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Endosome fission is required for the release of carrier vesicles and the recycling of receptors to the plasma membrane. Early events in endosome budding and fission rely on actin branching to constrict the endosomal membrane, ultimately leading to nucleotide hydrolysis and enzymatic fission. However, our current understanding of this process is limited, particularly regarding the coordination between the early and late steps of endosomal fission. Here we have identified a novel interaction between the endosomal scaffolding protein, MICAL-L1, and the human homologue of the Drosophila Nervous Wreck (Nwk) protein, FCH and double SH3 domains protein 2 (FCHSD2). We demonstrate that MICAL-L1 recruits FCHSD2 to the endosomal membrane, where it is required for ARP2/3-mediated generation of branched actin, endosome fission and receptor recycling to the plasma membrane. Because MICAL-L1 first recruits FCHSD2 to the endosomal membrane, and is subsequently responsible for recruitment of the ATPase and fission protein EHD1 to endosomes, our findings support a model in which MICAL-L1 orchestrates endosomal fission by connecting between the early actin-driven and subsequent nucleotide hydrolysis steps of the process., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

Dynamically unstable polymers capture and move cellular cargos in bacteria and eukaryotes, but regulation of their assembly remains poorly understood. Here we describe polymerization of Alp7A, a bacterial actin-like protein (ALP) that distributes copies of plasmid pLS20 among daughter cells in Bacillus subtilis . Purified ATP-Alp7A forms dynamically unstable polymers with a high critical concentration for net assembly (cc N = 10.3 µM), but a much lower critical concentration for filament elongation (cc E = 0.6 µM). Rapid nucleation and stabilization of Alp7A polymers by the accessory factor, Alp7R, decrease cc N into the physiological range. Stable populations of Alp7A filaments appear under two conditions: (i) when Alp7R slows catastrophe rates or (ii) when Alp7A concentrations are high enough to promote filament bundling. These results reveal how dynamic instability maintains high steady-state concentrations of monomeric Alp7A, and how accessory factors regulate Alp7A assembly by modulating cc N independently of cc E ., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

Formin HOmology Domain 2-containing (FHOD) proteins are a subfamily of actin-organizing formins important for striated muscle development in many animals. We showed previously that absence of the sole FHOD protein, FHOD-1, from Caenorhabditis elegans results in thin body wall muscles with misshapen dense bodies that serve as sarcomere Z-lines. We demonstrate here that mutations predicted to specifically disrupt actin polymerization by FHOD-1 similarly disrupt muscle development, and that FHOD-1 cooperates with profilin PFN-3 for dense body morphogenesis, and with profilins PFN-2 and PFN-3 to promote body wall muscle growth. We further demonstrate that dense bodies in worms lacking FHOD-1 or PFN-2/PFN-3 are less stable than in wild-type animals, having a higher proportion of dynamic protein, and becoming distorted by prolonged muscle contraction. We also observe accumulation of actin and actin depolymerization factor/cofilin homologue UNC-60B in body wall muscle of these mutants. Such accumulations may indicate targeted disassembly of thin filaments dislodged from unstable dense bodies, possibly accounting for the abnormally slow growth and reduced body wall muscle strength in fhod-1 mutants. Overall, these results implicate FHOD protein-mediated actin assembly in forming stable sarcomere Z-lines, and identify profilin as a new contributor to FHOD activity in striated muscle development., Competing Interests: Conflicts of interest: S.A.V. is a co-founder of NemaLife, Inc., which has licensed the microfluidic technology NemaFlex for commercialization.

Dysregulated actin cytoskeleton gives rise to aberrant cell motility and metastatic spread of tumor cells. This study evaluates the effect of overexpression of wild-type versus functional mutants of MRTF-A on migration and invasion of breast cancer (BC) cells. Our studies indicate that SRF's interaction is critical for MRTF-A-induced promotion of both two-dimensional and three-dimensional cell migration, while the SAP-domain function is important selectively for three-dimensional cell migration. Increased MRTF-A activity is associated with more effective membrane protrusion, a phenotype that is attributed predominantly to SRF's interaction with MRTF. We demonstrate formin-family protein mDia2 as an important mediator of MRTF-stimulated actin polymerization at the leading edge and cell migration. Multiplexed quantitative immunohistochemistry and transcriptome analyses of clinical BC specimens further demonstrate a positive correlation between nuclear localization of MRTF with malignant traits of cancer cells and enrichment of MRTF-SRF gene signature in pair-matched distant metastases versus primary tumors. In conclusion, this study establishes a novel mechanism of MRTF-dependent regulation of cell migration and provides evidence for the association between MRTF activity and increased malignancy in human BC, justifying future development of specific small molecule inhibitors of the MRTF-SRF transcriptional complex as potential therapeutic agents in BC., Competing Interests: Conflicts of interest: The authors declare no competing conflict of interest.

The regulation of maternal mRNAs is essential for proper oogenesis, the production of viable gametes, and to avoid birth defects and infertility. Many oogenic RNA-binding proteins have been identified with roles in mRNA metabolism, some of which localize to dynamic ribonucleoprotein granules and others that appear dispersed. Here, we use a combination of in vitro condensation assays and the in vivo Caenorhabditis elegans oogenesis model to characterize the properties of the conserved KH-domain MEX-3 protein and to identify novel regulators of MEX-3 and three other translational regulators. We demonstrate that MEX-3 undergoes phase separation and appears to have intrinsic gel-like properties in vitro. We also identify novel roles for the chaperonin-containing tailless complex polypeptide 1 (CCT) chaperonin and actin in preventing ectopic RNA-binding protein condensates in maturing oocytes that appear to be independent of MEX-3 folding. The CCT chaperonin and actin also oppose the expansion of endoplasmic reticulum sheets that may promote ectopic condensation of RNA-binding proteins. These novel regulators of condensation are also required for the translational repression of maternal mRNA which is essential for oocyte quality and fertility. The identification of this regulatory network may also have implications for understanding the role of hMex3 phase transitions in cancer., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

For over 20 years, the most effective probe for live imaging of yeast actin cables has been Abp140-GFP. Here, we report that endogenously-tagged Abp140-GFP poorly decorates actin patches and cables in the bud compartment of yeast cells, while robustly decorating these structures in the mother cell. Using mutagenesis, we found that asymmetric decoration by Abp140 requires F-actin binding. By expressing integrated Bni1-Bnr1 and Bnr1-Bni1 chimeras, we demonstrate that asymmetric cable decoration by Abp140 also does not depend on which formin assembles the cables in each compartment. In contrast, the short actin-binding fragment of Abp140 (known as "Lifeact"), fused to 1x or 3xmNeonGreen and expressed from the endogenous ABP140 promoter, uniformly decorates patches and cables in both compartments. Further, this probe dramatically improves live imaging detection of cables (and patches) without altering their in vivo dynamics or cell growth. Improved detection allows us to visualize cables growing inward from the cell cortex and dynamically interacting with the vacuole. This probe also robustly decorates the cytokinetic actomyosin ring. Because Lifeact-3xmNeon expressed at relatively low levels provides intense labeling of cellular F-actin structures, this tool may improve live imaging in other organisms where higher levels of Lifeact expression are detrimental., Competing Interests: Conflict of interest: The authors declare no financial conflict of interest.

