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Gastric cancer (GC) is one of the main causes of cancer-associated morbidity and mortality worldwide. The present study aimed to investigate the role of the gene encoding formin homology 2 domain containing 1 (FHOD1) protein in GC development. Data from The Cancer Genome Atlas were firstly analyzed, and immunohistochemistry was conducted on GC tissues. The results demonstrated that FHOD1 expression in GC tissues was significantly increased compared with adjacent non-tumor tissues. Furthermore, the expression level of FHOD1 was negatively associated with the overall survival of patients with GC. For the functional studies, lentivirus-mediated short hairpin RNA against FHOD1 and FHOD1-overexpression vectors were constructed to knockdown and overexpress the expression level of FHOD1 in human GC cell lines, respectively. The results indicated that FHOD1 knockdown inhibited the proliferation, colony formation and migratory and invasive abilities of GC cells. Conversely, overexpression of FHOD1 in GC cells promoted soft-agar colony formation and migratory and invasive abilities. In addition, it was demonstrated that genes of which expression levels were correlated with FHOD1 were enriched in the Gene Ontology term of 'extracellular matrix (ECM) structural constituent', suggesting that FHOD1 may serve an important role in the regulation of ECM. In conclusion, the present study demonstrated that FHOD1 may exert an oncogenic role in cultured GC cells and be inversely associated with the overall survival of patients with GC.

Background: As a new type of regulatory cell death, ferroptosis has been proven to be involved in cancer pathogenesis and therapeutic response. However, the detailed roles of ferroptosis or ferroptosis‐associated genes in glioma remain to be clarified. Methods: Here, we performed the TMT/iTRAQ‐Based Quantitative Proteomic Approach to identify the differentially expressed proteins between glioma specimens and adjacent tissues. Kaplan–Meier survival was used to estimate the survival values. We also explored the regulatory roles of abnormally expressed formin homology 2 domain‐containing protein 1 (FHOD1) in glioma ferroptosis sensitivity. Results: In our study, FHOD1 was identified to be the most significantly upregulated protein in glioma tissues. Multiple glioma datasets revealed that the glioma patients with low FHOD1 expression displayed favorable survival time. Functional analysis proved that the knockdown of FHOD1 inhibited cell growth and improved the cellular sensitivity to ferroptosis in glioma cells T98G and U251. Mechanically, we found the up‐regulation and hypomethylation of HSPB1, a negative regulator of ferroptosis, in glioma tissues. FHOD1 knockdown could enhance the ferroptosis sensitivity of glioma cells via up‐regulating the methylated heat‐shock protein B (HSPB1). Overexpression of HSPB1 significantly reversed FHOD1 knockdown‐mediated ferroptosis. Conclusions: In summary, this study demonstrated that the FHOD1‐HSPB1 axis exerts marked regulatory effects on ferroptosis, and might affect the prognosis and therapeutic response in glioma. [ABSTRACT FROM AUTHOR]

Hypertension-induced tunica media thickening (TMT) is the most important fundamental for the subsequent complications like stroke and cardiovascular diseases. Pathogenically, TMT originates from both vascular smooth muscle cells (VSMCs) hypertrophy due to synthesizing more amount of intracellular contractile proteins and excess secretion of extracellular matrix. However, what key molecules are involved in the pathogenesis of TMT is unknown. We hypothesize that formin homology 2 domain-containing protein 1 (FHOD1), an amply expressed mediator for assembly of thin actin filament in VSMCs, is a key regulator for the pathogenesis of TMT. In this study, we found that FHOD1 expression and its phosphorylation/activation were both upregulated in the arteries of three kinds of hypertensive rats. Ang-II induced actin filament formation and hypertrophy through activation and upregulation of FHOD1 in VSMCs. Active FHOD1-mediated actin filament assembly and secretions of collagen-1α/collagen-3α played crucial roles in Ang-II-induced VSMCs hypertrophy in vitro and hypertensive TMT in vivo. Proteomics demonstrated that activated FL-FHOD1 or its C-terminal diaphanous-autoregulatory domain significantly upregulated RNF213 (ring finger protein 213), a 591-kDa cytosolic E3 ubiquitin ligase with its loss-of-functional mutations being a susceptibility gene for Moyamoya disease which has prominent tunica media thinning in both intracranial and systemic arteries. Mechanistically, activated FHOD1 upregulated its downstream effector RNF213 independently of its classical pathway of decreasing G-actin/F-actin ratio, transcription, and translation, but dependently on its C-terminus-mediated stabilization of RNF213 protein. FHOD1-RNF213 signaling dramatically promoted collagen-1α/collagen-3α syntheses in VSMCs. Our results discovered a novel signaling axis of FHOD1-RNF213-collagen-1α/collagen-3α and its key role in the pathogenesis of hypertensive TMT. [Display omitted] • FHOD1 was activated in hypertensive rats and angiotensin-II-stimulated VSMCs. • Inactivated FHOD1 alleviated angiotensin-II-induced hypertension and TMT. • Activated FHOD1 promoted the hypertrophy, migration and fibrosis of VSMCs. • Activated FHOD1 upregulated RNF213 through a novel mechanism. • This study deepens our understanding of the pathogenesis of hypertensive TMT. [ABSTRACT FROM AUTHOR]

