FHODs: Nuclear tethered formins for nuclear mechanotransduction

In this review, we discuss FHOD formins with a focus on recent studies that reveal a new role for them as critical links for nuclear mechanotransduction. The FHOD family in vertebrates comprises two structurally related proteins, FHOD1 and FHOD3. Their similar biochemical properties suggest overlapping and redundant functions. FHOD1 is widely expressed, FHOD3 less so, with highest expression in skeletal (FHOD1) and cardiac (FHOD3) muscle where specific splice isoforms are expressed. Unlike other formins, FHODs have strong F-actin bundling activity and relatively weak actin polymerization activity. These activities are regulated by phosphorylation by ROCK and Src kinases; bundling is additionally regulated by ERK1/2 kinases. FHODs are unique among formins in their association with the nuclear envelope through direct, high affinity binding to the outer nuclear membrane proteins nesprin-1G and nesprin-2G. Recent crystallographic structures reveal an interaction between a conserved motif in one of the spectrin repeats (SRs) of nesprin-1G/2G and a site adjacent to the regulatory domain in the amino terminus of FHODs. Nesprins are components of the LINC (linker of nucleoskeleton and cytoskeleton) complex that spans both nuclear membranes and mediates bidirectional transmission of mechanical forces between the nucleus and the cytoskeleton. FHODs interact near the actin-binding calponin homology (CH) domains of nesprin-1G/2G enabling a branched connection to actin filaments that presumably strengthens the interaction. At the cellular level, the tethering of FHODs to the outer nuclear membrane mechanically couples perinuclear actin arrays to the nucleus to move and position it in fibroblasts, cardiomyocytes, and potentially other cells. FHODs also function in adhesion maturation during cell migration and in the generation of sarcomeres, activities distant from the nucleus but that are still influenced by it. Human genetic studies have identified multiple FHOD3 variants linked to dilated and hypertrophic cardiomyopathies, with many mutations mapping to “hot spots” in FHOD3 domains. We discuss how FHOD1/3’s role in reinforcing the LINC complex and connecting to perinuclear actin contributes to functions of mechanically active tissues such as striated muscle

Muscular Dystrophy-Associated SUN1 and SUN2 Variants Disrupt Nuclear-Cytoskeletal Connections and Myonuclear Organization

Proteins of the nuclear envelope (NE) are associated with a range of inherited disorders, most commonly involving muscular dystrophy and cardiomyopathy, as exemplified by Emery-Dreifuss muscular dystrophy (EDMD). EDMD is both genetically and phenotypically variable, and some evidence of modifier genes has been reported. Six genes have so far been linked to EDMD, four encoding proteins associated with the LINC complex that connects the nucleus to the cytoskeleton. However, 50% of patients have no identifiable mutations in these genes. Using a candidate approach, we have identified putative disease-causing variants in the SUN1 and SUN2 genes, also encoding LINC complex components, in patients with EDMD and related myopathies. Our data also suggest that SUN1 and SUN2 can act as disease modifier genes in individuals with co-segregating mutations in other EDMD genes. Five SUN1/SUN2 variants examined impaired rearward nuclear repositioning in fibroblasts, confirming defective LINC complex function in nuclear-cytoskeletal coupling. Furthermore, myotubes from a patient carrying compound heterozygous SUN1 mutations displayed gross defects in myonuclear organization. This was accompanied by loss of recruitment of centrosomal marker, pericentrin, to the NE and impaired microtubule nucleation at the NE, events that are required for correct myonuclear arrangement. These defects were recapitulated in C2C12 myotubes expressing exogenous SUN1 variants, demonstrating a direct link between SUN1 mutation and impairment of nuclear-microtubule coupling and myonuclear positioning. Our findings strongly support an important role for SUN1 and SUN2 in muscle disease pathogenesis and support the hypothesis that defects in the LINC complex contribute to disease pathology through disruption of nuclear-microtubule association, resulting in defective myonuclear positioning

Regulation of actin cytoskeleton remodeling and focal adhesion turnover by Abl, Crk, and Nck families of proteins

