauth CD, Yi Q, Mellad JA, Warren from murine myocardium results in cardiomyopathy, PMID:25555464; http://dx.doi.org/10.1016/j.ejcb. DT, et al. Nesprin-1 and -2 are involved in the patho- altered nuclear morphology and inhibition of the bio- 2014.11.003 genesis of Emery Dreifuss muscular dystrophy and are mechanical gene response. PLoS Genet 2014; 10: 41. Dwyer J, Pluess M, Iskratsch T, Dos Remedios CG, critical for nuclear envelope integrity. Hum Mol Genet e1004114; PMID:24586179; http://dx.doi.org/ Ehler E. The formin FHOD1 in cardiomyocytes. Anat 10.1371/journal.pgen.1004114 Record 2014; 297:1560-70