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Low-resolution coarse-grained (CG) models provide significant computational and conceptual advantages for simulating soft materials. However, the properties of CG models depend quite sensitively upon the mapping, M, that maps each atomic configuration, r, to a CG configuration, R. In particular, M determines how the configurational information of the atomic model is partitioned between the mapped ensemble of CG configurations and the lost ensemble of atomic configurations that map to each R. In this work, we investigate how the mapping partitions the atomic configuration space into CG and intra-site components. We demonstrate that the corresponding coordinate transformation introduces a nontrivial Jacobian factor. This Jacobian factor defines a labeling entropy that corresponds to the uncertainty in the atoms that are associated with each CG site. Consequently, the labeling entropy effectively transfers configurational information from the lost ensemble into the mapped ensemble. Moreover, our analysis highlights the possibility of resonant mappings that separate the atomic potential into CG and intra-site contributions. We numerically illustrate these considerations with a Gaussian network model for the equilibrium fluctuations of actin. We demonstrate that the spectral quality, Q , provides a simple metric for identifying high quality representations for actin. Conversely, we find that neither maximizing nor minimizing the information content of the mapped ensemble results in high quality representations. However, if one accounts for the labeling uncertainty, Q (M) correlates quite well with the adjusted configurational information loss, I ̂ map (M) , that results from the mapping. [ABSTRACT FROM AUTHOR]

Depletion interactions are thought to significantly contribute to the organization of intracellular structures in the crowded cytosol. The strength of depletion interactions depends on physical parameters such as the depletant number density and the depletant size ratio. Cells are known to dynamically regulate these two parameters by varying the copy number of proteins of a wide distribution of sizes. However, mammalian cells are also known to keep the total protein mass density remarkably constant, to within 0.5% throughout the cell cycle. We thus ask how the strength of depletion interactions varies when the total depletant mass is held fixed, a.k.a. fixed-mass depletion. We answer this question via scaling arguments, as well as by studying depletion effects on networks of reconstituted semiflexible actin in silico and in vitro. We examine the maximum strength of the depletion interaction potential U∗ as a function of q, the size ratio between the depletant and the matter being depleted. We uncover a scaling relation U∗ ∼ qζ for two cases: fixed volume fraction φ and fixed mass density ρ. For fixed volume fraction, we report ζ < 0. For the fixed mass density case, we report ζ > 0, which suggests that the depletion interaction strength increases as the depletant size ratio is increased. To test this prediction, we prepared our filament networks at fixed mass concentrations with varying sizes of the depletant molecule poly(ethylene glycol) (PEG). We characterize the depletion interaction strength in our simulations via the mesh size. In experiments, we observe two distinct actin network morphologies, which we call weakly bundled and strongly bundled. We identify a mass concentration where different PEG depletant sizes lead to weakly bundled or strongly bundled morphologies. For these conditions, we find that the mesh size and intra-bundle spacing between filaments across the different morphologies do not show significant differences, while the dynamic light scattering relaxation time and storage modulus between the two states do show significant differences. Our results demonstrate the ability to tune actin network morphology and mechanics by controlling depletant size and give insights into depletion interaction mechanisms under the fixed-depletant-mass constraint relevant to living cells. [ABSTRACT FROM AUTHOR]

Microtubule-associated protein, MAP1B, is crucial for neuronal morphogenesis and disruptions in MAP1B function are correlated with neurodevelopmental disorders. MAP1B encodes a single polypeptide that is processed into discrete proteins, a heavy chain (HC) and a light chain (LC); however, it is unclear if these two chains operate individually or as a complex within the cell. In vivo studies have characterized the contribution of MAP1B HC and LC to microtubule and actin-based processes, but their molecular mechanisms of action are unknown. Using in vitro reconstitution with purified proteins, we dissect the biophysical properties of the HC and LC and uncover distinct binding behaviors and functional roles for these MAPs. Our biochemical assays indicate that MAP1B HC and LC do not form a constitutive complex, supporting the hypothesis that these proteins operate independently within cells. Both HC and LC inhibit the microtubule motors, kinesin-3, kinesin-4, and dynein, and differentially affect the severing activity of spastin. Notably, MAP1B LC binds to actin filaments in vitro and can simultaneously bind and cross-link actin filaments and microtubules, a function not observed for MAP1B HC. Phosphorylation of MAP1B HC by dual-specificity, tyrosine phosphorylation-regulated kinase 1a negatively regulates its actin-binding activity without significantly affecting its microtubule-binding capacity, suggesting a dynamic contribution of MAP1B HC in cytoskeletal organization. Overall, our study provides new insights into the distinct functional properties of MAP1B HC and LC, underscoring their roles in coordinating cytoskeletal networks during neuronal development.

Our understanding of the transitions of human embryonic stem cells (hESCs) between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of hESCs as they transition from primed to naïve pluripotency which includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for formation of the actin ring, to establish uniform cell mechanics within naïve colonies, to promote nuclear translocation of the Hippo pathway effectors YAP and TAZ, and for effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. Moreover, expression of YAP-S127A partially restores the actin filament fence with Arp2/3 complex inhibition, suggesting that actin filament remodeling is both upstream and downstream of YAP activity. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states.

Inherited mutations in human beta-cardiac myosin (M2β) can lead to severe forms of heart failure. The E525K mutation in M2β is associated with dilated cardiomyopathy (DCM) and was found to stabilize the interacting heads motif (IHM) and autoinhibited super-relaxed (SRX) state in dimeric heavy meromyosin. However, in monomeric M2β subfragment 1 (S1) we found that E525K enhances (threefold) the maximum steady-state actin-activated ATPase activity (k cat) and decreases (eightfold) the actin concentration at which ATPase is one-half maximal (K ATPase). We also found a twofold to fourfold increase in the actin-activated power stroke and phosphate release rate constants at 30 μM actin, which overall enhanced the duty ratio threefold. Loaded motility assays revealed that the enhanced intrinsic motor activity translates to increased ensemble force in M2β S1. Glutamate 525, located near the actin binding region in the so-called activation loop, is highly conserved and predicted to form a salt bridge with another conserved residue (lysine 484) in the relay helix. Enhanced sampling molecular dynamics simulations predict that the charge reversal mutation disrupts the E525-K484 salt bridge, inducing conformations with a more flexible relay helix and a wide phosphate release tunnel. Our results highlight a highly conserved allosteric pathway associated with actin activation of the power stroke and phosphate release and suggest an important feature of the autoinhibited IHM is to prevent this region of myosin from interacting with actin. The ability of the E525K mutation to stabilize the IHM likely overrides the enhanced intrinsic motor properties, which may be key to triggering DCM pathogenesis.

In plant vegetative tissues, cell division employs a mitotic microtubule array called the preprophase band (PPB) that marks the cortical division site. This transient cytoskeletal array imprints the spatial information to be read by the cytokinetic phragmoplast at later stages of mitotic cell division. In Arabidopsis thaliana, we discovered that the PPB recruited the Myosin XI motor MYA1/Myo11F to the cortical division site, where it joined microtubule-associated proteins and motors to form a ring of prominent cytoskeletal assemblies that received the expanding phragmoplast. Such a myosin localization pattern at the cortical division site was dependent on the POK1/2 Kinesin-12 motors. This regulatory function of MYA1/Myo11F in phragmoplast guidance was dependent on intact actin filaments. The discovery of these cytoskeletal motor assemblies pinpoints a mechanism underlying how two dynamic cytoskeletal networks work in concert to govern PPB-dependent division plane orientation in flowering plants.