An organized and dynamic cytoskeleton is required for platelet formation and function. Formins are a large family of actin regulatory proteins which are also able to regulate microtubule dynamics. There are four formin family members expressed in human and mouse megakaryocytes and platelets. We have previously shown that the actin polymerization activity of formin proteins is required for cytoskeletal dynamics and platelet spreading using a small molecule inhibitor. In the current study, we analyze transgenic mouse models deficient in two of these proteins, mDia1 and Fhod1, along with a model lacking both proteins. We demonstrate that double knockout mice display macrothrombocytopenia which is due to aberrant megakaryocyte function and a small decrease in platelet lifespan. Platelet function is unaffected by the loss of these proteins. This data indicates a critical role for formins in platelet and megakaryocyte function.

Marta Hałas-Wiśniewska, Magdalena Izdebska, Wioletta Zielińska, Alina Grzanka Department of Histology and Embryology, Nicolaus Copernicus University in Toruń, Faculty of Medicine, Collegium Medicum in Bydgoszcz, Bydgoszcz 85-092, PolandCorrespondence: Marta Hałas-WiśniewskaDepartment of Histology and Embryology, Nicolaus Copernicus University in Toruń, Faculty of Medicine, Collegium Medicum in Bydgoszcz, 24 Karłowicza Street, Bydgoszcz 85-092, PolandTel +48525853725Fax +48525853734Email mhalas@cm.umk.plPurpose: Metastasis remains a serious clinical problem in which epithelial-to-mesenchymal transition is strictly involved. The change of cell phenotype is closely related to the dynamics of the cytoskeleton. Regarding the great interest in microfilaments, the manipulation of ABPs (actin-binding proteins) appears to be an interesting treatment strategy.Material: The research material was the highly aggressive A549 cells with FHOD1 (F FH1/FH2 domain-containing protein 1) downregulation. The metastatic potential of the cells and the sensitivity to treatment with alkaloids (piperlongumine, sanguinarine) were analyzed.Results: In comparison to A549 cells with naïve expression of FHOD1, those after manipulation were characterized by a reduced migratory potential. The obtained results were associated with microfilaments and vimentin reorganization induced by the manipulation of FHOD1 together with alkaloids treatment. The result was also an increase in the percentage of late apoptotic cells.Conclusion: Downregulation of FHOD1 induced reorganization of microfilament network followed by the reduction in the metastatic potential of the A549 cells, as well as their sensitization to selected compounds. The presented results and the analysis of clinical data indicate the possibility of transferring research from the basic level to in vivo models in the context of manipulation of ABPs as a new therapeutic target in oncology.Keywords: FHOD1, lung cancer, F-actin, migration, A549

Background: Gastric cancer (GC) is the third most common cause of cancer death. Intestinal type GC is a molecularly diverse disease. Formins control cytoskeletal processes and have been implicated in the progression of many cancers. Their clinical significance in GC remains unclear. Here, we characterize the expression of formin proteins FHOD1 and FMNL1 in intestinal GC tissue samples and investigate their association with clinical parameters, GC molecular subtypes and intratumoral T lymphocytes. Methods: The prognostic significance of FHOD1 and FMNL1 mRNA expression was studied with Kaplan–Meier analyses in an online database. The expression of FHOD1 and FMNL1 proteins was characterized in GC cells, and in non-neoplastic and malignant tissues utilizing tumor microarrays of intestinal GC representing different molecular subtypes. FHOD1 and FMNL1 expression was correlated with clinical parameters, molecular features and T lymphocyte infiltration. Immunohistochemical expression of neither formin correlated with survival. Results: Kaplan–Meier analysis associated high FHOD1 and FMNL1 mRNA expression with reduced overall survival (OS). Characterization of FHOD1 and FMNL1 in GC cells showed cytoplasmic expression along the actin filaments. Similar pattern was recapitulated in GC tissue samples. Elevated FMNL1 was associated with larger tumor size and higher disease stage. Downregulation of FHOD1 associated with TP53-mutated GC tumors. Tumor cell FHOD1 expression strongly correlated with high numbers of tumor-infiltrating CD8 + lymphocytes. Conclusions: FHOD1 and FMNL1 proteins are expressed in the tumor cells of intestinal GC and significantly associate with clinical parameters without direct prognostic significance. FHOD1 correlates with high intratumoral CD8 + T lymphocyte infiltration in this cohort. [ABSTRACT FROM AUTHOR]

