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Our knowledge of the assembly and dynamics of the cytokinetic contractile ring (CR) in animal cells remains incomplete. We have previously used super-resolution light microscopy and platinum replica electron microscopy to elucidate the ultrastructural organization of the CR in first division sea urchin embryos. To date, our studies indicate that the CR initiates as an equatorial band of clusters containing myosin II, actin, septin and anillin, which then congress over time into patches which coalesce into a linear array characteristic of mature CRs. In the present study, we applied super-resolution interferometric photoactivated localization microscopy to confirm the existence of septin filament-like structures in the developing CR, demonstrate the close associations between septin2, anillin, and myosin II in the CR, as well as to show that septin2 appears consistently submembranous, whereas anillin is more widely distributed in the early CR. We also provide evidence that the major actin cross-linking protein α-actinin only associates with the linearized, late-stage CR and not with the early CR clusters, providing further support to the idea that α-actinin associates with actomyosin structures under tension and can serve as a counterbalance. In addition, we show that inhibition of actomyosin contraction does not stop the assembly of the early CR clusters but does arrest the progression of these structures to the aligned arrays required for functional cytokinesis. Taken together our results reinforce and extend our model for a cluster to patch to linear structural progression of the CR in sea urchin embryos and highlight the evolutionary relationships with cytokinesis in fission yeast. [ABSTRACT FROM AUTHOR]

Our knowledge of the assembly and dynamics of the cytokinetic contractile ring (CR) in animal cells remains incomplete. We have previously used superresolution light microscopy and platinum replica electron microscopy to elucidate the ultrastructural organization of the CR in first division sea urchin embryos. To date, our studies indicate that the CR initiates as an equatorial band of clusters containing myosin II, actin, septin and anillin, which then congress over time into patches which coalesce into a linear array characteristic of mature CRs. In the present study, we applied super-resolution interferometric photoactivated localization microscopy to confirm the existence of septin filament-like structures in the developing CR, demonstrate the close associations between septin2, anillin, and myosin II in the CR, as well as to show that septin2 appears consistently submembranous, whereas anillin is more widely distributed in the early CR. We also provide evidence that the major actin cross-linking protein a-actinin only associates with the linearized, late-stage CR and not with the early CR clusters, providing further support to the idea that a-actinin associates with actomyosin structures under tension and can serve as a counterbalance. In addition, we show that inhibition of actomyosin contraction does not stop the assembly of the early CR clusters but does arrest the progression of these structures to the aligned arrays required for functional cytokinesis. Taken together our results reinforce and extend our model for a cluster to patch to linear structural progression of the CR in sea urchin embryos and highlight the evolutionary relationships with cytokinesis in fission yeast. [ABSTRACT FROM AUTHOR]

The actin cytoskeleton regulates the integrity and repair of epithelial barriers by mediating the assembly of tight junctions (TJs), and adherens junctions (AJs), and driving epithelial wound healing. Actin filaments undergo a constant turnover guided by numerous actin-binding proteins, however, the roles of actin filament dynamics in regulating intestinal epithelial barrier integrity and repair remain poorly understood. Coactosin-like protein 1 (COTL1) is a member of the ADF/ cofilin homology domain protein superfamily that binds and stabilizes actin filaments. COTL1 is essential for neuronal and cancer cell migration, however, its functions in epithelia remain unknown. The goal of this study is to investigate the roles of COTL1 in regulating the structure, permeability, and repair of the epithelial barrier in human intestinal epithelial cells (IEC). COTL1 was found to be enriched at apical junctions in polarized IEC monolayers in vitro. The knockdown of COTL1 in IEC significantly increased paracellular permeability, impaired the steady state TJ and AJ integrity, and attenuated junctional reassembly in a calcium-switch model. Consistently, downregulation of COTL1 expression in Drosophila melanogaster increased gut permeability. Loss of COTL1 attenuated collective IEC migration and decreased cell-matrix attachment. The observed junctional abnormalities in COTL1-depleted IEC were accompanied by the impaired assembly of the cortical actomyosin cytoskeleton. Overexpression of either wild-type COTL1 or its actin-binding deficient mutant tightened the paracellular barrier and activated junction-associated myosin II. Furthermore, the actin-uncoupled COTL1 mutant inhibited epithelial migration and matrix attachment. These findings highlight COTL1 as a novel regulator of the intestinal epithelial barrier integrity and repair. [ABSTRACT FROM AUTHOR]

Our knowledge of the assembly and dynamics of the cytokinetic contractile ring (CR) in animal cells remains incomplete. We have previously used super-resolution light microscopy and platinum replica electron microscopy to elucidate the ultrastructural organization of the CR in first division sea urchin embryos. To date, our studies indicate that the CR initiates as an equatorial band of clusters containing myosin II, actin, septin and anillin, which then congress over time into patches which coalesce into a linear array characteristic of mature CRs. In the present study, we applied super-resolution interferometric photoactivated localization microscopy to confirm the existence of septin filament-like structures in the developing CR, demonstrate the close associations between septin2, anillin, and myosin II in the CR, as well as to show that septin2 appears consistently submembranous, whereas anillin is more widely distributed in the early CR. We also provide evidence that the major actin cross-linking protein α-actinin only associates with the linearized, late-stage CR and not with the early CR clusters, providing further support to the idea that α-actinin associates with actomyosin structures under tension and can serve as a counterbalance. In addition, we show that inhibition of actomyosin contraction does not stop the assembly of the early CR clusters but does arrest the progression of these structures to the aligned arrays required for functional cytokinesis. Taken together our results reinforce and extend our model for a cluster to patch to linear structural progression of the CR in sea urchin embryos and highlight the evolutionary relationships with cytokinesis in fission yeast.

