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Formin is an important player in promoting apical actin polymerization in pollen tubes, but the mechanism regulating its activity remains unknown. We here identify REN1, a Rho GTPase-activating protein, as a negative regulator of formins in Arabidopsis pollen tubes. Specifically, we found that depletion of REN1 promotes apical actin polymerization and increases the amount of filamentous actin in pollen tubes. Interestingly, the effect of REN1 loss of function phenocopies the effect of formin gain of function, as it causes the formation of supernumerary membrane-derived actin bundles, which leads to tube swelling and membrane deformation. Importantly, inhibition of formins suppresses the phenotypic defects in ren1 mutant pollen tubes. We further demonstrate that REN1 physically interacts with the Arabidopsis formin protein AtFH5, predominantly with the C terminus, and inhibits the ability of AtFH5 to nucleate and assemble actin in vitro. Depletion of AtFH5 partially suppresses the phenotype in ren1 mutant pollen tubes, demonstrating that REN1 regulates apical actin polymerization at least partially through inhibiting AtFH5. We thus uncover a novel mechanism regulating formins and actin polymerization in plants. [Display omitted] • Depletion of REN1 promotes apical actin polymerization in pollen tubes • REN1 inhibits the ability of AtFH5 to nucleate actin assembly in vitro • Depletion of AtFH5 partially suppresses the phenotype in ren1 mutant pollen tubes • AtFH5 interacts with REN1 predominantly through its C terminus Xu et al. demonstrate that REN1, a Rho GTPase-activating protein, regulates apical actin polymerization through inhibiting Arabidopsis formin protein AtFH5 in pollen tubes. This study uncovers a novel mechanism regulating formins and actin polymerization in plants. [ABSTRACT FROM AUTHOR]

Abstract The patterns of Formin B and of the Arp2/3 complex formed during mitosis were studied in a mutant of Dictyostelium discoideum that produces multinucleate cells, which divide by the ingression of unilateral cleavage furrows. During cytokinesis the cells of this mutant remain spread on a glass surface where they generate a planar pattern based on the sorting-out of actin-binding proteins. During anaphase, Formin B and Arp2/3 became localized to the regions of microtubule asters around the centrosomes; Formin B in particular in the form of round, quite uniformly covered areas. These areas have been shown to be depleted of myosin II and the actin-filament crosslinker cortexillin, and to be avoided by cleavage furrows on their path into the cell.

The patterns of Formin B and of the Arp2/3 complex formed during mitosis were studied in a mutant of Dictyostelium discoideum that produces multinucleate cells, which divide by the ingression of unilateral cleavage furrows. During cytokinesis the cells of this mutant remain spread on a glass surface where they generate a planar pattern based on the sorting-out of actin-binding proteins. During anaphase, Formin B and Arp2/3 became localized to the regions of microtubule asters around the centrosomes; Formin B in particular in the form of round, quite uniformly covered areas. These areas have been shown to be depleted of myosin II and the actin-filament crosslinker cortexillin, and to be avoided by cleavage furrows on their path into the cell. [ABSTRACT FROM AUTHOR]

Transport of macromolecules from the nucleus to the cytoplasm is essential for nearly all cellular and developmental events, and when mis‐regulated, is associated with diseases, tumor formation/growth, and cancer progression. Nuclear Envelope (NE)‐budding is a newly appreciated nuclear export pathway for large macromolecular machineries, including those assembled to allow co‐regulation of functionally related components, that bypasses canonical nuclear export through nuclear pores. In this pathway, large macromolecular complexes are enveloped by the inner nuclear membrane, transverse the perinuclear space, and then exit through the outer nuclear membrane to release its contents into the cytoplasm. NE‐budding is a conserved process and shares many features with nuclear egress mechanisms used by herpesviruses. Despite its biological importance and clinical relevance, little is yet known about the regulatory and structural machineries that allow NE‐budding to occur in any system. Here we summarize what is currently known or proposed for this intriguing nuclear export process. [ABSTRACT FROM AUTHOR]