Summary: Centrosome separation in late G2/ early prophase requires precise spatial coordination that is determined by a balance of forces promoting and antagonizing separation. The major effector of centrosome separation is the kinesin Eg5. However, the identity and regulation of Eg5-antagonizing forces is less well characterized. By manipulating candidate components, we find that centrosome separation is reversible and that separated centrosomes congress toward a central position underneath the flat nucleus. This positioning mechanism requires microtubule polymerization, as well as actin polymerization. We identify perinuclear actin structures that form in late G2/early prophase and interact with microtubules emanating from the centrosomes. Disrupting these structures by breaking the interactions of the linker of nucleoskeleton and cytoskeleton (LINC) complex with perinuclear actin filaments abrogates this centrosome positioning mechanism and causes an increase in subsequent chromosome segregation errors. Our results demonstrate how geometrical cues from the cell nucleus coordinate the orientation of the emanating spindle poles before nuclear envelope breakdown.

Background: The prognosis of glioblastoma remains poor, related to its diffuse spread within the brain. There is an ongoing search for molecular regulators of this particularly invasive behavior. One approach is to look for actin regulating proteins that might be targeted by future anti-cancer therapy. The formin family of proteins orchestrates rearrangement of the actin cytoskeleton in multiple cellular processes. Recently, the formin proteins mDia1 and mDia2 were shown to be expressed in glioblastoma in vitro, and their function could be modified by small molecule agonists. This finding implies that the formins could be future therapeutic targets in glioblastoma.Methods: In cell studies, we investigated the changes in expression of the 15 human formins in primary glioblastoma cells and commercially available glioblastoma cell lines during differentiation from spheroids to migrating cells using transcriptomic analysis and qRT-PCR. siRNA mediated knockdown of selected formins was performed to investigate whether their expression affects glioblastoma migration. Using immunohistochemistry, we studied the expression of two formins, FHOD1 and INF2, in tissue samples from 93 IDH-wildtype glioblastomas. Associated clinicopathological parameters and follow-up data were utilized to test whether formin expression correlates with survival or has prognostic value.Results: We found that multiple formins were upregulated during migration. Knockdown of individual formins mDia1, mDia2, FHOD1 and INF2 significantly reduced migration in most studied cell lines. Among the studied formins, knockdown of INF2 generated the greatest reduction in motility in vitro. Using immunohistochemistry, we demonstrated expression of formin proteins FHOD1 and INF2 in glioblastoma tissues. Importantly, we found that moderate/high expression of INF2 was associated with significantly impaired prognosis.Conclusions: Formins FHOD1 and INF2 participate in glioblastoma cell migration. Moderate/high expression of INF2 in glioblastoma tissue is associated with worse outcome. Taken together, our in vitro and tissue studies suggest a pivotal role for INF2 in glioblastoma. When specific inhibiting compounds become available, INF2 could be a target in the search for novel glioblastoma therapies. [ABSTRACT FROM AUTHOR]

FHOD1 is a member of Diaphanous-related formins (DRFs) which belongs to the Formin family. Previous studies have shown that the DFRs might affect several cellular functions such as morphogenesis, cytokinesis, cell polarity, and embryonic differentiation. However, there is no evidence showing the functions of FHOD1 during oocyte meiosis. This study is aimed at exploring the roles of FHOD1 during the mammalian oocyte maturation. Immunofluorescent staining showed that FHOD1 was restricted to the nucleus in germinal vesicle (GV) stage of the oocytes, after the GV breakdown FHOD1 was primarily located at two poles of the spindle at both metaphases I and II stages. Knockdown of FHOD1 by siRNA injection did not affect polar body extrusion but generated the large polar bodies. In addition, we observed the spindle migration failure in metaphase I oocytes, with a large number of meiotic spindles anchoring in the center of cytoplasm. The expression level of cytoplasmic actin but not cortex actin was significantly reduced, indicating that FHOD1 regulates cytoplasmic actin distribution for the spindle movement. Furthermore, we found that the disruption of ROCK (the Rho-dependent protein kinase) with inhibitor Y-27632 caused the decreased FHOD1 protein expression. Therefore, our data indicate that FHOD1 is regulated by ROCK for cytoplasm actin assembly and spindle migration during mouse oocyte meiosis. [ABSTRACT FROM AUTHOR]

