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Eukaryotic cells depend on dynamic changes in shape to fulfill a wide range of cellular functions, maintain essential biological processes, and regulate cellular behavior. The single-celled, predatory ciliate Lacrymaria exhibits extraordinary dynamic shape-shifting using a flexible "neck" that can stretch 7-8 times the length of its body to capture prey. The molecular mechanism behind this morphological change remains a mystery. We have observed that when in an active state, Lacrymaria repeatedly extends and contracts its neck to enable 360-degree space search and prey capture. This remarkable morphological change involves a unique actin-myosin system rather than the Ca 2+ -dependent system found in other contractile ciliates. Two cytoskeletons are identified in the cortex of the Lacrymaria cell, namely the myoneme cytoskeleton and the microtubule cytoskeleton. The myoneme cytoskeleton is composed of centrin-myosin proteins, exhibiting distinct patterns between the neck and body, with their boundary seemingly associated with the position of the macronucleus. A novel giant protein forming a ladder-like structure was discovered as a component of the microtubule cytoskeleton. Thick centrin-myosin fibers are situated very close to the right side of the ladders in the neck but are far away from such structures in the body. This arrangement enables the decoupling of the neck and body. Plasmodium-like unconventional actin has been discovered in Lacrymaria, and this may form highly dynamic short filaments that could attach to the giant protein and myosin, facilitating coordination between the two cytoskeletons in the neck. In summary, this fascinating organism employs unconventional cytoskeletal components to accomplish its extraordinary dynamic shape-shifting., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Methylmercury chloride (MMC) is a persistent heavy metal contaminant that can bioaccumulate in humans via the food chain, exerting detrimental effects on health. Nevertheless, the specific influence of MMC on oocyte meiotic maturation has yet to be elucidated. This research demonstrated that MMC exposure during the in vitro cultivation of mouse oocytes did not influence germinal vesicle breakdown but markedly decreased oocyte maturation rates. Subsequent analysis indicated that MMC exposure resulted in aberrant spindle morphology and disorganized chromosome alignment, alongside continuous activation of the spindle assembly checkpoint (SAC). However, MMC exposure didn't alter the localization pattern of microtubule-organizing center-associated proteins. MMC exposure considerably diminished the acetylation level of α-tubulin, signifying reduced microtubule stability. Additionally, MMC exposure disrupted the dynamic alterations of F-actin. MMC exposure didn't affect mitochondrial localization, mitochondrial membrane potential, adenosine triphosphate content or the concentrations of reactive oxygen species. Nonetheless, MMC exposure triggered DNA damage and modified histone modification levels. Consequently, the defects in oocyte maturation induced by MMC exposure can be attributed to impaired cytoskeleton dynamics and DNA damage. This study offers the first comprehensive elucidation of the negative impacts of MMC on oocyte maturation, highlighting the potential reproductive health risks associated with MMC exposure., 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 Ltd. All rights reserved.)

Paracrine IL-2 signalling drives the CD8 + T cell expansion and differentiation that allow protection against viral infections, but the underlying molecular events are incompletely understood. Here we show that the transcription factor SRF, a master regulator of cytoskeletal gene expression, is required for effective IL-2 signalling during L. monocytogenes infection. Acting cell-autonomously with its actin-regulated cofactors MRTF-A and MRTF-B, SRF is dispensible for initial TCR-mediated CD8 + T cell proliferation, but is required for sustained IL-2 dependent CD8 + effector T cell expansion, and persistence of memory cells. Following TCR activation, Mrtfab-null CD8 + T cells produce IL-2 normally, but homotypic clustering is impaired both in vitro and in vivo. Expression of cytoskeletal structural and regulatory genes, most notably actins, is defective in Mrtfab-null CD8 + T cells. Activation-induced cell clustering in vitro requires F-actin assembly, and Mrtfab-null cell clusters are small, contain less F-actin, and defective in IL-2 retention. Clustering of Mrtfab-null cells can be partially restored by exogenous actin expression. IL-2 mediated CD8 + T cell proliferation during infection thus depends on the control of cytoskeletal dynamics and actin gene expression by MRTF-SRF signalling., (© 2024. The Author(s).)

Plasma membrane repair is a fundamental homeostatic process of eukaryotic cells. Here, we report a new function for the conserved cytoskeletal proteins known as septins in the repair of cells perforated by pore-forming toxins or mechanical disruption. Using a silencing RNA screen, we identified known repair factors (e.g. annexin A2, ANXA2) and novel factors such as septin 7 (SEPT7) that is essential for septin assembly. Upon plasma membrane injury, the septin cytoskeleton is extensively redistributed to form submembranous domains arranged as knob and loop structures containing F-actin, myosin IIA, S100A11, and ANXA2. Formation of these domains is Ca 2+ -dependent and correlates with plasma membrane repair efficiency. Super-resolution microscopy revealed that septins and F-actin form intertwined filaments associated with ANXA2. Depletion of SEPT7 prevented ANXA2 recruitment and formation of submembranous actomyosin domains. However, ANXA2 depletion had no effect on domain formation. Collectively, our data support a novel septin-based mechanism for resealing damaged cells, in which the septin cytoskeleton plays a key structural role in remodeling the plasma membrane by promoting the formation of SEPT/F-actin/myosin IIA/ANXA2/S100A11 repair domains., (© 2024. The Author(s).)

Tuft cells are a rare epithelial cell type that play important roles in sensing and responding to luminal antigens. A defining morphological feature of this lineage is the actin-rich apical "tuft," which contains large fingerlike protrusions. However, details of the cytoskeletal ultrastructure underpinning the tuft, the molecules involved in building this structure, or how it supports tuft cell biology remain unclear. In the context of the small intestine, we found that tuft cell protrusions are supported by long-core bundles that consist of F-actin crosslinked in a parallel and polarized configuration; they also contain a tuft cell-specific complement of actin-binding proteins that exhibit regionalized localization along the bundle axis. Remarkably, in the sub-apical cytoplasm, the array of core actin bundles interdigitates and co-aligns with a highly ordered network of microtubules. The resulting cytoskeletal superstructure is well positioned to support subcellular transport and, in turn, the dynamic sensing functions of the tuft cell that are critical for intestinal homeostasis., (© 2024 Silverman et al.)

