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Background: Human restricted genes contribute to human specific traits in the immune system. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR), the highest Ca 2+ conductor of the ACh receptors implicated in innate immunity. Understanding the mechanism of how CHRFAM7A affects the immune system remains unexplored., Methods: Two model systems are used, human induced pluripotent stem cells (iPSC) and human primary monocytes, to characterize α7 nAChR function, Ca 2+ dynamics and decoders to elucidate the pathway from receptor to phenotype., Findings: CHRFAM7A/α7 nAChR is identified as a hypomorphic receptor with mitigated Ca 2+ influx and prolonged channel closed state. This shifts the Ca 2+ reservoir from the extracellular space to the endoplasmic reticulum (ER) leading to Ca 2+ dynamic changes. Ca 2+ decoder small GTPase Rac1 is then activated, reorganizing the actin cytoskeleton. Observed actin mediated phenotypes include cellular adhesion, motility, phagocytosis and tissue mechanosensation., Interpretation: CHRFAM7A introduces an additional, human specific, layer to Ca 2+ regulation leading to an innate immune gain of function. Through the actin cytoskeleton it drives adaptation to the mechanical properties of the tissue environment leading to an ability to invade previously immune restricted niches. Human genetic diversity predicts profound translational significance as its understanding builds the foundation for successful treatments for infectious diseases, sepsis, and cancer metastasis., Funding: This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti) and in part by NIH grant R01HL163168 (Yongho Bae)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

During plant-pathogen interactions, cytoskeleton and calcium signaling work independently as well as in coordination with each other for developing preformed and induced defense responses. A cell wall (CW) - plasma membrane (PM) - cytoskeleton (CS) continuum is maintained by coordination of cytoskeleton and calcium signaling. The current review is focused on the current knowledge of cytoskeleton‑calcium cross-regulation during plant-pathogen interactions. Implications of recent technological developments in the existing toolkit that can address the outstanding questions of cytoskeleton‑calcium coordination plant immunity are also discussed., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

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

Hypertension is a pervasive and widespread health condition that poses a significant risk factor for cardiovascular disease, which includes conditions such as heart attack, stroke, and heart failure. Despite its widespread occurrence, the exact cause of hypertension remains unknown, and the mechanisms underlying the progression from prehypertension to hypertension require further investigation. Recent proteomic studies have shown promising results in uncovering potential biomarkers related to disease development. In this study, serum proteomic data collected from Qatar Biobank were analyzed to identify altered protein expression between individuals with normal blood pressure, prehypertension, and hypertension and to elucidate the biological pathways contributing to this disease. The results revealed a cluster of proteins, including the SRC family, CAMK2B, CAMK2D, TEC, GSK3, VAV, and RAC, which were markedly upregulated in patients with hypertension compared to those with prehypertension (fold change ≥ 1.6 or ≤-1.6, area under the curve ≥ 0.8, and q -value < 0.05). Pathway analysis showed that the majority of these proteins play a role in actin cytoskeleton remodeling. Actin cytoskeleton reorganization affects various biological processes that contribute to the maintenance of blood pressure, including vascular tone, endothelial function, cellular signaling, inflammation, fibrosis, and mechanosensing. Therefore, the findings of this study suggest a potential novel role of actin cytoskeleton-related proteins in the progression from prehypertension to hypertension. The present study sheds light on the underlying pathological mechanisms involved in hypertension and could pave the way for new diagnostic and therapeutic approaches for the treatment of this disease.

Actin dynamics are critical for plant cell morphogenesis, but the underlying signaling mechanisms regulating these dynamics are not well understood. Here, we established that PLEIOTROPIC REGULATORY LOCUS1 (PRL1) modulates leaf pavement cell (PC) morphogenesis in Arabidopsis (Arabidopsis thaliana) by maintaining the dynamic homeostasis of actin microfilaments (MF). Our previous studies indicated that PC shape was determined by antagonistic RHO-RELATED GTPase FROM PLANTS 2 (ROP2) and RHO-RELATED GTPase FROM PLANTS 6 (ROP6) signaling pathways that promote cortical MF and microtubule organization, respectively. Our genetic screen for additional components in ROP6-mediated signaling identified prl1 alleles. Genetic analysis confirmed that PRL1 plays a key role in PC morphogenesis. Mutations in PRL1 caused cortical MF depolymerization, resulting in defective PC morphogenesis. Further genetic analysis revealed that PRL1 is epistatic to ROP2 and ROP6 in PC morphogenesis. Mutations in PRL1 enhanced the effects of ROP2 and ROP6 in PC morphogenesis, leading to a synergistic phenotype in the PCs of ROP2 prl1 and ROP6 prl1. Furthermore, the activities of ROP2 and ROP6 were differentially altered in prl1 mutants, suggesting that ROP2 and ROP6 function downstream of PRL1. Additionally, cortical MF depolymerization in prl1 mutants occurred independently of ROP2 and ROP6, implying that these proteins impact PC morphogenesis in the prl1 mutant through other cellular processes. Our research indicates that PRL1 preserves the structural integrity of actin and facilitates pavement cell morphogenesis in Arabidopsis., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

There are three main classes of actin nucleation factors: Arp2/3 complexes, Spire and Formin. Spire assembles microfilaments by nucleating stable longitudinal tetramers and binding actin to the growing end of the microfilament. As early as 1999, Wellington et al. identified Spire as an actin nucleating agent, however, over the years, most studies have focused on Arp2/3 and Formin proteins; there has been relatively less research on Spire as a member of the actin nucleating factors. Recent studies have shown that Spire is involved in the vesicular transport through the synthesis of actin and plays an important role in neural development. In this paper, we reviewed the structure, expression and function of Spire, and its association with disease in order to identify meaningful potential directions for studies on Spire.