The functional properties of actin-regulating formin proteins are diverse and in many cases cell-type specific. FHOD1, a formin expressed predominantly in cells of mesenchymal lineage, bundles actin filaments and participates in maintenance of cell shape, migration and cellular protrusions. FHOD1 participates in cancer-associated epithelial to mesenchymal transition (EMT) in oral squamous cell carcinoma and breast cancer. The role of FHOD1 in melanomas has not been characterized. Here, we show that FHOD1 expression is typically strong in cutaneous melanomas and cultured melanoma cells while the expression is low or absent in benign nevi. By using shRNA to knockdown FHOD1 in melanoma cells, we discovered that FHOD1 depleted cells are larger, rounder and have smaller focal adhesions and inferior migratory capacity as compared to control cells. Importantly, we found FHOD1 depleted cells to have reduced colony-forming capacity and attenuated tumor growth in vivo , a finding best explained by the reduced proliferation rate caused by cell cycle arrest. Unexpectedly, FHOD1 depletion did not prevent invasive growth at the tumor margins. These results suggest that FHOD1 participates in key cellular processes that are dysregulated in malignancy, but may not be essential for melanoma cell invasion. [ABSTRACT FROM AUTHOR]

Podosomes are actin-rich adhesion structures that depend on Arp2/3-complex-based actin nucleation. We now report the identification of the formins FHOD1 and INF2 as novel components and additional actin-based regulators of podosomes in primary human macrophages. FHOD1 surrounds the podosome core and is also present at podosome-connecting cables, whereas INF2 localizes at the podosome cap structure. Using a variety of microscopy-based methods; including a semiautomated podosome reformation assay, measurement of podosome oscillations, FRAP analysis of single podosomes, and structured illumination microscopy, both formins were found to regulate different aspects of podosome-associated contractility, with FHOD1 mediating actomyosin contractility between podosomes, and INF2 regulating contractile events at individual podosomes. Moreover, INF2 was found to be a crucial regulator of podosome de novo formation and size. Collectively, we identify FHOD1 and INF2 as novel regulators of inter- and intra-structural contractility of podosomes. Podosomes thus present as one of the few currently identified structures which depend on the concerted activity of both Arp2/3 complex and specific formins and might serve as a model system for the analysis of complex actin architectures in cells. [ABSTRACT FROM AUTHOR]

Vaccinia virus dissemination relies on the recruitment of the nucleation promoting factor N-WASP underneath cell-associated extracellular virus (CEVs) and subsequent recruitment and activation of the ARP2/3 complex, a major actin nucleator of the host cell. We have recently discovered that, in addition to the N-WASP/ARP2/3 pathway, vaccinia actin-based motility also relies on the small GTPase Rac1 and its downstream effector the formin-type actin nucleator FHOD1. Here we discuss the potential signaling mechanisms supporting the integration of the N-WASP/ARP2/3 and Rac1/FHOD1 pathways. We suggest the existence of a receptor tyrosine kinase family member that would integrate the Src-dependent activation of the N-WASP/ARP2/3 pathway and the GTP exchange factor-dependent activation of the Rac1/FHOD1 pathway. [ABSTRACT FROM PUBLISHER]

The formin homology domain-containing protein1 (FHOD1) suppresses actin polymerization by inhibiting nucleation, but bundles actin filaments and caps filament barbed ends. Two polyclonal antibodies against FHOD1 were generated against (i) its N-terminal sequence (residues 1–339) and (ii) a peptide corresponding the sequence from position 358–371, which is unique for FHOD1 and does not occur in its close relative FHOD3. After affinity purification both antibodies specifically stain purified full length FHOD1 and a band of similar molecular mass in homogenates of cardiac muscle. The antibody against the N-terminus of FHOD1 was used for immunostaining cells of established lines, primary neonatal (NRC) and adult (ARC) rat cardiomyocytes and demonstrated the presence of FHOD1 in HeLa and fibroblastic cells along stress fibers and within presumed lamellipodia and actin arcs. In NRCs and ARCs we observed a prominent staining of presumed intercalated discs (ICD). Immunostaining of sections of hearts with both anti-FHOD1 antibodies confirmed the presence of FHOD1 in ICDs and double immunostaining demonstrated its colocalisation with cadherin, plakoglobin and a probably slightly shifted localization to connexin43. Similarly, immunostaining of isolated mouse or pig ICDs corroborated the presence of FHOD1 and its colocalisation with the mentioned cell junctional components. Anti-FHOD1 immunoblots of isolated ICDs demonstrated the presence of an immunoreactive band comigrating with purified FHOD1. Conversely, an anti-peptide antibody specific for FHOD3 with no cross-reactivity against FHOD1 immunostained on sections of cardiac muscle and ARCs the myofibrils in a cross-striated pattern but not the ICDs. In addition, the anti-peptide-FHOD1 antibody stained the lateral sarcolemma of ARCs in a banded pattern. Double immunostaining with anti-cadherin and -integrin-ß1 indicated the additional localization of FHOD1 in costameres. Immunostaining of cardiac muscle sections or ARCs with antibodies against mDia3-FH2-domain showed colocalisation with cadherin along the lateral border of cardiomyocytes suggesting also its presence in costameres. [ABSTRACT FROM AUTHOR]

