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Gelsolin is known as one of the actin-binding proteins capable of severing and capping filamentous actin, and of undergoing structural changes in the presence of calcium ions to interact with actin filaments. In this study, single-particle 3D reconstruction using electron microscopy (EM) revealed that, in the presence of calcium, the structure of gelsolin undergoes structural changes before interacting with actin. These differences are subtle with similarities, as confirmed by the EM map. According to the results of the molecular dynamics simulations, these nuanced structural differences primarily manifest at the domain level when calcium is present. These results provide structural evidence that, in the presence of calcium, gelsolin enters a phase of conformational preparation to transition into the active state. This process enables gelsolin to bind to actin, whereupon gelsolin undergoes more drastic structural changes upon interaction with actin filaments, which allows it to participate in binding and severing to regulate the cytoskeleton. This is the first visualization of full-length gelsolin, and helps to clarify crucial aspects of the as of yet incompletely understood interaction between gelsolin and actin., 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.)

The cytoskeleton of mammal oocytes provides structural support to the plasma membrane and contributes to critical cellular dynamic processes such as nuclear positioning, germinal vesicle breakdown, spindle orientation, chromosome segregation, polar body extrusion, and transmembrane signaling pathways. The ERM family (ezrin, radixin and moesin) well known as membrane-cytoskeletal crosslinkers play a crucial role in organizing plasma membrane domains through their capacity to interact with transmembrane proteins and the underlying cytoskeleton. Recent works mainly focused on the structural analysis of the ERM family members and their binding partners, together with multiple functions in cell mitosis, have significantly advanced our understanding of the importance of membrane-cytoskeletal interactions. In the present study, we documented that p-ERM was expressed and localized at cortical and nucleus during mouse oocyte meiosis. p-ERM and microfilaments were colocalized from GV to MII during mouse oocyte maturation. After being treated with cytochalasin B (CB), the F-actin was disassembled. Meanwhile, p-ERM exhibited a diffuse cytoplasmic distribution and no special staining was detected in either the oocyte membrane or condensed chromosomes. p-ERM depletion by trim-away caused the meiotic procedure arrest with a significantly lower polar body extrusion rate. Collectively, these data demonstrate that the subcellular distribution of p-ERM is correlated with microfilaments. Meanwhile, the p-ERM contributes to the first polar extrusion but does not regulate the microfilament assembly., 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. Published by Elsevier Inc.)

As a key planar cell polarity protein, Van Gogh-like 2 (Vangl2) is essential for mammalian spermatogenesis. As a decapod crustacean, Eriocheir sinensis exhibits distinct spermatogenic processes due to its unique seminiferous tubule morphology and hemolymph-testis barrier (HTB). To determine whether Vangl2 performs analogous functions in E. sinensis, we identified the Es-Vangl2. Es-Vangl2 exhibited high expression and wide distribution in the testes, indicating its crucial involvement in spermatogenesis. Following targeted knockdown of Es-Vangl2in vivo, the structure of seminiferous tubules was disrupted, characterized by vacuolization of the germinal zone and obstruction of spermatozoon release. Concurrently, the integrity of the HTB was compromised, accompanied by reduced expression and aberrant localization of junction proteins. More importantly, the regulatory influence of Es-Vangl2 was manifested through modulating the organization of microfilaments, a process mediated by epidermal growth factor receptor pathway substrate 8 (Eps8). Further studies demonstrated that these phenotypes resulting from Es-Vangl2 knockdown were attributed to the inhibition of Rock signaling pathway activity, which was verified by the Es-Rock interference and Y27632 inhibition assays. In summary, the findings highlight the pivotal role of Es-Vangl2 in stabilizing HTB integrity by regulating Eps8-mediated actin remodeling through the Rock signaling pathway in the spermatogenesis of E. sinensis., 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 B.V. All rights reserved.)