Synaptic plasticity is important for learning and memory formation; it describes the strengthening or weakening of connections between synapses. The postsynaptic part of excitatory synapses resides in dendritic spines, which are small protrusions on the dendrites. One of the key features of synaptic plasticity is its correlation with the size of these spines. A long-lasting synaptic strength increase [long-term potentiation (LTP)] is only possible through the reconfiguration of the actin spine cytoskeleton. Here, we develop an experimentally informed three-dimensional computational model in a moving boundary framework to investigate this reconfiguration. Our model describes the reactions between actin and actin-binding proteins leading to the cytoskeleton remodeling and their effect on the spine membrane shape to examine the spine enlargement upon LTP. Moreover, we find that the incorporation of perisynaptic elements enhances spine enlargement upon LTP, exhibiting the importance of accounting for these elements when studying structural LTP. Our model shows adaptation to repeated stimuli resulting from the interactions between spine proteins and mechanical forces.

Stroke, trauma, and neurodegenerative disorders cause loss of neurites (axons and dendrites) in addition to neuronal death. Neurite loss may result directly from a primary insult, secondary to parental neuron death, or secondary to a post-injury inflammatory response. Here, we use lipopolysaccharide and the alarmin S100β to selectively evaluate neurite loss caused by the inflammatory response. Activation of microglia and infiltrating macrophages by these stimuli causes neurite loss that far exceeds neuronal death, both in vitro and in vivo. Neurite loss is accompanied by the formation of cofilactin rods and aggregates (CARs), which are polymers of cofilin-1 and actin induced by oxidative stress and other factors. Mice deficient in either cofilin-1 or the superoxide-generating enzyme NADPH oxidase-2 show reduced CAR formation, neurite loss, and motor impairment. The findings identify a mechanism by which inflammation leads to neurite loss via CAR formation and highlight the relevance of neurite loss to functional impairment.

Skeletal muscle actin (ACTA1) mutations are a prevalent cause of skeletal myopathies consistent with ACTA1's high expression in skeletal muscle. Rare de novo mutations in ACTA1 associated with combined cardiac and skeletal myopathies have been reported, but ACTA1 represents only ~20% of the total actin pool in cardiomyocytes, making its role in cardiomyopathy controversial. Here we demonstrate how a mutation in an actin isoform expressed at low levels in cardiomyocytes can cause cardiomyopathy by focusing on a unique ACTA1 variant, R256H. We previously identified this variant in a family with dilated cardiomyopathy, who had reduced systolic function without clinical skeletal myopathy. Using a battery of multiscale biophysical tools, we show that R256H has potent effects on ACTA1 function at the molecular scale and in human cardiomyocytes. Importantly, we demonstrate that R256H acts in a dominant manner, where the incorporation of small amounts of mutant protein into thin filaments is sufficient to disrupt molecular contractility, and that this effect is dependent on the presence of troponin and tropomyosin. To understand the structural basis of this change in regulation, we resolved a structure of R256H filaments using cryoelectron microscopy, and we see alterations in actin's structure that have the potential to disrupt interactions with tropomyosin. Finally, we show that ACTA1R256H/+ human-induced pluripotent stem cell cardiomyocytes demonstrate reduced contractility and sarcomeric organization. Taken together, we demonstrate that R256H has multiple effects on ACTA1 function that are sufficient to cause reduced contractility and establish a likely causative relationship between ACTA1 R256H and clinical cardiomyopathy. [ABSTRACT FROM AUTHOR]

MICAL proteins play a crucial role in cellular dynamics by binding and disassembling actin filaments, impacting processes like axon guidance, cytokinesis, and cell morphology. Their cellular activity is tightly controlled, as dysregulation can lead to detrimental effects on cellular morphology. Although previous studies have suggested that MICALs are autoinhibited, and require Rab proteins to become active, the detailed molecular mechanisms remained unclear. Here, we report the cryo-EM structure of human MICAL1 at a nominal resolution of 3.1 Å. Structural analyses, alongside biochemical and functional studies, show that MICAL1 autoinhibition is mediated by an intramolecular interaction between its N-terminal catalytic and C-terminal coiled-coil domains, blocking F-actin interaction. Moreover, we demonstrate that allosteric changes in the coiled-coil domain and the binding of the tripartite assembly of CH-L2α1-LIM domains to the coiled-coil domain are crucial for MICAL activation and autoinhibition. These mechanisms appear to be evolutionarily conserved, suggesting a potential universality across the MICAL family. The study reveals the cryo-EM structure of human MICAL1, an enzyme that depolymerizes F-actin. Structural analyses show a conserved autoinhibition mechanism through intramolecular interactions between the N-terminal and C-terminal domains. [ABSTRACT FROM AUTHOR]

Membrane remodeling drives a broad spectrum of cellular functions, and it is regulated through mechanical forces exerted on the membrane by cytoplasmic complexes. Here, we investigate how actin filaments dynamically tune their structure to control the active transfer of membranes between cellular compartments with distinct compositions and biophysical properties. Using intravital subcellular microscopy in live rodents we show that a lattice composed of linear filaments stabilizes the granule membrane after fusion with the plasma membrane and a network of branched filaments linked to the membranes by Ezrin, a regulator of membrane tension, initiates and drives to completion the integration step. Our results highlight how the actin cytoskeleton tunes its structure to adapt to dynamic changes in the biophysical properties of membranes. [ABSTRACT FROM AUTHOR]

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

Focal adhesions (FAs) strengthen their link with the actin cytoskeleton to resist force. Talin-vinculin association could reinforce actin anchoring to FAs by controlling actin polymerization. However, the actin polymerization activity of the talin-vinculin complex is not known because it requires the reconstitution of the mechanical and biochemical activation steps that control the association of talin and vinculin. By combining kinetic and binding assays with single actin filament observations in TIRF microscopy, we show that the association of talin and vinculin mutants, mimicking mechanically stretched talin and activated vinculin, triggers a sequential mechanism in which filaments are nucleated, capped and released to elongate. In agreement with these observations, FRAP experiments in cells co-expressing the same constitutive mutants of talin and vinculin revealed accelerated growth of stress fibers. Our findings suggest a versatile mechanism for the regulation of actin assembly in FAs subjected to various combinations of biochemical and mechanical cues. In cells, focal adhesions (FAs) strengthen their connection with the actin cytoskeleton to resist force. By combining biochemistry and cell biology, the authors show that the proteins talin and vinculin control actin assembly, thus reinforcing the anchoring of actin to FAs. [ABSTRACT FROM AUTHOR]