The actin cortex is very dynamic during the migration of eukaryotes. In cells that use blebs as leading-edge protrusions, the cortex reforms beneath the cell membrane (bleb cortex) and completely disassembles at the site of bleb initiation (actin scar). The molecular factors that regulate the dynamic reorganization of the cortex are not fully understood. Myosin motor protein activity has been shown to be necessary for blebbing, with its major role associated with pressure generation to drive bleb expansion. Here, we examine the role of myosin in regulating cortex dynamics during bleb stabilization. Analysis of microscopy data from protein localization experiments in Dictyostelium discoideum cells reveal a rapid formation of the bleb cortex with a delay in myosin accumulation. In the degrading actin scar, myosin is observed to accumulate before active degradation of the cortex begins. Through a combination of mathematical modeling and data fitting, we identify that myosin helps to regulate the equilibrium concentration of actin in the bleb cortex during its reformation by increasing its disassembly rate. Our analysis also suggests that cortex degradation in the actin scar is driven primarily by an exponential decrease in the actin assembly rate rather than increased myosin activity. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is a major human pathogen that colonizes the gastric mucosa and plays a causative role in development of peptic ulcers and gastric cancer. Neutrophils are heavily infected with this organism in vivo and play a prominent role in tissue destruction and disease. Recently, we demonstrated that H. pylori exploits neutrophil plasticity as part of its virulence strategy eliciting N1-like subtype differentiation that is notable for profound nuclear hypersegmentation. We undertook this study to test the hypothesis that hypersegmentation may enhance neutrophil migratory capacity. However, EZ-TAXIScan™ video imaging revealed a previously unappreciated and progressive chemotaxis defect that was apparent prior to hypersegmentation onset. Cell speed and directionality were significantly impaired to fMLF as well as C5a and IL-8. Infected cells oriented normally in chemotactic gradients, but speed and direction were impaired because of a uropod retraction defect that led to cell elongation, nuclear lobe trapping in the contracted rear and progressive narrowing of the leading edge. In contrast, chemotactic receptor abundance, adhesion, phagocytosis and other aspects of cell function were unchanged. At the molecular level, H. pylori phenocopied the effects of Blebbistatin as indicated by aberrant accumulation of F-actin and actin spikes at the uropod together with enhanced ROCKII-mediated phosphorylation of myosin IIA regulatory light chains at S19. At the same time, RhoA and ROCKII disappeared from the cell rear and accumulated at the leading edge whereas myosin IIA was enriched at both cell poles. These data suggest that H. pylori inhibits the dynamic changes in myosin IIA contractility and front-to-back polarity that are essential for chemotaxis. Taken together, our data advance understanding of PMN plasticity and H. pylori pathogenesis. [ABSTRACT FROM AUTHOR]

A recently developed variational Bayesian analysis using pattern recognition and machine learning of single viral ribonucleoprotein (RNP) particle tracks in the cytoplasm of living cells provides a quantitative molecular explanation for active diffusion, a concept previously "explained" largely by hypothetical models based on indirect analyses such as continuum microrheology. Machine learning shows that vesicular stomatitis virus (VSV) RNP particles are temporarily confined to dynamic traps or pores made up of cytoskeletal elements. Active diffusion occurs when the particles escape from one trap to a nearby trap. In this paper, we demonstrate that actin filament disruption increased RNP mobility by increasing trap size. Inhibition of nonmuscle myosin II ATPase decreased mobility by decreasing trap size. Trap sizes were observed to fluctuate with time, dependent on nonmuscle myosin II activity. This model for active diffusion is likely to account for the dominant motion of other viral and cellular elements. IMPORTANCE RNA virus ribonucleoproteins (RNPs) are too large to freely diffuse in the host cytoplasm, yet their dominant motions consist of movements in random directions that resemble diffusion. We show that vesicular stomatitis virus (VSV) RNPs overcome limitations on diffusion in the host cytoplasm by hopping between traps formed in part by actin filaments and that these traps expand and contract by nonmuscle myosin II ATPase activity. ATP-dependent random motion of cellular particles has been termed "active diffusion." Thus, these mechanisms are applicable to active diffusion of other cellular and viral elements. [ABSTRACT FROM AUTHOR]

Cytokinetic abscission leads to the physical cut of the intercellular bridge (ICB) connecting the daughter cells and concludes cell division. In different animal cells, it is well established that the ESCRT-III machinery is responsible for the constriction and scission of the ICB. Here, we review the mechanical context of abscission. We first summarize the evidence that the ICB is initially under high tension and explain why, paradoxically, this can inhibit abscission in epithelial cells by impacting on ESCRT-III assembly. We next detail the different mechanisms that have been recently identified to release ICB tension and trigger abscission. Finally, we discuss whether traction-induced mechanical cell rupture could represent an ancient alternative mechanism of abscission and suggest future research avenues to further understand the role of mechanics in regulating abscission.