Integrin and growth factor signaling pathways regulate multiple cellular behaviors including cellular migration for various biological events such as animal development and survival. Abl family non-receptor tyrosine kinases, and Crk and Nck families of Src Homology 2 (SH2)/SH3 adaptor proteins are known to regulate actin cytoskeleton rearrangement and focal adhesion turnover to modulate cellular migration. However, the functional interactions among these three families of proteins remain elusive in these two important signaling pathways. ^ In a 1st part of my thesis project, I showed that the c-Abl PxxP motifs, which bind SH3 domains, are indispensable for the coordinated regulation of filopodium and focal adhesion formation and cell spreading dynamics during cell attachment. Candidate Abl PxxP motif binding partners were identified by screening a comprehensive SH3 domain phage display library. A combination of protein overexpression, silencing, pharmacological manipulation and mutational analysis demonstrated that the PxxP motifs of c-Abl exert their effects on actin organization by two distinct mechanisms, involving the inhibition of Crk signaling and the engagement of Nck. ^ In a 2nd part, I studied the roles of Crk family adaptors in platelet-growth factor receptor β (PDGFβR) signaling using siRNA-based gene silencing combined with mutational analysis. I showed that Crk family adaptors are essential for coordination of actin cytoskeleton rearrangement and focal adhesion turnover to promote PDGF-mediated cell migration. Interestingly, Crk adaptors were required only during the early period of PDGFβR signaling, and inhibited in large part by Abl family tyrosine kinases shortly after PDGF stimulation. Our data also suggest that Crk adaptors regulate cytoskeletal rearrangements through the selective activation of the small GTPases Rac1 or Rap1, and that the C-terminal SH3 domain of Crk determines which GTPase is activated. ^ My studies revealed previously unappreciated roles of c-Abl PxxP motifs in regulation of cell spreading and essential roles of Crk adaptors in PDGFβR signaling. The new insights gained from my thesis project provided molecular details for how these proteins work together to orchestrate actin cytoskeleton rearrangement and focal adhesion dynamics. These will help to elucidate developmental and pathological events in where these proteins play critical roles.

Distinct roles for Crk adaptor isoforms in actin reorganization induced by extracellular signals

Crk family adaptors, consisting of Src homology 2 (SH2) and SH3 protein-binding domains, mediate assembly of protein complexes in signaling. CrkI, an alternately spliced form of Crk, lacks the regulatory phosphorylation site and C-terminal SH3 domain present in CrkII and CrkL. We used gene silencing combined with mutational analysis to probe the role of Crk adaptors in platelet-derived growth-factor receptor β (PDGFβR) signaling. We demonstrate that Crk adaptors are required for formation of focal adhesions, and for PDGF-stimulated remodeling of the actin cytoskeleton and cell migration. Crk-dependent signaling is crucial during the early stages of PDGFβR activation, whereas its termination by Abl family tyrosine kinases is important for turnover of focal adhesions and progression of dorsal-membrane ruffles. CrkII and CrkL preferentially activate the small GTPase Rac1, whereas variants lacking a functional C-terminal SH3 domain, including CrkI, preferentially activate Rap1. Thus, differences in the activity of Crk isoforms, including their effectors and their ability to be downregulated by phosphorylation, are important for coordinating dynamic changes in the actin cytoskeleton in response to extracellular signals

A Crucial Role in Cell Spreading for the Interaction of Abl PxxP Motifs with Crk and Nck Adaptors

The dynamic reorganization of actin structures helps to mediate the interaction of cells with their environment. The Abl non-receptor tyrosine kinase can modulate actin rearrangement during cell attachment. Here we report that the Abl PxxP motifs, which bind Src homology 3 (SH3) domains, are indispensable for the coordinated regulation of filopodium and focal adhesion formation and cell-spreading dynamics during attachment. Candidate Abl PxxP-motif-binding partners were identified by screening a comprehensive SH3-domain phage-display library. A combination of protein overexpression, silencing, pharmacological manipulation and mutational analysis demonstrated that the PxxP motifs of Abl exert their effects on actin organization by two distinct mechanisms, involving the inhibition of Crk signaling and the engagement of Nck. These results uncover a previously unappreciated role for Abl PxxP motifs in the regulation of cell spreading

The UL12.5 Gene Product of Herpes Simplex Virus Type 1 Exhibits Nuclease and Strand Exchange Activities but Does Not Localize to the Nucleus