In assisted fertility protocols, in vitro culture conditions mimic physiological conditions to preserve gametes in the best conditions. After collection, oocytes are maintained in a culture medium inside the incubator until in vitro fertilization (IVF) is performed. This time outside natural and physiological conditions exposes oocytes to an oxidative stress that renders in vitro aging. It has been described that in vitro aging produces a spontaneous cortical granule (CG) release decreasing the fertilization rate of oocytes. Nevertheless, this undesirable phenomenon has not been investigated, let alone prevented. In this work, we characterized the spontaneous CG secretion in in vitro aged oocytes. Using immunofluorescence indirect, quantification, and functional assays, we showed that the expression of regulatory proteins of CG exocytosis was affected. Our results demonstrated that in vitro oocyte aging by 4 and 8 h altered the expression and localization of alpha-SNAP and reduced the expression of NSF and Complexin. These alterations were prevented by supplementing culture medium with dithiothreitol (DTT), which in addition to having a protective effect on those proteins, also had an unexpected effect on the actin cytoskeleton. Indeed, DTT addition thickened the cortical layer of fibrillar actin. Both DTT effects, together, prevented the spontaneous secretion of CG and recovered the IVF rate in in vitro aged oocytes. We propose the use of DTT in culture media to avoid the spontaneous CG secretion and to improve the success rate of IVF protocols in in vitro aged oocytes. • DTT restores the normal cortical granules density in in vitro aged oocytes • DTT has a protective effect on alpha-SNAP, NSF, and Complexin • In vitro aged oocytes respond to SrCl 2 as control oocytes in presence of DTT • DTT increases the polymerization of F-actin in the cortical region of mouse oocytes • DTT recovers the in vitro fertilization rate in in vitro aged oocytes. [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. [ABSTRACT FROM AUTHOR]

Both the pollen tube and hyphae of filamentous pathogens penetrate the outer layer of the host and then grow within host tissues. Early epidermal responses are decisive for the outcome of these two-cell interaction processes. We identified a single cell type, the papilla in the stigma of Arabidospis, as a tool to conduct a comprehensive comparative analysis on how an epidermal cell responds to the invasion of an unwanted pathogen or a welcome pollen tube. We showed that Phytophtora parasitica , a root oomycete, effectively breaches the stigmatic cell wall and develops as a biotroph within the papilla cytoplasm. These invasive features resemble the behaviour exhibited by the pathogen within its natural host cell, but diverge from the manner in which the pollen tube progresses, being engulfed within the papilla cell wall. Quantitative analysis revealed that both invaders trigger reorganization of the stigmatic endomembrane system and the actin cytoskeleton. While some remodelling processes are shared between the two interactions, others appear more specific towards the respective invader. These findings underscore the remarkable ability of an epidermal cell to differentiate between two types of invaders, thereby enabling it to trigger the most suitable response during the onset of invasion. [ABSTRACT FROM AUTHOR]

The ACTA1 gene encodes skeletal muscle alpha‐actin, which forms the core of the sarcomeric thin filament in adult skeletal muscle. ACTA1 represents one of six highly conserved actin proteins that have all been associated with human disease. The first 15 pathogenic variants in ACTA1 were reported in 1999, which expanded to 177 in 2009. Here, we update on the now 607 total variants reported in LOVD, HGMD, and ClinVar, which includes 343 reported pathogenic/likely pathogenic (P/LP) variants. We also provide suggested ACTA1‐specific modifications to ACMG variant interpretation guidelines based on our analysis of known variants, gnomAD reports, and pathogenicity in other actin isoforms. Using these criteria, we report a total of 447 P/LP ACTA1 variants. From a clinical perspective, the number of reported ACTA1 disease phenotypes has grown from five to 20, albeit with some overlap. The vast majority (74%) of ACTA1 variants cause nemaline myopathy (NEM), but there are increasing numbers that cause cardiomyopathy and novel phenotypes such as distal myopathy. We highlight challenges associated with identifying genotype–phenotype correlations for ACTA1. Finally, we summarize key animal models and review the current state of preclinical treatments for ACTA1 disease. This update provides important resources and recommendations for the study and interpretation of ACTA1 variants. [ABSTRACT FROM AUTHOR]

The editorial "Editors' showcase 2023: insights in cell adhesion and migration" published in Frontiers in Cell & Developmental Biology focuses on the importance of cell adhesion for migration, with a particular emphasis on the extracellular matrix and adjacent cell interactions. The research topic includes reviews and original research papers that delve into the mechanisms of cell movement in multicellular organisms, both individually and collectively. Various aspects of cell migration, such as cytoskeleton remodeling, molecular motor activity, and chemoattractant gradients, are explored in the editorial. [Extracted from the article]

Introduction: Cells generate mechanical forces mainly through myosin motor activity on the actin cytoskeleton. In C. elegans, actomyosin stress fibers drive contractility of the smooth muscle-like cells of the spermatheca, a distensible, tube-shaped tissue in the hermaphrodite reproductive system and the site of oocyte fertilization. Stretching of the spermathecal cells by oocyte entry triggers activation of the small GTPase Rho. In this study, we asked how forces are distributed in vivo, and explored how spermathecal tissue responds to alterations in myosin activity. Methods: In animals expressing GFP labeled actin or apical membrane complexes, we severed these structures using femtosecond laser ablation and quantified retractions. RNA interference was used to deplete key contractility regulators. Results: We show that the basal actomyosin fibers are under tension in the occupied spermatheca. Reducing actomyosin contractility by depletion of the phospholipase C-e/PLC-1 or non-muscle myosin II/NMY-1, leads to distended spermathecae occupied by one or more embryos, but does not alter tension on the basal actomyosin fibers. However, activating myosin through depletion of the Rho GAP SPV-1 increases tension on the actomyosin fibers, consistent with earlier studies showing Rho drives spermathecal contractility. On the inner surface of the spermathecal tube, tension on the apical junctions is decreased by depletion of PLC-1 and NMY-1. Surprisingly, when basal contractility is increased through SPV-1 depletion, the tension on apical junctions also decreases, with the most significant effect on the junctions aligned in perpendicular to the axis of the spermatheca. Discussion: Our results suggest that much of the tension on the basal actin fibers in the occupied spermatheca is due to the presence of the embryo. Additionally, increased tension on the outer basal surface may compress the apical side, leading to lower tensions apically. The three dimensional shape of the spermatheca plays a role in force distribution and contractility during ovulation. [ABSTRACT FROM AUTHOR]

Membranes undergo various patterns of deformation during vesicle fusion, but how this membrane deformation is regulated and contributes to fusion remains unknown. In this study, we developed a new method of observing the fusion of individual late endosomes and lysosomes by using mouse yolk sac visceral endoderm cells that have huge endocytic vesicles. We found that there were two distinct fusion modes that were differently regulated. In homotypic fusion, two late endosomes fused quickly, whereas in heterotypic fusion they fused to lysosomes slowly. Mathematical modeling showed that vesicle size is a critical determinant of these fusion types and that membrane fluctuation forces can overcome the vesicle size effects. We found that actin filaments were bound to late endosomes and forces derived from dynamic actin remodeling were necessary for quick fusion during homotypic fusion. Furthermore, cofilin played a role in endocytic fusion by regulating actin turnover. These data suggest that actin promotes vesicle fusion for efficient membrane trafficking in visceral endoderm cells. [ABSTRACT FROM AUTHOR]