In assisted fertility protocols, in vitro culture conditions mimic physiological conditions to preserve gametes in the best conditions. After collection, oocytes are maintained in a culture medium inside the incubator until in vitro fertilization (IVF) is performed. This time outside natural and physiological conditions exposes oocytes to an oxidative stress that renders in vitro aging. It has been described that in vitro aging produces a spontaneous cortical granule (CG) release decreasing the fertilization rate of oocytes. Nevertheless, this undesirable phenomenon has not been investigated, let alone prevented. In this work, we characterized the spontaneous CG secretion in in vitro aged oocytes. Using immunofluorescence indirect, quantification, and functional assays, we showed that the expression of regulatory proteins of CG exocytosis was affected. Our results demonstrated that in vitro oocyte aging by 4 and 8 h altered the expression and localization of alpha-SNAP and reduced the expression of NSF and Complexin. These alterations were prevented by supplementing culture medium with dithiothreitol (DTT), which in addition to having a protective effect on those proteins, also had an unexpected effect on the actin cytoskeleton. Indeed, DTT addition thickened the cortical layer of fibrillar actin. Both DTT effects, together, prevented the spontaneous secretion of CG and recovered the IVF rate in in vitro aged oocytes. We propose the use of DTT in culture media to avoid the spontaneous CG secretion and to improve the success rate of IVF protocols in in vitro aged oocytes., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Daam1 protein regulates Wnt-induced cytoskeletal changes during vertebrate gastrulation though its full mode of action and binding partners remain unresolved. Here we identify Reversion Induced LIM domain protein (RIL) as a new interacting protein of Daam1. Interaction studies uncover binding of RIL to the C-terminal actin-nucleating portion of Daam1 in a Wnt-responsive manner. Immunofluorescence studies showed subcellular localization of RIL to actin fibers and co-localization with Daam1 at the plasma membrane. RIL gain- and loss-of-function approaches in Xenopus produced severe gastrulation defects in injected embryos. Additionally, a simultaneous loss of Daam1 and RIL synergized to produce severe gastrulation defects indicating RIL and Daam1 may function in the same signaling pathway. RIL further synergizes with another novel Daam1-interacting protein, Formin Binding Protein 1 (FNBP1), to regulate gastrulation. Our studies altogether show RIL mediates Daam1-regulated non-canonical Wnt signaling that is required for vertebrate gastrulation., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The dynamic actin cytoskeleton contributes to many critical biological processes by providing the structural support underlying the morphology of most cells, facilitating intracellular transport, and generating forces required for cell motility and division. To execute many of these functions, actin monomers polymerize into polarized filaments that display different structural and biochemical properties at each end. Filament dynamics are regulated by diverse regulatory proteins which collaborate to dictate rates of elongation and disassembly, particularly at the fast-growing barbed (plus) end. This review highlights the biochemical mechanisms of six barbed end regulatory proteins: formin, profilin, capping protein, IQGAP1, cyclase-associated protein, and twinfilin. We discuss how individual proteins influence actin dynamics and how several intriguing complex assemblies influence the polymerization fate of actin filaments. Understanding these mechanisms offers insights into how actin is regulated in essential cell processes and dysregulated in disease., 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 Ltd.. All rights reserved.)

Background: Endocytosis is the process by which platelets incorporate extracellular molecules into their secretory granules. Endocytosis is mediated by the actin cytoskeleton in nucleated cells; however, the endocytic mechanisms in platelets are undefined., Objectives: To better understand platelet endocytosis, we studied gelsolin (Gsn), an actin-severing protein that promotes actin assembly., Methods: Mouse platelets from Gsn-null (Gsn -/- ) and wild-type (WT) controls were used. The uptake of fluorescent cargo molecules was compared as a measure of their endocytic efficiency. Receptor-mediated endocytosis was measured by the uptake of fibrinogen and transferrin; fluid-phase endocytosis was monitored by the uptake of fluorescent dextrans., Results: Adenosine diphosphate (ADP)-stimulated WT platelets readily internalized both receptor-mediated and fluid-phase cargoes. In contrast, Gsn -/- platelets showed a severe defect in the endocytosis of both types of cargo. The treatment of WT platelets with the actin-disrupting drugs cytochalasin D and jasplakinolide also reduced endocytosis. Notably, the individual and combined effects of Gsn deletion and drug treatment were similar for both receptor-mediated and fluid-phase endocytosis, indicating that Gsn mediates endocytosis via its action on the actin cytoskeleton., Conclusion: Our study demonstrates that Gsn plays a key role in the uptake of bioactive mediators by platelets., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Cell polarization can be guided by substrate topology through space constraints and adhesion induction, which are part of cellular mechanosensing pathways. Here, we demonstrated that protein tyrosine phosphatase Shp2 plays a crucial role in mediating the response of cells to substrate spatial cues. When compared to cells spreading on surfaces coated uniformly with fibronectin (FN), cells attached to 10 μm-width FN-strip micropattern (MP), which provides spatial cues for uniaxial spreading, exhibited elongated focal adhesions (FAs) and aligned stress fibers in the direction of the MP. As a result of uniaxial cell spreading, nuclei became elongated, dependent on ROCK-mediated actomyosin contractility. Additionally, intracellular viscoelasticity also increased. Shp2-deficient cells did not display elongated FAs mediated by MP, well-aligned stress fibers, or changes in nuclear shape and intracellular viscoelasticity. Overall, our data suggest that Shp2 is involved in regulating FAs and the actin cytoskeleton to modulate nuclear shape and intracellular physical properties in response to substrate spatial cues., 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.)