The leiomodin1 (LMOD1) gene, encoding a potent actin nucleator, was recently reported as a potential pathogenic gene of megacystis‐microcolon‐intestinal hypoperistalsis syndrome (MMIHS, OMIM 619362). However, only a single patient has been reported to have LMOD1 mutations, and the underlying pathogenic mechanism remains unknown. Here, we described a male infant with LMOD1 mutations presenting typical symptoms of pediatric intestinal pseudo‐obstruction (PIPO) but without megacystis and microcolon. Two compound heterozygous missense variants (c.1106C>T, p.T369M; c.1262G>A, p.R421H) were identified, both affecting highly conserved amino acid residues within the second actin‐binding site (ABS2) domain of LMOD1. Expression analysis showed that both variants resulted in significantly reduced protein amounts, especially for p.T369M, which was almost undetectable. The reduction was only partially rescued by the proteasome inhibitor MG‐132, indicating that there might be proteasome‐independent pathways involved in the degradation of the mutant proteins. Molecular modeling showed that variant p.T369M impaired the local protein conformation of the ABS2 domain, while variant p.R421H directly impaired the intermolecular interaction between ABS2 and actin. Accordingly, both variants significantly damaged LMOD1‐mediated actin nucleation. These findings provide further human genetic evidence supporting LMOD1 as a pathogenic gene underlying visceral myopathy including PIPO and MMIHS, strengthen the critical role of ABS2 domain in LMOD1‐mediated actin nucleation, and moreover, reveal an unrecognized role of ABS2 in protein stability. [ABSTRACT FROM AUTHOR]

Weibel–Palade bodies (WPB) are endothelial cell-specific storage granules that regulate vascular hemostasis by releasing the platelet adhesion receptor von Willebrand factor (VWF) following stimulation. Fusion of WPB with the plasma membrane is accompanied by the formation of actin rings or coats that support the expulsion of large multimeric VWF fibers. However, factor(s) organizing these actin ring structures have remained elusive. We now identify the actin-binding proteins Spire1 and Myosin Vc (MyoVc) as cytosolic factors that associate with WPB and are involved in actin ring formation at WPB-plasma membrane fusion sites. We show that both, Spire1 and MyoVc localize only to mature WPB and that upon Ca2+ evoked exocytosis of WPB, Spire1 and MyoVc together with F-actin concentrate in ring-like structures at the fusion sites. Depletion of Spire1 or MyoVc reduces the number of these actin rings and decreases the amount of VWF externalized to the cell surface after histamine stimulation. [ABSTRACT FROM AUTHOR]

Summary: Plants respond to bacterial infection acutely with actin remodeling during plant immune responses. The mechanisms by which bacterial virulence factors (VFs) modulate plant actin polymerization remain enigmatic. Here, we show that plant-type-I formin serves as the molecular sensor for actin remodeling in response to two bacterial VFs: Xanthomonas campestris pv. campestris (Xcc) diffusible signal factor (DSF), and pathogen-associated molecular pattern (PAMP) flagellin in pattern-triggered immunity (PTI). Both VFs regulate actin assembly by tuning the clustering and nucleation activity of formin on the plasma membrane (PM) at the nano-sized scale. By being integrated within the cell-wall-PM-actin cytoskeleton (CW-PM-AC) continuum, the dynamic behavior and function of formins are highly dependent on each scaffold layer’s composition within the CW-PM-AC continuum during both DSF and PTI signaling. Our results reveal a central mechanism for rapid actin remodeling during plant-bacteria interactions, in which bacterial signaling molecules fine tune plant formin nanoclustering in a host mechanical-structure-dependent manner.

Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation.

The ARP2/3 complex and formins are the only known plant actin nucleators. Besides their actin-related functions, both systems also modulate microtubule organization and dynamics. Loss of the main housekeeping Arabidopsis thaliana Class I membrane-targeted formin FH1 (At3g25500) is known to increase cotyledon pavement cell lobing, while mutations affecting ARP2/3 subunits exhibit an opposite effect. Here we examine the role of FH1 and the ARP2/3 complex subunit ARPC5 (At4g01710) in epidermal cell morphogenesis with focus on pavement cells and trichomes using a model system of single fh1 and arpc5, as well as double fh1 arpc5 mutants. While cotyledon pavement cell shape in double mutants mostly resembled single arpc5 mutants, analysis of true leaf epidermal morphology, as well as actin and microtubule organization and dynamics, revealed a more complex relationship between the two systems and similar, rather than antagonistic, effects on some parameters. Both fh1 and arpc5 mutations increased actin network density and increased cell shape complexity in pavement cells and trichomes of first true leaves, in contrast to cotyledons. Thus, while the two actin nucleation systems have complementary roles in some aspects of cell morphogenesis in cotyledon pavement cells, they may act in parallel in other cell types and developmental stages.