Formin proteins were recognized as effectors of Rho GTPases some 15 years ago. They contribute to different cellular actin cytoskeleton structures by their ability to polymerize straight actin filaments at the barbed end. While not all formins necessarily interact with Rho GTPases, a subgroup of mammalian formins, termed Diaphanous-related formins or DRFs, were shown to be activated by small GTPases of the Rho superfamily. DRFs are autoinhibited in the resting state by an N- to C-terminal interaction that renders the central actin polymerization domain inactive. Upon the interaction with a GTP-bound Rho, Rac, or Cdc42 GTPase, the C-terminal autoregulation domain is displaced from its N-terminal recognition site and the formin becomes active to polymerize actin filaments. In this review we discuss the current knowledge on the structure, activation, and function of formin-GTPase interactions for the mammalian formin families Dia, Daam, FMNL, and FHOD. We describe both direct and indirect interactions of formins with GTPases, which lead to formin activation and cytoskeletal rearrangements. The multifaceted function of formins as effector proteins of Rho GTPases thus reflects the diversity of the actin cytoskeleton in cells. [ABSTRACT FROM PUBLISHER]

The formin FHOD1 (formin homology 2 domain containing protein 1) can act as a capping and bundling protein in vitro. In cells, active FHOD1 stimulates the formation of ventral stress fibers. However, the cellular mechanisms by which this phenotype is produced and the physiological relevance of FHOD1 function are not currently understood. Here, we first show that FHOD1 controls the formation of two distinct stress fiber precursors differentially. On the one hand, it inhibits dorsal fiber growth, which requires the polymerization of parallel bundles of long actin filaments. On the other hand, it stimulates transverse arcs that are formed by the fusion of short antiparallel actin filaments. This combined action is crucial for the maturation of stress fibers and their spatio-temporal organization, and a lack of FHOD1 function perturbs dynamic cell behavior during cell migration. Furthermore, we show that the GTPase-binding and formin homology 3 domains (GBD and FH3) are responsible for stress fiber association and colocalization with myosin. Surprisingly, a version of FHOD1 that lacks these domains nevertheless retains its full capacity to stimulate arc and ventral stress fiber formation. Based on our findings, we propose a mechanism in which FHOD1 promotes the formation of short actin filaments and transiently associates with transverse arcs, thus providing tight temporal and spatial control of the formation and turnover of transverse arcs into mature ventral stress fibers during dynamic cell behavior. [ABSTRACT FROM AUTHOR]

Spatiotemporal regulation of mitotic kinase activity underlies the extensive rearrangement of cellular components required for cell division. One highly dynamic mitotic kinase is Aurora-B (AurB), which has multiple roles defined by the changing localisation of the chromosome passenger complex (CPC) as cells progress through mitosis, including regulation of cytokinesis and abscission. Like other mitotic kinases, AurB is a target of the anaphase-promoting complex (APC/C) ubiquitin ligase during mitotic exit, but it is not known if APC/C-mediated destruction plays any specific role in controlling AurB activity. We have examined the contribution of the Cdh1 coactivator-associated APC/CCdh1 to the organization of AurB activity as cells exit mitosis and re-enter interphase. We report that APC/ CCdh1-dependent proteolysis restricts a cell-cortex-associated pool of active AurB in space and time. In early G1 phase this pool of AurB is found at protrusions associated with cell spreading. AurB retention at the cortex depends on a formin, FHOD1, critically required to organize the cytoskeleton after division. We identify AurB phosphorylation sites in FHOD1 and show that phosphomutant FHOD1 is impaired in post-mitotic assembly of oriented actin cables. We propose that Cdh1 contributes to spatiotemporal organization of AurB activity and that organization of FHOD1 activity by AurB contributes to daughter cell spreading after mitosis. [ABSTRACT FROM AUTHOR]