The actin cortex, commonly described as a thin 2-dimensional layer of actin filaments beneath the plasma membrane, is beginning to be recognized as part of a more dynamic and three-dimensional composite material. In this review, we focus on the elements that contribute to the three-dimensional architecture of the actin cortex. We also argue that actin-rich structures such as filopodia and stress fibers can be viewed as specialized integral parts of the 3D actin cortex. This broadens our definition of the cortex, shifting from its simplified characterization as a thin, two-dimensional layer of actin filaments., 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.)

Actin organization is crucial for establishing cell polarity, which influences processes such as directed cell motility and division. Despite its critical role in living organisms, achieving similar polarity in synthetic cells remains challenging. In this study, we employ a bottom-up approach to investigate how molecular crowders facilitate the formation of cortex-like actin networks and how these networks localize and organize based on membrane shape. Using giant unilamellar vesicles (GUVs) as models for cell membranes, we show that actin filaments can arrange along the membrane to form cortex-like structures. Notably, this organization is achieved using only actin and crowders as a minimal set of components. We utilize surface micropatterning to examine actin filament organization in deformed GUVs adhered to various pattern shapes. Our findings indicate that at the periphery of spherical GUVs, actin bundles align along the membrane. However, in highly curved regions of adhered GUVs, actin bundles avoid crossing the highly curved edges perpendicular to the adhesion site and instead remain in the lower curved regions by aligning parallel to the micropatterned surface. Furthermore, the actin bundles increase the stiffness of the GUVs, effectively counteracting strong deformations when GUVs adhere to micropatterns. This finding is corroborated by real-time deformability cytometry on GUVs with synthetic actin cortices. By precisely manipulating the shape of GUVs, our study provides a minimal system to investigate the interplay between actin structures and the membrane. Our findings provide insights into the spatial organization of actin structures within crowded environments, specifically inside GUVs that resemble the size and shape of cells. This study advances our understanding of actin network organization and functionality within cell-sized compartments., 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 GmbH.. All rights reserved.)

At the cell surface, the actin cytoskeleton and the plasma membrane interact reciprocally in a variety of processes related to the remodeling of the cell surface. The actin cytoskeleton has been known to modulate membrane organization and reshape the membrane. To this end, actin-membrane linking molecules play a major role in regulating actin assembly and spatially direct the interaction between the actin cytoskeleton and the membrane. While studies in cells have provided a wealth of knowledge on the molecular composition and interactions of the actin-membrane interface, the complex molecular interactions make it challenging to elucidate the precise actions of the actin-membrane linkers at the interface. Synthetic reconstituted systems, consisting of model membranes and purified proteins, have been a powerful approach to elucidate how actin-membrane linkers direct actin assembly to drive membrane shape changes. In this review, we will focus only on several actin-membrane linkers that have been studied by using reconstitution systems. We will discuss the design principles of these reconstitution systems and how they have contributed to the understanding of the cellular functions of actin-membrane linkers. Finally, we will provide a perspective on future research directions in understanding the intricate actin-membrane interaction., 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 GmbH.. All rights reserved.)