SummaryStabilization of long-term potentiation (LTP) depends on reorganization of the dendritic spine actin cytoskeleton. The present studies tested if this involves activity-driven effects on the actin-regulatory protein cortactin, and if such effects are disturbed in the Fmr1-knockout (KO) model of Fragile X Syndrome in which stabilization of both actin filaments and LTP is impaired. LTP induced by theta burst stimulation (TBS) in hippocampal slices from wild-type mice was associated with rapid, broadly distributed, and NMDA receptor-dependent decreases in synapse-associated cortactin. The reduction in cortactin content was blocked by blebbistatin while basal levels were reduced by nocodazole, indicating that cortactin’s movements into and away from synapses are respectively regulated by microtubule and actomyosin motors. These results further suggest that synapse-specific LTP influences cytoskeletal elements at distant connections. The rapid effects of TBS on synaptic cortactin content were absent in Fmr1-KOs as was evidence for activity-driven phosphorylation of the protein or its upstream kinase, ERK1/2. Phosphorylation regulates cortactin’s interactions with actin and co-precipitation of the two proteins was reduced in the KOs. We propose that, in the KOs, excessive basal phosphorylation of ERK1/2 disrupts its interactions with cortactin, thereby blocking the latter protein’s use of actomyosin transport systems. These impairments are predicted to compromise the response of the subsynaptic cytoskeleton to learning-related afferent activity, both locally and at distant sites.

Abstract Nonmuscle myosin II is an actin-based motor that executes numerous mechanical tasks in cells including spatiotemporal organization of the actin cytoskeleton, adhesion, migration, cytokinesis, tissue remodeling, and membrane trafficking. Nonmuscle myosin II is ubiquitously expressed in mammalian cells as a tissue-specific combination of three paralogs. Recent studies reveal novel specific aspects of their kinetics, intracellular regulation and functions. On the other hand, the three paralogs also can copolymerize and cooperate in cells. Here we review the recent advances from the prospective of how distinct features of the three myosin II paralogs adapt them to perform specialized and joint tasks in the cell. Highlights • Nonmuscle myosin II executes numerous mechanical tasks in cells including cytoskeleton organization and cell migration. • Three mammalian nonmuscle myosin II paralogs have distinct kinetic and dynamic properties in vitro and in vivo. • Mammalian nonmuscle myosin II paralogs mix and match their abilities to perform both individual and joint tasks in the cell. [ABSTRACT FROM AUTHOR]

Abstract: Actin nucleators and their binding partners play crucial roles during Salmonella invasion, but how these factors are dynamically coordinated remains unclear. Here, we show that septins, a conserved family of GTP binding proteins, play a role during the early stages of Salmonella invasion. We demonstrate that septins are rapidly enriched at sites of bacterial entry and contribute to the morphology of invasion ruffles. We found that SEPTIN2, SEPTIN7, and SEPTIN9 are required for efficient bacterial invasion. Septins contributed to the recruitment of ROCK2 kinase during Salmonella invasion, and the downstream activation of the actin nucleating protein FHOD1. In contrast, activation of the ROCK2 substrate myosin II, which is known to be required for Salmonella enterica serovar Typhimurium invasion, did not require septins. Collectively, our studies provide new insight into the mechanisms involved in Salmonella invasion of host cells. [ABSTRACT FROM AUTHOR]

Cellular communication relies on fusion of secretory vesicles with the plasma membrane, following dynamic events that change the micro- and nanoscale environment of the approaching vesicles in the vicinity of docking sites. Visualization of fine cortical actin network structures and their interactions with vesicle and plasma membrane has recently been facilitated by the development of new imaging technologies. Consequently, a greater understanding is emerging of the role of the cortical actin network on controlling secretory vesicles as they undergo docking, priming, and fusion in exocytic hot spots. In this review, we propose a mechanistic framework underpinning the mesoscopic properties of the cortical actin and discuss how molecular coupling of these pleiotropic effects orchestrate every single step of regulated exocytosis. [ABSTRACT FROM AUTHOR]

Summary Conventional type 1 DCs (cDC1s) excel at cross-presentation of dead cell-associated antigens partly because they express DNGR-1, a receptor that recognizes exposed actin filaments on dead cells. In vitro polymerized F-actin can be used as a synthetic ligand for DNGR-1. However, cellular F-actin is decorated with actin-binding proteins, which could affect DNGR-1 recognition. Here, we demonstrate that myosin II, an F-actin-associated motor protein, greatly potentiates the binding of DNGR-1 to F-actin. Latex beads coated with F-actin and myosin II are taken up by DNGR-1 + cDC1s, and antigen associated with those beads is efficiently cross-presented to CD8 + T cells. Myosin II-deficient necrotic cells are impaired in their ability to stimulate DNGR-1 or to serve as substrates for cDC1 cross-presentation to CD8 + T cells. These results provide insights into the nature of the DNGR-1 ligand and have implications for understanding immune responses to cell-associated antigens and for vaccine design. [ABSTRACT FROM AUTHOR]

Many actin filaments in animal cells are co-polymers of actin and tropomyosin. In many cases, non-muscle myosin II associates with these co-polymers to establish a contractile network. However, the temporal relationship of these three proteins in the de novo assembly of actin filaments is not known. Intravital subcellular microscopy of secretory granule exocytosis allows the visualisation and quantification of the formation of an actin scaffold in real time, with the added advantage that it occurs in a living mammal under physiological conditions. We used this model system to investigate the de novo assembly of actin, tropomyosin Tpm3.1 (a short isoform of TPM3) and myosin IIA (the form of non-muscle myosin II with its heavy chain encoded by Myh9) on secretory granules in mouse salivary glands. Blocking actin polymerization with cytochalasin D revealed that Tpm3.1 assembly is dependent on actin assembly. We used time-lapse imaging to determine the timing of the appearance of the actin filament reporter LifeAct-RFP and of Tpm3.1-mNeonGreen on secretory granules in LifeAct-RFP transgenic, Tpm3.1-mNeonGreen and myosin IIA-GFP (GFP-tagged MYH9) knock-in mice. Our findings are consistent with the addition of tropomyosin to actin filaments shortly after the initiation of actin filament nucleation, followed by myosin IIA recruitment. [ABSTRACT FROM AUTHOR]