AbstractIn February 2021, elongated grey lesions with reddish margin were observed on sudangrass (Sorghum × drummondii) leaves, in the municipality of Guasave, Sinaloa, Mexico. These symptoms resembled those of leaf blight caused by Bipolaris and Exserohilum on other grasses. Two fungal isolates were recovered from symptomatic leaves, and their identities were confirmed based on a polyphasic approach. The morphological and phylogenetic analysis of the actin gene (act) and internal transcribed spacer region (ITS) of both isolates revealed their identity as Exserohilum turcicum. A pathogenicity test with a representative isolate confirmed this fungus as the causal agent of leaf blight on sudangrass, as symptoms previously observed in the field were developed on the inoculated plants. The E. turcicum isolate was recovered from the diseased sudangrass plants, fulfilling Koch’s postulates. This is the first report of E. turcicum causing leaf blight on sudangrass in Mexico. [ABSTRACT FROM AUTHOR]

The high turgor pressure across the plasma membrane of yeasts creates a requirement for substantial force production by actin polymerization and myosin motor activity for clathrin-mediated endocytosis (CME). Endocytic internalization is severely impeded in the absence of fimbrin, an actin filament crosslinking protein called Sac6 in budding yeast. Here, we combine live-cell imaging and mathematical modeling to gain insights into the role of actin filament crosslinking proteins in force generation. Genetic manipulation showed that CME sites with more crosslinking proteins are more effective at internalization under high load. Simulations of an experimentally constrained, agent-based mathematical model recapitulate the result that endocytic networks with more double-bound fimbrin molecules internalize the plasma membrane against elevated turgor pressure more effectively. Networks with large numbers of crosslinks also have more growing actin filament barbed ends at the plasma membrane, where the addition of new actin monomers contributes to force generation and vesicle internalization. Our results provide a richer understanding of the crucial role played by actin filament crosslinking proteins during actin network force generation, highlighting the contribution of these proteins to the self-organization of the actin filament network and force generation under increased load. [ABSTRACT FROM AUTHOR]

Aim Methods Results Conclusion Whole‐exome sequencing (WES) studies have revealed that germline de novo variants (gDNVs) contribute to the genetic etiology of schizophrenia. However, the contribution of mosaic DNVs (mDNVs) to the risk of schizophrenia remains to be elucidated. In the present study, we systematically investigated the gDNVs and mDMVs that contribute to the genetic etiology of schizophrenia in a Japanese population.We performed deep WES (depth: 460×) of 73 affected offspring and WES (depth: 116×) of 134 parents from 67 families with schizophrenia. Prioritized rare nonsynonymous gDNV and mDNV candidates were validated using Sanger sequencing and ultra‐deep targeted amplicon sequencing (depth: 71,375×), respectively. Subsequently, we performed a Gene Ontology analysis of the gDNVs and mDNVs to obtain biological insights. Lastly, we selected DNVs in known risk genes for psychiatric and neurodevelopmental disorders.We identified 62 gDNVs and 98 mDNVs. The Gene Ontology analysis of mDNVs implicated actin filament and actin cytoskeleton as candidate biological pathways. There were eight DNVs in known risk genes: splice region gDNVs in AKAP11 and CUL1; a frameshift gDNV in SHANK1; a missense gDNV in SRCAP; missense mDNVs in CTNNB1, GRIN2A, and TSC2; and a nonsense mDNV in ZFHX4.Our results suggest the potential contributions of rare nonsynonymous gDNVs and mDNVs to the genetic etiology of schizophrenia. This is the first report of the mDNVs in schizophrenia trios, demonstrating their potential relevance to schizophrenia pathology. [ABSTRACT FROM AUTHOR]

Apical cell-cell junctions, including adherens junctions and tight junctions, adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing the embryonic epithelium of gastrula-stage Xenopus laevis embryos, we define a mechanism by which the mechanosensitive protein Vinculin helps anchor the actomyosin network at TCJs, thus maintaining TCJ integrity and barrier function. Using an optogenetic approach to acutely increase junctional tension, we find that Vinculin is mechanosensitively recruited to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that Vinculin KD reduces actin stability at TCJs and destabilizes Angulin-1, a key tricellular tight junction protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJ integrity is not maintained, filamentous actin (F-actin) morphology at TCJs is disrupted, and breaks in the signal of the tight junction protein ZO-1 signal are detected. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs. [Display omitted] • Vinculin is mechanosensitively recruited to tricellular junctions • Vinculin's actin-binding function is needed for tricellular actomyosin organization • Vinculin reinforces tricellular junction integrity in response to increased tension • Vinculin is required to maintain barrier function at tricellular junctions Epithelial tricellular junctions are vulnerable to disruption. Here, van den Goor et al. reveal that Vinculin is mechanosensitively recruited to vertebrate tricellular junctions, where it regulates actomyosin organization and barrier function. Vinculin reinforces tricellular junction integrity when epithelia experience increased mechanical force. [ABSTRACT FROM AUTHOR]

The multicellular haploid stage of land plants develops from a single haploid cell produced by meiosis - the spore. Starting from a nonpolar state, these spores develop polarity, divide asymmetrically and establish the first axis of symmetry. Here, we show that the nucleus migrates from the cell centroid to the basal pole during polarisation of the Marchantia polymorpha spore cell. A microtubule organising centre on the leading edge of the nucleus initiates a microtubule array between the nuclear surface and the cortex at the basal pole. Simultaneously, cortical microtubules disappear from the apical hemisphere but persist in the basal hemisphere. This is accompanied by the formation a dense network of fine actin filaments between the nucleus and the basal pole cortex. Experimental depolymerisation of either microtubules or actin filaments disrupts cellular asymmetry. These data demonstrate that the cytoskeleton reorganises during spore polarisation and controls the directed migration of the nucleus to the basal pole. The presence of the nucleus at the basal pole provides the cellular asymmetry for the asymmetric cell division that establishes the apical-basal axis of the plant. [ABSTRACT FROM AUTHOR]

Reelin, a secreted glycoprotein, plays a crucial role in guiding neocortical neuronal migration, dendritic outgrowth and arborization, and synaptic plasticity in the adult brain. Reelin primarily operates through the canonical lipoprotein receptors apolipoprotein E receptor 2 (Apoer2) and very low-density lipoprotein receptor (Vldlr). Reelin also engages with noncanonical receptors and unidentified coreceptors; however, the effects of which are less understood. Using high-throughput tandem mass tag (TMT) liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomics and gene set enrichment analysis (GSEA), we identified both shared and unique intracellular pathways activated by Reelin through its canonical and noncanonical signaling in primary murine neurons of either sex during dendritic growth and arborization. We observed pathway cross talk related to regulation of cytoskeleton, neuron projection development, protein transport, and actin filament-based process. We also found enriched gene sets exclusively by the noncanonical Reelin pathway including protein translation, mRNA metabolic process, and ribonucleoprotein complex biogenesis suggesting Reelin fine-tunes neuronal structure through distinct signaling pathways. A key discovery is the identification of aldolase A, a glycolytic enzyme and actin-binding protein, as a novel effector of Reelin signaling. Reelin induced de novo translation and mobilization of aldolase A from the actin cytoskeleton. We demonstrated that aldolase A is necessary for Reelin-mediated dendrite growth and arborization in primary murine neurons and mouse brain cortical neurons. Interestingly, the function of aldolase A in dendrite development is independent of its known role in glycolysis. Altogether, our findings provide new insights into the Reelin-dependent signaling pathways and effector proteins that are crucial for dendritic development. [ABSTRACT FROM AUTHOR]

