Differing and isoform-specific roles for the formin DIAPH3 in plasma membrane blebbing and filopodia formation

Plasma membrane (PM) blebs are dynamic actin-rich cell protrusions that occur, e.g., during cytokinesis, amoeboid cell motility and cell attachment. Using a targeted siRNA screen against 21 actin nucleation factors, we identify a novel and essential role of the human diaphanous formin DIAPH3 in PM blebbing during cell adhesion. Suppression of DIAPH3 inhibited blebbing to promote rapid cell spreading involving β1-integrin. Multiple isoforms of DIAPH3 were detected on the mRNA and protein level of which isoforms 3 and 7 were the largest and most abundant isoforms that however did not induce formation of actin-rich protrusions. Rather, PM blebbing specifically involved the low abundance isoform 1 of DIAPH3 and activation of isoform 7 by deletion of the diaphanous-autoregulatory domain caused the formation of filopodia. Dimerization and actin assembly activity were essential for induction of specific cell protrusions by DIAPH3 isoforms 1 and 7. Our data suggest that the N-terminal region comprising the GTPase-binding domain determined the subcellular localization of the formin as well as its protrusion activity between blebs and filopodia. We propose that isoform-selective actin assembly by DIAPH3 exerts specific and differentially regulated functions during cell adhesion and motility.     

FHOD1 coordinates actin filament and microtubule alignment to mediate cell elongation

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.     

The formin FHOD1 and the small GTPase Rac1 promote vaccinia virus actin–based motility

Vaccinia virus dissemination relies on the N-WASP–ARP2/3 pathway, which mediates actin tail formation underneath cell-associated extracellular viruses (CEVs). Here, we uncover a previously unappreciated role for the formin FHOD1 and the small GTPase Rac1 in vaccinia actin tail formation. FHOD1 depletion decreased the number of CEVs forming actin tails and impaired the elongation rate of the formed actin tails. Recruitment of FHOD1 to actin tails relied on its GTPase binding domain in addition to its FH2 domain. In agreement with previous studies showing that FHOD1 is activated by the small GTPase Rac1, Rac1 was enriched and activated at the membrane surrounding actin tails. Rac1 depletion or expression of dominant-negative Rac1 phenocopied the effects of FHOD1 depletion and impaired the recruitment of FHOD1 to actin tails. FHOD1 overexpression rescued the actin tail formation defects observed in cells overexpressing dominant-negative Rac1. Altogether, our results indicate that, to display robust actin-based motility, vaccinia virus integrates the activity of the N-WASP–ARP2/3 and Rac1–FHOD1 pathways.     

Cellular Regulation of Extension and Retraction of Pseudopod-Like Blebs Produced by Nanosecond Pulsed Electric Field (nsPEF)

Recently we described a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells exposed to nanosecond pulsed electric field (nsPEF). In Ca²⁺-free buffer such exposure initiates formation of pseudopod-like blebs (PLBs), protrusive cylindrical cell extensions that are distinct from apoptotic and necrotic blebs. PLBs nucleate predominantly on anode-facing cell pole and extend toward anode during nsPEF exposure. Bleb extension depends on actin polymerization and availability of actin monomers. Inhibition of intracellular Ca²⁺, cell contractility, and RhoA produced no effect on PLB initiation. Meanwhile, inhibition of WASP by wiskostatin causes dose-dependent suppression of PLB growth. Soon after the end of nsPEF exposure PLBs lose directionality of growth and then retract. Microtubule toxins nocodazole and paclitaxel did not show immediate effect on PLBs; however, nocodazole increased mobility of intracellular components during PLB extension and retraction. Retraction of PLBs is produced by myosin activation and the corresponding increase in PLB cortex contractility. Inhibition of myosin by blebbistatin reduces retraction while inhibition of RhoA–ROCK pathway by Y-27632 completely prevents retraction. Contraction of PLBs can produce cell translocation resembling active cell movement. Overall, the formation, properties, and life cycle of PLBs share common features with protrusions associated with ameboid cell migration. PLB life cycle may be controlled through activation of WASP by its upstream effectors such as Cdc42 and PIP2, and main ROCK activator—RhoA. Parallels between pseudopod-like blebbing and motility blebbing may provide new insights into their underlying mechanisms.     