We repurposed micropillar arrays to quantify spatiotemporal inter-adhesion communication. Following the observation that integrin adhesions formed around pillar tops we relied on the precise repetitive spatial control of the pillars to reliably monitor F-actin dynamics in mouse embryonic fibroblasts as a model for spatiotemporal adhesion-related intracellular signaling. Using correlation-based analyses, we revealed localized information flows propagating between adjacent pillars that were integrated over space and time to synchronize the adhesion dynamics within the entire cell. Probing the mechanical regulation, we discovered that stiffer pillars or partial actomyosin contractility inhibition enhances inter-adhesion F-actin synchronization, and that inhibition of Arp2/3, but not formin, reduces synchronization. Our results suggest that adhesions can communicate and highlight the potential of using micropillar arrays as a tool to measure spatiotemporal intracellular signaling., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

The organization of cytoskeletal elements is pivotal for coordinating intracellular transport in eukaryotic cells. Several quantitative measures based on image analysis have been proposed to characterize morphometric features of fluorescently labeled actin networks. While helpful in detecting differences in actin organization between treatments or genotypes, the accuracy of these measures could not be rigorously assessed due to a lack of ground-truth data to which they could be compared. To overcome this limitation, we utilized coarse-grained computer simulations of actin filaments and cross-linkers to generate synthetic actin networks with varying levels of bundling. We converted the simulated networks into pseudofluorescence images similar to images obtained using confocal microscopy. Using both published and novel analysis procedures, we extracted a series of morphometric parameters and benchmarked them against analogous measures based on the ground-truth actin configurations. Our analysis revealed a set of parameters that reliably reports on actin network density, orientation, ordering, and bundling. Application of these morphometric parameters to root epidermal cells of Arabidopsis thaliana revealed subtle changes in network organization between wild-type and mutant cells. This work provides robust measures that can be used to quantify features of actin networks and characterize changes in actin organization for different experimental conditions., Competing Interests: Conflicts of interests: The authors declare no financial conflict of interest.

Mesenchymal stem cells (MSCs) are self-renewing, multipotent cells, which can be used in cellular and tissue therapeutics. MSCs cell number can be expanded in vitro, but premature differentiation results in reduced cell number and compromised therapeutic efficacies. Current techniques fail to discriminate the "stem-like" population from early stages (12 h) of differentiated MSC population. Here, we imaged nuclear structure and actin architecture using immunofluorescence and used deep learning-based computer vision technology to discriminate the early stages (6-12 h) of MSC differentiation. Convolutional neural network models trained by nucleus and actin images have high accuracy in reporting MSC differentiation; nuclear images alone can identify early stages of differentiation. Concurrently, we show that chromatin fluidity and heterochromatin levels or localization change during early MSC differentiation. This study quantifies changes in cell architecture during early MSC differentiation and describes a novel image-based diagnostic tool that could be widely used in MSC culture, expansion and utilization., Competing Interests: Conflicts of interests: The authors declare no financial conflict of interest.

Idiopathic pulmonary fibrosis (IPF), one of the most common forms of interstitial lung disease, is a poorly understood, chronic, and often fatal fibroproliferative condition with only two FDA-approved medications. Understanding the pathobiology of the fibroblast in IPF is critical to evaluating and discovering novel therapeutics. Using a decellularized lung matrix derived from patients with IPF, we generate three-dimensional hydrogels as in vitro models of lung physiology and characterize the phenotype of fibroblasts seeded into the hydrogels. When cultured in IPF extracellular matrix hydrogels, IPF fibroblasts display differential contractility compared with their normal counterparts, lose the classical myofibroblast marker α-smooth muscle actin, and increase expression of proinflammatory cytokines compared with fibroblasts seeded two-dimensionally on tissue culture dishes. We validate this proinflammatory state in fibroblast-conditioned media studies with monocytes and monocyte-derived macrophages. These findings add to a growing understanding of the lung microenvironment effect on fibroblast phenotypes, shed light on the potential role of fibroblasts as immune signaling hubs during lung fibrosis, and suggest intervention in fibroblast-immune cell cross-talk as a possible novel therapeutic avenue.

Animal cell cytokinesis, or the physical division of one cell into two, is thought to be driven by constriction of an actomyosin contractile ring at the division plane. The mechanisms underlying cell type-specific differences in cytokinesis remain unknown. Germ cells are totipotent cells that pass genetic information to the next generation. Previously, using formin cyk-1 (ts) mutant Caenorhabditis elegans 4-cell embryos, we found that the P2 germ precursor cell is protected from cytokinesis failure and can divide with greatly reduced F-actin levels at the cell division plane. Here, we identified two canonical germ fate determinants required for P2-specific cytokinetic protection: PIE-1 and POS-1. Neither has been implicated previously in cytokinesis. These germ fate determinants protect P2 cytokinesis by reducing the accumulation of septin UNC-59 and anillin ANI-1 at the division plane, which here act as negative regulators of cytokinesis. These findings may provide insight into the regulation of cytokinesis in other cell types, especially in stem cells with high potency.

In response to pheromone Saccharomyces cerevisiae extend a mating projection. This process depends on the formation of polarized actin cables which direct secretion to the mating tip and translocate the nucleus for karyogamy. Here, we demonstrate that proper mating projection formation requires the formin Bni1, as well as the actin nucleation promoting activities of Bud6, but not the formin Bnr1. Further, Bni1 is required for pheromone gradient tracking. Our work also reveals unexpected new functions for Bil2 in the pheromone response. Previously we identified Bil2 as a direct inhibitor of Bnr1 during vegetative cell growth. Here, we show that Bil2 has Bnr1-independent functions in spatially focusing Bni1-GFP at mating projection tips, and in vitro Bil2 and its binding partner Bud6 organize Bni1 into clusters that nucleate actin assembly. bil2∆ cells also display entangled Bni1-generated actin cable arrays and defects in secretory vesicle transport and nuclear positioning. At low pheromone concentrations, bil2∆ cells are delayed in establishing a polarity axis, and at high concentrations they prematurely form a second and a third mating projection. Together, these results suggest that Bil2 promotes the proper formation and timing of mating projections by organizing Bni1 and maintaining a persistent axis of polarized growth.