Electron microscope studies have shown that the switched-off state of myosin II in muscle involves intramolecular interaction between the two heads of myosin and between one head and the tail. The interaction, seen in both myosin filaments and isolated molecules, inhibits activity by blocking actin-binding and ATPase sites on myosin. This interacting-heads motif is highly conserved, occurring in invertebrates and vertebrates, in striated, smooth, and nonmuscle myosin IIs, and in myosins regulated by both Ca2+ binding and regulatory light-chain phosphorylation. Our goal was to determine how early this motif arose by studying the structure of inhibited myosin II molecules from primitive animals and from earlier, unicellular species that predate animals. Myosin II from Cnidaria (sea anemones, jellyfish), themost primitive animalswithmuscles, and Porifera (sponges), the most primitive of all animals (lacking muscle tissue) showed the same interacting-heads structure as myosins fromhigher animals, confirming the early origin of themotif. The social amoeba Dictyostelium discoideum showed a similar, but modified, version of the motif, while the amoeba Acanthamoeba castellanii and fission yeast (Schizosaccharomyces pombe) showed no head-head interaction, consistentwith the different sequences and regulatory mechanisms of these myosins compared with animal myosin IIs. Our results suggest that head-head/head-tail interactions have been conserved, with slight modifications, as a mechanism for regulating myosin II activity from the emergence of the first animals and before. The early origins of these interactions highlight their importance in generating the inhibited (relaxed) state of myosin in muscle and nonmuscle cells. [ABSTRACT FROM AUTHOR]

The actin cytoskeleton and its response to external chemical stimuli is fundamental to the mechano-biology of eukaryotic cells and their functions. One of the key players that governs the dynamics of the actin network is the motor protein myosin II. Based on a phase space embedding we have identified from experiments three phases in the cytoskeletal dynamics of starved Dictyostelium discoideum in response to a precisely controlled chemotactic stimulation. In the first two phases the dynamics of actin and myosin II in the cortex is uncoupled, while in the third phase the time scale for the recovery of cortical actin is determined by the myosin II dynamics. We report a theoretical model that captures the experimental observations quantitatively. The model predicts an increase in the optimal response time of actin with decreasing myosin II-actin coupling strength highlighting the role of myosin II in the robust control of cell contraction.

T-cell receptor (TCR) triggering and subsequent T-cell activation are essential for the adaptive immune response. Recently, multiple lines of evidence have shown that force transduction across the TCR complex is involved during TCR triggering, and that the T cell might use its force-generation machinery to probe the mechanical properties of the opposing antigen-presenting cell, giving rise to different signaling and physiological responses. Mechanistically, actin polymerization and turnover have been shown to be essential for force generation by T cells, but how these actin dynamics are regulated spatiotemporally remains poorly understood. Here, we report that traction forces generated by T cells are regulated by dynamic microtubules (MTs) at the interface. These MTs suppress Rho activation, nonmuscle myosin II bipolar filament assembly, and actin retrograde flow at the T-cell-substrate interface. Our results suggest a novel role of the MT cytoskeleton in regulating force generation during T-cell activation. [ABSTRACT FROM AUTHOR]

Pyramidal neurons are a major cell type of the forebrain, consisting of a pyramidally shaped soma with axonal and apicobasal dendritic processes. It is poorly understood how the neuronal soma develops its pyramidal morphology, while generating neurites of the proper shape and orientation. Here, we discovered that the spherical somata of immature neurite-less neurons possess a circumferential wreath-like network of septin filaments, which promotes neuritogenesis by balancing the protrusive activity of lamellipodia and filopodia. In embryonic rat hippocampal and mouse cortical neurons, the septin wreath network consists of curvilinear filaments that contain septins 5, 7, and 11 (Sept5/7/11). The Sept5/7/11 wreath network demarcates a zone of myosin II enrichment and Arp2/3 diminution at the base of filopodial actin bundles. In Sept7-depleted neurons, cell bodies are enlarged with hyperextended lamellae and abnormally shaped neurites that originate from lamellipodia. This phenotype is accompanied by diminished myosin II and filopodia lifetimes and increased Arp2/3 and lamellipodial activity. Inhibition of Arp2/3 rescues soma and neurite phenotypes, indicating that the septin wreath network suppresses the extension of lamellipodia, facilitating the formation of neurites from the filopodia of a consolidated soma. We show that this septin function is critical for developing a pyramidally shaped soma with properly distributed and oriented dendrites in cultured rat hippocampal neurons and in vivo in mouse perinatal cortical neurons. Therefore, the somatic septin cytoskeleton provides a key morphogenetic mechanism for neuritogenesis and the development of pyramidal neurons. [Display omitted] • A septin wreath-like network controls the shape of neuronal somata and neurites • Septins promote and suppress filopodial and lamellipodial protrusions, respectively • Septins scaffold myosin II and exclude Arp2/3 at the base of filopodial actin • Development of pyramidally shaped neurons requires septins in vitro and in vivo Radler et al. report a septin wreath-like cytoskeleton in the soma of immature neurons that promotes neurite formation by balancing the protrusive activity of lamellipodia and filopodia. The septin network is critical for developing a pyramidally shaped soma with properly distributed and oriented dendrites. [ABSTRACT FROM AUTHOR]