Cell adhesion requires linkage of transmembrane receptors to the cytoskeleton through intermediary linker proteins. Integrin-based adhesion to the extracellular matrix (ECM) involves large adhesion complexes that contain multiple cytoskeletal adapters that connect to the actin cytoskeleton. Many of these adapters, including the essential cytoskeletal linker Talin, have been shown to contain multiple actin-binding sites (ABSs) within a single protein. To investigate the possible role of having such a variety of ways of linking integrins to the cytoskeleton, we generated mutations in multiple actin binding sites in Drosophila talin. Using this approach, we have been able to show that different actin-binding sites in talin have both unique and complementary roles in integrin-mediated adhesion. Specifically, mutations in either the C-terminal ABS3 or the centrally located ABS2 result in lethality showing that they have unique and non-redundant function in some contexts. On the other hand, flies simultaneously expressing both the ABS2 and ABS3 mutants exhibit a milder phenotype than either mutant by itself, suggesting overlap in function in other contexts. Detailed phenotypic analysis of ABS mutants elucidated the unique roles of the talin ABSs during embryonic development as well as provided support for the hypothesis that talin acts as a dimer in in vivo contexts. Overall, our work highlights how the ability of adhesion complexes to link to the cytoskeleton in multiple ways provides redundancy, and consequently robustness, but also allows a capacity for functional specialization., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Camp et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In eukaryotic cells, organelle and vesicle transport, positioning, and interactions play crucial roles in cytoplasmic organization and function. These processes are governed by intracellular trafficking mechanisms. At the core of that trafficking, the cytoskeleton and directional transport by motor proteins stand out as its key regulators. Plant cell tip growth is a well-studied example of cytoplasm organization by polarization. This polarization, essential for the cell's function, is driven by the cytoskeleton and its associated motors. This review will focus on myosin XI, a molecular motor critical for vesicle trafficking and polarized plant cell growth. We will center our discussion on recent data from the moss Physcomitrium patens and the liverwort Marchantia polymorpha. The biochemical properties and structure of myosin XI in various plant species are discussed, highlighting functional conservation across species. We further explore this conservation of myosin XI function in the process of vesicle transport in tip-growing cells. Existing evidence indicates that myosin XI actively organizes actin filaments in tip-growing cells by a mechanism based on vesicle clustering at their tips. A hypothetical model is presented to explain the essential function of myosin XI in polarized plant cell growth based on vesicle clustering at the tip. The review also provides insight into the in vivo localization and dynamics of myosin XI, emphasizing its role in cytosolic calcium regulation, which influences the polymerization of F-actin. Lastly, we touch upon the need for additional research to elucidate the regulation of myosin function., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Mechanical work serves as the foundation for dynamic cellular processes, ranging from cell division to migration. A fundamental driver of cellular mechanical work is the actin cytoskeleton, composed of filamentous actin (F-actin) and myosin motors, where force generation relies on adenosine triphosphate (ATP) hydrolysis. F-actin architectures, whether bundled by crosslinkers or branched via nucleators, have emerged as pivotal regulators of myosin II force generation. However, it remains unclear how distinct F-actin architectures impact the conversion of chemical energy to mechanical work. Here, we employ in vitro reconstitution of distinct F-actin architectures with purified components to investigate their influence on myosin ATP hydrolysis (consumption). We find that F-actin bundles composed of mixed polarity F-actin hinder network contraction compared to non-crosslinked network and dramatically decelerate ATP consumption rates. Conversely, linear-nucleated networks allow network contraction despite reducing ATP consumption rates. Surprisingly, branched-nucleated networks facilitate high ATP consumption without significant network contraction, suggesting that the branched network dissipates energy without performing work. This study establishes a link between F-actin architecture and myosin energy consumption, elucidating the energetic principles underlying F-actin structure formation and the performance of mechanical work., (© 2024. The Author(s).)

Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck region to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a myosin VIII. Here, we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among CMLs tested for binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with red fluorescent protein-myosin fusion proteins containing IQ and tail domains of myosin VIIIs. In vitro actin motility assays using recombinant myosin VIIIs demonstrated that CaM, CML13, and CML14 function as light chains. Suppression of CML13 or CML14 expression using RNA silencing resulted in a shortened-hypocotyl phenotype, similar to that observed in a quadruple myosin mutant, myosin viii4KO. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Podocyte detachment due to mechanical stress is a common issue in hypertension-induced kidney disease. This study highlights the role of zyxin for podocyte stability and function. We have found that zyxin is significantly up-regulated in podocytes after mechanical stretch and relocalizes from focal adhesions to actin filaments. In zyxin knockout podocytes, we found that the loss of zyxin reduced the expression of vinculin and VASP as well as the expression of matrix proteins, such as fibronectin. This suggests that zyxin is a central player in the translation of mechanical forces in podocytes. In vivo, zyxin is highly up-regulated in patients suffering from diabetic nephropathy and in hypertensive DOCA-salt treated mice. Furthermore, zyxin loss in mice resulted in proteinuria and effacement of podocyte foot processes that was measured by super resolution microscopy. This highlights the essential role of zyxin for podocyte maintenance in vitro and in vivo, especially under mechanical stretch., (© 2024. The Author(s).)

Several actin-binding proteins (ABPs) phase separate to form condensates capable of curating the actin network shapes. Here, we use computational modeling to understand the principles of actin network organization within VASP condensate droplets. Our simulations reveal that the different actin shapes, namely shells, rings, and mixture states are highly dependent on the kinetics of VASP-actin interactions, suggesting that they arise from kinetic trapping. Specifically, we show that reducing the residence time of VASP on actin filaments reduces degree of bundling, thereby promoting assembly of shells rather than rings. We validate the model predictions experimentally using a VASP-mutant with decreased bundling capability. Finally, we investigate the ring opening within deformed droplets and found that the sphere-to-ellipsoid transition is favored under a wide range of filament lengths while the ellipsoid-to-rod transition is only permitted when filaments have a specific range of lengths. Our findings highlight key mechanisms of actin organization within phase-separated ABPs., (© 2024. The Author(s).)