The Ras superfamily of proteins consists of five branches: Ras, Rho, Arf, Rab and Ran subfamilies. These proteins are involved in a plethora of biological functions spanning cytoskeletal organization, cell proliferation, transcription and intracellular trafficking. Ras-Binding Domains (RBDs) have classically been identified as autonomous ubiquitin-like folded regions that bind certain activated Ras GTPases of the Ras subfamily. In general, RBDs in many proteins have been tagged with membrane-targeting functions as in the case of the well-characterized c-Raf-RBD/Ras interaction. However, it is becoming apparent that the definition and functions of RBDs need to be revamped in order to reflect the new discoveries associated with this domain. Here, we discuss in more detail the recent advances associated with these RBDs. we highlight research identifying RBDs in formins, eLMOs and the RhoGeF, Syx and discuss the emerging role for RBDs in controlling autoinhibition relief and the newly recognized versatility of RBDs to interact with Rho and Arf family GTPases. In addition, these recent findings raise the exciting hypothesis that functional RBDs remain hidden in the proteome and are ready to be uncovered. [ABSTRACT FROM AUTHOR]

Formins are actin polymerization factors that are known to nucleate and elongate actin filaments at the barbed end. In the present study we show that human FHOD1 lacks actin nucleation and elongation capacity, but acts as an actin bundling factor with capping activity toward the filament barbed end. Constitutively active FHOD1 associates with actin filaments in filopodia and lamellipodia at the leading edge, where it moves with the actin retrograde flow. At the base of lamellipodia, FHOD1 is enriched in nascent, bundled actin arcs as well as in more mature stress fibers. This function requires actin-binding domains located N-terminally to the canonical FH1-FH2 element. The bundling phenotype is maintained in the presence of tropomyosin, confirmed by electron microscopy showing assembly of 5 to 10 actin filaments into parallel, closely spaced filament bundles. Taken together, our data suggest a model in which FHOD1 stabilizes actin filaments by protecting barbed ends from depolymerization with its dimeric FH2 domain, whereas the region N-terminal to the FH1 domain mediates F-actin bundling by simultaneously binding to the sides of adjacent F-actin filaments. [ABSTRACT FROM AUTHOR]

Formin-family proteins, in the active state, form actin-based structures such as stress fibres. Their activation mechanisms, however, are largely unknown except that mDia and its closely related formins can be activated by direct binding of the small GTPase Rho or Cdc42. Here we show that the Rho-dependent protein kinase ROCK phosphorylates the C-terminal residues Ser1131, Ser1137, and Thr1141 of formin homology domain protein 1 (FHOD1), a major endothelial formin that is normally autoinhibited by intramolecular interaction between the N- and C-terminal regions. Phosphorylation of FHOD1 at the three residues fully disrupts the autoinhibitory interaction, which culminates in formation of stress fibres. We also demonstrate that, in vascular endothelial cells, thrombin, a vasoactive substance leading to Rho activation, elicits both FHOD1 phosphorylation and stress fibre formation in a ROCK-dependent manner, and that FHOD1 depletion by RNA interference impairs thrombin-induced stress fibre formation. Based on these findings we propose a novel mechanism for activation of formin-family proteins: ROCK, activated by G protein-coupled receptor ligands such as thrombin, directly phosphorylates FHOD1 at the C-terminal region, which renders this formin in the active form, leading to stress fibre formation. [ABSTRACT FROM AUTHOR]

Formins are key regulators of actin cytoskeletal dynamics that constitute a diverse protein family that is present in all eukaryotes examined. They typically consist of more than 1000 amino acids and are defined by the presence of two conserved regions, namely the formin homology 1 and 2 domains. Additional conserved domains comprise a GTPase-binding domain for activation, a C-terminal autoregulation motif and an N-terminal recognition domain. In this study, the N-terminal region (residues 1–339) of the human formin homology domain-containing protein 1 (FHOD1) was purified and crystallized from 20%( w/ v) PEG 4000, 10%( v/ v) glycerol, 0.3 M magnesium chloride and 0.1 M Tris–HCl pH 8.0. Native crystals belong to space group P1, with unit-cell parameters a = 35.4, b = 73.9, c = 78.7 Å, α = 78.2, β = 86.2, γ = 89.7°. They contain two monomers of FHOD1 in the asymmetric unit and diffract to a resolution of 2.3 Å using a synchrotron-radiation source. [ABSTRACT FROM AUTHOR]

The formin homology (FH) proteins play a crucial role in cytoskeleton remodelling during many essential processes. In this study, we demonstrate for the first time that the formin-homology-domain-containing protein FHOD1 is cleaved by caspase-3 at the SVPD616 site during apoptosis. Using confocal microscopy, we further demonstrate that while full length FHOD1 is mostly cytoplasmic, the FHOD1 N-terminal cleavage product is diffusely localized throughout the cytoplasm and the nucleoplasm, whereas the C-terminal cleavage product is almost exclusively nuclear with some nucleolar localization. Finally, using a run-on transcription assay we show that the C-terminal FHOD1 cleavage product has the ability to inhibit RNA polymerase I transcription when overexpressed in HeLa cells as shown by blockage of BrUTP incorporation. [ABSTRACT FROM AUTHOR]