The herpes simplex virus type 1 (HSV-1) alkaline nuclease, encoded by the UL12 gene, plays an important role in HSV-1 replication, as a null mutant of UL12 displays a severe growth defect. Although the precise in vivo role of UL12 has not yet been determined, several in vitro activities have been identified for the protein, including endo- and exonuclease activities, interaction with the HSV-1 single-stranded DNA binding protein ICP8, and an ability to promote strand exchange in conjunction with ICP8. In this study, we examined a naturally occurring N-terminally truncated version of UL12 called UL12.5. Previous studies showing that UL12.5 exhibits nuclease activity but is unable to complement a UL12 null virus posed a dilemma and suggested that UL12.5 may lack a critical activity possessed by the full-length protein, UL12. We constructed a recombinant baculovirus capable of expressing UL12.5 and purified soluble UL12.5 from infected insect cells. The purified UL12.5 exhibited both endo- and exonuclease activities but was less active than UL12. Like UL12, UL12.5 could mediate strand exchange with ICP8 and could also be coimmunoprecipitated with ICP8. The primary difference between the two proteins was in their intracellular localization, with UL12 localizing to the nucleus and UL12.5 remaining in the cytoplasm. We mapped a nuclear localization signal to the N terminus of UL12, the domain absent from UL12.5. In addition, when UL12.5 was overexpressed so that some of the enzyme leaked into the nucleus, it was able to partially complement the UL12 null mutant

Structures of FHOD1-Nesprin1/2 complexes reveal alternate binding modes for the FH3 domain of formins

© 2020 Elsevier Ltd The nuclear position in eukaryotes is controlled by a nucleo-cytoskeletal network, critical in cell differentiation, division, and movement. Forces are transmitted through conserved Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes that traverse the nuclear envelope and engage on either side of the membrane with diverse binding partners. Nesprin-2-giant (Nes2G), a LINC element in the outer nuclear membrane, connects to the actin directly as well as through FHOD1, a formin primarily involved in actin bundling. Here, we report the crystal structure of Nes2G bound to FHOD1 and show that the presumed G-binding domain of FHOD1 is rather a spectrin repeat (SR) binding enhancer for the neighboring FH3 domain. The structure reveals that SR binding by FHOD1 is likely not regulated by the diaphanous-autoregulatory domain helix of FHOD1. Finally, we establish that Nes1G also has one FHOD1 binding SR, indicating that these abundant, giant Nesprins have overlapping functions in actin-bundle recruitment for nuclear movement

Fascin Regulates Nuclear Movement and Deformation in Migrating Cells

Fascin is an F-actin-bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells, and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers. Here, we identified the nuclear envelope protein nesprin-2 as a binding partner for fascin in a range of cell types in vitro and in vivo. Nesprin-2 interacts with fascin through a direct, F-actin-independent interaction, and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery. Moreover, disrupting the interaction between fascin and nesprin-2 C-terminal domain leads to specific defects in F-actin coupling to the nuclear envelope, nuclear movement, and the ability of cells to deform their nucleus to invade through confined spaces. Together, our results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion

Expression of LINC complex proteins is increased in patient MD-1 (<i>SUN1</i> p.G68D/p.G338S) myoblasts.

<p>(<b>A</b>) Control and MD-1 myoblasts were fixed in methanol and analysed by immunofluorescence microscopy using SUN1, SUN2, emerin, nesprin-2G and lamin A/C antibodies, as indicated, together with DAPI staining of DNA. Scale bar, 22 µm. (<b>B</b>) Mean fluorescence intensity of SUN1, SUN2, emerin, nesprin 2 and lamin A/C was measured in individual DAPI-stained nuclei using an Olympus Scan∧R screening station and analysed using Scan∧R analysis software. The results are presented as mean ± S.E. of 1000 cells taken from at least 3 independent experiments. **<i>P</i>≤0.05. Significant <i>P</i>-value for SUN1 was <i>P</i> = 0.009. (<b>C</b>) Total protein extracts from control (C) and patient MD-1 myoblasts were Western blotted using antibodies against LINC complex-associated proteins, as indicated. (<b>D</b>) Samples prepared as in A were Western blotted using nesprin-2 (N2–N3) antibodies. (<b>E–F</b>) Protein expression was quantified by densitometric analysis of at least 3 independent experiments. The results are presented as mean ± S.E. *<i>P</i>≤0.05 and **<i>P</i>≤0.01. Each significant <i>P</i>- values are as follows: SUN1 <i>P</i> = 0.05, α-tubulin <i>P</i> = 0.003, nesprin-2 <i>P</i> = 0.002. <i>P</i>- value for emerin was <i>P</i> = 0.06.</p