The drebrin/EB3/Cdk5 intracellular signalling pathway couples actin filaments to dynamic microtubules in cellular settings where cells are changing shape. The pathway has been most intensively studied in neuronal development, particularly neuritogenesis and neuronal migration, and in synaptic plasticity at dendritic spines in mature neurons. Drebrin is an actin filament side-binding and bundling protein that stabilises actin filaments. The end-binding (EB) proteins are microtubule plus-end tracking proteins (+TIPs) that localise to the growing plus-ends of dynamic microtubules and regulate their behavior and the binding of other +TIP proteins. EB3 binds specifically to drebrin when drebrin is bound to actin filaments, for example at the base of a growth cone filopodium, and EB3 is located at the plus-end of a growing microtubule inserting into the filopodium. This interaction therefore forms the basis for coupling dynamic microtubules to actin filaments in growth cones of developing neurons. Appropriate responses to growth cone guidance cues depend on actin filament/microtubule co-ordination in the growth cone, although the role of the drebrin/EB3/Cdk5 pathway in this context has not been directly tested. A similar cytoskeleton coupling pathway operates in dendritic spines in mature neurons where the activity-dependent insertion of dynamic microtubules into dendritic spines is facilitated by drebrin binding to EB3. Microtubule insertion into dendritic spines drives spine maturation during long-term potentiation and therefore has a role in synaptic plasticity and memory formation. In Alzheimer’s disease and related chronic neurodegenerative diseases, there is an early and dramatic loss of drebrin from dendritic spines that precedes synapse loss and neurodegeneration and might contribute to a failure of synaptic plasticity and hence to cognitive decline. [ABSTRACT FROM AUTHOR]

The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled viamembrane-associated actin-binding proteins to dynamic actin filaments andmyosinmotors. Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponent membrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase-segregating domains. These results show how local membrane composition can be driven by active processes arising from actomyosin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization. [ABSTRACT FROM AUTHOR]

Cucurbitacins are cytotoxic triterpenoid sterols isolated from plants. One of their earliest cellular effect is the aggregation of actin associated with blockage of cell migration and division that eventually lead to apoptosis. We unravel here that cucurbitacin I actually induces the co-aggregation of actin with phospho-myosin II. This co-aggregation most probably results from the stimulation of the Rho/ROCK pathway and the direct inhibition of the LIMKinase. We further provide data that suggest that the formation of these co-aggregates is independent of a putative pro-oxidant status of cucurbitacin I. The results help to understand the impact of cucurbitacins on signal transduction and actin dynamics and open novel perspectives to use it as drug candidates for cancer research. [ABSTRACT FROM AUTHOR]

Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect onWT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly thanWT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD. [ABSTRACT FROM AUTHOR]

Similar to memory formation, memory extinction is also a new learning process that requires synaptic plasticity. Actin rearrangement is fundamental for synaptic plasticity, however, whether actin rearrangement in the infralimbic cortex (IL) plays a role in memory extinction, as well as the mechanisms underlying it, remains unclear. Here, using a conditioned taste aversion (CTA) paradigm, we demonstrated increased synaptic density and actin rearrangement in the IL during the extinction of CTA. Targeted infusion of an actin rearrangement inhibitor, cytochalasin D, into the IL impaired memory extinction and de novo synapse formation. Notably, we also found increased myosin II phosphorylation in the IL during the extinction of CTA. Microinfusion of a specific inhibitor of the myosin II ATPase, blebbistatin (Blebb), into the IL impaired memory extinction as well as the related actin rearrangement and changes in synaptic density. Moreover, the extinction deficit and the reduction of synaptic density induced by Blebb could be rescued by the actin polymerization stabilizer jasplakinolide (Jasp), suggesting that myosin II acts via actin filament polymerization to stabilize synaptic plasticity during the extinction of CTA. Taken together, we conclude that myosin II may regulate the plasticity of actin-related synaptic structure during memory extinction. Our studies provide a molecular mechanism for understanding the plasticity of actin rearrangement-associated synaptic structure during memory extinction. [ABSTRACT FROM AUTHOR]

Activity-dependent bulk endocytosis allows neurons to internalize large portions of the plasma membrane in response to stimulation. However, whether this critical type of compensatory endocytosis is unique to neurons or also occurs in other excitable cells is currently unknown. Here we used fluorescent 70 kDa dextran to demonstrate that secretagogue-induced bulk endocytosis also occurs in bovine chromaffin cells. The relatively large size of the bulk endosomes found in this model allowed us to investigate how the neck of the budding endosomes constricts to allow efficient recruitment of the fission machinery. Using time-lapse imaging of Lifeact-GFP-transfected chromaffin cells in combination with fluorescent 70 kDa dextran, we detected acto-myosin II rings surrounding dextran-positive budding endosomes. Importantly, these rings were transient and contracted before disappearing, suggesting that they might be involved in restricting the size of the budding endosome neck. Based on the complete recovery of dextran fluorescence after photobleaching, we demonstrated that the actin ring-associated budding endosomes were still connected with the extracellular fluid. In contrast, no such recovery was observed following the constriction and disappearance of the actin rings, suggesting that these structures were pinched-off endosomes. Finally, we showed that the rings were initiated by a circular array of phosphatidylinositol(4,5)bisphosphate microdomains, and that their constriction was sensitive to both myosin II and dynamin inhibition. The acto-myosin II rings therefore play a key role in constricting the neck of budding bulk endosomes before dynamin-dependent fission from the plasma membrane of neurosecretory cells. [ABSTRACT FROM AUTHOR]