Many cytoskeletal networks consist of individual filaments that are organized into elaborate higher-order structures. While it is appreciated that the size and architecture of these networks are critical for their biological functions, much of the work investigating control over their assembly has focused on mechanisms that regulate the turnover of individual filaments through size-dependent feedback. Here, we propose a very different, feedback-independent mechanism to explain how yeast cells control the length of their actin cables. Our findings, supported by quantitative cell imaging and mathematical modeling, indicate that actin cable length control is an emergent property that arises from the cross-linked and bundled organization of the filaments within the cable. Using this model, we further dissect the mechanisms that allow cables to grow longer in larger cells and propose that cell length-dependent tuning of formin activity allows cells to scale cable length with cell length. This mechanism is a significant departure from prior models of cytoskeletal filament length control and presents a different paradigm to consider how cells control the size, shape, and dynamics of higher-order cytoskeletal structures., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background/aim: Cold physical plasma (CPP) has emerged as an effective therapy in oncology by inducing cytotoxic effects in various cancer cells, including chondrosarcoma (CS), Ewing's sarcoma (ES), and osteosarcoma (OS). The current study investigated the impact of CPP on cell motility in CS (CAL-78), ES (A673), and OS (U2-OS) cell lines, focusing on the actin cytoskeleton., Materials and Methods: The CASY Cell Counter and Analyzer was used to study cell proliferation and determine the optimal concentrations of fetal calf serum to maintain viability without stimulation of cell proliferation. CellTiter-BlueCell viability assay was used to determine the effects of CPP on the viability of bone sarcoma cells. The Radius assay was used to determine cell migration. Staining for Deoxyribonuclease I, G-actin, and F-actin was used to assay for the effects on the cytoskeleton., Results: Reductions in cell viability and motility were observed across all cell lines following CPP treatment. CPP induced changes in the actin cytoskeleton, leading to decreased cell motility., Conclusion: CPP effectively reduces the motility of bone sarcoma cells by altering the actin cytoskeleton. These findings underscore CPP's potential as a therapeutic tool for bone sarcomas and highlight the need for further research in this area., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The microgeometry of the cellular microenvironment profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains unclear. Herein, we describe a fluorescent micropipette aspiration assay that allows for simultaneous visualization of intracellular calcium dynamics and cytoskeletal architecture in real-time, under varied micropipette geometries. By integrating elastic shell finite element analysis with fluorescent lifetime imaging microscopy and employing PIEZO1-specific transgenic red blood cells and HEK cell lines, we demonstrate a direct correlation between the microscale geometry of aspiration and PIEZO1-mediated calcium signaling. We reveal that increased micropipette tip angles and physical constrictions lead to a significant reorganization of F-actin, accumulation at the aspirated cell neck, and subsequently amplify the tension stress at the dome of the cell to induce more PIEZO1's activity. Disruption of the F-actin network or inhibition of its mobility leads to a notable decline in PIEZO1 mediated calcium influx, underscoring its critical role in cellular mechanosensing amidst geometrical constraints., (© 2024. The Author(s).)

Due to its central role in cell biology, the cytoskeleton is a key regulator of viral infection, influencing nearly every step of the viral life cycle. In this review, we will discuss the role of two key components of the cytoskeleton, namely the actin and microtubule networks in early HIV-1 infection. We will discuss key contributions to processes ranging from the attachment and entry of viral particles at the cell surface to their arrival and import into the nucleus and identify areas where further research into this complex relationship may yield new insights into HIV-1 pathogenesis., (© 2022 Federation of European Biochemical Societies.)

Epidemiological studies show that cardiovascular events related to platelet hyperactivity remain the leading causes of death among multiple sclerosis (MS) patients. Quantitative or structural changes of platelet cytoskeleton alter their morphology and function. Here, we demonstrated, for the first time, the structural changes in MS platelets that may be related to their hyperactivity. MS platelets were found to form large aggregates compared to control platelets. In contrast to the control, the images of overactivated, irregularly shaped MS platelets show changes in the cytoskeleton architecture, fragmented microtubule rings. Furthermore, MS platelets have long and numerous pseudopodia rich in actin filaments. We showed that MS platelets and megakaryocytes, overexpress β1-tubulin and β-actin mRNAs and proteins and have altered post-translational modification patterns. Moreover, we identified two previously undisclosed mutations in the gene encoding β1-tubulin in MS. We propose that the demonstrated structural changes of platelet cytoskeleton enhance their ability to adhere, aggregate, and degranulate fueling the risk of adverse cardiovascular events in MS., (Copyright © 2024. Published by Elsevier Ltd.)

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.)

The Rho GTPases - RHOA, RAC1 and CDC42 - are small GTP binding proteins that regulate basic biological processes such as cell locomotion, cell division and morphogenesis by promoting cytoskeleton-based changes in the cell cortex. This regulation results from active (GTP-bound) Rho GTPases stimulating target proteins that, in turn, promote actin assembly and myosin 2-based contraction to organize the cortex. This basic regulatory scheme, well supported by in vitro studies, led to the natural assumption that Rho GTPases function in vivo in an essentially linear matter, with a given process being initiated by GTPase activation and terminated by GTPase inactivation. However, a growing body of evidence based on live cell imaging, modelling and experimental manipulation indicates that Rho GTPase activation and inactivation are often tightly coupled in space and time via signalling circuits and networks based on positive and negative feedback. In this Review, we present and discuss this evidence, and we address one of the fundamental consequences of coupled activation and inactivation: the ability of the Rho GTPases to self-organize, that is, direct their own transition from states of low order to states of high order. We discuss how Rho GTPase self-organization results in the formation of diverse spatiotemporal cortical patterns such as static clusters, oscillatory pulses, travelling wave trains and ring-like waves. Finally, we discuss the advantages of Rho GTPase self-organization and pattern formation for cell function., (© 2024. Springer Nature Limited.)

The human kidney includes ~1 million nephrons which are long U-shaped tubules with convoluted segments that serve as filtration units. During the passage of the ultrafiltrate through a nephron, electrolytes and nutrients are re-absorbed into peritubular capillaries. The fluid remaining in the distal end of the renal tubules flows through the collecting ducts into the ureter. In this study, we generated high-resolution images of mouse kidney sections using confocal microscopy with only two fluorescently tagged biomarkers, F-actin binding phalloidin and CD34 antibodies as a marker for blood vessels. In tile-scan images of entire sections of mouse kidney (composed of >1000 images), the tubule segments are easily identifiable by their F-actin bundles on cell borders and the outlines of the peritubular capillaries by CD34 immunofluorescence. In the inner stripe of the medulla, the vascular bundles composed of vasa recta (straight vessels) could be easily distinguished from the peritubular capillaries by their full circular shapes. The highly vascular inner medulla and the papilla similarly have straight capillaries. About 95% of kidney volume is composed of renal tubules and blood vessels. Thus, our results show that relatively simple cytoskeletal mapping can be used to visualize the structural organization of the kidney. This method can also be applied to examine pathological changes in the kidney., (© 2023 Wiley Periodicals LLC.)