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1&#95;KO human hepatic cells, liver organoids from ICAM-1&#95;KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress., Competing Interests: CC, CL, NR, NC, AC, JC, SB, Gd, SC, AP, GD, CM, JF, GA, FM, PM, IC, JC, SN, MH, JM No competing interests declared, (© 2023, Cacho-Navas et al.)

Quantum dots (QDs) are a novel type of nanomaterial that has unique optical and physical characteristics. As such, QDs are highly desired because of their potential to be used in both biomedical and industrial applications. However, the mass adoption of QDs usage has raised concerns among the scientific community regarding QDs' toxicity. Although many papers have reported the negative impact of QDs on a cellular level, the exact mechanism of the QDs' toxicity is still unclear. In this investigation, we study the adverse effects of QDs by focusing on one of the most important cellular processes: actin polymerization and depolymerization. Our results showed that QDs act in a biphasic manner where lower concentrations of QDs stimulate the polymerization of actin, while high concentrations of QDs inhibit actin polymerization. Furthermore, we found that QDs can bind to filamentous actin (F-actin) and cause bundling of the filament while also promoting actin depolymerization. Through this study, we found a novel mechanism in which QDs negatively influence cellular processes and exert toxicity.

Cellular vesicle long-distance transport along the cytoplasmic actin network has recently been uncovered in several cell systems. In metaphase mouse oocytes, the motor protein myosin-5b (Myo5b) and the actin nucleation factor Spire are recruited to the Rab11a-positive vesicle membrane, forming a ternary complex of Myo5b/Spire/Rab11a that drives the vesicle long-distance transport to the oocyte cortex. However, the mechanism underlying the intermolecular regulation of the Myo5b/Spire/Rab11a complex remains unknown. In this study, we expressed and purified Myo5b, Spire2, and Rab11a proteins, and performed ATPase activity measurements, pulldown and single-molecule motility assays. Our results demonstrate that both Spire2 and Rab11a are required to activate Myo5b motor activity under physiological ionic conditions. The GTBM fragment of Spire2 stimulates the ATPase activity of Myo5b, while Rab11a enhances this activation. This activation occurs by disrupting the head-tail interaction of Myo5b. Furthermore, at the single-molecule level, we observed that the GTBM fragment of Spire2 and Rab11a coordinate to stimulate the Myo5b motility activity. Based on our results, we propose that upon association with the vesicle membrane, Myo5b, Spire2 and Rab11a form a ternary complex, and the inhibited Myo5b is synergistically activated by Spire2 and Rab11a, thereby triggering the long-distance transport of vesicles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Myosin family proteins are ATP-driven, actin filament-based motor proteins that generate force along actin filaments. In in vitro actin filament gliding assays, certain myosins generate rotation of gliding actin filaments around their long axes. In this study, we assessed the effects of temperature on the corkscrewing motion of actin filaments, including factors like gliding and rotational velocities and corkscrewing pitch. The corkscrewing motion was driven by a nonprocessive, full-length single-headed Drosophila myosin IC attached to an antibody adsorbed onto a cover glass. We performed an in vitro actin filament corkscrewing assay at temperatures ranging from 25 °C to 35 °C. We found that the gliding and rotational velocities and the pitch of corkscrewing actin filaments generated by myosin IC molecules increased with increasing temperature. Since the pitch is determined by dividing the gliding velocity by the rotational velocity, an increase in the pitch indicates that the gliding velocity increased faster than the rotational velocity with increasing temperature. These results suggest that temperature has distinct effects on the gliding and rotational forces produced by myosin IC, with implications for interpreting the temperature effect on torque-generation mechanisms driven by myosins on actin filaments at physiological temperatures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Actomyosin networks constrict cell area and junctions to alter cell and tissue shape. However, during cell expansion under mechanical stress, actomyosin networks are strengthened and polarized to relax stress. Thus, cells face a conflicting situation between the enhanced actomyosin contractile properties and the expansion behaviour of the cell or tissue. To address this paradoxical situation, we study late Drosophila oogenesis and reveal an unusual epithelial expansion wave behaviour. Mechanistically, Rac1 and Rho1 integrate basal pulsatile actomyosin networks with ruffles and focal adhesions to increase and then stabilize basal area of epithelial cells allowing their flattening and elongation. This epithelial expansion behaviour bridges cell changes to oocyte growth and extension, while oocyte growth in turn deforms the epithelium to drive cell spreading. Basal pulsatile actomyosin networks exhibit non-contractile mechanics, non-linear structures and F-actin/Myosin-II spatiotemporal signal separation, implicating unreported expanding properties. Biophysical modelling incorporating these expanding properties well simulates epithelial cell expansion waves. Our work thus highlights actomyosin expanding properties as a key mechanism driving tissue morphogenesis., (© 2024. The Author(s).)

Actin is one of the most abundant proteins in eukaryotic cells and is a key component of the cytoskeleton. A range of small molecules has emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Among these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin ( OptoLat ), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390-490 nm pulsed light and rapidly relaxes to its inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated via live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of the microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