Effect of Fgd1 on cortactin in Arp2/3 complex-mediated actin assembly

Mutations in faciogenital dysplasia protein (Fgd1) result in the human disease faciogenital dysplasia (FGDY). Fgd1 contains a RhoGEF domain specific for Cdc42. Fgd1 also contains a Src homology (SH3) binding domain (SH3-BD) that binds directly to the SH3 domain of cortactin, which promotes actin assembly by actin-related protein (Arp)2/3 complex. Here, we report the effect of ligation of cortactin's SH3 domain by the Fgd1 SH3-BD on actin polymerization in vitro. Glutathione S-transferase (GST)-fused Fgd1 SH3-BD enhanced the ability of cortactin to stimulate Arp2/3-mediated actin polymerization. However, a synthetic peptide containing only the SH3-BD sequence had no effect. The SH3-BD peptide bound to cortactin and inhibited the effect of GST-Fgd1 SH3-BD, suggesting that GST dimerization was responsible for the stimulating effect of GST-Fgd1 SH3-BD. When GST-Fgd1 SH3-BD was prepared as a heterodimer with a control GST fusion protein (GST-Pac1), no stimulatory effect on actin polymerization was observed. In addition, when cortactin was dimerized via its N-terminus, away from the C-terminal SH3 domain, actin polymerization with Arp2/3 complex increased markedly, compared to free cortactin. Thus, cortactin ligated by Fgd1 is fully active, indicating that the cell can use Fgd1 to target actin assembly. Moreover, if Fgd1 is multimerized, then cortactin's activity should be enhanced. Fgd1 and cortactin may participate as scaffolds and signal transducers in a positive feedback cycle to promote actin assembly at the cell cortex.     

Src-mediated cortactin phosphorylation regulates actin localization and injurious blebbing in acinar cells

Suprastimulation of pancreatic acini is a well-known model for pancreatitis, and it is characterized by actin reorganization and cell blebbing. Currently, however, the mechanisms underlying regulation of these aberrant cytoskeletal and membrane dynamics and how they contribute to cell injury are unclear. We observed that suprastimulation results in a rapid activation of Src and relocalization of the actin-binding protein cortactin from the apical to the basolateral domain at the necks of membrane blebs. Furthermore, Src-mediated cortactin tyrosine phosphorylation was markedly increased after suprastimulation. Pretreatment of acini with Src inhibitors or expression of a cortactin tyrosine phospho-inhibitory mutant reduced actin redistribution and bleb formation induced by suprastimulation in vitro. Importantly, inhibition of Src activity in rat models of suprastimulation-induced pancreatitis substantially reduced disease severity, as indicated by a reduction in serum amylase and pancreatic edema and a striking improvement in tissue histology. These findings indicate a novel, disease-relevant role for Src-mediated cortactin phosphorylation in aberrant reorganization of the actin cytoskeleton, a mechanism that is likely to have implications in other types of cell injury. In addition, they suggest a potential use for Src inhibitors as an approach to reduce cell injury.     

Formin‐mediated actin polymerization promotes Salmonella invasion

Salmonella invade host cells using Type 3 secreted effectors, which modulate host cellular targets to promote actin rearrangements at the cell surface that drive bacterial uptake. The Arp2/3 complex contributes to Salmonella invasion but is not essential, indicating other actin regulatory factors are involved. Here, we show a novel role for FHOD1, a formin family member, in Salmonella invasion. FHOD1 and Arp2/3 occupy distinct microdomains at the invasion site and control distinct aspects of membrane protrusion formation. FHOD1 is phosphorylated during infection and this modification is required for promoting bacterial uptake by host cells. ROCK II, but not ROCK I, is recruited to the invasion site and is required for FHOD1 phosphorylation and for Salmonella invasion. Together, our studies revealan important phospho‐dependent FHOD1 actin polymerization pathway in Salmonella invasion.     

A Highlights from MBoC Selection: Regulation of the formin Bnr1 by septins anda MARK/Par1-family septin-associated kinase

Formin-family proteins promote the assembly of linear actin filaments and are required to generate cellular actin structures, such as actin stress fibers and the cytokinetic actomyosin contractile ring. Many formin proteins are regulated by an autoinhibition mechanism involving intramolecular binding of a Diaphanous inhibitory domain and a Diaphanous autoregulatory domain. However, the activation mechanism for these Diaphanous-related formins (DRFs) is not completely understood. Although small GTPases play an important role in relieving autoinhibition, other factors likely contribute. Here we describe a requirement for the septin Shs1 and the septin-associated kinase Gin4 for the localization and in vivo activity of the budding yeast DRF Bnr1. In budding yeast strains in which the other formin, Bni1, is conditionally inactivated, the loss of Gin4 or Shs1 results in the loss of actin cables and cell death, similar to the loss of Bnr1. The defects in these strains can be suppressed by constitutive activation of Bnr1. Gin4 is involved in both the localization and activation of Bnr1, whereas the septin Shs1 is required for Bnr1 activation but not its localization. Gin4 promotes the activity of Bnr1 independently of the Gin4 kinase activity, and Gin4 lacking its kinase domain binds to the critical localization region of Bnr1. These data reveal novel regulatory links between the actin and septin cytoskeletons.     

Src-dependent phosphorylation of ASAP1 regulates podosomes

Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.     