LAMB3, a major extracellular matrix and basal membrane component, is involved in wound healing. We aimed to understand its role in Asherman's syndrome (AS), which is associated with infertility, by using bioinformatics analysis and cultured endometrial stromal cells (ESCs). MRNAs extracted from tissues obtained from control subjects and patients with severe intrauterine adhesion were sequenced and subjected to bioinformatics analysis and the RhoA/ROCK1/MYL9 pathway was implicated and this subsequently studied using cultured primary ESCs. The effects of overexpression and knockdown and activation and inhibition of LAMB3 on the mesenchymal to myofibroblastic phenotypic transformation of ECCs were assessed using PCR and western blot analysis. Phalloidin was used to localize the actin cytoskeletal proteins. Silencing of LAMB3 reversed the TGF-β-induced ESC myofibroblast phenotype conversion, whereas overexpression of LAMB3 promoted this process. Activation and silencing of LAMB3 led to remodeling of the ESC cytoskeleton. Overexpression and silencing of LAMB3 caused activation and inhibition of ESCs, respectively. Y-27632 and LPA reversed the activation and inhibition of the RhoA/ROCK1/MYL9 pathway after overexpression and silencing, respectively. These results suggest that LAMB3 can regulate ESC fibrosis transformation and cytoskeleton remodeling via the RhoA/ROCK1/MYL9 pathway. This study provides a potential new target for gene therapy and drug intervention of AS., (© 2023. The Author(s).)

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.

Epithelial cells undergoing EMT experience significant alterations at transcriptional and morphological levels. However, changes in the cytoskeleton, especially cytoskeleton dynamics are poorly described. Addressing the question we induced EMT in three cell lines (MCF-7, HaCaT and A-549) and analyzed morphological and cytoskeletal changes there using immunostaining and life cell imaging of cells transfected with microtubule and focal adhesion markers. In all studied cell lines, cell area after EMT increased, MCF-7 and A-549 cells became elongated, while HaCaT cells kept the aspect ratio the same. We next analyzed three components of the cytoskeleton: microtubules, stress fibers and focal adhesions. The following changes were observed after EMT in cultured cells: (i) Organization of microtubules becomes more radial; and the growth rate of microtubule plus ends was accelerated; (ii) Actin stress fibers become co-aligned forming the longitudinal cell axis; and (iii) Focal adhesions had decreased area in all cancer cell lines studied and became more numerous in HaCaT cells. We conclude that among dynamic components of the cytoskeleton, the most significant changes during EMT happen in the regulation of microtubules., (© 2023. The Author(s).)

Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been conclusively established. Here, we used cryogenic electron tomography of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca 2+ ATPase with the small molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, we observed cofilin dephosphorylation, an activating modification, in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNA interference knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen., (© 2023 MRC Laboratory of Molecular Biology and The Authors. Published under the terms of the CC BY 4.0 license.)

In healthy tissues, cells are in mechanical homeostasis. During cancer progression, this equilibrium is disrupted. Cancer cells alter their mechanical phenotype to a softer and more fluid-like one than that of healthy cells. This is connected to cytoskeletal remodeling, changed adhesion properties, faster cell proliferation and increased cell motility. In this work, we investigated the mechanical properties of breast cancer cells representative of different breast cancer subtypes, using MCF-7, tamoxifen-resistant MCF-7, MCF10A and MDA-MB-231 cells. We derived viscoelastic properties from atomic force microscopy force spectroscopy measurements and showed that the mechanical properties of the cells are associated with cancer cell malignancy. MCF10A are the stiffest and least fluid-like cells, while tamoxifen-resistant MCF-7 cells are the softest ones. MCF-7 and MDA-MB-231 show an intermediate mechanical phenotype. Confocal fluorescence microscopy on cytoskeletal elements shows differences in actin network organization, as well as changes in focal adhesion localization. These findings provide further evidence of distinct changes in the mechanical properties of cancer cells compared to healthy cells and add to the present understanding of the complex alterations involved in tumorigenesis.

Live staining of actin brings valuable information in the field of mechanobiology. Gene transfer of GFP-actin has been reported to disturb cell rheological properties while gene transfer of fluorescent actin binding proteins was not. However the influence of gene transfer on cellular forces in adhered cells has never been investigated. This would provide a more complete picture of mechanical disorders induced by actin live staining for mechanobiology studies. Indeed, most of these techniques were shown to alter cell morphology. Change in cell morphology may in itself be sufficient to perturb cellular forces. Here we focus on quantifying the alterations of cellular stresses that result from baculoviral transduction of GFP-actin in MDCK cell line. We report that GFP-actin transduction increases the proportion of cells with large intracellular or surface stresses, especially in epithelia with low cell density. We show that the enhancement of the mechanical stresses is accompanied by small perturbations of cell shape, but not by a significant change in cell size. We thus conclude that this live staining method enhances the cellular forces but only brings subtle shape alterations., Competing Interests: Conflict of interest A.N. is co-founder, shareholder and scientific advisor of Cell&Soft company that sells pre-coated soft hydrogels for cell culture with uniform or textured stiffness., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Cells are continuously exposed to dynamic environmental cues that influence their behavior. Mechanical cues can influence cellular and genomic architecture, gene expression, and intranuclear mechanics, providing evidence of mechanosensing by the nucleus, and a mechanoreciprocity between the nucleus and environment. Force disruption at the tissue level through aging, disease, or trauma, propagates to the nucleus and can have lasting consequences on proper functioning of the cell and nucleus. While the influence of mechanical cues leading to axonal damage has been well studied in neuronal cells, the mechanics of the nucleus following high impulse loading is still largely unexplored. Using an in vitro model of traumatic neural injury, we show a dynamic nuclear behavioral response to impulse stretch (up to 170% strain per second) through quantitative measures of nuclear movement, including tracking of rotation and internal motion. Differences in nuclear movement were observed between low and high strain magnitudes. Increased exposure to impulse stretch exaggerated the decrease in internal motion, assessed by particle tracking microrheology, and intranuclear displacements, assessed through high-resolution deformable image registration. An increase in F-actin puncta surrounding nuclei exposed to impulse stretch additionally demonstrated a corresponding disruption of the cytoskeletal network. Our results show direct biophysical nuclear responsiveness in neuronal cells through force propagation from the substrate to the nucleus. Understanding how mechanical forces perturb the morphological and behavioral response can lead to a greater understanding of how mechanical strain drives changes within the cell and nucleus, and may inform fundamental nuclear behavior after traumatic axonal injury. STATEMENT OF SIGNIFICANCE: The nucleus of the cell has been implicated as a mechano-sensitive organelle, courting molecular sensors and transmitting physical cues in order to maintain cellular and tissue homeostasis. Disruption of this network due to disease or high velocity forces (e.g., trauma) can not only result in orchestrated biochemical cascades, but also biophysical perturbations. Using an in vitro model of traumatic neural injury, we aimed to provide insight into the neuronal nuclear mechanics and biophysical responses at a continuum of strain magnitudes and after repetitive loads. Our image-based methods demonstrate mechanically-induced changes in cellular and nuclear behavior after high intensity loading and have the potential to further define mechanical thresholds of neuronal cell injury., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)