Highlights: [•] Cytokinesis in epithelial tissues needs to counteract the tension imposed by surrounding epithelial cells. [•] Cytokinetic ring constriction is sensed by neighboring cells and acts as a mechanical signal to remodel AJs. [•] Apical midbody formation on asymmetric or symmetric furrowing regulates the planar and apical–basal organization of epithelia. [•] Cytokinesis in epithelia is a multicellular process. [Copyright &y& Elsevier]

Variable requirements for actin during clathrin-mediated endocytosis ( CME) may be related to regional or cellular differences in membrane tension. To compensate, local regulation of force generation may be needed to facilitate membrane curving and vesicle budding. Force generation is assumed to occur primarily through actin polymerization. Here we examine the role of myosin II using loss of function experiments. Our results indicate that myosin II acts on cortical actin scaffolds primarily in the plane of the plasma membrane (bottom arrow) to generate changes that are critical for enhancing CME progression. [ABSTRACT FROM AUTHOR]

Cell shape dynamics, motility, and cell proliferation all depend on the actin cytoskeleton. Malignant cancer cells hijack the actin network to grow and migrate to secondary sites. Understanding the function of actin regulators is therefore of major interest. In the present study, we identify the actin cross-linking protein Filamin/Cheerio (Cher) as a mediator of malignancy in genetically defined Drosophila tumors. We show that in invasive tumors, resulting from cooperation of activated Ras with disrupted epithelial cell polarity, Cher is upregulated in a Jun N-terminal kinase (JNK)-dependent manner. Although dispensable in normal epithelium, Cher becomes required in the tumor cells for their growth and invasiveness. When deprived of Cher, these tumor clones lose their full potential to proliferate and breach tissue boundaries. Instead, the Cher-deficient clones remain confined within the limits of their source epithelium, permitting survival of the host animal. Through interaction with the myosin II heavy chain subunit, Cher is likely to strengthen the cortical actomyosin network and reinforce mechanical tension within the invasive tumors. Accordingly, Cher is required for aberrant expression of genes downstream of the Hippo/Yorkie signaling in the tumor tissue. Our study identifies Cher as a new target of JNK signaling that links cytoskeleton dynamics to tumor progression. [ABSTRACT FROM AUTHOR]

Abstract: Fcγ receptor (FcγR)-mediated phagocytosis requires myosin II activity. Here we show that myosin II contributes to FcγR activation and subsequent F-actin assembly at the nascent phagocytic cup. Inhibition of myosin II attenuates phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of FcγR and binding of Syk to the ITAM. Furthermore, FcγR clusters independently of myosin II activity at the phagocytic cup, from which the receptor-like protein tyrosine phosphatase CD45 is excluded depending on myosin II activity. These findings suggest that myosin II-dependent segregation of CD45 from FcγR facilitates phosphorylation of the ITAM and triggers phagocytosis. [Copyright &y& Elsevier]

It is well known that actin can associate with intracellular membranes to drive endocytosis and the rocketing motion of bacteria, virions and some organelles and to regulate synaptic vesicle plasticity. Actin also has been extensively reported to be involved at several steps of exocytosis; however, it has typically been described as functioning either within the actin cortex or by providing actin tracks for organelle transport. Increasingly, actin filament coats or rings have been directly localized on the surface of the exocytic organelle. Here, we suggest a common mechanism for actin-based regulation of large secretory granules whereby organelle-associated actomyosin II contraction either directly expels secretory content or stabilizes the exocytosing organelle. [ABSTRACT FROM AUTHOR]

Abstract: Dynamics of the actomyosin cytoskeleton regulate cellular processes such as secretion, cell division, cell motility, and shape change. Actomyosin dynamics are themselves regulated by proteins that control actin filament polymerization and depolymerization, and myosin motor contractility. Previous theoretical work has focused on translational movement of actin filaments but has not considered the role of filament rotation. Since filament rotational movements are likely sources of forces that direct cell shape change and movement we explicitly model the dynamics of actin filament rotation as myosin II motors traverse filament pairs, drawing them into alignment. Using Monte Carlo simulations we find an optimal motor velocity for alignment of actin filaments. In addition, when we introduce polymerization and depolymerization of actin filaments, we find that alignment is reduced and the filament arrays exist in a stable, asynchronous state. Further analysis with continuum models allows us to investigate factors contributing to the stability of filament arrays and their ability to generate force. Interestingly, we find that two different morphologies of F-actin arrays generate the same amount of force. We also identify a phase transition to alignment which occurs when either polymerization rates are reduced or motor velocities are optimized. We have extended our analysis to include a maximum allowed stretch of the myosin motors, and a non-uniform length for filaments leading to little change in the qualitative results. Through the integration of simulations and continuum analysis, we are able to approach the problem of understanding rotational alignment of actin filaments by myosin II motors. [Copyright &y& Elsevier]