Recent findings indicate that Solo, a RhoGEF, is involved in cellular mechanical stress responses. However, the mechanism of actin cytoskeletal remodeling via Solo remains unclear. Therefore, this study aimed to identify Solo-interacting proteins using the BioID, a proximal-dependent labeling method, and elucidate the molecular mechanisms of function of Solo. We identified PDZ-RhoGEF (PRG) as a Solo-interacting protein. PRG colocalized with Solo in the basal area of cells, depending on Solo localization, and enhanced actin polymerization at the Solo accumulation sites. Additionally, Solo and PRG interaction was necessary for actin cytoskeletal remodeling. Furthermore, the purified Solo itself had little or negligible GEF activity, even its GEF-inactive mutant directly activated the GEF activity of PRG through interaction. Moreover, overexpression of the Solo and PRG binding domains, respectively, had a dominant-negative effect on actin polymerization and actin stress fiber formation in response to substrate stiffness. Therefore, Solo restricts the localization of PRG and regulates actin cytoskeletal remodeling in synergy with PRG in response to the surrounding mechanical environment.

The actin cytoskeleton is essential for many functions of eukaryotic cells, but the factors that nucleate actin assembly are not well understood at the organismal level or in the context of disease. To explore the function of the actin nucleation factor WHAMM in mice, we examined how Whamm inactivation impacts kidney physiology and cellular proteostasis. We show that male WHAMM knockout mice excrete elevated levels of albumin, glucose, phosphate, and amino acids, and display structural abnormalities of the kidney proximal tubule, suggesting that WHAMM activity is important for nutrient reabsorption. In kidney tissue, the loss of WHAMM results in the accumulation of the lipidated autophagosomal membrane protein LC3, indicating an alteration in autophagy. In mouse fibroblasts and human proximal tubule cells, WHAMM and its binding partner the Arp2/3 complex control autophagic membrane closure and cargo receptor recruitment. These results reveal a role for WHAMM-mediated actin assembly in maintaining kidney function and promoting proper autophagosome membrane remodeling.

Dendritic spines, the mushroom-shaped extensions along dendritic shafts of excitatory neurons, are critical for synaptic function and are one of the first neuronal structures disrupted in neurodevelopmental and neurodegenerative diseases. Microtubule (MT) polymerization into dendritic spines is an activity-dependent process capable of affecting spine shape and function. Studies have shown that MT polymerization into spines occurs specifically in spines undergoing plastic changes. However, discerning the function of MT invasion of dendritic spines requires the specific inhibition of MT polymerization into spines, while leaving MT dynamics in the dendritic shaft, synaptically connected axons and associated glial cells intact. This is not possible with the unrestricted, bath application of pharmacological compounds. To specifically disrupt MT entry into spines we coupled a MT elimination domain (MTED) from the Efa6 protein to the actin filament-binding peptide LifeAct. LifeAct was chosen because actin filaments are highly concentrated in spines and are necessary for MT invasions. Temporally controlled expression of this LifeAct-MTED construct inhibits MT entry into dendritic spines, while preserving typical MT dynamics in the dendrite shaft. Expression of this construct will allow for the determination of the function of MT invasion of spines and more broadly, to discern how MT-actin interactions affect cellular processes.

Natural killer (NK) cells patrol tissue to mediate lysis of virally infected and tumorigenic cells. Human NK cells are typically identified by their expression of neural cell adhesion molecule (NCAM, CD56), yet despite its ubiquitous expression on NK cells, CD56 remains a poorly understood protein on immune cells. CD56 has been previously demonstrated to play roles in NK cell cytotoxic function and cell migration. Specifically, CD56-deficient NK cells have impaired cell migration on stromal cells and CD56 is localized to the uropod of NK cells migrating on stroma. Here, we show that CD56 is required for NK cell migration on ICAM-1 and is required for the establishment of persistent cell polarity and unidirectional actin flow. The intracellular domain of CD56 (NCAM-140) is required for its function and the loss of CD56 leads to enlarged actin foci and sequestration of phosphorylated Pyk2 accompanied by increased size and frequency of activated LFA-1 clusters. Together, these data identify a role for CD56 in regulating human NK cell migration through modulation of actin dynamics and integrin turnover.

During neuronal development, dynamic filopodia emerge from dendrites and mature into functional dendritic spines during synaptogenesis. Dendritic filopodia and spines respond to extracellular cues, influencing dendritic spine shape and size as well as synaptic function. Previously, the E3 ubiquitin ligase TRIM9 was shown to regulate filopodia in early stages of neuronal development, including netrin-1-dependent axon guidance and branching. Here, we demonstrate that TRIM9 also localizes to dendritic filopodia and spines of murine cortical and hippocampal neurons during synaptogenesis and is required for synaptic responses to netrin. In particular, TRIM9 is enriched in the postsynaptic density (PSD) within dendritic spines and loss of Trim9 alters the PSD proteome, including the actin cytoskeleton landscape. While netrin exposure induces accumulation of the Arp2/3 complex and filamentous actin in dendritic spine heads, this response is disrupted by genetic deletion of Trim9. In addition, we document changes in the synaptic receptors associated with loss of Trim9. These defects converge on a loss of netrin-dependent increases in neuronal firing rates, indicating TRIM9 is required downstream of synaptic netrin-1 signaling. We propose that TRIM9 regulates cytoskeletal dynamics in dendritic spines and is required for the proper response to synaptic stimuli.

α-catenin (α-cat) displays force-dependent unfolding and binding to actin filaments through direct and indirect means, but features of adherens junction structure and function most vulnerable to loss of these allosteric mechanisms have not been directly compared. By reconstituting an α-cat F-actin-binding domain unfolding mutant known to exhibit enhanced binding to actin (α-cat-H0-FABD + ) into α-cat knockout Madin Darby Canine Kidney (MDCK) cells, we show that partial loss of the α-cat catch bond mechanism (via an altered H0 α-helix) leads to stronger epithelial sheet integrity with greater colocalization between the α-cat-H0-FABD + mutant and actin. α-cat-H0-FABD + -expressing cells are less efficient at closing scratch-wounds, suggesting reduced capacity for more dynamic cell-cell coordination. Evidence that α-cat-H0-FABD + is equally accessible to the conformationally sensitive α18 antibody epitope as WT α-cat and shows similar vinculin recruitment suggests this mutant engages lower tension cortical actin networks, as its M-domain is not persistently open. Conversely, α-cat-M-domain salt-bridge mutants with persistent recruitment of vinculin and phosphorylated myosin light chain show only intermediate monolayer adhesive strengths, but display less directionally coordinated and thereby slower migration speeds during wound-repair. These data show α-cat M- and FABD-unfolding mutants differentially impact cell-cell cohesion and migration properties, and suggest signals favoring α-cat-cortical actin interaction without persistent M-domain opening may improve epithelial monolayer strength through enhanced coupling to lower tension actin networks.