The cytoskeleton, comprising actin microfilaments, microtubules, and intermediate filaments, is crucial for cell motility and tissue integrity. While prior studies largely focused on individual cytoskeletal networks, recent research underscores the interconnected nature of these systems in fundamental cellular functions like adhesion, migration, and division. Understanding the coordination of these distinct networks in both time and space is essential. This review synthesizes current findings on the intricate interplay between these networks, emphasizing the pivotal role of intermediate filaments. Notably, these filaments engage in extensive crosstalk with microfilaments and microtubules through direct molecular interactions, cytoskeletal linkers, and molecular motors that form molecular bridges, as well as via more complex regulation of intracellular signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Substance use disorders (SUD) are chronic relapsing disorders governed by continually shifting cycles of positive drug reward experiences and drug withdrawal-induced negative experiences. A large body of research points to plasticity within systems regulating emotional, motivational, and cognitive processes as drivers of continued compulsive pursuit and consumption of substances despite negative consequences. This plasticity is observed at all levels of analysis from molecules to networks, providing multiple avenues for intervention in SUD. The cytoskeleton and its regulatory proteins within neurons and glia are fundamental to the structural and functional integrity of brain processes and are potentially the major drivers of the morphological and behavioral plasticity associated with substance use. In this review, we discuss preclinical studies that provide support for targeting the brain cytoskeleton as a therapeutic approach to SUD. We focus on the interplay between actin cytoskeleton dynamics and exposure to cocaine, methamphetamine, alcohol, opioids, and nicotine and highlight preclinical studies pointing to a wide range of potential therapeutic targets, such as nonmuscle myosin II, Rac1, cofilin, prosapip 1, and drebrin. These studies broaden our understanding of substance-induced plasticity driving behaviors associated with SUD and provide new research directions for the development of SUD therapeutics., Competing Interests: Declaration of Competing Interest CAM holds equity in Myosin Therapeutics., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Actin remodeling is a critical regulator of mast cell secretion. In previous work, we have shown that dehydroleucodine and xanthatin, two natural α,β-unsaturated lactones, exhibit anti-inflammatory and mast cell stabilizing properties. Based on this background, this study aimed to determine whether the mast cell stabilizing action of these lactones is associated with changes in the actin cytoskeleton. Rat peritoneal mast cells were preincubated in the presence of dehydroleucodine or xanthatin before incubation with compound 48/80. Comparative studies with sodium cromoglycate and latrunculin B were also made. After treatments, different assays were performed on mast cell samples: β-hexosaminidase release, cell viability studies, quantification of mast cells and their state of degranulation by light microscopy, transmission electron microscopy, and actin staining for microscopy observation. Results showed that dehydroleucodine and xanthatin inhibited mast cell degranulation, evidenced by the inhibition of β-hexosaminidase release and decreased degranulated mast cell percentage. At the same time, both lactones altered the F-actin cytoskeleton in mast cells resulting, similarly to Latrunculin B, in a higher concentration of nuclear F-actin when activated by compound 48/80. For the first time, this study describes the biological properties of dehydroleucodine and xanthatin concerning to the rearrangement of actin filaments during stimulated exocytosis in mast cells. These data have important implications for developing new anti-inflammatory and mast cell stabilizing drugs and for designing new small molecules that may interact with the actin cytoskeleton., (© 2023 Wiley Periodicals LLC.)

The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens 1-3 , including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure 4,5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells 6,7 , this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid L-tryptophan protects the host against Citrobacter rodentium, a mouse AE pathogen that is widely used as a model for EHEC infection 8,9 . We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Mitochondria are the powerhouse of the cell and play important roles in multiple cellular processes including cell metabolism, proliferation, and programmed cell death. Mitochondria are double-membrane organelles with the inner membrane folding inward to form cristae. Mitochondria networks undergo dynamic fission and fusion. Deregulation of mitochondrial structure has been linked to perturbed mitochondrial membrane potential and disrupted metabolism, as evidenced in tumorigenesis, neurodegenerative diseases, etc. Actin and its motors-myosins have long been known to generate mechanical forces and participate in short-distance cargo transport. Accumulating knowledge from biochemistry and live cell/electron microscope imaging has demonstrated the role of actin filaments in pre-constricting the mitochondria during fission. Recent studies have suggested the involvement of myosins in cristae maintenance and mitochondria quality control. Here, we review current findings and discuss future directions in the emerging fields of cytoskeletal regulation in cristae formation, mitochondrial dynamics, intracellular transport, and mitocytosis, with focus on the actin cytoskeleton and its motor proteins., (© 2023 Wiley Periodicals LLC.)

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

The ability to dynamically assemble contractile networks is required throughout cell physiology, yet direct biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here, we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the static actin architecture plays a less clear role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin-driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes filament stacks prior to partitioning into clusters that feed higher-order networks. Together, these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology., (© 2024 Quintanilla et al.)

Alveolar septation increases gas-exchange surface area and requires coordinated cytoskeletal rearrangement in lung fibroblasts (LFs) to balance the demands of contraction and cell migration. We hypothesized that DBN (drebrin), a modulator of the actin cytoskeleton in neuronal dendrites, regulates the remodeling of the LF cytoskeleton. Using mice bearing a transgelin-Cre-targeted deletion of Dbn in pulmonary fibroblasts and pericytes, we examined alterations in alveolar septal outgrowth, LF spreading and migration, and actomyosin function. The alveolar surface area and number of alveoli were reduced, whereas alveolar ducts were enlarged, in mice bearing the dbn deletion (DBNΔ) compared with their littermates bearing only one dbn -Flox allele (control). Cultured DBNΔ LFs were deficient in their responses to substrate rigidity and migrated more slowly. Drebrin was abundant in the actin cortex and lamella, and the actin fiber orientation was less uniform in lamella of DBNΔ LFs, which limited the development of traction forces and altered focal adhesion dynamics. Actin fiber orientation is regulated by contractile NM2 (nonmuscle myosin-2) motors, which help arrange actin stress fibers into thick ventral actin stress fibers. Using fluorescence anisotropy, we observed regional intracellular differences in myosin regulatory light chain phosphorylation in control LFs that were altered by dbn deletion. Using perturbations to induce and then release stalling of NM2 on actin in LFs from both genotypes, we made predictions explaining how DBN interacts with actin and NM2. These studies provide new insight for diseases such as emphysema and pulmonary fibrosis, in which fibroblasts inappropriately respond to mechanical cues in their environment.