The ARP2/3 complex and formins are the only known plant actin nucleators. Besides their actin-related functions, both systems also modulate microtubule organization and dynamics. Loss of the main housekeeping Arabidopsis thaliana Class I membrane-targeted formin FH1 (At3g25500) is known to increase cotyledon pavement cell lobing, while mutations affecting ARP2/3 subunits exhibit an opposite effect. Here we examine the role of FH1 and the ARP2/3 complex subunit ARPC5 (At4g01710) in epidermal cell morphogenesis with focus on pavement cells and trichomes using a model system of single fh1 and arpc5 , as well as double fh1 arpc5 mutants. While cotyledon pavement cell shape in double mutants mostly resembled single arpc5 mutants, analysis of true leaf epidermal morphology, as well as actin and microtubule organization and dynamics, revealed a more complex relationship between the two systems and similar, rather than antagonistic, effects on some parameters. Both fh1 and arpc5 mutations increased actin network density and increased cell shape complexity in pavement cells and trichomes of first true leaves, in contrast to cotyledons. Thus, while the two actin nucleation systems have complementary roles in some aspects of cell morphogenesis in cotyledon pavement cells, they may act in parallel in other cell types and developmental stages. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) serve a key function in host defense, linking innate detection of microbes to activation of pathogen-specific adaptive immune responses. DCs express cell surface receptors for HIV-1 entry, but are relatively resistant to productive viral replication. They do, however, facilitate infection of co-cultured T-helper cells through a process referred to as trans-infection. We previously showed that tetraspanin 7 (TSPAN7), a transmembrane protein, is involved, through positive regulation of actin nucleation, in the transfer of HIV-1 from the dendrites of immature monocyte-derived DCs (iMDDCs) to activated CD4+ T lymphocytes. Various molecular mechanisms have been described regarding HIV-1 trans-infection and seem to depend on DC maturation status. We sought to investigate the crosstalk between DC maturation status, TSPAN7 expression and trans-infection. We followed trans-infection through co-culture of iMDDCs with CD4+ T lymphocytes, in the presence of CXCR4-tropic replicative-competent HIV-1 expressing GFP. T cell infection, DC maturation status and dendrite morphogenesis were assessed through time both by flow cytometry and confocal microscopy. Our previously described TSPAN7/actin nucleation-dependent mechanism of HIV-1 transfer appeared to be mostly observed during the first 20 h of co-culture experiments and to be independent of HIV replication. In the course of co-culture experiments, we observed a progressive maturation of MDDCs, correlated with a decrease in TSPAN7 expression, a drastic loss of dendrites and a change in the shape of DCs. A TSPAN7 and actin nucleation-independent mechanism of trans-infection, relying on HIV-1 replication, was then at play. We discovered that TSPAN7 expression is downregulated in response to different innate immune stimuli driving DC maturation, explaining the requirement for a TSPAN7/actin nucleation-independent mechanism of HIV transfer from mature MDDCs (mMDDCs) to T lymphocytes. As previously described, this mechanism relies on the capture of HIV-1 by the I-type lectin CD169/Siglec-1 on mMDDCs and the formation of a “big invaginated pocket” at the surface of DCs, both events being tightly regulated by DC maturation. Interestingly, in iMDDCs, although CD169/Siglec-1 can capture HIV-1, this capture does not lead to HIV-1 transfer to T lymphocytes.

