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Reinforcing the LINC Complex Connection to Actin Filaments: The EXTRA VIEW Cell Cycle 14:14, 2200--2205; July 15, 2015; © 2015 Taylor & Francis Group, LLC 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 centro- some 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
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