Plants have developed fine-tuned cellular mechanisms to respond to a variety of intracellular and extracellular signals. These responses often necessitate the rearrangement of the plant cytoskeleton to modulate cell shape and/or to guide vesicle trafficking. At the cell periphery, both actin filaments and microtubules associate with the plasma membrane that acts as an integrator of the intrinsic and extrinsic environments. At this membrane, acidic phospholipids such as phosphatidic acid, and phosphoinositides contribute to the selection of peripheral proteins and thereby regulate the organization and dynamic of the actin and microtubules. After recognition of the importance of phosphatidic acid on cytoskeleton dynamics and rearrangement, it became apparent that the other lipids might play a specific role in shaping the cytoskeleton. This review focuses on the emerging role of the phosphatidylinositol 4,5-bisphosphate for the regulation of the peripherical cytoskeleton during cellular processes such as cytokinesis, polar growth, biotic and abiotic responses., 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 article., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Uncontrolled cell proliferation leads to several pathologies, including cancer. Thus, this process must be tightly regulated. The cell cycle accounts for cell proliferation, and its progression is coordinated with changes in cell shape, for which cytoskeleton reorganization is responsible. Rearrangement of the cytoskeleton allows for its participation in the precise division of genetic material and cytokinesis. One of the main cytoskeletal components is filamentous actin-based structures. Mammalian cells have at least six actin paralogs, four of which are muscle-specific, while two, named β- and γ-actin, are abundantly present in all types of cells. This review summarizes the findings that establish the role of non-muscle actin paralogs in regulating cell cycle progression and proliferation. We discuss studies showing that the level of a given non-muscle actin paralog in a cell influences the cell's ability to progress through the cell cycle and, thus, proliferation. Moreover, we elaborate on the non-muscle actins' role in regulating gene transcription, interactions of actin paralogs with proteins involved in controlling cell proliferation, and the contribution of non-muscle actins to different structures in a dividing cell. The data cited in this review show that non-muscle actins regulate the cell cycle and proliferation through varying mechanisms. We point to the need for further studies addressing these mechanisms., 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.)

Actin, one of the most abundant proteins in nature and a key component of the cytoskeleton, undergoes a unique multistep N-terminal (Nt) maturation. In a recent report, Haahr et al. identified actin maturation protease (ACTMAP) as the dedicated actin aminopeptidase and showed that its absence is associated with abnormal muscle physiology., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The spectrin-based membrane skeleton is a ubiquitous membrane-associated two-dimensional cytoskeleton underneath the lipid membrane of metazoan cells. Mutations of skeleton proteins impair the mechanical strength and functions of the membrane, leading to several different types of human diseases. Here, we report the cryo-EM structures of the native spectrin-actin junctional complex (from porcine erythrocytes), which is a specialized short F-actin acting as the central organizational unit of the membrane skeleton. While an α-/β-adducin hetero-tetramer binds to the barbed end of F-actin as a flexible cap, tropomodulin and SH3BGRL2 together create an absolute cap at the pointed end. The junctional complex is strengthened by ring-like structures of dematin in the middle actin layers and by patterned periodic interactions with tropomyosin over its entire length. This work serves as a structural framework for understanding the assembly and dynamics of membrane skeleton and offers insights into mechanisms of various ubiquitous F-actin-binding factors in other F-actin systems., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Many cytoskeletal systems are now sufficiently well known to permit their precise quantitative modeling. Microtubule and actin filaments are well characterized, and the associated proteins are often known, as well as their abundance and the interactions between these elements. Thus, computer simulations can be used to investigate the collective behavior of the system precisely, in a way that is complementary to experiments. Cytosim is an Open Source cytoskeleton simulation suite designed to handle large systems of flexible filaments with associated proteins such as molecular motors. It also offers the possibility to simulate passive crosslinkers, diffusible crosslinkers, nucleators, cutters, and discrete versions of the motors that only step on unoccupied lattice sites on a filament. Other objects complement the filaments by offering spherical or more complicated geometry that can be used to represent chromosomes, the nucleus, or vesicles in the cell. Cytosim offers simple command-line tools for running a simulation and displaying its results, which are versatile and do not require programming skills. In this workflow, step-by-step instructions are given to i) install the necessary environment on a new computer, ii) configure Cytosim to simulate the contraction of a 2D actomyosin network, and iii) produce a visual representation of the system. Next, the system is probed by systematically varying a key parameter: the number of crosslinkers. Finally, the visual representation of the system is complemented by the numerical quantification of contractility to view, in a graph, how contractility depends on the composition of the system. Overall, these different steps constitute a typical workflow that can be applied with few modifications to tackle many other problems in the cytoskeletal field.

Cells can actively change their shapes and become motile, a property that depends on their ability to actively reorganize their internal structure. This feature is attributed to the mechanical and dynamic properties of the cell cytoskeleton, notably, the actomyosin cytoskeleton, which is an active gel of polar actin filaments, myosin motors, and accessory proteins that exhibit intrinsic contraction properties. The usually accepted view is that the cytoskeleton behaves as a viscoelastic material. However, this model cannot always explain the experimental results, which are more consistent with a picture describing the cytoskeleton as a poroelastic active material-an elastic network embedded with cytosol. Contractility gradients generated by the myosin motors drive the flow of the cytosol across the gel pores, which infers that the mechanics of the cytoskeleton and the cytosol are tightly coupled. One main feature of poroelasticity is the diffusive relaxation of stresses in the network, characterized by an effective diffusion constant that depends on the gel elastic modulus, porosity, and cytosol (solvent) viscosity. As cells have many ways to regulate their structure and material properties, our current understanding of how cytoskeleton mechanics and cytosol flow dynamics are coupled remains poorly understood. Here, an in vitro reconstitution approach is employed to characterize the material properties of poroelastic actomyosin gels as a model system for the cell cytoskeleton. Gel contraction is driven by myosin motor contractility, which leads to the emergence of a flow of the penetrating solvent. The paper describes how to prepare these gels and run experiments. We also discuss how to measure and analyze the solvent flow and gel contraction both at the local and global scales. The various scaling relations used for data quantification are given. Finally, the experimental challenges and common pitfalls are discussed, including their relevance to cell cytoskeleton mechanics.