The actin cytoskeleton and reactive oxygen species (ROS) both play crucial roles in various cellular processes. Previous research indicated a direct interaction between two key components of these systems: the WAVE1 subunit of the WAVE regulatory complex (WRC), which promotes actin polymerization and the p47 phox subunit of the NADPH oxidase 2 complex (NOX2), which produces ROS. Here, using carefully characterized recombinant proteins, we find that activated p47 phox uses its dual Src homology 3 domains to bind to multiple regions within the WAVE1 and Abi2 subunits of the WRC, without altering WRC's activity in promoting Arp2/3-mediated actin polymerization. Notably, contrary to previous findings, p47 phox uses the same binding pocket to interact with both the WRC and the p22 phox subunit of NOX2, albeit in a mutually exclusive manner. This observation suggests that when activated, p47 phox may separately participate in two distinct processes: assembling into NOX2 to promote ROS production and engaging with WRC to regulate the actin cytoskeleton., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The orchestrated assembly of actin and actin-binding proteins into cytoskeletal structures coordinates cell morphology changes during migration, cytokinesis, and adaptation to external stimuli. The accurate and unbiased visualization of the diverse actin assemblies within cells is an ongoing challenge. We describe here the identification and use of designed ankyrin repeat proteins (DARPins) as synthetic actin binders. Actin-binding DARPins were identified through ribosome display and validated biochemically. When introduced or expressed inside living cells, fluorescently labeled DARPins accumulated at actin filaments, validated through phalloidin colocalization on fixed cells. Nevertheless, different DARPins displayed different actin labeling patterns: some DARPins labeled efficiently dynamic structures, such as filopodia, lamellipodia, and blebs, while others accumulated primarily in stress fibers. This differential intracellular distribution correlated with DARPin-actin binding kinetics, as measured by fluorescence recovery after photobleaching experiments. Moreover, the rapid arrest of actin dynamics induced by pharmacological treatment led to the fast relocalization of DARPins. Our data support the hypothesis that the localization of actin probes depends on the inherent dynamic movement of the actin cytoskeleton. Compared to the widely used LifeAct probe, one DARPin exhibited enhanced signal-to-background ratio while retaining a similar ability to label stress fibers. In summary, we propose DARPins as promising actin-binding proteins for labeling or manipulation in living cells.

Actin cytoskeleton remodeling sustains the ability of cytotoxic T cells to search for target cells and eliminate them. We here investigated the relationship between energetic status, actin remodeling, and functional fitness in human CD8 + effector T cells. Cell spreading during migration or immunological synapse assembly mirrored cytotoxic activity. Morphological and functional fitness were boosted by interleukin-2 (IL-2), which also stimulated the transcription of glycolytic enzymes, actin isoforms, and actin-related protein (ARP)2/3 complex subunits. This molecular program scaled with F-actin content and cell spreading. Inhibiting glycolysis impaired F-actin remodeling at the lamellipodium, chemokine-driven motility, and adhesion, while mitochondrial oxidative phosphorylation blockade impacted cell elongation during confined migration. The severe morphological and functional defects of ARPC1B-deficient T cells were only partially corrected by IL-2, emphasizing ARP2/3-mediated actin polymerization as a crucial energy state integrator. The study therefore underscores the tight coordination between metabolic and actin remodeling programs to sustain the cytotoxic activity of CD8 + T cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Focal adhesions (FAs) connect inner workings of cell to the extracellular matrix to control cell adhesion, migration and mechanosensing. Previous studies demonstrated that FAs contain three vertical layers, which connect extracellular matrix to the cytoskeleton. By using super-resolution iPALM microscopy, we identify two additional nanoscale layers within FAs, specified by actin filaments bound to tropomyosin isoforms Tpm1.6 and Tpm3.2. The Tpm1.6-actin filaments, beneath the previously identified α-actinin cross-linked actin filaments, appear critical for adhesion maturation and controlled cell motility, whereas the adjacent Tpm3.2-actin filament layer beneath seems to facilitate adhesion disassembly. Mechanistically, Tpm3.2 stabilizes ACF-7/MACF1 and KANK-family proteins at adhesions, and hence targets microtubule plus-ends to FAs to catalyse their disassembly. Tpm3.2 depletion leads to disorganized microtubule network, abnormally stable FAs, and defects in tail retraction during migration. Thus, FAs are composed of distinct actin filament layers, and each may have specific roles in coupling adhesions to the cytoskeleton, or in controlling adhesion dynamics., (© 2024. The Author(s).)

Actin is well known for its cytoskeletal functions, where it helps to control and maintain cell shape and architecture, as well as regulating cell migration and intracellular cargo transport, among others. However, actin is also prevalent in the nucleus, where genome-regulating roles have been described, including it being part of chromatin-remodeling complexes. More recently, with the help of advances in microscopy techniques and specialized imaging probes, direct visualization of nuclear actin filament dynamics has helped elucidate new roles for nuclear actin, such as in cell cycle regulation, DNA replication and repair, chromatin organization and transcriptional condensate formation. In this Cell Science at a Glance article, we summarize the known signaling events driving the dynamic assembly of actin into filaments of various structures within the nuclear compartment for essential genome functions. Additionally, we highlight the physiological role of nuclear F-actin in meiosis and early embryonic development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Contractile rings are formed from cytoskeletal filaments during cell division. Ring formation is induced by specific crosslinkers, while contraction is typically associated with motor protein activity. Here, we engineer DNA nanotubes and peptide-functionalized starPEG constructs as synthetic crosslinkers to mimic this process. The crosslinker induces bundling of ten to hundred DNA nanotubes into closed micron-scale rings in a one-pot self-assembly process yielding several thousand rings per microliter. Molecular dynamics simulations reproduce the detailed architectural properties of the DNA rings observed in electron microscopy. Theory and simulations predict DNA ring contraction - without motor proteins - providing mechanistic insights into the parameter space relevant for efficient nanotube sliding. In agreement between simulation and experiment, we obtain ring contraction to less than half of the initial ring diameter. DNA-based contractile rings hold promise for an artificial division machinery or contractile muscle-like materials., (© 2024. The Author(s).)

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., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Bonilla-Quintana and Rangamani.)