Intracellular cargo transport is a ubiquitous cellular process in all eukaryotes. In many cell types, membrane bound cargo is associated with molecular motors which transport cargo along microtubule and actin tracks. In Toxoplasma gondii ( T. gondii ), an obligate intracellular parasite in the phylum Apicomplexa, organization of the endomembrane pathway depends on actin and an unconventional myosin motor, myosin F (MyoF). Loss of MyoF and actin disrupts vesicle transport, organelle positioning, and division of the apicoplast, a nonphotosynthetic plastid organelle. How this actomyosin system contributes to these cellular functions is still unclear. Using live-cell imaging, we observed that MyoF-EmeraldFP (MyoF-EmFP) displayed a dynamic and filamentous-like organization in the parasite cytosol, reminiscent of cytosolic actin filament dynamics. MyoF was not associated with the Golgi, apicoplast or dense granule surfaces, suggesting that it does not function using the canonical cargo transport mechanism. Instead, we found that loss of MyoF resulted in a dramatic rearrangement of the actin cytoskeleton in interphase parasites accompanied by significantly reduced actin dynamics. However, actin organization during parasite replication and motility was unaffected by the loss of MyoF. These findings revealed that MyoF is an actin organizing protein in Toxoplasma and facilitates cargo movement using an unconventional transport mechanism.

Neuronal growth cones sense a variety of cues including chemical and mechanical ones to establish functional connections during nervous system development. Substrate-cytoskeletal coupling is an established model for adhesion-mediated growth cone advance; however, the detailed molecular and biophysical mechanisms underlying the mechanosensing and mechanotransduction process remain unclear. Here, we adapted a motor-clutch model to better understand the changes in clutch and cytoskeletal dynamics, traction forces, and substrate deformation when a growth cone interacts with adhesive substrates of different stiffnesses. Model parameters were optimized using experimental data from Aplysia growth cones probed with force-calibrated glass microneedles. We included a reinforcement mechanism at both motor and clutch level. Furthermore, we added a threshold for retrograde F-actin flow that indicates when the growth cone is strongly coupled to the substrate. Our modeling results are in strong agreement with experimental data with respect to the substrate deformation and the latency time after which substrate-cytoskeletal coupling is strong enough for the growth cone to advance. Our simulations show that it takes the shortest time to achieve strong coupling when substrate stiffness was low at 4 pN/nm. Taken together, these results suggest that Aplysia growth cones respond faster and more efficiently to soft than stiff substrates.

Phagocytosis by macrophages is a highly polarized process to destroy large target cells. Binding to particles induces extensive cortical actin-generated forces that drive the formation of elaborate pseudopods around the target particle. Postinternalization, the resultant phagosome is driven toward the cell interior on microtubules (MTs) by cytoplasmic dynein. However, it is unclear whether dynein and cargo-adaptors contribute to the earlier steps of particle internalization and phagosome formation. Here we reveal that ninein, a MT minus-end-associated protein that localizes to the centrosome, is also present at the phagocytic cup in macrophages. Ninein depletion impairs particle internalization by delaying the early F-actin recruitment to sites of particle engagement and cup formation, with no impact on F-actin dynamics beyond this initial step. Ninein forms membrane-bound clusters on phagocytic cups that do not nucleate acentrosomal MTs but instead mediate the assembly of dynein-dynactin complex at active phagocytic membranes. Both ninein depletion and pharmacological inhibition of dynein activity reduced inward displacement of bound particles into macrophages. We found that ninein and dynein motor activity were required for timely retrograde movement of phagosomes and for phagolysosome formation. Taken together, these data show that ninein, alone and with dynein, play significant roles during phagocytosis.

Myristoylated, alanine-rich C-kinase substrate (MARCKS) is an F-actin and phospholipid binding protein implicated in numerous cellular activities, including the regulation of morphology in neuronal dendrites and dendritic spines. MARCKS contains a lysine-rich effector domain that mediates its binding to plasma membrane phosphatidylinositol-4,5-biphosphate (PI(4,5)P 2 ) in a manner controlled by PKC and calcium/calmodulin. In neurons, manipulations of MARCKS concentration and membrane targeting strongly affect the numbers, shapes, and F-actin properties of dendritic spines, but the mechanisms remain unclear. Here, we tested the hypothesis that the effects of MARCKS on dendritic spine morphology are due to its capacity to regulate the availability of plasma membrane PI(4,5)P 2 . We observed that the concentration of free PI(4,5)P 2 on the dendritic plasma membrane was inversely proportional to the concentration of MARCKS. Endogenous PI(4,5)P 2 levels were increased or decreased, respectively, by acutely overexpressing either phosphatidylinositol-4-phosphate 5-kinase (PIP5K) or inositol polyphosphate 5-phosphatase (5ptase). PIP5K, like MARCKS depletion, induced severe spine shrinkage; 5ptase, like constitutively membrane-bound MARCKS, induced aberrant spine elongation. These phenotypes involved changes in actin properties driven by the F-actin severing protein cofilin. Collectively, these findings support a model in which neuronal activity regulates actin-dependent spine morphology through antagonistic interactions of MARCKS and PI(4,5)P 2 .

The capping of barbed filament ends is a fundamental mechanism for actin regulation. Capping protein controls filament growth and actin turnover in cells by binding to the barbed ends of the filaments with high affinity and slow off-rate. The interaction between capping protein and actin is regulated by capping protein interaction (CPI) motif proteins. We identified a novel CPI motif protein, Bsp1, which is involved in cytokinesis and endocytosis in budding yeast. We demonstrate that Bsp1 is an actin binding protein with a high affinity for capping protein via its CPI motif. In cells, Bsp1 regulates capping protein at endocytic sites and is a major recruiter of capping protein to the cytokinetic actin ring. Lastly, we define Bsp1-related proteins as a distinct fungi-specific CPI protein group. Our results suggest that Bsp1 promotes actin filament capping by the capping protein. This study establishes Bsp1 as a new capping protein regulator and promising candidate to regulate actin networks in fungi.

The nucleus is a mechanically stable compartment of the cell that contains the genome and performs many essential functions. Nuclear mechanical components chromatin and lamins maintain nuclear shape, compartmentalization, and function by resisting antagonistic actin contraction and confinement. Studies have yet to compare chromatin and lamins perturbations side-by-side as well as modulated actin contraction while holding confinement constant. To accomplish this, we used nuclear localization signal green fluorescent protein to measure nuclear shape and rupture in live cells with chromatin and lamin perturbations. We then modulated actin contraction while maintaining actin confinement measured by nuclear height. Wild type, chromatin decompaction, and lamin B1 null present bleb-based nuclear deformations and ruptures dependent on actin contraction and independent of actin confinement. Actin contraction inhibition by Y27632 decreased nuclear blebbing and ruptures while activation by CN03 increased rupture frequency. Lamin A/C null results in overall abnormal shape also reliant on actin contraction, but similar blebs and ruptures as wild type. Increased DNA damage is caused by nuclear blebbing or abnormal shape which can be relieved by inhibition of actin contraction which rescues nuclear shape and decreases DNA damage levels in all perturbations. Thus, actin contraction drives nuclear blebbing, bleb-based ruptures, and abnormal shape independent of changes in actin confinement.