Abstract: Cyclic uniaxial stretching of adherent nonmuscle cells induces the gradual reorientation of their actin stress fibers perpendicular to the stretch direction to an extent dependent on stretch frequency. By subjecting cells to various temporal waveforms of cyclic stretch, we revealed that stress fibers are much more sensitive to strain rate than strain frequency. By applying asymmetric waveforms, stress fibers were clearly much more responsive to the rate of lengthening than the rate of shortening during the stretch cycle. These observations were interpreted using a theoretical model of networks of stress fibers with sarcomeric structure. The model predicts that stretch waveforms with fast lengthening rates generate greater average stress fiber tension than that generated by fast shortening. This integrated approach of experiment and theory provides new insight into the mechanisms by which cells respond to matrix stretching to maintain tensional homeostasis. [Copyright &y& Elsevier]

Abstract: Recently, a growing number of reports have reported that micro- or nanoscale topography enhances cellular functions such as cell adhesion and stem cell differentiation, but the mechanisms responsible for this topography-mediated cell behavior are not fully understood. In this study, we examine the underlying processes and mechanisms behind specific topography-mediated cellular functions. Formation of focal adhesions (FA) was studied by culturing cells on different kinds of topographies, including a flat surface and surfaces with a micropatterned topography (2 μm lattice pattern with 3 μm intervals). We found that the formation and maturation of focal adhesions were highly dependent on the topography of the substrate although the shape, morphology and spreading of cells on the different substrates were not significantly affected. Focal adhesion maturation and actin polymerization were also promoted in cells cultured on the micropatterned substrate. These differences in cell adhesion led us to focus on the Rho GTPases, RhoA and downstream pathways since a number of reports have demonstrated that RhoA-activated cells have highly enhanced focal adhesions and actin activation such as polymerization. By inhibiting the Rho-associated kinase (ROCK) and downstream myosin II, we found that the FA formation, actin organization, and FAK phosphorylation were dramatically decreased. The topographical dependency of FA formation was also highly decreased. These results show that the FA formation and actin cytoskeleton organization of cells on the microtopography is regulated by the RhoA/ROCK pathway. [Copyright &y& Elsevier]

It is becoming increasingly evident that the micromechanics of cells and their environment determine cell fate and function as much as soluble molecular factors do. We hypothesized that extracellular matrix proteolysis by membrane type 1 matrix metalloproteinase (MT1-MMP) depends on adhesion, force generation and rigidity sensing of the cell. Melanoma cells (MV3 clone) stably transfected with MT1-MMP, or the empty vector as a control, served as the model system. α2β1 integrins (cell adhesion), actin and myosin II (force generation and rigidity sensing) were blocked by their corresponding inhibitors (α2β1 integrin antibodies, Cytochalasin D, blebbistatin). A novel, anisotropic matrix array of parallel, fluorescently labeled collagen-I fibrils was used. Cleavage and bundling of the collagen-I fibrils, and spreading and durotaxis of the cells on this matrix array could be readily discerned and quantified by a combined set-up for fluorescence and atomic force microscopy. In short, expression of the protease resulted in the generation of structural matrix defects, clearly indicated by gaps in the collagen lattice and loose fiber bundles. This key feature of matrix remodeling depended essentially on the functionality of α2β1 integrin, the actin filament network and myosin II motor activity. Interference with any of these negatively impacted matrix cleavage and three-dimensional matrix entanglement of cells. [ABSTRACT FROM AUTHOR]

Abstract: Cytokinesis is emerging as a control system defined by interacting biochemical and mechanical modules, which form a system of feedback loops. This integrated system accounts for the regulation and kinetics of cytokinesis furrowing and demonstrates that cytokinesis is a whole-cell process in which the global and equatorial cortices and cytoplasm are active players in the system. Though originally defined in Dictyostelium, features of the control system are recognizable in other organisms, suggesting a universal mechanism for cytokinesis regulation and contractility. [ABSTRACT FROM AUTHOR]

The dynamics of the myosin molecular motor as it binds to actin filaments during muscle contraction are still not clearly understood. In this paper, we focus on the coupling mechanism of multi-force interactions in the myosin molecule during its interaction with actin. These forces include the electrostatic force, the van der Waals force and the Casimir force in molecular dynamic simulations of the molecules in solvent with thermal fluctuations. Based on the Hamaker approach, van der Waals and Casimir potentials and forces are calculated between myosin and actin. We have developed a Monte Carlo method to simulate the dynamic activity of the molecular motor. We have shown that because of the retardation effect, the van der Waals force falls into the Casimir force when the distance between the surfaces is larger than 3 nm. When the distance is smaller than 3 nm, the electrostatic force and the van der Waals force increase until the myosin becomes attached to the actin. Over the distances studied in the present work, the electrostatic force dominates the attractive interactions. Our calculations are in good agreement with recently reported experimental results. [ABSTRACT FROM AUTHOR]

We describe an active polymer network in which processive molecular motors control network elasticity. This system consists of actin filaments cross-linked by filamin A (FLNa) and contracted by bipolar filaments of muscle myosin II. The myosin motors stiffen the network by more than two orders of magnitude by pulling on actin filaments anchored in the network by FLNa cross-links, thereby generating internal stress. The stiffening response closely mimics the effects of external stress applied by mechanical shear. Both internal and external stresses can drive the network into a highly nonlinear, stiffened regime. The active stress reaches values that are equivalent to an external stress of 14 Pa. consistent with a 1-pN force per myosin head. This active network mimics many mechanical properties of cells and suggests that adherent cells exert mechanical control by operating in a nonlinear regime where cell stiffness is sensitive to changes in motor activity. This design principle may be applicable to engineering novel biologically inspired, active materials that adjust their own stiffness by internal catalytic control. [ABSTRACT FROM AUTHOR]

The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces. [ABSTRACT FROM AUTHOR]