Biomolecular condensation of proteins contributes to the organization of the cytoplasm and nucleoplasm. A number of condensation processes appear to be directly involved in regulating the structure, function and dynamics of the cytoskeleton. Liquid-liquid phase separation of cytoskeleton proteins, together with polymerization modulators, promotes cytoskeletal fiber nucleation and branching. Furthermore, the attachment of protein condensates to the cytoskeleton can contribute to cytoskeleton stability and organization, regulate transport, create patterns of functional reaction containers, and connect the cytoskeleton with membranes. Surface-bound condensates can exert and buffer mechanical forces that give stability and flexibility to the cytoskeleton, thus, may play a large role in cell biology. In this review, we introduce the concept and role of cellular biomolecular condensation, explain its special function on cytoskeletal fiber surfaces, and point out potential definition and experimental caveats. We review the current literature on protein condensation processes related to the actin, tubulin, and intermediate filament cytoskeleton, and discuss some of them in the context of neurobiology. In summary, we provide an overview about biomolecular condensation in relation to cytoskeleton structure and function, which offers a base for the exploration and interpretation of cytoskeletal condensates in neurobiology., (Copyright © 2023. Published by Elsevier Inc.)

A common adverse response to the clinical use of glucocorticoids (GCs) is elevated intraocular pressure (IOP) which is a major risk factor for glaucoma. Elevated IOP arises due to impaired outflow of aqueous humor (AH) through the trabecular meshwork (TM). Although GC-induced changes in actin cytoskeletal dynamics, contractile characteristics, and cell adhesive interactions of TM cells are believed to influence AH outflow and IOP, the molecular mechanisms mediating changes in these cellular characteristics are poorly understood. Our studies focused on evaluating changes in the cytoskeletal and cytoskeletal-associated protein (cytoskeletome) profile of human TM cells treated with dexamethasone (Dex) using label-free mass spectrometric quantification, identified elevated levels of specific proteins known to regulate actin stress fiber formation, contraction, actin networks crosslinking, cell adhesion, and Wnt signaling, including LIMCH1, ArgBP2, CNN3, ITGBL1, CTGF, palladin, FAT1, DIAPH2, EPHA4, SIPA1L1, and GPC4. Several of these proteins colocalized with the actin cytoskeleton and underwent alterations in distribution profile in TM cells treated with Dex, and an inhibitor of Abl/Src kinases. Wnt/Planar Cell Polarity (PCP) signaling agonists-Wnt5a and 5b were detected prominently in the cytoskeletome fraction of TM cells, and studies using siRNA to suppress expression of glypican-4 (GPC4), a known modulator of the Wnt/PCP pathway revealed that GPC4 deficiency impairs Dex induced actin stress fiber formation, and activation of c-Jun N-terminal Kinase (JNK) and Rho kinase. Additionally, while Dex augmented, GPC4 deficiency suppressed the formation of actin stress fibers in TM cells in the presence of Dex and Wnt5a. Taken together, these results identify the GPC4-dependent Wnt/PCP signaling pathway as one of the crucial upstream regulators of Dex induced actin cytoskeletal reorganization and cell adhesion in TM cells, opening an opportunity to target the GPC4/Wnt/PCP pathway for treatment of ocular hypertension in glaucoma., (© 2023 Wiley Periodicals LLC.)

Recently, researches have revealed the key roles of the cytoskeleton in the occurrence and development of multiple diseases, suggesting that targeting the cytoskeleton is a viable approach for treating numerous refractory diseases. The cytoskeleton is a highly structured and complex network composed of actin filaments, microtubules, and intermediate filaments. In normal cells, these three cytoskeleton components are highly integrated and coordinated. However, the cytoskeleton undergoes drastic remodeling in cytoskeleton-related diseases, causing changes in cell polarity, affecting the cell cycle, leading to senescent diseases, and influencing cell migration to accelerate cancer metastasis. Additionally, mutations or abnormalities in cytoskeletal proteins and their related proteins are closely associated with several congenital diseases. Therefore, this review summarizes the roles of the cytoskeleton in cytoskeleton-related diseases as well as its potential roles in disease treatment to provide insights regarding the physiological functions and pathological roles of the cytoskeleton., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

β-arrestins, which have multiple cellular functions, were initially described as proteins that desensitize rhodopsin and other G protein-coupled receptors. The cytoskeletal system plays a role in various cellular processes, including intracellular transport, cell division, organization of organelles, and cell cycle. The interactome of β-arrestins includes the major proteins of the three main cytoskeletal systems: tubulins for microtubules, actins for the actin filaments, and vimentin for intermediate filaments. β-arrestins bind to microtubules and regulate their activity by recruiting signaling proteins and interacting with assembly proteins that regulate the actin cytoskeleton and the intermediate filaments. Altered regulation of the cytoskeletal system plays an essential role in the development of Alzheimer's, Parkinson's and other neurodegenerative diseases. Thus, β-arrestins, which interact with the cytoskeleton, were implicated in the pathogenesis progression of these diseases and are potential targets for the treatment of neurodegenerative disorders in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Szénási, Turu and Hunyady.)

Cell adhesion systems commonly operate in close partnership with the cytoskeleton. Adhesion receptors bind to the cortex and regulate its dynamics, organization and mechanics; conversely, the cytoskeleton influences aspects of adhesion, including strength, stability and ductility. In this review we consider recent advances in elucidating such cooperation, focusing on interactions between classical cadherins and actomyosin. The evidence presents an apparent paradox. Molecular mechanisms of mechanosensation by the cadherin-actin apparatus imply that adhesion strengthens under tension. However, this does not always translate to the broader setting of confluent tissues, where increases in fluctuations of tension can promote intercalation due to the shrinkage of adherens junctions. Emerging evidence suggests that understanding of timescales may be important in resolving this issue, but that further work is needed to understand the role of adhesive strengthening across scales., Competing Interests: Conflict of interest Nothing declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Actin cytoskeleton force generation, sensing, and adaptation are dictated by the bending and twisting mechanics of filaments. Here, we use magnetic tweezers and microfluidics to twist and pull individual actin filaments and evaluate their response to applied loads. Twisted filaments bend and dissipate torsional strain by adopting a supercoiled plectoneme. Pulling prevents plectoneme formation, which causes twisted filaments to sever. Analysis over a range of twisting and pulling forces and direct visualization of filament and single subunit twisting fluctuations yield an actin filament torsional persistence length of ~10 µm, similar to the bending persistence length. Filament severing by cofilin is driven by local twist strain at boundaries between bare and decorated segments and is accelerated by low pN pulling forces. This work explains how contractile forces generated by myosin motors accelerate filament severing by cofilin and establishes a role for filament twisting in the regulation of actin filament stability and assembly dynamics.