An active Src kinase-beta-actin association is linked to actin dynamics at the periphery of colon cancer cells

Src controls the dynamic actin cytoskeleton in fibroblasts and in cancer cells, although it is not known how direct its effects are. Using FRET/FLIM imaging, we found that wild type Src associates directly, or indirectly, with peripheral beta-actin at integrin adhesions after serum stimulation, and that an active Src kinase domain is essential. Beta-actin can be directly tyrosine-phosphorylated by Src in vitro, and in a Src-dependent manner in cells. Moreover, beta-actin dynamics are suppressed when Src is rendered kinase-inactive. Surprisingly, debilitating mutations in the Src SH2 or SH3 domains do not suppress association of Src with beta-actin. This may therefore be an example of a spatially regulated Src kinase/substrate interaction that is controlling peripheral actin dynamics. Interestingly, there is no FRET between Src and beta-actin at cadherin-mediated cell-cell contacts, despite apparent co-localization there, demonstrating precise spatial specificity of Src/beta-actin complexes.     

Silencing of TBC1D15 promotes RhoA activation and membrane blebbing

Membrane blebs are round-shaped dynamic membrane protrusions that occur under many physiological conditions. Membrane bleb production is primarily controlled by actin cytoskeletal rearrangements mediated by RhoA. Tre2–Bub2–Cdc16 (TBC) domain-containing proteins are negative regulators of the Rab family of small GTPases and contain a highly conserved TBC domain. In this report, we show that the expression of TBC1D15 is associated with the activity of RhoA and the production of membrane blebs. Depletion of TBC1D15 induced activation of RhoA and membrane blebbing, which was abolished by the addition of an inhibitor for RhoA signaling. In addition, we show that TBC1D15 is required for the accumulation of RhoA at the equatorial cortex for the ingression of the cytokinetic furrow during cytokinesis. Our results demonstrate a novel role for TBC1D15 in the regulation of RhoA during membrane blebbing and cytokinesis.     

Mobility and invasiveness of metastatic esophageal cancer are potentiated by shear stress in a ROCK- and Ras-dependent manner

To metastasize, tumor cells must adopt different morphological responses to resist shear forces encountered in circulating blood and invade through basement membranes. The Rho and Ras GTPases play a critical role in regulating this dynamic behavior. Recently, we demonstrated shear-induced activation of adherent esophageal metastatic cells, characterized by formation of dynamic membrane blebs. Although membrane blebbing has only recently been characterized as a rounded mode of cellular invasion promoted through Rho kinase (ROCK), the role of shear forces in modulating membrane blebbing activity is unknown. To further characterize membrane blebbing in esophageal metastatic cells (OC-1 cell line), we investigated the role of shear in cytoskeletal remodeling and signaling through ROCK and Ras. Our results show that actin and tubulin colocalize to the cortical ring of the OC-1 cell under static conditions. However, under shear, actin acquires a punctuate distribution and tubulin localizes to the leading edge of the OC-1 cell. We show for the first time that dynamic bleb formation is induced by shear alone independent of integrin-mediated adhesion (P < 0.001, compared with OC-1 cells). Y-27632, a specific inhibitor of ROCK, causes a significant reduction in shear-induced bleb formation and inhibits integrin _v3-Ras colocalization at the leading edge of the cell. Direct measurement of Ras activation shows that the level of GTP-bound Ras is elevated in sheared OC-1 cells and that the shear-induced increase in Ras activity is inhibited by Y-27632. Finally, we show that shear stress significantly increases OC-1 cell invasion (P < 0.007), an effect negated by the presence of Y-27632. Together our findings suggest a novel physiological role for ROCK and Ras in metastatic cell behavior. cytoskeletal remodeling; dynamic blebs     

The human formin FHOD1 contains a bipartite structure of FH3 and GTPase-binding domains required for activation

Formins induce the nucleation and polymerization of unbranched actin filaments. They share three homology domains required for profilin binding, actin polymerization, and regulation. Diaphanous-related formins (DRFs) are activated by GTPases of the Rho/Rac family, whose interaction with the N-terminal formin domain is thought to displace a C-terminal Diaphanous-autoregulatory domain (DAD). We have determined the structure of the N-terminal domains of FHOD1 consisting of a GTPase-binding domain (GBD) and the DAD-recognition domain FH3. In contrast to the formin mDia1, the FHOD1-GBD reveals a ubiquitin superfold as found similarly in c-Raf1 or PI3 kinase. This GBD is recruited by Rac and Ras GTPases in cells and plays an essential role for FHOD1-mediated actin remodeling. The FHOD1-FH3 domain is composed of five armadillo repeats, similarly to other formins. Mutation of one residue in the predicted DAD-interaction surface efficiently activates FHOD1 in cells. These results demonstrate that DRFs have evolved different molecular solutions to govern their autoregulation and GTPase specificity.