Mammals have 6 highly conserved actin isoforms with nonredundant biological functions. The molecular basis of isoform specificity, however, remains elusive due to a lack of tools. Here, we describe the development of IntAct, an internal tagging strategy to study actin isoforms in fixed and living cells. We identified a residue pair in β-actin that permits tag integration and used knock-in cell lines to demonstrate that IntAct β-actin expression and filament incorporation is indistinguishable from wild type. Furthermore, IntAct β-actin remains associated with common actin-binding proteins (ABPs) and can be targeted in living cells. We demonstrate the usability of IntAct for actin isoform investigations by showing that actin isoform-specific distribution is maintained in human cells. Lastly, we observed a variant-dependent incorporation of tagged actin variants into yeast actin patches, cables, and cytokinetic rings demonstrating cross species applicability. Together, our data indicate that IntAct is a versatile tool to study actin isoform localization, dynamics, and molecular interactions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 van Zwam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In autophagy, autophagosomes deliver the lumenal contents to lysosomes for degradation via autophagosome-lysosome fusion. In contrast, autophagosome outer membrane components were recycled via autophagosomal components recycling (ACR), which is mediated by the recycler complex. The recycler complex, composed of SNX4, SNX5, and SNX17, cooperate with the dynein-dynactin complex to mediate ACR. However, how ACR is regulated remains unknown. Here, we found that Rab32 family proteins localize to autolysosomes and are required for ACR, rather than other autophagosomal or lysosomal Rab proteins. The GTPase activity of Rab32 family proteins, governed by their guanine nucleotide exchange factor and GTPase-activating protein, plays a key role in regulating ACR. This regulation occurs through the control of recycler complex formation, as well as the connection between the recycler-cargo and dynactin complex. Together, our study reveals an unidentified Rab32 family-dependent regulatory mechanism for ACR., (© 2024 Wu et al.)

Microglia dynamically reorganize their cytoskeleton to perform essential functions such as phagocytosis of toxic protein aggregates, surveillance of the brain parenchyma, and regulation of synaptic plasticity during neuronal activity bursts. Recent studies have shed light on the critical role of the microtubule cytoskeleton in microglial reactivity and function, revealing key regulators like cyclin-dependent kinase 1 and centrosomal nucleation in the remodeling of microtubules in activated microglia. Concurrently, the role of the actin cytoskeleton is also pivotal, particularly in the context of small GTPases like RhoA, Rac1, and Cdc42 and actin-binding molecules such as profilin-1 and cofilin. This article delves into the intricate molecular landscape of actin and microtubules, exploring their synergistic roles in driving microglial cytoskeletal dynamics. We propose a more integrated view of actin and microtubule cooperation, which is fundamental to understanding the functional coherence of the microglial cytoskeleton and its pivotal role in propelling brain homeostasis. Furthermore, we discuss how alterations in microglial cytoskeleton dynamics during aging and in disease states could have far-reaching implications for brain function. By unraveling the complexities of microglia cytoskeletal dynamics, we can deepen our understanding of microglial functional states and their implications in health and disease, offering insights into potential therapeutic interventions for neurologic disorders., Competing Interests: Declaration of Competing Interest The authors declare no competing interests related to this manuscript., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The organization of actin filaments (F-actin) into crosslinked networks determines the transmission of mechanical stresses within the cytoskeleton and subsequent changes in cell and tissue shape. Principally mediated by proteins such as α-actinin, F-actin crosslinking increases both network connectivity and rigidity, thereby facilitating stress transmission at low crosslinking yet attenuating transmission at high crosslinker concentration. Here, we engineer a two-dimensional model of the actomyosin cytoskeleton, in which myosin-induced mechanical stresses are controlled by light. We alter the extent of F-actin crosslinking by the introduction of oligomerized cofilin. At pH 6.5, F-actin severing by cofilin is weak, but cofilin bundles and crosslinks filaments. Given its effect of lowering the F-actin bending stiffness, cofilin- crosslinked networks are significantly more flexible and softer in bending than networks crosslinked by α-actinin. Thus, upon local activation of myosin-induced contractile stress, the network bends out-of-plane in contrast to the in-plane compression as observed with networks crosslinked by α-actinin. Here, we demonstrate that local effects on filament mechanics by cofilin introduces novel large-scale network material properties that enable the sculpting of complex shapes in the cell cytoskeleton., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Apicomplexans, such as Plasmodium and Toxoplasma are obligate intracellular parasites that invade, replicate and finally EXIT their host cell. During replication within a parasitophorous vacuole (PV), the parasites establish an extensive F-actin-containing network that connects individual parasites and is required for material exchange, recycling and the final steps of daughter cell assembly. After multiple rounds of replication, the parasites exit the host cell involving multiple signalling cascades, disassembly of the network, secretion of microneme proteins and activation of the acto-myosin motor. Blocking the host cell EXIT process leads to the formation of large PVs, making the screening for genes involved in exiting the cell relatively straightforward. Given that apicomplexans are highly diverse from other eukaryotes, approximately 30% of all genes are annotated as hypothetical, some apicomplexan-specific factors are likely to be critical during EXIT. This motivated several labs to design and perform forward genetic and phenotypic screens using various approaches, such as random insertion mutagenesis, temperature-sensitive mutants and, more recently, CRISPR/Cas9-mediated targeted editing and conditional mutagenesis. Here we will provide an overview of the technological developments over recent years and the most successful stories that led to the identification of new critical factors in Toxoplasma gondii., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Arp2/3 complex nucleates branched actin filaments that drive membrane invagination during endocytosis and leading-edge protrusion in lamellipodia. Arp2/3 complex is maximally activated in vitro by binding of a WASP family protein to two sites-one on the Arp3 subunit and one spanning Arp2 and ARPC1-but the importance of each site in the regulation of force-producing actin networks is unclear. Here, we identify mutations in budding yeast Arp2/3 complex that decrease or block engagement of Las17, the budding yeast WASP, at each site. As in the mammalian system, both sites are required for maximal activation in vitro. Dimerization of Las17 partially restores activity of mutations at both CA-binding sites. Arp2/3 complexes defective at either site assemble force-producing actin networks in a bead motility assay, but their reduced activity hinders motility by decreasing actin assembly near the bead surface and by failing to suppress actin filament bundling within the networks. While even the most defective Las17-binding site mutants assembled actin filaments at endocytic sites, they showed significant internalization defects, potentially because they lack the proper architecture to drive plasma membrane remodeling. Together, our data indicate that both Las17-binding sites are important to assemble functional endocytic actin networks in budding yeast, but Arp2/3 complex retains some activity in vitro and in vivo even with a severe defect at either Las17-binding site., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Spectrins function together with actin as obligatory subunits of the submembranous cytoskeleton. Spectrins maintain cell shape, resist mechanical forces, and stabilize ion channel and transporter protein complexes through binding to scaffolding proteins. Recently, pathogenic variants of SPTBN4 (β4 spectrin) were reported to cause both neuropathy and myopathy. Although the role of β4 spectrin in neurons is mostly understood, its function in skeletal muscle, another excitable tissue subject to large forces, is unknown. Here, using a muscle specific β4 spectrin conditional knockout mouse, we show that β4 spectrin does not contribute to muscle function. In addition, we show β4 spectrin is not present in muscle, indicating the previously reported myopathy associated with pathogenic SPTBN4 variants is neurogenic in origin. More broadly, we show that α2, β1 and β2 spectrins are found in skeletal muscle, with α2 and β1 spectrins being enriched at the postsynaptic neuromuscular junction (NMJ). Surprisingly, using muscle specific conditional knockout mice, we show that loss of α2 and β2 spectrins had no effect on muscle health, function or the enrichment of β1 spectrin at the NMJ. Muscle specific deletion of β1 spectrin also had no effect on muscle health, but, with increasing age, resulted in the loss of clustered NMJ Na + channels. Together, our results suggest that muscle β1 spectrin functions independently of an associated α spectrin to maintain Na + channel clustering at the postsynaptic NMJ. Furthermore, despite repeated exposure to strong forces and in contrast to neurons, muscles do not require spectrin cytoskeletons to maintain cell shape or integrity. KEY POINTS: The myopathy found in pathogenic human SPTBN4 variants (where SPTBN4 is the gene encoding β4 spectrin) is neurogenic in origin. β1 spectrin plays essential roles in maintaining the density of neuromuscular junction Nav1.4 Na + channels. By contrast to the canonical view of spectrin organization and function, we show that β1 spectrin can function independently of an associated α spectrin. Despite the large mechanical forces experienced by muscle, we show that spectrins are not required for muscle cell integrity. This is in stark contrast to red blood cells and the axons of neurons., (© 2024 The Authors. The Journal of Physiology © 2024 The Physiological Society.)