Abstract: The mammalian formin homology domain containing protein FHOD1 influences a variety of cellular events including cell migration, cytoskeletal arrangement, signal transduction, and gene expression. In this paper, we show that Src regulates a variety of FHOD1-associated effects. FHOD1 distribution to lamellipodia was prevented by the absence of Src. However, stress fiber formation induced by a C-terminal truncated form of FHOD1 was unaffected. Gene expression from an SRE-dependent promoter and from the skeletal actin promoter was induced by two truncated forms of FHOD1 and in both instances, inhibition of Src tyrosine kinase activity abrogated induction of gene expression. Furthermore, Src activity was necessary to maintain mRNA levels of FHOD1 itself, and as such, this finding represents the first description of mechanisms involved in the regulation of formin gene expression in mammals. In summary, we have identified Src as a key regulator of FHOD1 biology. [Copyright &y& Elsevier]

Abstract: Formin homology 2 domain containing protein (FHOD1), a mammalian formin, regulates cytoskeletal architecture, enhances cell migration, and induces gene expression from the serum response element. In this study, we describe co-precipitation of FHOD1 with components of the ERK MAP kinase pathway while co-precipitation of FHOD1 with p38 MAP kinase and JNK was not observed. In addition, FHOD1 co-localized to lamellipodia with Raf-1 and to stress fibers with MEK. FHOD1-induced gene expression from the serum response element was dependent on ERK MAP kinase activation, and the native skeletal actin promoter were activated by FHOD1 through the SRF site. However, FHOD1-induced stress-fiber formation and gene expression from the skeletal actin promoter was independent of ERK activation. These novel data demonstrate that FHOD1-ERK MAP kinase interaction regulates key aspects of FHOD1 biology. [Copyright &y& Elsevier]

Abstract: Diaphanous-related formins (DRFs) are actin nucleators that mediate rearrangements of the actin cytoskeleton downstream of specific Rho GTPases. The DRF Formin Homology 2 Domain containing 1 (FHOD1) interacts with the Rac1 GTPase and induces the formation of and associates with bundled actin stress fibers. Here we report that active FHOD1 also coordinates microtubules with these actin stress fibers. Expression of a constitutive active FHOD1 variant in HeLa cells not only resulted in pronounced formation of FHOD1-actin fibers but also caused marked cell elongation and parallel alignment of microtubules without affecting cytokinesis of these cells. The analysis of deletions in the FH1 and FH2 functional regions revealed that the integrity of both domains was strictly required for FHOD1''s effects on the cytoskeleton. Dominant-negative approaches demonstrated that filament coordination and cell elongation depended on the activity of the Rho-ROCK cascade, but did not involve Rac or Cdc42 activity. Experimental depolymerization of actin filaments or microtubules revealed that the formation of FHOD1-actin fibers was a prerequisite for the polarization of microtubules. However, only simultaneous disruption of both filament systems reversed the cell elongation induced by activated FHOD1. Thus, sustained cell elongation was a consequence of FHOD1-mediated actin–microtubule coordination. These results suggest filament coordination as a conserved function of mammalian DRFs. [Copyright &y& Elsevier]

An organized and dynamic cytoskeleton is required for platelet formation and function. Formins are a large family of actin regulatory proteins which are also able to regulate microtubule dynamics. There are four formin family members expressed in human and mouse megakaryocytes and platelets. We have previously shown that the actin polymerization activity of formin proteins is required for cytoskeletal dynamics and platelet spreading using a small molecule inhibitor. In the current study, we analyze transgenic mouse models deficient in two of these proteins, mDia1 and Fhod1, along with a model lacking both proteins. We demonstrate that double knockout mice display macrothrombocytopenia which is due to aberrant megakaryocyte function and a small decrease in platelet lifespan. Platelet function is unaffected by the loss of these proteins. This data indicates a critical role for formins in platelet and megakaryocyte function.