Adherens junctions are cadherin-based structures critical for cellular architecture. E-cadherin junctions in mature epithelial cell monolayers tether to an apical actomyosin ring to form the zonula adherens (ZA). We have previously shown that the adherens junction protein PLEKHA7 associates with and regulates the function of the core RNA interference (RNAi) component AGO2 specifically at the ZA. However, the mechanism mediating AGO2 recruitment to the ZA remained unexplored. Here, we reveal that this ZA-specific recruitment of AGO2 depends on both the structural and tensile integrity of the actomyosin cytoskeleton. We found that depletion of not only PLEKHA7, but also either of the three PLEKHA7-interacting, LIM-domain family proteins, namely LMO7, LIMCH1, and PDLIM1, results in disruption of actomyosin organization and tension, as well as disruption of AGO2 junctional localization and of its miRNA-binding ability. We also show that AGO2 binds Myosin IIB and that PLEKHA7, LMO7, LIMCH1, and PDLIM1 all disrupt interaction of AGO2 with Myosin IIB at the ZA. These results demonstrate that recruitment of AGO2 to the ZA is sensitive to actomyosin perturbations, introducing the concept of mechanosensitive RNAi machinery, with potential implications in tissue remodeling and in disease.

Most eukaryotic cells utilize clathrin-mediated endocytosis as well as multiple clathrin-independent pathways to internalize proteins and membranes. Although clathrin-mediated endocytosis has been studied extensively and many machinery proteins have been identified, clathrin-independent pathways remain poorly characterized by comparison. We previously identified the first known yeast clathrin-independent endocytic pathway, which relies on the actin-modulating GTPase Rho1, the formin Bni1 and unbranched actin filaments, but does not require the clathrin coat or core clathrin machinery proteins. In this study, we sought to better understand clathrin-independent endocytosis in yeast by exploring the role of myosins as actin-based motors, because actin is required for endocytosis in yeast. We find that Myo2, which transports secretory vesicles, organelles and microtubules along actin cables to sites of polarized growth, participates in clathrin-independent endocytosis. Unexpectedly, the ability of Myo2 to transport microtubule plus ends to the cell cortex appears to be required for its role in clathrin-independent endocytosis. In addition, dynein, dynactin, and proteins involved in cortical microtubule capture are also required. Thus, our results suggest that interplay between actin and microtubules contributes to clathrin-independent internalization in yeast.

Many eukaryotic cells, including animal cells and unicellular amoebae, use dynamic-actin networks to crawl across solid surfaces. Recent discoveries of actin-dependent crawling in additional lineages have sparked interest in understanding how and when this type of motility evolved. Tracing the evolution of cell crawling requires understanding the molecular mechanisms underlying motility. Here we outline what is known about the diversity and evolution of the molecular mechanisms that drive cell motility, with a focus on actin-dependent crawling. Classic studies and recent work have revealed a surprising number of distinct mechanical modes of actin-dependent crawling used by different cell types and species to navigate different environments. The overlap in actin network regulators driving multiple types of actin-dependent crawling, along with cortical-actin networks that support the plasma membrane in these cells, suggest that actin motility and cortical actin networks might have a common evolutionary origin. The rapid development of additional evolutionarily diverse model systems, advanced imaging technologies, and CRISPR-based genetic tools, is opening the door to testing these and other new ideas about the evolution of actin-dependent cell crawling.

Directional cell migration is driven by the conversion of oscillating edge motion into lasting periods of leading edge protrusion. Actin polymerization against the membrane and adhesions control edge motion, but the exact mechanisms that determine protrusion period remain elusive. We addressed this by developing a computational model in which polymerization of actin filaments against a deformable membrane and variable adhesion dynamics support edge motion. Consistent with previous reports, our model showed that actin polymerization and adhesion lifetime power protrusion velocity. However, increasing adhesion lifetime decreased the protrusion period. Measurements of adhesion lifetime and edge motion in migrating cells confirmed that adhesion lifetime is associated with and promotes protrusion velocity, but decreased duration. Our model showed that adhesions' control of protrusion persistence originates from the Brownian ratchet mechanism for actin filament polymerization. With longer adhesion lifetime or increased-adhesion density, the proportion of actin filaments tethered to the substrate increased, maintaining filaments against the cell membrane. The reduced filament-membrane distance generated pushing force for high edge velocity, but limited further polymerization needed for protrusion duration. We propose a mechanism for cell edge protrusion in which adhesion strength regulates actin filament polymerization to control the periods of leading edge protrusion.

Myosin-1s are monomeric actin-based motors that function at membranes. Myo1 is the single myosin-1 isoform in Schizosaccharomyces pombe that works redundantly with Wsp1-Vrp1 to activate the Arp2/3 complex for endocytosis. Here, we identified Ank1 as an uncharacterized cytoplasmic Myo1 binding partner. We found that in ank1Δ cells, Myo1 dramatically redistributed from endocytic patches to decorate the entire plasma membrane and endocytosis was defective. Biochemical analysis and structural predictions suggested that the Ank1 ankyrin repeats bind the Myo1 lever arm and the Ank1 acidic tail binds the Myo1 TH1 domain to prevent TH1-dependent Myo1 membrane binding. Indeed, Ank1 overexpression precluded Myo1 membrane localization and recombinant Ank1 reduced purified Myo1 liposome binding in vitro. Based on biochemical and cell biological analyses, we propose budding yeast Ank1 and human OSTF1 are functional Ank1 orthologs and that cytoplasmic sequestration by small ankyrin repeat proteins is a conserved mechanism regulating myosin-1s in endocytosis.

Ena/VASP proteins are processive actin polymerases that are required throughout animal phylogeny for many morphogenetic processes, including axon growth and guidance. Here we use in vivo live imaging of morphology and actin distribution to determine the role of Ena in promoting the growth of the TSM1 axon of the Drosophila wing. Altering Ena activity causes stalling and misrouting of TSM1. Our data show that Ena has a substantial impact on filopodial morphology in this growth cone but exerts only modest effects on actin distribution. This is in contrast to the main regulator of Ena, Abl tyrosine kinase, which was shown previously to have profound effects on actin and only mild effects on TSM1 growth cone morphology. We interpret these data as suggesting that the primary role of Ena in this axon may be to link actin to the morphogenetic processes of the plasma membrane, rather than to regulate actin organization itself. These data also suggest that a key role of Ena, acting downstream of Abl, may be to maintain consistent organization and reliable evolution of growth cone structure, even as Abl activity varies in response to guidance cues in the environment.