Dendritic cells (DCs) serve a key function in host defense, linking innate detection of microbes to activation of pathogen-specific adaptive immune responses. DCs express cell surface receptors for HIV-1 entry, but are relatively resistant to productive viral replication. They do, however, facilitate infection of co-cultured T-helper cells through a process referred to as trans-infection. We previously showed that tetraspanin 7 (TSPAN7), a transmembrane protein, is involved, through positive regulation of actin nucleation, in the transfer of HIV-1 from the dendrites of immature monocyte-derived DCs (iMDDCs) to activated CD4+ T lymphocytes. Various molecular mechanisms have been described regarding HIV-1 trans-infection and seem to depend on DC maturation status. We sought to investigate the crosstalk between DC maturation status, TSPAN7 expression and trans-infection. We followed trans-infection through co-culture of iMDDCs with CD4+ T lymphocytes, in the presence of CXCR4-tropic replicative-competent HIV-1 expressing GFP. T cell infection, DC maturation status and dendrite morphogenesis were assessed through time both by flow cytometry and confocal microscopy. Our previously described TSPAN7/actin nucleation-dependent mechanism of HIV-1 transfer appeared to be mostly observed during the first 20 h of co-culture experiments and to be independent of HIV replication. In the course of co-culture experiments, we observed a progressive maturation of MDDCs, correlated with a decrease in TSPAN7 expression, a drastic loss of dendrites and a change in the shape of DCs. A TSPAN7 and actin nucleation-independent mechanism of trans-infection, relying on HIV-1 replication, was then at play. We discovered that TSPAN7 expression is downregulated in response to different innate immune stimuli driving DC maturation, explaining the requirement for a TSPAN7/actin nucleation-independent mechanism of HIV transfer from mature MDDCs (mMDDCs) to T lymphocytes. As previously described, this mechanism relies on the capture of HIV-1 by the I-type lectin CD169/Siglec-1 on mMDDCs and the formation of a "big invaginated pocket" at the surface of DCs, both events being tightly regulated by DC maturation. Interestingly, in iMDDCs, although CD169/Siglec-1 can capture HIV-1, this capture does not lead to HIV-1 transfer to T lymphocytes. [ABSTRACT FROM AUTHOR]

Owing to the local enrichment of factors that influence its dynamics and organization, the actin cytoskeleton displays different shapes and functions within the same cell. In yeast cells, post-Golgi vesicles ride on long actin cables to the bud tip. The proteins Boi1 and Boi2 (Boi1/2) participate in tethering and docking these vesicles to the plasma membrane. Here, we show in Saccharomyces cerevisiae that Boi1/2 also recruit nucleation and elongation factors to form actin filaments at sites of exocytosis. Disrupting the connection between Boi1/2 and the nucleation factor Bud6 impairs filament formation, reduces the directed movement of the vesicles to the tip and shortens the vesicles' tethering time at the cortex. Transplanting Boi1 from the bud tip to the peroxisomal membrane partially redirects the actin cytoskeleton and the vesicular flow towards the peroxisome, and creates an alternative, rudimentary vesicle-docking zone. We conclude that Boi1/2, through interactions with Bud6 and Bni1, induce the formation of a cortical actin structure that receives and aligns incoming vesicles before fusion with the membrane. [ABSTRACT FROM AUTHOR]

Spir actin nucleators and myosin V motor proteins were recently discovered to coexist in a protein complex. The direct interaction allows the coordinated activation of actin motor proteins and actin filament track generation at vesicle membranes. By now the cooperation of myosin V (MyoV) motors and Spir actin nucleation function has only been shown in the exocytic transport of Rab11 vesicles in metaphase mouse oocytes. Next to Rab11, myosin V motors however interact with a variety of Rab GTPases including Rab3, Rab8 and Rab10. As a common theme most of the MyoV interacting Rab GTPases function at different steps along the exocytic transport routes. We here summarize the different transport functions of class V myosins and provide as proof of principle data showing a colocalization of Spir actin nucleators and MyoVa at Rab8a vesicles. This suggests that besides Rab11/MyoV transport also the Rab8/MyoV and possibly other MyoV transport processes recruit Spir actin filament nucleation function. [ABSTRACT FROM AUTHOR]

The brain encompasses a complex network of neurons with exceptionally elaborated morphologies of their axonal (signal-sending) and dendritic (signal-receiving) parts. De novo actin filament formation is one of the major driving and steering forces for the development and plasticity of the neuronal arbor. Actin filament assembly and dynamics thus require tight temporal and spatial control. Such control is particularly effective at the level of regulating actin nucleation-promoting factors, as these are key components for filament formation. Arginine methylation represents an important post-translational regulatory mechanism that had previously been mainly associated with controlling nuclear processes. We will review and discuss emerging evidence from inhibitor studies and loss-of-function models for protein arginine methyltransferases (PRMTs), both in cells and whole organisms, that unveil that protein arginine methylation mediated by PRMTs represents an important regulatory mechanism in neuritic arbor formation, as well as in dendritic spine induction, maturation and plasticity. Recent results furthermore demonstrated that arginine methylation regulates actin cytosolic cytoskeletal components not only as indirect targets through additional signaling cascades, but can also directly control an actin nucleation-promoting factor shaping neuronal cells—a key process for the formation of neuronal networks in vertebrate brains.