Alterations in the organization of the cytoskeleton precede the escape of adherent cells from the framework of cell-cell and cell-matrix interactions into suspension. With cytoskeletal dynamics being linked to cell mechanical properties, many studies elucidated this relationship under either native adherent or suspended conditions. In contrast, tethered cells that mimic the transition between both states have not been the focus of recent research. Using human embryonic kidney 293 T cells we investigated all three conditions in the light of alterations in cellular shape, volume, as well as mechanical properties and relate these findings to the level, structure, and intracellular localization of filamentous actin (F-actin). For cells adhered to a substrate, our data shows that seeding density affects cell size but does not alter their elastic properties. Removing surface contacts leads to cell stiffening that is accompanied by changes in cell shape, and a reduction in cellular volume but no alterations in F-actin density. Instead, we observe changes in the organization of F-actin indicated by the appearance of blebs in the semi-adherent state. In summary, our work reveals an interplay between molecular and mechanical alterations when cells detach from a surface that is mainly dominated by cell morphology., (© 2022 The Authors. Cytoskeleton published by Wiley Periodicals LLC.)

The plant cytoskeleton, consisting of actin filaments and microtubules, is a highly dynamic filamentous framework involved in plant growth, development, and stress responses. Recently, research has demonstrated that the plant cytoskeleton undergoes rapid remodeling upon sensing pathogen attacks, coordinating the formation of microdomain immune complexes, the dynamic and turnover of pattern-recognizing receptors (PRRs), the movement and aggregation of organelles, and the transportation of defense compounds, thus serving as an important platform for responding to pathogen infections. Meanwhile, pathogens produce effectors targeting the cytoskeleton to achieve pathogenicity. Recent findings have uncovered several cytoskeleton-associated proteins mediating cytoskeletal remodeling and defense signaling. Furthermore, the reorganization of the actin cytoskeleton is revealed to further feedback-regulate reactive oxygen species (ROS) production and trigger salicylic acid (SA) signaling, suggesting an extremely complex role of the cytoskeleton in plant immunity. Here, we describe recent advances in understanding the host cytoskeleton dynamics upon sensing pathogens and summarize the effectors that target the cytoskeleton. We highlight advances in the regulation of cytoskeletal remodeling associated with the defense response and assess the important function of the rearrangement of the cytoskeleton in the immune response. Finally, we propose suggestions for future research in this area.

The endocrine disrupting activity of bisphenol compounds is well documented, but less is known regarding their impact on cell division and early embryo formation. Here, we tested the effects of acute in vitro exposure to bisphenol A (BPA) and its common substitute, bisphenol F (BPF), during critical stages of mouse pre-implantation embryo development, including the first mitotic division, cell polarization, as well as morula and blastocyst formation. Timing of initial cleavage was determined by live-cell imaging, while subsequent divisions, cytoskeletal organization and lineage marker labeling were assessed by high-resolution fluorescence microscopy. Our analysis reveals that brief culture with BPA or BPF impeded cell division and disrupted embryo development at all stages tested. Surprisingly, BPF was more detrimental to the early embryo than BPA. Notably, poor embryo development was associated with cytoskeletal disruptions of the actomyosin network, apical domain formation during cell polarization, actin ring zippering for embryo sealing and altered cell lineage marker profiles. These results underscore that bisphenols can disrupt cytoskeletal integrity and remodeling that is vital for early embryo development and raise concerns regarding the use of BPF as a 'safe' BPA substitute.

To cope with continuous physiological and environmental stresses, cells of all sizes require an effective wound repair process to seal breaches to their cortex. Once a wound is recognized, the cell must rapidly plug the injury site, reorganize the cytoskeleton and the membrane to pull the wound closed, and finally remodel the cortex to return to homeostasis. Complementary studies using various model organisms have demonstrated the importance and complexity behind the formation and translocation of an actin ring at the wound periphery during the repair process. Proteins such as actin nucleators, actin bundling factors, actin-plasma membrane anchors, and disassembly factors are needed to regulate actin ring dynamics spatially and temporally. Notably, Rho family GTPases have been implicated throughout the repair process, whereas other proteins are required during specific phases. Interestingly, although different models share a similar set of recruited proteins, the way in which they use them to pull the wound closed can differ. Here, we describe what is currently known about the formation, translocation, and remodeling of the actin ring during the cell wound repair process in model organisms, as well as the overall impact of cell wound repair on daily events and its importance to our understanding of certain diseases and the development of therapeutic delivery modalities.

Many cells can generate complementary traveling waves of actin filaments (F-actin) and cytoskeletal regulators. This phenomenon, termed cortical excitability, results from coupled positive and negative feedback loops of cytoskeletal regulators. The nature of these feedback loops, however, remains poorly understood. We assessed the role of the Rho GAP RGA-3/4 in the cortical excitability that accompanies cytokinesis in both frog and starfish. RGA-3/4 localizes to the cytokinetic apparatus, "chases" Rho waves in an F-actin-dependent manner, and when coexpressed with the Rho GEF Ect2, is sufficient to convert the normally quiescent, immature Xenopus oocyte cortex into a dramatically excited state. Experiments and modeling show that changing the ratio of RGA-3/4 to Ect2 produces cortical behaviors ranging from pulses to complex waves of Rho activity. We conclude that RGA-3/4, Ect2, Rho, and F-actin form the core of a versatile circuit that drives a diverse range of cortical behaviors, and we demonstrate that the immature oocyte is a powerful model for characterizing these dynamics., (© 2022 Michaud et al.)