Actin cytoskeleton predominantly regulates the formation and maintenance of synapses by controlling dendritic spine morphology and motility. To visualize actin dynamics, actin molecules can be labeled by genetically fusing fluorescent proteins to actin monomers, actin-binding proteins, or single-chain anti-actin antibodies. In the present study, we compared the dendritic effect of EGFP-actin, LifeAct-TagGFP2 (LifeAct-GFP), and Actin-Chromobody-TagGFP2 (AC-GFP) in mouse cultured hippocampal neurons using unbiased quantitative methods. The actin-binding probes LifeAct-GFP and AC-GFP showed similar affinity to F-actin, but in contrast to EGFP-actin, they did not reveal subtle changes in actin remodeling between mushroom-shaped spines and filopodia. All tested actin probes colocalized with phalloidin similarly; however, the enrichment of LifeAct-GFP in dendritic spines was remarkably lower compared with the other constructs. LifeAct-GFP expression was tolerated at a higher expression level compared with EGFP-actin and AC-GFP with only subtle differences identified in dendritic spine morphology and protrusion density. While EGFP-actin and LifeAct-GFP expression did not alter dendritic arborization, AC-GFP-expressing neurons displayed a reduced dendritic tree. Thus, although all tested actin probes may be suitable for actin imaging studies, certain limitations should be considered before performing experiments with a particular actin-labeling probe in primary neurons.

Apical constriction results in apical surface reduction in epithelial cells and is a widely used mechanism for epithelial morphogenesis. Both medioapical and junctional actomyosin remodeling are involved in apical constriction, but the deployment of medial versus junctional actomyosin and their genetic regulation in vertebrate embryonic development have not been fully described. In this study, we investigate actomyosin dynamics and their regulation by the RhoGEF protein Plekhg5 in Xenopus bottle cells. Using live imaging and quantitative image analysis, we show that bottle cells assume different shapes, with rounding bottle cells constricting earlier in small clusters followed by fusiform bottle cells forming between the clusters. Both medioapical and junctional actomyosin signals increase as surface area decreases, though correlation of apical constriction with medioapical actomyosin localization appears to be stronger. F-actin bundles perpendicular to the apical surface form in constricted cells, which may correspond to microvilli previously observed in the apical membrane. Knockdown of plekhg5 disrupts medioapical and junctional actomyosin activity and apical constriction but does not affect initial F-actin dynamics. Taking the results together, our study reveals distinct cell morphologies, uncovers actomyosin behaviors, and demonstrates the crucial role of a RhoGEF protein in controlling actomyosin dynamics during apical constriction of bottle cells in Xenopus gastrulation.

Cell migration on soft surfaces occurs in both physiological and pathological processes such as corticogenesis during embryonic development and cancer invasion and metastasis. The Arp2/3 complex in neural progenitor cells was previously demonstrated to be necessary for cell migration on soft elastic substrate but not on stiff surfaces, but the underlying mechanism was unclear. Here, we integrate computational and experimental approaches to elucidate how the Arp2/3 complex enables cell migration on soft surfaces. We found that lamellipodia comprised of a branched actin network nucleated by the Arp2/3 complex distribute forces over a wider area, thus decreasing stress in the substrate. Additionally, we found that interactions between parallel focal adhesions within lamellipodia prolong cell-substrate interactions by compensating for the failure of neighboring adhesions. Together with decreased substrate stress, this leads to the observed improvements in migratory ability on soft substrates in cells utilizing lamellipodia-dependent mesenchymal migration when compared with filopodia-based migration. These results show that the Arp2/3 complex-dependent lamellipodia provide multiple distinct mechanical advantages to gliomas migrating on soft 2D substrates, which can contribute to their invasive potential.

Mammalian cell migration in open spaces requires F-actin polymerization and myosin contraction. While many studies have focused on myosin's coupling to focal adhesion and stress fibers, the indirect effect of myosin contraction on cell migration through actin depolymerization is not well studied. In this work, we quantified how cell velocity and effective power output are influenced by the rate of actin depolymerization, which is affected by myosin contraction. In addition, we derived scaling laws to provide physical insights into cell migration. Model analysis shows that the cell migration velocity displays a biphasic dependence on the rate of actin depolymerization and myosin contraction. Our model further predicts that the effective cell energy output depends not only on the cell velocity but also on myosin contractility. The work has implications on in vivo processes such as immune response and cancer metastasis, where cells overcome barriers imposed by the physical environment.

Two very polarized views exist for understanding the cellular plasma membrane (PM). For some, it is the simple fluid described by the original Singer-Nicolson fluid mosaic model. For others, due to the presence of thousands of molecular species that extensively interact with each other, the PM forms various clusters and domains that are constantly changing and therefore, no simple rules exist that can explain the structure and molecular dynamics of the PM. In this article, we propose that viewing the PM from its two predominant components, cholesterol and actin filaments, provides an excellent and transparent perspective of PM organization, dynamics, and mechanisms for its functions. We focus on the actin-induced membrane compartmentalization and lipid raft domains coexisting in the PM and how they interact with each other to perform PM functions. This view provides an important update of the fluid mosaic model.

Given the role of E-cadherin (E-cad) in holding epithelial cells together, an inverse relationship between E-cad levels and cell invasion during the epithelial-mesenchymal transition and cancer metastasis has been well recognized. Here we report that E-cad is necessary for the invasiveness of Ras V12 -transformed intestinal epithelial cells in Drosophila . E-cad/β-catenin disassembles at adherens junctions and assembles at invasive protrusions--the actin- and cortactin-rich invadopodium-like protrusions associated with the breach of the extracellular matrix (ECM)--during dissemination of Ras V12 -transformed intestinal epithelial cells. Loss of E-cad impairs the elongation of invasive protrusions and attenuates the ability of Ras V12 -transformed cells to compromise the ECM. Notably, E-cad and cortactin affect each other's localization to invasive protrusions. Given the essential roles of cortactin in cell invasion, our observations indicate that E-cad plays a role in the invasiveness of Ras V12 -transformed intestinal epithelial cells by controlling cortactin localization to invasive protrusions. Thus our study demonstrates that E-cad is a component of invasive protrusions and provides molecular insights into the unconventional role of E-cad in cell dissemination in vivo.

By acting both upstream of and downstream from biochemical organizers of the cytoskeleton, physical forces function as central integrators of cell shape and movement. Here we use a combination of genetic, pharmacological, and optogenetic perturbations to probe the role of the conserved mechanosensitive mTOR complex 2 (mTORC2) programs in neutrophil polarity and motility. We find that the tension-based inhibition of leading-edge signals (Rac, F-actin) that underlies protrusion competition is gated by the kinase-independent role of the complex, whereas the regulation of RhoA and myosin II-based contractility at the trailing edge depend on mTORC2 kinase activity. mTORC2 is essential for spatial and temporal coordination of the front and back polarity programs for persistent migration under confinement. This mechanosensory pathway integrates multiple upstream signals, and we find that membrane stretch synergizes with biochemical co-input phosphatidylinositol (3,4,5)-trisphosphate to robustly amplify mTORC2 activation. Our results suggest that different signaling arms of mTORC2 regulate spatially and molecularly divergent cytoskeletal programs for efficient coordination of neutrophil shape and movement.