The transcriptional co-activators of the Hippo pathway, YAP and TAZ, are regulated by mechanotransduction, which depends on dynamic actin cytoskeleton remodeling. Here, we identified SEPTIN10 as a novel cytoskeletal protein, which is transcriptionally regulated by YAP/TAZ and whose overexpression correlates with poor survival and vascular invasion in hepatocellular carcinoma (HCC) patients. Functional characterization demonstrated that SEPTIN10 promotes YAP/TAZ-dependent cell viability, migration and invasion of liver cancer cells. Mechanistically, SEPTIN10 interacts with actin and microtubule filaments supporting actin stress fiber formation and intracellular tension through binding to CAPZA2 while concurrently inhibiting microtubule polymerization through the blockage of MAP4 function. This functional antagonism is important for cytoskeleton-dependent feedback activation of YAP/TAZ, as microtubule depolymerization induces actin stress fiber formation and subsequently YAP/TAZ activity. Importantly, the crosstalk between microfilaments and microtubules is mediated by SEPTIN10 as its loss abrogates actin stress fiber formation after microtubule disruption. Together, the YAP/TAZ target gene SEPTIN10 controls the dynamic interplay between actin and microtubule filaments, which feeds back on Hippo pathway activity in HCC cells and thus acts as molecular switch with impact on oncogenic signaling and cancer cell biology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