ABSTRACT Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro. Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility. IMPORTANCE Structures containing branched actin filaments require the Arp2/3 complex. One of the most commonly used systems to study intracellular movement generated by Arp2/3-based actin motility exploits actin-rich comet tails made by Listeria. Using these infections together with live imaging and cell-free protein reconstitution experiments, we show that another protein, palladin, can be used in place of Arp2/3 to form actin-rich structures. Additionally, we show that palladin is needed for the structural integrity of comet tails as its depletion or mutation of critical regions causes dramatic changes to comet tail organization. These findings are the first to identify a protein that can functionally replace the Arp2/3 complex and have implications for all actin-based structures thought to exclusively use that complex.

The mammalian Sorting Nexin 9 (Snx9) family consists of three paralogs: Snx9, Snx18 and Snx33. Most of the published literature to date has centered on the role of Snx9 in clathrin-mediated endocytosis (CME). Snx9 contains an Sh3 domain at its N-terminus and has been shown to interact with Dynamin and actin nucleation factors via this domain. In addition to the Sh3 domain, Snx9 also contains a C-terminal BAR domain. BAR domains are known to sense and/or induce membrane curvature. In addition to endocytosis, recent studies have implicated the Snx9 family in diverse processes such as autophagy, macropinocytosis, phagocytosis and mitosis. The Snx9 family is encoded by a single gene in Drosophila called sh3px1. In this report, we present our initial characterization of sh3px1. We found that depletion of Sh3px1 from Drosophila Schneider 2 (S2) cells resulted in defective lamellipodia formation. A similar phenotype has been reported upon depletion of Scar, the actin nucleation factor implicated in forming lamellipodia. In addition, we demonstrate that over-expression of Sh3px1 in S2 cells results in the formation of tubules as well as long protrusions. Formation of these structures required the C-terminal BAR domain as well as the adjacent Phox homology (PX) domain of Sh3px1. Furthermore, efficient protrusion formation by Sh3px1 required the actin nucleation factor Wasp. Tubules and protrusions were also generated upon over-expressing the mammalian orthologs Snx18 and Snx33 in S2 cells. By contrast, over-expressing Snx9 mostly induced long tubules.

Abstract Fibroblasts in the extra-cellular matrix (ECM) often adopt a predominantly one-dimensional fibrillar geometry by virtue of their adhesion to the fibrils in the ECM. How much forces such fibrillar fibroblasts exert and how they respond to the extended stiffness of their micro-environment comprising of other ECM components and cells are not clear. We use fibroblasts adherent on fibronectin lines micropatterned onto soft polyacrylamide gels as an in vitro experimental model that maintains fibrillar cell morphology while still letting the cell mechanically interact with a continuous micro-environment of specified stiffness. We find that the exerted traction, quantified as the strain energy or the maximum exerted traction stress, is not a function of cell length. Both the strain energy and the maximum traction stress exerted by fibrillar cells are similar for low (13 kPa) or high (45 kPa) micro-environmental stiffness. Furthermore, we find that fibrillar fibroblasts exhibit prominent linear actin structures. Accordingly, inhibition of the formin family of nucleators strongly decreases the exerted traction forces. Interestingly, fibrillar cell migration is, however, not affected under formin inhibition. Our results suggest that fibrillar cell migration in such soft microenvironments is not dependent on high cellular force exertion in the absence of other topological constraints. Highlights • Fibroblasts in a fibrillar geometry exert forces in a stiffness-insensitive manner. • Fibrillar force exertion depends on formin-based actin nucleation. • Fibrillar cell migration speed remains unaffected upon formin inhibition. [ABSTRACT FROM AUTHOR]

Cell polarity in the adult mammalian testis refers to the polarized alignment of developing spermatids during spermiogenesis and the polarized organization of organelles (e.g., phagosomes, endocytic vesicles, Sertoli cell nuclei, Golgi apparatus) in Sertoli cells and germ cells to support spermatogenesis. Without these distinctive features of cell polarity in the seminiferous epithelium, it is not possible to support the daily production of millions of sperm in the limited space provided by the seminiferous tubules in either rodent or human males through the adulthood. In short, cell polarity provides a novel mean to align spermatids and the supporting organelles (e.g., phagosomes, Golgi apparatus, endocytic vesicles) in a highly organized fashion spatially in the seminiferous epithelium during the epithelial cycle of spermatogenesis. This is analogous to different assembling units in a manufacturing plant such that as developing spermatids move along the “assembly line” conferred by Sertoli cells, different structural/functional components can be added to (or removed from) the developing spermatids during spermiogenesis, so that functional spermatozoa are produced at the end of the assembly line. Herein, we briefly review findings regarding the regulation of cell polarity in the testis with specific emphasis on developing spermatids, supported by an intriguing network of regulatory proteins along a local functional axis. Emerging evidence has suggested that cell cytoskeletons provide the tracks which in turn confer the unique assembly lines in the seminiferous epithelium. We also provide some thought-provoking concepts based on which functional experiments can be designed in future studies. [ABSTRACT FROM AUTHOR]