ABSTRACT Actin depolymerizing factors (ADFs), like other actin‐binding proteins (ABPs), are modified by phosphorylation to regulate the dynamics of the actin filaments, thereby functioning in various processes throughout the plant lifecycle. In this study, we found that the Arabidopsis thaliana cytoplasmic kinase AGC1.7 interacts with ADF7 in vitro and in vivo. AGC1.7 phosphorylates ADF7 at its Ser‐6, Ser‐103 and Ser‐104 residues in vitro, while replacing these residues with alanine promotes ADF7‐mediated actin depolymerization in vitro. Expression of the phosphorylation‐mimetic mutant protein ADF7S6/103/104D driven by the pollen‐specific LAT52 promoter fully rescues the defects in germination rate, silique length and seeds per silique in both adf7‐2 and agc1.5 agc1.7 (agcdm) mutants. Our data establish a model whereby AGC1.7‐mediated ADF7 phosphorylation plays an important role in pollen germination and pollen tube growth. [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]

Stress granules (SGs) are non-membranous organelles composed of mRNA and proteins that assemble in the cytosol when the cell is under stress. Although the composition of mammalian SGs is both cell-type and stress-dependent, they consistently contain core components, such as Ras GTPase activating protein SH3 domain binding protein 1 (G3BP1). Upon stress, living cells rapidly assemble micrometric SGs, sometimes within a few minutes, suggesting that SG components may be actively transported by the microtubule and/or actin cytoskeleton. Indeed, SG assembly has been shown to depend on the microtubule cytoskeleton and the associated motor proteins. However, the role of the actin cytoskeleton and associated myosin motor proteins remains controversial. Here, we identified G3BP1 as a novel binding protein of unconventional myosin-5a (Myo5a). G3BP1 uses its C-terminal RNA-binding domain to interact with the middle portion of Myo5a tail domain (Myo5a-MTD). Suppressing Myo5a function in mammalian cells, either by overexpressing Myo5a-MTD, eliminating Myo5a gene expression, or treatment with myosin-5 inhibitor, inhibits the arsenite-induced formation of both small and large SGs. This is different from the effect of microtubule disruption, which abolishes the formation of large SGs but enhances the formation of small SGs under stress conditions. We therefore propose that, under stress conditions, Myo5a facilitates the formation of SGs at an earlier stage than the microtubule-dependent process. [ABSTRACT FROM AUTHOR]

Regulated cell shape change requires the induction of cortical cytoskeletal domains. Often, local changes to plasma membrane (PM) topography are involved. Centrosomes organize cortical domains and can affect PM topography by locally pulling the PM inward. Are these centrosome effects coupled? At the syncytial Drosophila embryo cortex, centrosome-induced actin caps grow into dome-like compartments for mitoses. We found the nascent cap to be a collection of PM folds and tubules formed over the astral centrosomal MT array. The localized infoldings require centrosome and dynein activities, and myosin-based surface tension prevents them elsewhere. Centrosome-engaged PM infoldings become specifically enriched with an Arp2/3 induction pathway. Arp2/3 actin network growth between the infoldings counterbalances centrosomal pulling forces and disperses the folds for actin cap expansion. Abnormal domain topography with either centrosome or Arp2/3 disruption correlates with decreased exocytic vesicle association. Together, our data implicate centrosome-organized PM infoldings in coordinating Arp2/3 network growth and exocytosis for cortical domain assembly. [ABSTRACT FROM AUTHOR]

Cell migration requires the constant modification of cellular shape by reorganization of the actin cytoskeleton. Fine-tuning of this process is critical to ensure new actin filaments are formed only at specific times and in defined regions of the cell. The Scar/WAVE complex is the main catalyst of pseudopod and lamellipodium formation during cell migration. It is a pentameric complex highly conserved through eukaryotic evolution and composed of Scar/WAVE, Abi, Nap1/NCKAP1, Pir121/CYFIP, and HSPC300/Brk1. Its function is usually attributed to activation of the Arp2/3 complex through Scar/WAVE's VCA domain, while other parts of the complex are expected to mediate spatial-temporal regulation and have no direct role in actin polymerization. Here, we show in both B16-F1 mouse melanoma and Dictyostelium discoideum cells that Scar/WAVE without its VCA domain still induces the formation of morphologically normal, actin-rich protrusions, extending at comparable speeds despite a drastic reduction of Arp2/3 recruitment. However, the proline-rich regions in Scar/WAVE and Abi subunits are essential, though either is sufficient for the generation of actin protrusions in B16-F1 cells. We further demonstrate that N-WASP can compensate for the absence of Scar/WAVE's VCA domain and induce lamellipodia formation, but it still requires an intact WAVE complex, even if without its VCA domain. We conclude that the Scar/WAVE complex does more than directly activating Arp2/3, with proline-rich domains playing a central role in promoting actin protrusions. This implies a broader function for the Scar/WAVE complex, concentrating and simultaneously activating many actin-regulating proteins as a lamellipodium-producing core. [Display omitted] • Scar/WAVE, without its VCA domain, promotes the formation of actin-rich protrusions • Scar/WAVEΔVCA's function is conserved in both B16-F1 and D. dictyostelium cells • At least one of the WRC's polyproline domains is required for actin protrusions • N-WASP compensates for the absence of WRC's VCA domain and promotes lamellipodia Buracco et al. show that the WRC, without its VCA domain, still promotes actin-rich protrusions. However, WRC's proline-rich regions and N-WASP are essential, with the latter compensating for the absence of Scar/WAVE's VCA domain. This proves that the WRC has a broader function than activating Arp2/3. [ABSTRACT FROM AUTHOR]

Tropomyosins (Tpms) are rod-shaped proteins that interact head-to-tail to form a continuous polymer along both sides of most cellular actin filaments. Head-to-tail interaction between adjacent Tpm molecules and the formation of an overlap complex between them leads to the assembly of actin filaments with one type of Tpm isoform in time and space. Variations in the affinity of tropomyosin isoforms for different actin structures are proposed as a potential sorting mechanism. However, the detailed mechanisms of the spatio-temporal sorting of Tpms remain elusive. In this study, we investigated the early intermediates during actin–tropomyosin filament assembly, using a skeletal/cardiac Tpm isoform (Tpm1.1) and a cytoskeletal isoform (Tpm1.6) that differ only in the last 27 amino acids. We investigated how the muscle isoform Tpm1.1 and the cytoskeletal isoform Tpm1.6 nucleate domains on the actin filament, and tested whether (1) recruitment is affected by the actin isoform (muscle vs. cytoskeletal) and (2) whether there is specificity in recruiting the same isoform to a domain at these early stages. To address these questions, actin filaments were exposed to low concentrations of fluorescent tropomyosins in solution. The filaments were immobilized onto glass coverslips and the pattern of decoration was visualized by TIRF microscopy. We show that at the early assembly stage, tropomyosins formed multiple distinct fluorescent domains (here termed "cluster") on the actin filaments. An automated image analysis algorithm was developed and validated to identify clusters and estimate the number of tropomyosins in each cluster. The analysis showed that tropomyosin isoform sorting onto an actin filament is unlikely to be driven by a preference for nucleating on the corresponding muscle or cytoskeletal actin isoforms, but rather is facilitated by a higher probability of incorporating the same tropomyosin isoforms into an early assembly intermediate. We showed that the 27 amino acids at the end of each tropomyosin seem to provide enough molecular information for the attachment of the same tropomyosin isoforms adjacent to each other on an actin filament. This results in the formation of homogeneous clusters composed of the same isoform rather than clusters with mixed isoforms. [ABSTRACT FROM AUTHOR]