When B cells are exposed to antigens, they use their B-cell receptors (BCRs) to transduce this external signal into internal signaling cascades and uptake antigen, which activate transcriptional programs. Signaling activation requires complex cytoskeletal remodeling initiated by BCR signaling. The actin cytoskeletal remodeling drives B-cell morphological changes, such as spreading, protrusion, contraction, and endocytosis of antigen by mechanical forces, which in turn affect BCR signaling. Therefore, the relationship between the actin cytoskeleton and BCR signaling is a two-way feedback loop. These morphological changes represent the indirect ways by which the actin cytoskeleton regulates BCR signaling. Recent studies using high spatiotemporal resolution microscopy techniques have revealed that actin also can directly influence BCR signaling. Cortical actin networks directly affect BCR mobility, not only during the resting stage by serving as diffusion barriers, but also at the activation stage by altering BCR diffusivity through enhanced actin flow velocities. Furthermore, the actin cytoskeleton, along with myosin, enables B cells to sense the physical properties of its environment and generate and transmit forces through the BCR. Consequently, the actin cytoskeleton modulates the signaling threshold of BCR to antigenic stimulation. This review discusses the latest research on the relationship between BCR signaling and actin remodeling, and the research techniques. Exploration of the role of actin in BCR signaling will expand fundamental understanding of the relationship between cell signaling and the cytoskeleton and the mechanisms underlying cytoskeleton-related immune disorders and cancer., (© 2021 Federation of European Biochemical Societies.)

Wound healing requires a complex cascade of highly controlled and conserved cellular and molecular processes. These involve numerous cell types and extracellular matrix molecules regulated by the actin cytoskeleton. This microscopic network of filaments is present within the cytoplasm of all cells and provides the shape and mechanical support required for cell movement and proliferation. Here, an overview of the processes of wound healing are described from the perspective of the cell in relation to the actin cytoskeleton. Key points of discussion include the role of actin, its binding proteins, signaling pathways, and events that play significant roles in the phases of wound healing. The identification of cytoskeletal targets that can be used to manipulate and improve wound healing is included as an emerging area of focus that may inform future therapeutic approaches to improve healing of complex wounds., (Copyright © 2022 Cold Spring Harbor Laboratory Press; all rights reserved.)

The cell nucleus is a dynamic structure that changes locales during cellular processes such as proliferation, differentiation, or migration, and its mispositioning is a hallmark of several disorders. As with most mechanobiological activities of adherent cells, the repositioning and anchoring of the nucleus are presumed to be associated with the organization of the cytoskeleton, the network of protein filaments providing structural integrity to the cells. However, demonstrating this correlation between cytoskeleton organization and nuclear position requires the parameterization of the extraordinarily intricate cytoskeletal fiber arrangements. Here, we show that this parameterization and demonstration can be achieved outside the limits of human conceptualization, using generative network and raw microscope images, relying on machine-driven interpretation and selection of parameterizable features. The developed transformer-based architecture was able to generate high-quality, completed images of more than 8,000 cells, using only information on actin filaments, predicting the presence of a nucleus and its exact localization in more than 70 per cent of instances. Our results demonstrate one of the most basic principles of mechanobiology with a remarkable level of significance. They also highlight the role of deep learning as a powerful tool in biology beyond data augmentation and analysis, capable of interpreting-unconstrained by the principles of human reasoning-complex biological systems from qualitative data., Competing Interests: The authors have declared that no competing interests exist.

Single molecule imaging has shown that part of actin disassembles within a few seconds after incorporation into the dendritic filament network in lamellipodia, suggestive of frequent destabilization near barbed ends. To investigate the mechanisms behind network remodeling, we created a stochastic model with polymerization, depolymerization, branching, capping, uncapping, severing, oligomer diffusion, annealing, and debranching. We find that filament severing, enhanced near barbed ends, can explain the single molecule actin lifetime distribution, if oligomer fragments reanneal to free ends with rate constants comparable to in vitro measurements. The same mechanism leads to actin networks consistent with measured filament, end, and branch concentrations. These networks undergo structural remodeling, leading to longer filaments away from the leading edge, at the +/-35° orientation pattern. Imaging of actin speckle lifetimes at sub-second resolution verifies frequent disassembly of newly-assembled actin. We thus propose a unified mechanism that fits a diverse set of basic lamellipodia phenomenology., Competing Interests: DH, AH, EU, SY, NW, DV No competing interests declared, (© 2022, Holz et al.)

The role of the actin cytoskeleton during receptor-mediated endocytosis (RME) has been well characterized in yeast for many years. Only more recently has the interplay between the actin cytoskeleton and RME been extensively explored in mammalian cells. These studies have revealed the central roles of BAR proteins in RME, and have demonstrated significant roles of BAR proteins in linking the actin cytoskeleton to this cellular process. The actin cytoskeleton generates and transmits mechanical force to promote the extension of receptor-bound endocytic vesicles into the cell. Many adaptor proteins link and regulate the actin cytoskeleton at the sites of endocytosis. This review will cover key effectors, adaptors and signalling molecules that help to facilitate the invagination of the cell membrane during receptor-mediated endocytosis, including recent insights gained on the roles of BAR proteins. The final part of this review will explore associations of alterations to genes encoding BAR proteins with cancer., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Adhesion between cells and the extracellular matrix (ECM) is one of the prerequisites for multicellularity, motility, and tissue specialization. Focal adhesions (FAs) are defined as protein complexes that mediate signals from the ECM to major components of the cytoskeleton (microtubules, actin, and intermediate filaments), and their mutual communication determines a variety of cellular processes. In this study, human cytoskeletal crosstalk proteins were identified by comparing datasets with experimentally determined cytoskeletal proteins. The spectraplakin dystonin was the only protein found in all datasets. Other proteins (FAK, RAC1, septin 9, MISP, and ezrin) were detected at the intersections of FAs, microtubules, and actin cytoskeleton. Homology searches for human crosstalk proteins as queries were performed against a predefined dataset of proteomes. This analysis highlighted the importance of FA communication with the actin and microtubule cytoskeleton, as these crosstalk proteins exhibit the highest degree of evolutionary conservation. Finally, phylogenetic analyses elucidated the early evolutionary history of spectraplakins and cortical microtubule stabilization complexes (CMSCs) as model representatives of the human cytoskeletal crosstalk. While spectraplakins probably arose at the onset of opisthokont evolution, the crosstalk between FAs and microtubules is associated with the emergence of metazoans. The multiprotein complexes contributing to cytoskeletal crosstalk in animals gradually gained in complexity from the onset of metazoan evolution.