How cells tightly control the formation and turnover of branched actin filament arrays to drive cell motility, endocytosis, and other cellular processes is still not well understood. Here, we investigated the mechanistic relationship between two binding partners of the Arp2/3 complex, glia maturation factor (GMF) and cortactin. Individually, GMF and cortactin have opposite effects on the stability of actin filament branches, but it is unknown how they work in concert with each other to govern branch turnover. Using TIRF microscopy, we observe that GMF's branch destabilizing activities are potently blocked by cortactin (IC 50 = 1.3 nM) and that this inhibition requires direct interactions of cortactin with Arp2/3 complex. The simplest model that would explain these results is competition for binding Arp2/3 complex. However, we find that cortactin and GMF do not compete for free Arp2/3 complex in solution. Further, we use single molecule analysis to show that cortactin's on-rate (3 ×10 7 s -1 M -1 ) and off-rate (0.03 s -1 ) at branch junctions are minimally affected by excess GMF. Together, these results show that cortactin binds with high affinity to branch junctions, where it blocks the destabilizing effects of GMF, possibly by a mechanism that is allosteric in nature. In addition, the affinities we measure for cortactin at actin filament branch junctions (K d = 0.9 nM) and filament sides (K d = 206 nM) are approximately 20-fold stronger than previously reported. These observations contribute to an emerging view of molecular complexity in how Arp2/3 complex is regulated through the integration of multiple inputs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Cell shape changes mainly rely on the remodeling of the actin cytoskeleton. Multiciliated cells (MCCs) of the mucociliary epidermis of Xenopus laevis embryos, as they mature, dramatically reshape their apical domain to grow cilia, in coordination with the underlying actin cytoskeleton. Crumbs (Crb) proteins are multifaceted transmembrane apical polarity proteins known to recruit actin linkers and promote apical membrane growth. Here, we identify the homeolog Crb3.L as an important player for the migration of centrioles or basal bodies (collectively centrioles/BBs) and apical domain morphogenesis in MCCs. Crb3.L is present in cytoplasmic vesicles close to the ascending centrioles/BBs, where it partially colocalizes with Rab11a. Crb3.L morpholino-mediated depletion in MCCs caused abnormal migration of centrioles/BBs, a reduction of their apical surface, disorganization of their apical actin meshwork and defective ciliogenesis. Rab11a morpholino-mediated depletion phenocopied Crb3.L loss-of-function in MCCs. Thus, the control of centrioles/BBs migration by Crb3.L might be mediated by Rab11a-dependent apical trafficking. Furthermore, we show that both phospho-activated ERM (pERM; Ezrin-Radixin-Moesin) and Crb3.L are recruited to the growing apical domain of MCCs, where Crb3.L likely anchors pERM, allowing actin-dependent expansion of the apical membrane., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The WRAMP structure is a protein network associated with tail-end actomyosin contractility, membrane retraction, and directional persistence during cell migration. A marker of WRAMP structures is melanoma cell adhesion molecule (MCAM) which dynamically polarizes to the cell rear. However, factors that mediate MCAM polarization are still unknown. In this study, BioID using MCAM as bait identifies the ERM family proteins, moesin, ezrin, and radixin, as WRAMP structure components. We also present a novel image analysis pipeline, Protein Polarity by Percentile ("3P"), which classifies protein polarization using machine learning and facilitates quantitative analysis. Using 3P, we find that depletion of moesin, and to a lesser extent ezrin, decreases the proportion of cells with polarized MCAM. Furthermore, although copolarized MCAM and ERM proteins show high spatial overlap, 3P identifies subpopulations with ERM proteins closer to the cell periphery. Live-cell imaging confirms that MCAM and ERM protein polarization is tightly coordinated, but ERM proteins enrich at the cell edge first. Finally, deletion of a juxtamembrane segment in MCAM previously shown to promote ERM protein interactions impedes MCAM polarization. Our findings highlight the requirement for ERM proteins in recruitment of MCAM to WRAMP structures and an advanced computational tool to characterize protein polarization.

Cytoskeletal protein filaments such as actin and microtubules confer mechanical support to cells and facilitate many cellular functions such as motility and division. Recent years have witnessed the development of a variety of molecular scaffolds that mimic such filaments. Indeed, filaments that are programmable and compatible with biological systems may prove useful in studying or substituting such proteins. Here, we explore the use of ssRNA tiles to build and modify filaments in vitro . We engineer a number of functionalities that are crucial to the function of natural proteins filaments into the ssRNA tiles, including the abilities to assemble or disassemble filaments, to tune the filament stiffness, to induce membrane binding, and to bind proteins. This work paves the way for building dynamic cytoskeleton-mimicking systems made out of rationally designed ssRNA tiles that can be transcribed in natural or synthetic cells.

The Arp2/3 complex, which generates both branched but also linear actin filaments via activation of SPIN90, is evolutionarily conserved in eukaryotes. Several factors regulate the stability of filaments generated by the Arp2/3 complex to maintain the dynamics and architecture of actin networks. In this review, we summarise recent studies on the molecular mechanisms governing the tuning of Arp2/3 complex nucleated actin filaments, which includes investigations using microfluidics and single-molecule imaging to reveal the mechanosensitivity, dissociation and regeneration of actin branches. We also discuss the high-resolution cryo-EM structure of cortactin bound to actin branches, as well as the differences and similarities between the stability of Arp2/3 complex nucleated branches and linear filaments. These new studies provide a clearer picture of the stabilisation of Arp2/3 nucleated filaments at the molecular level. We also identified gaps in our understanding of how different factors collectively contribute to the stabilisation of Arp2/3 complex-generated actin networks., (© 2024 The Author(s).)

The cytoskeletal protein actin plays a critical role in the pathogenicity of the intracellular parasite, Toxoplasma gondii, mediating invasion and egress, cargo transport, and organelle inheritance. Advances in live cell imaging have revealed extensive filamentous actin networks in the Apicomplexan parasite, but there are conflicting data regarding the biochemical and biophysical properties of Toxoplasma actin. Here, we imaged the in vitro assembly of individual Toxoplasma actin filaments in real time, showing that native, unstabilized filaments grow tens of microns in length. Unlike skeletal muscle actin, Toxoplasma filaments intrinsically undergo rapid treadmilling due to a high critical concentration, fast monomer dissociation, and rapid nucleotide exchange. Cryo-EM structures of jasplakinolide-stabilized and native (i.e. unstabilized) filaments show an architecture like skeletal actin, with differences in assembly contacts in the D-loop that explain the dynamic nature of the filament, likely a conserved feature of Apicomplexan actin. This work demonstrates that evolutionary changes at assembly interfaces can tune the dynamic properties of actin filaments without disrupting their conserved structure., (© 2024. The Author(s).)

Precise control over how and where actin filaments are created leads to the construction of unique cytoskeletal arrays within a common cytoplasm. Actin filament nucleators are key players in this activity and include the conserved actin-related protein 2/3 (Arp2/3) complex as well as a large family of formins. In some eukaryotic cells, these nucleators compete for a common pool of actin monomers and loss of one favors the activity of the other. To test whether this mechanism is conserved, we combined the ability to image single filament dynamics in the homeostatic cortical actin array of living Arabidopsis (Arabidopsis thaliana) epidermal cells with genetic and/or small molecule inhibitor approaches to stably or acutely disrupt nucleator activity. We found that Arp2/3 mutants or acute CK-666 treatment markedly reduced the frequency of side-branched nucleation events as well as overall actin filament abundance. We also confirmed that plant formins contribute to side-branched filament nucleation in vivo. Surprisingly, simultaneous inhibition of both classes of nucleator increased overall actin filament abundance and enhanced the frequency of de novo nucleation events by an unknown mechanism. Collectively, our findings suggest that multiple actin nucleation mechanisms cooperate to generate and maintain the homeostatic cortical array of plant epidermal cells., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)