Chromaffin cells of the adrenal medulla are innervated by the sympathetic nervous system. Stimulation causes chromaffin cells to fire action potentials, leading to the exocytosis of various classes of transmitters into the circulation. Low-frequency electrical stimulation (action potentials delivered at 0.5 Hz) causes adrenal chromaffin cells to selectively release catecholamines through a kiss-and-run fusion event. Elevated electrical stimulation (action potentials at 15 Hz) evokes fusion pore dilation, full granule collapse, and additional release of the neuropeptide-containing proteinaceous granule core. Here we apply single-cell electrophysiological, electrochemical, and fluorescence measurements to investigate the cellular mechanism for this shift in exocytic behavior. We show that at low-frequency stimulation, a filamentous-actin cell cortex plays a key role in stabilizing the kiss-and-run fusion event. Increased stimulation disrupts the actin cortex, driving full granule collapse. We show that pharmacological perturbation of the actin cortex supersedes stimulus frequency in controlling exocytic mode. Finally, we show that nonmuscle myosin II activation contributes to the cytoskeleton-dependent control of the fusion event. Inhibition of myosin II or myosin light chain kinase under elevated stimulation frequencies inhibits fusion pore dilation and maintains the granule in a kiss-and-run mode of exocytosis. These results demonstrate an essential role for activity-evoked cytoskeletal rearrangement and the action of myosin II in the regulation of catecholamine and neuropeptide exocytosis and represent an essential element of the sympathetic stress response. [ABSTRACT FROM AUTHOR]

A shortening muscle is a machine that converts metabolic energy into mechanical work, but, when a muscle is stretched, it acts as a brake, generating a high resistive force at low metabolic cost. The braking action of muscle can be activated with remarkable speed, as when the leg extensor muscles rapidly decelerate the body at the end of a jump. Here we used time-resolved x-ray and mechanical measurements on isolated muscle cells to elucidate the molecular basis of muscle braking and its rapid control. We show that a stretch of only 5 nm between each overlapping set of myosin and actin filaments in a muscle sarcomere is sufficient to double the number of myosin motors attached to actin within a few milliseconds. Each myosin molecule has two motor domains, only one of which is attached to actin during shortening or activation at constant length. A stretch strains the attached motor domain, and we propose that combined steric and mechanical coupling between the two domains promotes attachment of the second motor domain. This mechanism allows skeletal muscle to resist external stretch without increasing the force per motor and provides an answer to the longstanding question of the functional role of the dimeric structure of muscle myosin. [ABSTRACT FROM AUTHOR]

Abstract: The function of the src-homology 3 (SH3) domain in class II myosins, a distinct β-barrel structure, remains unknown. Here, we provide evidence, using electron cryomicroscopy, in conjunction with light-scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41 residue extension contains four conserved lysine residues followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the ∼9 nm distance between the myosin lever arm and the thin filament. In order to localize the N terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle. [Copyright &y& Elsevier]

Abstract: The adherens junction (AJ) densely associated with actin filaments is a major cell–cell adhesion structure. To understand the importance of actin filament association in AJ formation, we first analyzed punctate AJs in NRK fibroblasts where one actin cable binds to one AJ structure unit. The accumulation of AJ components such as the cadherin/catenin complex and vinculin, as well as the formation of AJ-associated actin cables depended on Rho activity. Inhibitors for the Rho target, ROCK, which regulates myosin II activity, and for myosin II ATPase prevented the accumulation of AJ components, indicating that myosin II activity is more directly involved than Rho activity. Depletion of myosin II by RNAi showed similar results. The inhibition of myosin II activity in polarized epithelial MTD-1A cells affected the accumulation of vinculin to circumferential AJ (zonula adherens). Furthermore, correct zonula occludens (tight junction) formation along the apicobasal axis that requires cadherin activity was also impaired. Although MDCK cells which are often used as typical epithelial cells do not have a typical zonula adherens, punctate AJs formed dependently on myosin II activity by inducing wound closure in a MDCK cell sheet. These findings suggest that tension generated by actomyosin is essential for correct AJ assembly. [Copyright &y& Elsevier]

Myosins are a large family of actin-based motor proteins that are involved in a variety of cellular processes. Class II, or conventional, myosins are organized into a number of multi-component structures such as muscle thick filaments, non-muscle filaments and the actomyosin ring during cell division. A number of conditions must be met for the proper assembly and organization of myosin II-containing structures, including the correct stoichiometry of myosin and its associated proteins, and the conformation and regulation of the myosin molecule itself by molecular chaperones and protein kinases. In this review we discuss the use of model organisms in the genetic analysis of the assembly and organization of myosin-containing structures. [ABSTRACT FROM AUTHOR]

Discoveries from human and mouse genetics have identified cytoskeletal and signaling proteins that are essential for neuronal migration in the developing brain. To provide a meaningful context for these studies, we took an unbiased approach of correlative electron microscopy of neurons migrating through a three-dimensional matrix, and characterized the cytoskeletal events that occur as migrating neurons initiate saltatory forward movements of the cell nucleus. The formation of a cytoplasmic dilation in the proximal leading process precedes nuclear translocation. Cell nuclei translocate into these dilations in saltatory movements. Time-lapse imaging and pharmacological perturbation suggest that nucleokinesis requires stepwise or hierarchical interactions between microtubules, myosin II, and cell adhesion. We hypothesize that these interactions couple leading process extension to nuclear translocation during neuronal migration. [ABSTRACT FROM AUTHOR]