Skeletal muscle fibers need to have mechanisms to decrease energy consumption during intense physical exercise to avoid devastatingly low ATP levels, with the formation of rigor cross bridges and defective ion pumping. These protective mechanisms inevitably lead to declining contractile function in response to intense exercise, characterizing fatigue. Through our work, we have gained insights into cellular and molecular mechanisms underlying the decline in contractile function during acute fatigue. Key mechanistic insights have been gained from studies performed on intact and skinned single muscle fibers and more recently from studies performed and single myosin molecules. Studies on intact single fibers revealed several mechanisms of impaired sarcoplasmic reticulum Ca2+ release and experiments on single myosin molecules provide direct evidence of how putative agents of fatigue impact myosin's ability to generate force and motion. We conclude that changes in metabolites due to an increased dependency on anaerobic metabolism (e.g., accumulation of inorganic phosphate ions and H+) act to directly and indirectly (via decreased Ca2+ activation) inhibit myosin's force and motion-generating capacity. These insights into the acute mechanisms of fatigue may help improve endurance training strategies and reveal potential targets for therapies to attenuate fatigue in chronic diseases. [ABSTRACT FROM AUTHOR]

Neuronal development is a highly regulated process that is dependent on the correct coordination of cellular responses to extracellular cues. In response to semaphorin axon guidance proteins, the MICAL1 protein is stimulated to produce reactive oxygen species that oxidize actin on specific methionine residues, leading to filamentous actin depolymerization and consequent changes in neuronal growth cone dynamics. Crossing genetically modified mice homozygous for floxed Mical1 (Mical1fl/fl) alleles with transgenic mice expressing Cre recombinase under the control of a tyrosinase gene enhancer/promoter (Tyr::Cre) enabled conditional Mical1 deletion. Immunohistochemical analysis showed Mical1 expression in the cerebellum, which plays a prominent role in the coordination of motor movements, with reduced Mical1 expression in Mical1fl/fl mice co‐expressing Tyr::Cre. Analysis of the gaits of mice running on a treadmill showed that both male and female Mical1fl/fl, Tyr::Cre mutant mice had significant alterations to their striding patterns relative to wild‐type mice, although the specific aspects of their altered gaits differed between the sexes. Additional motor tests that involved movement on a rotating rod, descending a vertical pole, or crossing a balance beam did not show significant differences between the genotypes, suggesting that the effect of the Mical1fl/fl, Tyr::Cre genetic modifications was only manifested during specific highly coordinated movements that contribute to running. These findings indicate that there is a behavioral consequence in Mical1fl/fl, Tyr::Cre mutant mice that affects motor control as manifested by alterations in their gait. [ABSTRACT FROM AUTHOR]

We study a discrete-time interacting particle system with continuous state space, which is motivated by a mathematical model for turnover through branching in actin filament networks. It gives rise to transient clusters reminiscent of actin filament assemblies in the cortex of living cells. We reformulate the process in terms of the inter-particle distances and characterize their marginal and joint distributions. We construct a recurrence relation for the associated characteristic functions and pass to the large population limit, reminiscent of the Fleming-Viot super processes. The precise characterization of all marginal distributions established in this work opens the way to a detailed analysis of cluster dynamics. We also obtain a recurrence relation that enables us to compute the moments of the asymptotic single particle distribution characterizing the transient aggregates. Our results indicate that aggregates have a fat-tailed distribution. [ABSTRACT FROM AUTHOR]

This review highlights the complexity of renal epithelial cell membrane architectures and organelles through careful review of ultrastructural and physiological studies published over the past several decades. We also showcase the vital roles played by the actin cytoskeleton and actin-associated myosin motor proteins in regulating cell type-specific physiological functions within the cells of the renal epithelium. The purpose of this review is to provide a fresh conceptual framework to explain the structure-function relationships that exist between the actin cytoskeleton, organelle structure, and cargo transport within the mammalian kidney. With recent advances in technologies to visualize the actin cytoskeleton and associated proteins within intact kidneys, it has become increasingly imperative to reimagine the functional roles of these proteins in situ to provide a rationale for their unique, cell type-specific functions that are necessary to establish and maintain complex physiological processes. [ABSTRACT FROM AUTHOR]

The hair bundle, or stereocilia bundle, is the mechanosensory compartment of hair cells (HCs) in the inner ear. To date, most mechanistic studies have focused on stereocilia bundle morphogenesis, and it remains unclear how this organelle critical for hearing preserves its precise dimensions during life in mammals. The GPSM2-GNAI complex occupies the distal tip of stereocilia in the tallest row and is required for their elongation during development. Here, we ablate GPSM2-GNAI in adult mouse HCs after normal stereocilia elongation is completed. We observe a progressive height reduction of the tallest row stereocilia totaling ~600 nm after 12 wk in Gpsm2 mutant inner HCs. To measure GPSM2 longevity at tips, we generated a HaloTag-Gpsm2 mouse strain and performed pulse-chase experiments in vivo. Estimates using pulse-chase or tracking loss of GPSM2 immunolabeling following Gpsm2 inactivation suggest that GPSM2 is relatively long-lived at stereocilia tips with a half-life of 9 to 10 d. Height reduction coincides with dampened auditory brainstem responses evoked by low-frequency stimuli in particular. Finally, GPSM2 is required for normal tip enrichment of elongation complex (EC) partners MYO15A, WHRN, and EPS8, mirroring their established codependence during development. Taken together, our results show that the EC is also essential in mature HCs to ensure precise and stable stereocilia height and for sensitive detection of a full range of sound frequencies. [ABSTRACT FROM AUTHOR]

Summary: The effect of dry aging process (15 and 30 days) on some quality parameters, protein profile and formation of protein oxidation products of beef was investigated. The maximum weight and trim loss were calculated to be 47.07% and 23.06%, respectively. The meat dry‐aged for 30 days had higher dry matter, pH and protein content, but lower L* and a* values than those of the unaged meats. There was a significant decrease in actin protein band density in the meat dry‐aged for 30 days and a new protein band of 32 kDa appeared in the myofibrillar protein fraction. The dry aging process caused an insignificant increase in the total carbonyl content (P > 0.05). However, the γ‐glutamic semialdehyde (GGS), α‐aminoadipic semialdehyde (AAS) and total GGS and AAS contents were higher in the dry‐aged meats for 30 days. Total GGS and AAS contents ranged from 15.36% to 31.87% of the total carbonyl content. It was found that there was no significant difference between control group (unaged) meats and the meat dry‐aged for 15 days in terms of protein oxidation products. [ABSTRACT FROM AUTHOR]