The actin cytoskeleton is a ubiquitous participant in cellular functions that maintain viability, but how it controls programmed cell death is not well understood. Here we show that in response to DNA damage, human cells form a juxtanuclear F-actin-rich territory that coordinates the organized progression of apoptosome assembly to caspase activation. This cytoskeletal compartment is created by the actin nucleation factors JMY, WHAMM, and the Arp2/3 complex, and it excludes proteins that inhibit JMY and WHAMM activity. Within the territory, mitochondria undergo outer membrane permeabilization and JMY localization overlaps with punctate structures containing the core apoptosome components cytochrome c and Apaf-1. The F-actin-rich area also encompasses initiator caspase-9 and clusters of a cleaved form of executioner caspase-3 but restricts accessibility of the caspase inhibitor XIAP. The clustering and potency of caspase-3 activation are positively regulated by the amount of actin polymerized by JMY and WHAMM. These results indicate that JMY-mediated actin reorganization functions in apoptotic signaling by coupling the biogenesis of apoptosomes to the localized processing of caspases.

Ciliary assembly, trafficking, and regulation are dependent on microtubules, but the mechanisms of ciliary assembly also require the actin cytoskeleton. Here, we dissect subcellular roles of actin in ciliogenesis by focusing on actin networks nucleated by the Arp2/3 complex in the powerful ciliary model, Chlamydomonas . We find that the Arp2/3 complex is required for the initial stages of ciliary assembly when protein and membrane are in high demand but cannot yet be supplied from the Golgi complex. We provide evidence for Arp2/3 complex-dependent endocytosis of ciliary proteins, an increase in endocytic activity upon induction of ciliary growth, and relocalization of plasma membrane proteins to newly formed cilia.

Microvilli are defining morphological features of the apical surfaces in diverse epithelial tissues. To develop our understanding of microvillus biogenesis, we used a biotin proximity-labeling approach to uncover new molecules enriched near EPS8, a well-studied marker of the microvillus distal tip compartment. Mass spectrometry of biotinylated hits identified KIAA1671, a large (∼200 kDa), disordered, and previously uncharacterized protein. Based on immunofluorescent staining and expression of fluorescent protein-tagged constructs, we found that KIAA1671 localizes to the base of the brush border in native intestinal tissue and polarized epithelial-cell culture models, as well as dynamic actin-rich structures in unpolarized, nonepithelial cell types. Live imaging also revealed that during the early stages of microvillar growth, KIAA1671 colocalizes with EPS8 in diffraction-limited puncta. However, once elongation of the core bundle begins, these two factors separate, with EPS8 tracking the distal end and KIAA1671 remaining behind at the base of the structure. These results suggest that KIAA1671 cooperates with EPS8 and potentially other assembly factors to initiate growth of microvilli on the apical surface. These findings offer new details on how transporting epithelial cells builds the brush border and may inform our understanding of how apical specializations are assembled in other epithelial contexts.

The Rho family of small GTPases is a key regulator of cytoskeletal actin polymerization. Although the ubiquitination of Rho proteins is reported to control their activity, the mechanisms by which the ubiquitination of Rho family proteins is controlled by ubiquitin ligases have yet to be elucidated. In this study, we identified BAG6 as the first factor needed to prevent the ubiquitination of RhoA, a critical Rho family protein in F-actin polymerization. We found that BAG6 is necessary for stress fiber formation by stabilizing endogenous RhoA. BAG6 deficiency enhanced the association between RhoA and Cullin-3-based ubiquitin ligases, thus promoting its polyubiquitination and subsequent degradation, leading to the abrogation of actin polymerization. In contrast, the restoration of RhoA expression through transient overexpression rescued the stress fiber formation defects induced by BAG6 depletion. BAG6 was also necessary for the appropriate assembly of focal adhesions as well as cell migration events. These findings reveal a novel role for BAG6 in maintaining the integrity of actin fiber polymerization and establish BAG6 as a RhoA-stabilizing holdase, which binds to and supports the function of RhoA.

Coordination between the microtubule and actin networks is essential for cell motility, neuronal growth cone guidance, and wound healing. Members of the CLASP (cytoplasmic linker-associated protein) family of proteins have been implicated in the cytoskeletal cross-talk between microtubules and actin networks; however, the molecular mechanisms underlying the role of CLASP in cytoskeletal coordination are unclear. Here, we investigate CLASP2α's cross-linking function with microtubules and F-actin. Our results demonstrate that CLASP2α cross-links F-actin to the microtubule lattice in vitro. We find that the cross-linking ability is retained by L-TOG2-S, a minimal construct containing the TOG2 domain and serine-arginine-rich region of CLASP2α. Furthermore, CLASP2α promotes the accumulation of multiple actin filaments along the microtubule, supporting up to 11 F-actin landing events on a single microtubule lattice region. CLASP2α also facilitates the dynamic organization of polymerizing actin filaments templated by the microtubule network, with F-actin forming bridges between individual microtubules. Finally, we find that depletion of CLASPs in vascular smooth muscle cells results in disorganized actin fibers and reduced coalignment of actin fibers with microtubules, suggesting that CLASP and microtubules contribute to higher-order actin structures. Taken together, our results indicate that CLASP2α can directly cross-link F-actin to microtubules and that this microtubule-CLASP-actin interaction may influence overall cytoskeletal organization in cells.

Clathrin-mediated endocytosis is a conserved eukaryotic membrane trafficking pathway that is driven by a sequentially assembled molecular machinery that contains over 60 different proteins. SH3 domains are the most abundant protein-protein interaction domain in this process, but the function of most SH3 domains in protein dynamics remains elusive. Using mutagenesis and live-cell fluorescence microscopy in the budding yeast Saccharomyces cerevisiae , we dissected SH3-mediated regulation of the endocytic pathway. Our data suggest that multiple SH3 domains regulate the actin nucleation-promoting Las17-Vrp1 complex, and that the network of SH3 interactions coordinates both Las17-Vrp1 assembly and dissociation. Furthermore, most endocytic SH3 domain proteins use the SH3 domain for their own recruitment, while a minority use the SH3 domain to recruit other proteins and not themselves. Our results provide a dynamic map of SH3 functions in yeast endocytosis and a framework for SH3 interaction network studies across biology.

Cells are subjected to a barrage of daily insults that often lead to their cortices being ripped open and requiring immediate repair. An important component of the cell's repair response is the formation of an actomyosin ring at the wound periphery to mediate its closure. Here we show that inhibition of myosin or the linear actin nucleation factors Diaphanous and/or dishevelled associated activator of morphogenesis results in a disrupted contractile apparatus and delayed wound closure. We also show that the branched actin nucleators WASp and SCAR function nonredundantly as scaffolds to assemble and maintain this contractile actomyosin cable. Removing branched actin leads to the formation of smaller circular actin-myosin structures at the cell cortex and to slow wound closure. Removing linear and branched actin simultaneously results in failed wound closure. Surprisingly, removal of branched actin and myosin results in the formation of parallel linear F-actin filaments that undergo a chiral swirling movement to close the wound, uncovering a new mechanism of cell wound closure. Taken together, we demonstrate the roles of different actin substructures that are required for optimal actomyosin ring formation and the extraordinary resilience of the cell to undergo wound repair when it is unable to form different subsets of these substructures.