Centrosome separation in late G2/ early prophase requires precise spatial coordination that is determined by a balance of forces promoting and antagonizing separation. The major effector of centrosome separation is the kinesin Eg5. However, the identity and regulation of Eg5-antagonizing forces is less well characterized. By manipulating candidate components, we find that centrosome separation is reversible and that separated centrosomes congress toward a central position underneath the flat nucleus. This positioning mechanism requires microtubule polymerization, as well as actin polymerization. We identify perinuclear actin structures that form in late G2/early prophase and interact with microtubules emanating from the centrosomes. Disrupting these structures by breaking the interactions of the linker of nucleoskeleton and cytoskeleton (LINC) complex with perinuclear actin filaments abrogates this centrosome positioning mechanism and causes an increase in subsequent chromosome segregation errors. Our results demonstrate how geometrical cues from the cell nucleus coordinate the orientation of the emanating spindle poles before nuclear envelope breakdown. • Dynein and the actin/MT network coordinate centrosome positioning and separation • The MT/actin network antagonizes Eg5-dependent separation • LINC-complex-dependent perinuclear actin is critical for this mechanism • Disrupting the LINC/actin interaction results in centrosome mis-positioning Stiff et al. describe how prophase-specific perinuclear actin in connection with polymerizing microtubules regulates the positioning of centrosome separation before nuclear envelope breakdown. This depends on the interaction of the LINC complex with actin. Breaking this link results in centrosome mispositioning and an increase in sister chromatid segregation errors. [ABSTRACT FROM AUTHOR]

Summary Mechanical properties are cues for many biological processes in health or disease. In the heart, changes to the extracellular matrix composition and cross-linking result in stiffening of the cellular microenvironment during development. Moreover, myocardial infarction and cardiomyopathies lead to fibrosis and a stiffer environment, affecting cardiomyocyte behavior. Here, we identify that single cardiomyocyte adhesions sense simultaneous (fast oscillating) cardiac and (slow) non-muscle myosin contractions. Together, these lead to oscillating tension on the mechanosensitive adaptor protein talin on substrates with a stiffness of healthy adult heart tissue, compared with no tension on embryonic heart stiffness and continuous stretching on fibrotic stiffness. Moreover, we show that activation of PKC leads to the induction of cardiomyocyte hypertrophy in a stiffness-dependent way, through activation of non-muscle myosin. Finally, PKC and non-muscle myosin are upregulated at the costameres in heart disease, indicating aberrant mechanosensing as a contributing factor to long-term remodeling and heart failure. [ABSTRACT FROM AUTHOR]

Basal-like breast cancer is an aggressive form of breast cancer with limited treatment options. The subgroup can be identified immunohistochemically, by lack of hormone receptor expression combined with expression of basal markers such as CK5/6 and/or epidermal growth factor receptor (EGFR). In vitro, several regulators of the actin cytoskeleton are essential for efficient invasion of basal-like breast cancer cell lines. Whether these proteins are expressed in vivo determines the applicability of these findings in clinical settings. The actin-regulating formin protein FHOD1 participates in invasion of the triple-negative breast cancer cell line MDA-MB-231. Here, we measure the expression of FHOD1 protein in clinical triple-negative breast cancers by using immunohistochemistry and further characterize the expression of another formin protein, INF2. We report that basal-like breast cancers frequently overexpress formin proteins FHOD1 and INF2. In cell studies using basal-like breast cancer cell lines, we show that knockdown of FHOD1 or INF2 interferes with very similar processes: maintenance of cell shape, migration, invasion, and proliferation. Inhibition of EGFR, PI3K, or mitogen-activated protein kinase activity does not alter the expression of FHOD1 and INF2 in these cell lines. We conclude that the experimental studies on these formins have implications in the clinical behavior of basal-like breast cancer. [ABSTRACT FROM AUTHOR]

Nuclei are connected to the actin cytoskeleton for controlling its position in the cell and for mechanochemical signaling. Nesprin-2G is one of the major outer nuclear membrane proteins that links the nucleus to the actin cytoskeleton. In addition to its paired calponin homology (CH) domains, nesprin-2G interacts with actin filaments by binding the actin-bundling proteins FHOD1 and fascin. We describe methods to measure the interaction of nesprin-2G with actin filaments using an actin co-sedimentation assay and with its binding partner FHOD1 using a GST pull-down method.

Vaccinia virus dissemination relies on the recruitment of the nucleation promoting factor N-WASP underneath cell-associated extracellular virus (CEVs) and subsequent recruitment and activation of the ARP2/3 complex, a major actin nucleator of the host cell. We have recently discovered that, in addition to the N-WASP/ARP2/3 pathway, vaccinia actin-based motility also relies on the small GTPase Rac1 and its downstream effector the formin-type actin nucleator FHOD1. Here we discuss the potential signaling mechanisms supporting the integration of the N-WASP/ARP2/3 and Rac1/FHOD1 pathways. We suggest the existence of a receptor tyrosine kinase family member that would integrate the Src-dependent activation of the N-WASP/ARP2/3 pathway and the GTP exchange factor-dependent activation of the Rac1/FHOD1 pathway. [ABSTRACT FROM PUBLISHER]