Previous work from us and others has suggested that cholesterol is an important lipid in the context of the organization of the actin cytoskeleton. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol is rarely addressed in the literature. In this work, we explored the signaling crosstalk between cholesterol and the actin cytoskeleton by using a high-resolution confocal microscopic approach to quantitatively measure changes in F-actin content upon cholesterol depletion. Our results show that F-actin content significantly increases upon chronic cholesterol depletion, but not during acute cholesterol depletion. In addition, utilizing inhibitors targeting the cholesterol biosynthetic pathway at different steps, we show that reorganization of the actin cytoskeleton could occur due to the synergistic effect of multiple pathways, including prenylated Rho GTPases and availability of membrane phosphatidylinositol 4,5-bisphosphate. These results constitute one of the first comprehensive dissections of the mechanistic basis underlying the interplay between cellular actin levels and cholesterol biosynthesis. We envision these results will be relevant for future understating of the remodeling of the actin cytoskeleton in pathological conditions with altered cholesterol., Competing Interests: Conflicts of interest The authors declare that they have no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Inversin is an integrated component of the Frizzled (Fzd)/Dishevelled (Dvl)/Diversin planar cell polarity (PCP) complex that is known to work in concert with the Van Gogh-like protein (eg, Vangl2)/Prickle PCP complex to support tissue and organ development including the brain, kidney, pancreas, and others. These PCP protein complexes are also recently shown to confer developing haploid spermatid PCP to support spermatogenesis in adult rat testes. However, with the exception of Dvl3 and Vangl2, other PCP proteins have not been investigated in the testis. Herein, we used the technique of RNA interference (RNAi) to examine the role of inversin (Invs) in Sertoli cell (SC) and testis function by corresponding studies in vitro and in vivo. When inversin was silenced by RNAi using specific small interfering RNA duplexes by transfecting primary cultures of SCs in vitro or testes in vivo, it was shown that inversin knockdown (KD) perturbed the SC tight junction-barrier function in vitro and in vivo using corresponding physiological and integrity assays. More important, inversin exerted its regulatory effects through changes in the organization of the actin and microtubule cytoskeletons, including reducing the ability of their polymerization. These changes, in turn, induced defects in spermatogenesis by loss of spermatid polarity, disruptive distribution of blood-testis barrier-associated proteins at the SC-cell interface, appearance of multinucleated round spermatids, and defects in the release of sperm at spermiation., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Avian hepatitis E virus (HEV) causes liver diseases and multiple extrahepatic disorders in chickens. However, the mechanisms involved in avian HEV entry remain elusive. Herein, we identified the RAS-related protein 1b (Rap1b) as a potential HEV-ORF2 protein interacting candidate. Experimental infection of chickens and cells with an avian HEV isolate from China (CaHEV) led to upregulated expression and activation of Rap1b both in vivo and in vitro . By using CaHEV capsid as mimic of virion to treat cell in vitro , it appears that the interaction between the viral capsid and Rap1b promoted cell membrane recruitment of the downstream effector Rap1-interacting molecule (RIAM). In turn, RIAM further enhanced Talin-1 membrane recruitment and retention, which led to the activation of integrin α5/β1, as well as integrin-associated membrane protein kinases, including focal adhesion kinase (FAK). Meanwhile, FAK activation triggered activation of downstream signaling molecules, such as Ras-related C3 botulinum toxin substrate 1 RAC1 cell division cycle 42 (CDC42), p21-activated kinase 1 (PAK1), and LIM domain kinase 1 (LIMK1). Finally, F-actin rearrangement induced by Cofilin led to the formation of lamellipodia, filopodia, and stress fibers, contributes to plasma membrane remodeling, and might enhance CaHEV virion internalization. In conclusion, our data suggested that Rap1b activation was triggered during CaHEV infection and appeared to require interaction between CaHEV-ORF2 and Rap1b, thereby further inducing membrane recruitment of Talin-1. Membrane-bound Talin-1 then activates key Integrin-FAK-Cofilin cascades involved in modulation of actin kinetics, and finally leads to F-actin rearrangement and membrane remodeling to potentially facilitate internalization of CaHEV virions into permissive cells. IMPORTANCE Rap1b is a multifunctional protein that is responsible for cell adhesion, growth, and differentiation. The inactive form of Rap1b is phosphorylated and distributed in the cytoplasm, while active Rap1b is prenylated and loaded with GTP to the cell membrane. In this study, the activation of Rap1b was induced during the early stage of avian HEV infection under the regulation of PKA and SmgGDS. Continuously activated Rap1b recruited its effector RIAM to the membrane, thereby inducing the membrane recruitment of Talin-1 that led to the activation of membrane α5/β1 integrins. The triggering of the signaling pathway-associated Integrin α5/β1-FAK-CDC42&RAC1-PAK1-LIMK1-Cofilin culminated in F-actin polymerization and membrane remodeling that might promote avian HEV virion internalization. These findings suggested a novel mechanism that is potentially utilized by avian HEV to invade susceptible cells.

The coordinated interplay of cytoskeletal networks critically determines tissue biomechanics and structural integrity. Here, we show that plectin, a major intermediate filament-based cytolinker protein, orchestrates cortical cytoskeletal networks in epithelial sheets to support intercellular junctions. By combining CRISPR/Cas9-based gene editing and pharmacological inhibition, we demonstrate that in an F-actin-dependent context, plectin is essential for the formation of the circumferential keratin rim, organization of radial keratin spokes, and desmosomal patterning. In the absence of plectin-mediated cytoskeletal cross-linking, the aberrant keratin-desmosome (DSM)-network feeds back to the actin cytoskeleton, which results in elevated actomyosin contractility. Also, by complementing a predictive mechanical model with Förster resonance energy transfer-based tension sensors, we provide evidence that in the absence of cytoskeletal cross-linking, major intercellular junctions (adherens junctions and DSMs) are under intrinsically generated tensile stress. Defective cytoarchitecture and tensional disequilibrium result in reduced intercellular cohesion, associated with general destabilization of plectin-deficient sheets upon mechanical stress., (© 2022 Prechova et al.)

Membrane-cytoskeleton interactions (MCIs) are mediated by actin-binding proteins (ABPs). Ezrin is a crucial ABP that links membranes to actin filaments during lamellipodia formation, cell polarization, and migration. We discuss the concept of MCI and the potential of ezrin as a druggable target for treating inflammatory diseases and cancers., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Plasma membrane tension is known to regulate many cell functions, such as motility and membrane trafficking. Membrane tether pulling is an effective method for measuring the apparent membrane tension of cells and exploring membrane-cytoskeleton interactions. In this article, the mechanical properties of HP1α-depleted MCF7 breast cancer cells are explored in comparison to controls, by pulling membrane tethers using optical tweezers. These studies were inspired by previous findings that a loss of HP1α correlates with an increase in the invasive potential of malignant cancer cells. Specifically, the membrane tension and force relaxation curves for tethers pulled from MCF7 breast cancer cells with HP1α knockdown and their matched controls were measured, and shown to be significantly different., 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 © 2021 Elsevier Inc. All rights reserved.)