The fine regulation of actin polymerization is essential to control cell motility and architecture and to perform essential cellular functions. Formins are key regulators of actin filament assembly, known to processively elongate filament barbed ends and increase their polymerization rate. Different models have been extrapolated to describe the molecular mechanism governing the processive motion of formin FH2 domains at polymerizing barbed ends. Using negative stain electron microscopy, we directly identified for the first time two conformations of the mDia1 formin FH2 domains in interaction with the barbed ends of actin filaments. These conformations agree with the speculated open and closed conformations of the "stair-stepping" model. We observed the FH2 dimers to be in the open conformation for 79% of the data, interacting with the two terminal actin subunits of the barbed end while they interact with three actin subunits in the closed conformation. In addition, we identified and characterized the structure of single FH2 dimers encircling the core of actin filaments, and reveal their ability to spontaneously depart from barbed ends.

The contractile ring must anchor to the plasma membrane and cell wall to transmit its tension. F-BAR domain containing proteins including Imp2p and Cdc15p in fission yeast are likely candidate anchoring proteins based on their mutant phenotypes. Cdc15p is a node component, links the actin bundle to the plasma membrane, recruits Bgs1p to the division plane, prevents contractile ring sliding, and contributes to the stiffness of the contractile ring. Less is known about Imp2p. We found that similarly to Cdc15p, Imp2p contributes to the stiffness of the contractile ring and assembles into protein clusters. Imp2p clusters contain approximately eight Imp2p dimers and depend on the actin network for their stability at the division plane. Importantly, Imp2p and Cdc15p reciprocally affect the amount of each other in the contractile ring, indicating that the two proteins influence each other during cytokinesis, which may partially explain their similar phenotypes.

Phagocytosis requires rapid remodeling of the actin cytoskeleton for extension of membrane protrusions and force generation to ultimately drive the engulfment of targets. The detailed mechanisms of phagocytosis have almost exclusively been studied in immortalized cell lines. Here, we make use of high-resolution imaging and novel biophysical approaches to determine the structural and mechanical features of phagocytosis by primary bone marrow-derived macrophages. We find that the signature behavior of these primary cells is distinct from macrophage-like cell lines; specifically, it is gentle, with only weak target constriction and modest polarization of the F-actin distribution inside the phagocytic cup. We show that long-tailed myosins 1e/f are critical for this organization. Deficiency of myo1e/f causes dramatic shifts in F-actin localization, reducing F-actin at the phagocytic cup base and enhancing F-actin-mediated constriction at the cup rim. Surprisingly, these changes can be almost fully reverted upon inhibition of another myosin motor protein, myosin-II. Hence, we show that the biomechanics and large-scale organization of phagocytic cups is tightly regulated through competing contributions from myosin-Ie/f and myosin-II.

Actin is a central mediator between mechanical force and cellular phenotype. In tendons, it is speculated that mechanical stress deprivation regulates gene expression by reducing filamentous (F)-actin. However, the mechanisms regulating tenocyte F-actin remain unclear. Tropomyosins (Tpms) are master regulators of F-actin. There are more than 40 Tpm isoforms, each having the unique capability to stabilize F-actin subpopulations. We investigated F-actin polymerization in stress-deprived tendons and tested the hypothesis that stress fiber-associated Tpm(s) stabilize F-actin to regulate cellular phenotype. Stress deprivation of mouse tail tendon down-regulated tenogenic and up-regulated protease (matrix metalloproteinase-3) mRNA levels. Concomitant with mRNA modulation were increases in G/F-actin, confirming reduced F-actin by tendon stress deprivation. To investigate the molecular regulation of F-actin, we identified that tail, Achilles, and plantaris tendons express three isoforms in common: Tpm1.6, 3.1, and 4.2. Tpm3.1 associates with F-actin in native and primary tenocytes. Tpm3.1 inhibition reduces F-actin, leading to decreases in tenogenic expression, increases in chondrogenic expression, and enhancement of protease expression in mouse and human tenocytes. These expression changes by Tpm3.1 inhibition are consistent with tendinosis progression. A further understanding of F-actin regulation in musculoskeletal cells could lead to new therapeutic interventions to prevent alterations in cellular phenotype during disease progression.

Cells migrating through physiologically relevant three-dimensional (3D) substrates such as cell-derived matrix (CDM) use actomyosin and vimentin intermediate filaments to pull the nucleus forward and pressurize the front of the cell as part of the nuclear piston mechanism of 3D migration. In this study, we tested the role of the cytoskeleton cross-linking protein plectin in facilitating the movement of the nucleus through 3D matrices. We find that the interaction of F-actin and vimentin filaments in cells on 2D glass and in 3D CDM requires actomyosin contractility. Plectin also facilitated these interactions and interacts with vimentin in response to NMII contractility and substrate stiffness, suggesting that the association of plectin and vimentin is mechanosensitive. We find that this mechanosensitive plectin complex slows down 2D migration but is critical for pulling the nucleus forward and generating compartmentalized intracellular pressure in 3D CDM, as well as low-pressure lamellipodial migration in 3D collagen. Finally, plectin expression helped to polarize NMII to in front of the nucleus and to localize the vimentin network around the nucleus. Together, our data suggest that plectin cross-links vimentin and actomyosin filaments, organizes the vimentin network, and polarizes NMII to facilitate the nuclear piston mechanism of 3D cell migration.

Fission of transport vesicles from endosomes is a crucial step in the recycling of lipids and receptors to the plasma membrane, but this process remains poorly understood. Although key components of the fission machinery, including the actin cytoskeleton and the ATPase Eps15 homology domain protein 1 (EHD1), have been implicated in endosomal fission, how this process is coordinately regulated is not known. We have identified the actin regulatory protein Coronin2A (CORO2A) as a novel EHD1 interaction partner. CORO2A localizes to stress fibers and actin microfilaments but also can be observed in partial overlap with EHD1 on endosomal structures. siRNA knockdown of CORO2A led to enlarged lamellae-like actin-rich protrusions, consistent with a role of other Coronin family proteins in attenuating actin-branching. Moreover, CORO2A depletion also caused a marked decrease in the internalization of clathrin-dependent cargo but had little impact on the uptake of clathrin-independent cargo, highlighting key differences in the role of branched actin for different modes of endocytosis. However, CORO2A was required for recycling of clathrin-independent cargo, and its depletion led to enlarged endosomes, supporting a role for CORO2A in the fission of endosomal vesicles. Our data support a novel role for CORO2A in coordinating endosomal fission and recycling with EHD1. [Media: see text].