Human β-cardiac myosin exists in an ON-state where both myosin heads are accessible for interaction with actin, and an OFF-state where the heads are folded back onto their own coiled-coil tail, interacting with each other via an interacting-heads motif (IHM). Hypertrophic cardiomyopathy (HCM) mutations in β-cardiac myosin cause hypercontractility of the heart. Nine years ago, a unifying hypothesis proposed that hypercontractility caused by myosin HCM-associated mutations is primarily due to an increase in the number of ON-state myosin molecules, rather than altered fundamental alterations of functional myosin parameters such as intrinsic motor force, its velocity of movement along actin, or its ATPase turnover rate, all of which impact power output. We here revisit this unifying hypothesis in light of accumulated data measuring all these parameters, and the recent availability of a 3.6 Å-resolution structure of the human β-cardiac myosin IHM. Biochemical measurements show that nearly all myosin HCM mutations examined exhibit more ON-state myosin regardless of where they occur in the myosin head domain, consistent with the unifying hypothesis. Significant advances in structural and biochemical research validate the 9-year-old hypothesis that cardiac hypercontractility seen in patients with hypertrophic cardiomyopathy is primarily caused by sarcomeric mutations that increase the number of myosin molecules available for actin interaction. [ABSTRACT FROM AUTHOR]

Diffusion and immobilization of molecules in biomembranes are essential for life. Understanding it is crucial for biomimetic approaches where well-defined substrates are created for live cell assays or biomaterial development. Here, we present biomimetic model systems consisting of a supported lipid bilayer and membrane coupled proteins to study the influence of lipid–lipid and lipid–protein interactions on membrane mobility. To characterize the diffusion of lipids or proteins, the continuous photobleaching technique is used. Either Neutravidin coupled to DOPE-cap-Biotin lipids or GFP coupled to DOGS-NTA lipids is studied at 0.005–0.5 mol% concentration of the linker lipid. Neutravidin creates mobile obstacles in the membrane, while GFP coupling results in immobile obstacles. By actin filament coupling to Neutravidin-lipid complexes, obstacles are crosslinked, resulting in lipid mobility reduction along with the appearance of a membrane texture. Theoretical considerations accurately describe lipid diffusion changes at high obstacle concentration as a function of obstacle size and viscous effects. The mobility of membrane lipids depends on the concentration of protein-binding lipids and on the concentration and charge of the coupled protein. Next to diffusion and friction coefficients, we determine the effective obstacle size as well as a charge-dependent effect that dominates the decrease in lipid mobility. [ABSTRACT FROM AUTHOR]

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by low levels of the Survival of Motoneuron (SMN) protein. SMN interacts with and regulates the actin‐binding protein profilin2a, thereby influencing actin dynamics. Dysfunctional actin dynamics caused by SMN loss disrupts neurite outgrowth, axonal pathfinding, and formation of functional synapses in neurons. Whether the SMN protein directly interacts with and regulates filamentous (F‐) and monomeric globular (G‐) actin is still elusive. In a quantitative single cell approach, we show that SMN loss leads to dysregulated F‐/G‐actin fractions. Furthermore, quantitative assessment of cell morphology suggests an F‐actin organizational defect. Interestingly, this is mediated by an interaction of SMN with G‐ and F‐actin. In co‐immunoprecipitation, in‐vitro pulldown and co‐localization assays, we elucidated that this interaction is independent of the SMN‐profilin2a interaction. Therefore, we suggest two populations being relevant for functional actin dynamics in healthy neurons: SMN‐profilin2a‐actin and SMN‐actin. Additionally, those two populations may influence each other and therefore regulate binding of SMN to actin. In SMA, we showed a dysregulated co‐localization pattern of SMN‐actin which could only partially rescued by SMN restoration. However, dysregulation of F‐/G‐actin fractions was reduced by SMN restoration. Taken together, our results suggest a novel molecular function of SMN in binding to actin independent from SMN‐profilin2a interaction. [ABSTRACT FROM AUTHOR]

Raphid diatoms are one of the few eukaryotes capable of gliding motility, which is remarkably fast and allows for quasi-instantaneous directional reversals. Besides other mechanistic models, it has been suggested that an actomyosin system provides the force for diatom gliding. However, in vivo data on the dynamics of actin and myosin in diatoms are lacking. In this study, we demonstrate that the raphe-associated actin bundles required for diatom movement do not exhibit a directional turnover of subunits and thus their dynamics do not contribute directly to force generation. By phylogenomic analysis, we identified four raphid diatom-specific myosins in Craspedostauros australis (CaMyo51A-D) and investigated their in vivo localization and dynamics through GFP-tagging. Only CaMyo51B-D but not CaMyo51A exhibited coordinated movement during gliding, consistent with a role in force generation. The characterization of raphid diatom-specific myosins lays the foundation for unraveling the molecular mechanisms that underlie the gliding motility of diatoms. Phylogenomic analysis and TIRF microscopy were used to investigate the role of actin and myosin in diatom gliding. The findings support the involvement of three raphid diatom-specific myosins in force generation during cell movement. [ABSTRACT FROM AUTHOR]

Class I myosins (myosin-Is) colocalize with Arp2/3 complex-nucleated actin networks at sites of membrane protrusion and invagination, but the mechanisms by which myosin-I motor activity coordinates with branched actin assembly to generate force are unknown. We mimicked the interplay of these proteins using the "comet tail" bead motility assay, where branched actin networks are nucleated by the Arp2/3 complex on the surface of beads coated with myosin-I and nucleation-promoting factor. We observed that myosin-I increased bead movement efficiency by thinning actin networks without affecting growth rates. Myosin-I triggered symmetry breaking and comet tail formation in dense networks resistant to spontaneous fracturing. Even with arrested actin assembly, myosin-I alone could break the network. Computational modeling recapitulated these observations, suggesting myosin-I acts as a repulsive force shaping the network's architecture and boosting its force-generating capacity. We propose that myosin-I leverages its power stroke to amplify the forces generated by Arp2/3 complex-nucleated actin networks. [ABSTRACT FROM AUTHOR]