The actin cytoskeleton is of fundamental importance for numerous cellular processes, including intracellular transport, cell plasticity, and cell migration. However, functions of filamentous actin (F-actin) in the nucleus remain understudied due to the comparatively low abundance of nuclear actin and the resulting experimental limitations to its visualization. Owing to recent technological advances such as super-resolution microscopy and the development of nuclear-specific actin probes, essential roles of the actin cytoskeleton in the context of genome maintenance are now emerging. In addition to the contributions of monomeric actin as a component of multiple important nuclear protein complexes, nuclear actin has been found to undergo polymerization in response to DNA damage and DNA replication stress. Consequently, nuclear F-actin plays important roles in the regulation of intra-nuclear mobility of repair and replication foci as well as the maintenance of nuclear shape, two important aspects of efficient stress tolerance. Beyond actin itself, there is accumulating evidence for the participation of multiple actin-binding proteins (ABPs) in the surveillance of genome integrity, including nucleation factors and motor proteins of the myosin family. Here we summarize recent findings highlighting the importance of actin cytoskeletal factors within the nucleus in key genome maintenance pathways. • The nuclear actin cytoskeleton fulfills multiple functions during the maintenance of genome stability. • Nuclear actin filaments and myosins support double-strand break mobility and repair. • DNA replication stress responses rely on nuclear actin cytoskeletal proteins. • Nuclear F-actin and associated factors are implicated in carcinogenesis. [ABSTRACT FROM AUTHOR]

The actin cytoskeleton and associated motor proteins provide the driving forces for establishing the astonishing morphological diversity and dynamics of mammalian cells. Aside from functions in protruding and contracting cell membranes for motility, differentiation or cell division, the actin cytoskeleton provides forces to shape and move intracellular membranes of organelles and vesicles. To establish the many different actin assembly functions required in time and space, actin nucleators are targeted to specific subcellular compartments, thereby restricting the generation of specific actin filament structures to those sites. Recent research has revealed that targeting and activation of actin filament nucleators, elongators and myosin motors are tightly coordinated by conserved protein complexes to orchestrate force generation. In this Cell Science at a Glance article and the accompanying poster, we summarize and discuss the current knowledge on the corresponding protein complexes and their modes of action in actin nucleation, elongation and force generation. [ABSTRACT FROM AUTHOR]

There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes.

The evolutionarily conserved Arp2/3 complex plays a central role in nucleating the branched actin filament arrays that drive cell migration, endocytosis, and other processes. To better understand Arp2/3 complex regulation, we used single-particle electron microscopy to compare the structures of Arp2/3 complex bound to three different inhibitory ligands: glia maturation factor (GMF), Coronin, and Arpin. Although the three inhibitors have distinct binding sites on Arp2/3 complex, they each induced an “open” nucleation-inactive conformation. Coronin promoted a standard (previously described) open conformation of Arp2/3 complex, with the N-terminal β-propeller domain of Coronin positioned near the p35/ARPC2 subunit of Arp2/3 complex. GMF induced two distinct open conformations of Arp2/3 complex, which correlated with the two suggested binding sites for GMF. Furthermore, GMF synergized with Coronin in inhibiting actin nucleation by Arp2/3 complex. Arpin, which uses VCA-related acidic (A) motifs to interact with the Arp2/3 complex, induced the standard open conformation, and two new masses appeared at positions near Arp2 and Arp3. Furthermore, Arpin showed additive inhibitory effects on Arp2/3 complex with Coronin and GMF. Together, these data suggest that Arp2/3 complex conformation is highly polymorphic and that its activities can be controlled combinatorially by different inhibitory ligands. [ABSTRACT FROM AUTHOR]