The failure of intracellular zinc accumulation is a key process in prostate carcinogenesis. Although prostate cancer cells can accumulate zinc after long-term exposure, chronic zinc oversupply may accelerate prostate carcinogenesis or chemoresistance. Because cancer progression is associated with energetically demanding cytoskeletal rearrangements, we investigated the effect of long-term zinc presence on biophysical parameters, ATP production, and EMT characteristics of two prostate cancer cell lines (PC-3, 22Rv1). Prolonged exposure to zinc increased ATP production, spare respiratory capacity, and induced a response in PC-3 cells, characterized by remodeling of vimentin and a shift of cell dry mass density and caveolin-1 to the perinuclear region. This zinc-induced remodeling correlated with a greater tendency to maintain actin architecture despite inhibition of actin polymerization by cytochalasin. Zinc partially restored epithelial characteristics in PC-3 cells by decreasing vimentin expression and increasing E-cadherin. Nevertheless, the expression of E-cadherin remained lower than that observed in predominantly oxidative, low-invasive 22Rv1 cells. Following long-term zinc exposure, we observed an increase in cell stiffness associated with an increased refractive index in the perinuclear region and an increased mitochondrial content. The findings of the computational simulations indicate that the mechanical response cannot be attributed exclusively to alterations in cytoskeletal composition. This observation suggests the potential involvement of an additional, as yet unidentified, mechanical contributor. These findings indicate that long-term zinc exposure alters a group of cellular parameters towards an invasive phenotype, including an increase in mitochondrial number, ATP production, and cytochalasin resistance. Ultimately, these alterations are manifested in the biomechanical properties of the cells. [ABSTRACT FROM AUTHOR]

Here, we report the generation of a transgenic Lifeact-EGFP quail line for the investigation of actin organization and dynamics during morphogenesis in vivo. This transgenic avian line allows for the high-resolution visualization of actin structures within the living embryo, from the subcellular filaments that guide cell shape to the supracellular assemblies that coordinate movements across tissues. The unique suitability of avian embryos to live imaging facilitates the investigation of previously intractable processes during embryogenesis. Using high-resolution live imaging approaches, we present the dynamic behaviors and morphologies of cellular protrusions in different tissue contexts. Furthermore, through the integration of live imaging with computational segmentation, we visualize cells undergoing apical constriction and large-scale actin structures such as multicellular rosettes within the neuroepithelium. These findings not only enhance our understanding of tissue morphogenesis but also demonstrate the utility of the Lifeact-EGFP transgenic quail as a new model system for live in vivo investigations of the actin cytoskeleton. [ABSTRACT FROM AUTHOR]

Macropinocytosis mediates the non-selective bulk uptake of extracellular fluid, enabling cells to survey the environment and obtain nutrients. A conserved set of signaling proteins orchestrates the actin dynamics that lead to membrane ruffling and macropinosome formation across various eukaryotic organisms. At the center of this signaling network are Ras GTPases, whose activation potently stimulates macropinocytosis. However, how Ras signaling is initiated and spatiotemporally regulated during macropinocytosis is not well understood. By using the model system Dictyostelium and a proteomics-based approach to identify regulators of macropinocytosis, we uncovered Leep2, consisting of Leep2A and Leep2B, as a RasGAP complex. The Leep2 complex specifically localizes to emerging macropinocytic cups and nascent macropinosomes, where it modulates macropinosome formation by regulating the activities of three Ras family small GTPases. Deletion or overexpression of the complex, as well as disruption or sustained activation of the target Ras GTPases, impairs macropinocytic activity. Our data reveal the critical role of fine-tuning Ras activity in directing macropinosome formation. [ABSTRACT FROM AUTHOR]

We previously identified talin rod domain-containing protein 1 (TLNRD1) as a potent actin-bundling protein in vitro. Here, we report that TLNRD1 is expressed in the vasculature in vivo. Its depletion leads to vascular abnormalities in vivo and modulation of endothelial cell monolayer integrity in vitro. We demonstrate that TLNRD1 is a component of the cerebral cavernous malformations (CCM) complex through its direct interaction with CCM2, which is mediated by a hydrophobic C-terminal helix in CCM2 that attaches to a hydrophobic groove on the four-helix domain of TLNRD1. Disruption of this binding interface leads to CCM2 and TLNRD1 accumulation in the nucleus and actin fibers. Our findings indicate that CCM2 controls TLNRD1 localization to the cytoplasm and inhibits its actin-bundling activity and that the CCM2-TLNRD1 interaction impacts endothelial actin stress fiber and focal adhesion formation. Based on these results, we propose a new pathway by which the CCM complex modulates the actin cytoskeleton and vascular integrity. [ABSTRACT FROM AUTHOR]

Cell processes require precise regulation of actin polymerization that is mediated by plus-end regulatory proteins. Detailed mechanisms that explain plus-end dynamics involve regulators with opposing roles, including factors that enhance assembly, e.g., the formin mDia1, and others that stop growth (capping protein, CP). We explore IQGAP1's roles in regulating actin filament plus-ends and the consequences of perturbing its activity in cells. We confirm that IQGAP1 pauses elongation and interacts with plus ends through two residues (C756 and C781). We directly visualize the dynamic interplay between IQGAP1 and mDia1, revealing that IQGAP1 displaces the formin to influence actin assembly. Using four-color TIRF, we show that IQGAP1's displacement activity extends to formin-CP "decision complexes," promoting end-binding protein turnover at plus-ends. Loss of IQGAP1 or its plus-end activities disrupts morphology and migration, emphasizing its essential role. These results reveal a new role for IQGAP1 in promoting protein turnover on filament ends and provide new insights into how plus-end actin assembly is regulated in cells. [ABSTRACT FROM AUTHOR]

Myelin is an insulator that forms around axons that enhance the conduction velocity of nerve fibers. Oligodendrocytes dramatically change cell morphology to produce myelin throughout the central nervous system (CNS). Cytoskeletal alterations are critical for the morphogenesis of oligodendrocytes, and actin is involved in cell differentiation and myelin wrapping via polymerization and depolymerization, respectively. Various protein members of the myosin superfamily are known to be major binding partners of actin filaments and have been intensively researched because of their involvement in various cellular functions, including differentiation, cell movement, membrane trafficking, organelle transport, signal transduction, and morphogenesis. Some members of the myosin superfamily have been found to play important roles in the differentiation of oligodendrocytes and in CNS myelination. Interestingly, each member of the myosin superfamily expressed in oligodendrocyte lineage cells also shows specific spatial and temporal expression patterns and different distributions. In this review, we summarize previous findings related to the myosin superfamily and discuss how these molecules contribute to myelin formation and regeneration by oligodendrocytes. [ABSTRACT FROM AUTHOR]

Cytokinesis is driven in part by the constriction of a ring of actin filaments, myosin motors and other proteins. In fission yeast, three myosins contribute to cytokinesis including a Myosin-V Myo51. As Myosin-Vs typically carry cargo along actin filaments, the role of Myo51 in cytokinesis remains unclear. The previous work suggests that Myo51 may crosslink actin filaments. We hypothesized that if Myo51 crosslinks actin filaments, cells carrying double deletions of ain1, which encodes the crosslinker alpha-actinin, and myo51 (∆ain1 ∆myo51 cells) will exhibit more severe cytokinesis phenotypes than cells with the single ∆ain1 mutation. Contrary to our expectations, we found that the loss of Myo51 in ∆ain1 cells partially rescued the severity of the node clumping phenotype measured in ∆ain1 cells. Furthermore, we describe a normal process of contractile ring "shedding", the appearance of fragments of ring material extending away from the contractile ring along the ingressing septum that occurs in the second half of constriction. We measured that ∆ain1 ∆myo51 cells exhibit premature and exaggerated shedding. Our work suggests that Myo51 is not a simple actin filament crosslinker. Instead, a role in effective node motion better recapitulates its function during ring assembly and disassembly. [ABSTRACT FROM AUTHOR]