We analyzed actin cytoskeleton alterations during NET extrusion by neutrophil-like dHL-60 cells and human neutrophils in the absence of DNase1 containing serum to avoid chromatin degradation and microfilament disassembly. NET-formation by dHL-60 cells and neutrophils was induced by Ionomycin or phorbol-12-myristat-13-acetate (PMA). Subsequent staining with anti-actin and TRITC-phalloidin showed depolymerization of the cortical F-actin at spatially confined areas, the NET extrusion sites, effected by transient activation of the monooxygenase MICAL-1 supported by the G-actin binding proteins cofilin, profilin, thymosin ß4 and probably the F-actin fragmenting activity of gelsolin and/or its fragments, which also decorated the formed NETs. MICAL-1 itself appeared to be proteolyzed by neutrophil elastase possibly to confine its activity to the NET-extrusion area. The F-actin oxidization activity of MICAL-1 is inhibited by Levosimendan leading to reduced NET-formation. Anti-gasdermin-D immunohistochemistry showed a cytoplasmic distribution in non-stimulated cells. After stimulation the NET-extrusion pore displayed reduced anti-gasdermin-D staining but accumulated underneath the plasma membrane of the remaining cell body. A similar distribution was observed for myosin that concentrated together with cortical F-actin along the periphery of the remaining cell body suggesting force production by acto-myosin interactions supporting NET expulsion as indicated by the inhibitory action of the myosin ATPase inhibitor blebbistatin. Isolated human neutrophils displayed differences in their content of certain cytoskeletal proteins. After stimulation neutrophils with high gelsolin content preferentially formed "cloud"-like NETs, whereas those with low or no gelsolin formed long "filamentous" NETs., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Cryo-electron tomography (cryo-ET) has begun to provide intricate views of cellular architecture at unprecedented resolutions. Considerable efforts are being made to further optimize and automate the cryo-ET workflow, from sample preparation to data acquisition and analysis, to enable visual proteomics inside of cells. Here, we will discuss the latest advances in cryo-ET that go hand in hand with their application to the actin cytoskeleton. The development of deep learning tools for automated annotation of tomographic reconstructions and the serial lift-out sample preparation procedure will soon make it possible to perform high-resolution structural biology in a whole new range of samples, from multicellular organisms to organoids and tissues., 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.)

First identified in dividing cells as revolving clusters of actin filaments, these are now understood as mitochondrially-associated actin waves that are active throughout the cell cycle. These waves are formed from the polymerization of actin onto a subset of mitochondria. Within minutes, this F-actin depolymerizes while newly formed actin filaments assemble onto neighboring mitochondria. In interphase, actin waves locally fragment the mitochondrial network, enhancing mitochondrial content mixing to maintain organelle homeostasis. In dividing cells actin waves spatially mix mitochondria in the mother cell to ensure equitable partitioning of these organelles between daughter cells. Progress has been made in understanding the consequences of actin cycling as well as the underlying molecular mechanisms, but many questions remain, and here we review these elements. Also, we draw parallels between mitochondrially-associated actin cycling and cortical actin waves. These dynamic systems highlight the remarkable plasticity of the actin 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 © 2024 Elsevier Ltd. All rights reserved.)

Intracellular actin networks assemble through the addition of ATP-actin subunits at the growing barbed ends of actin filaments. This is followed by "aging" of the filament via ATP hydrolysis and subsequent phosphate release. Aged ADP-actin subunits thus "treadmill" through the filament before being released back into the cytoplasmic monomer pool as a result of depolymerization at filament pointed ends. The necessity for aging before filament disassembly is reinforced by preferential binding of cofilin to aged ADP-actin subunits over newly-assembled ADP-P i actin subunits in the filament. Consequently, investigations into how cofilin influences pointed-end depolymerization have, thus far, focused exclusively on aged ADP-actin filaments. Using microfluidics-assisted Total Internal Reflection Fluorescence (mf-TIRF) microscopy, we reveal that, similar to their effects on ADP filaments, cofilin and cyclase-associated protein (CAP) also promote pointed-end depolymerization of ADP-P i filaments. Interestingly, the maximal rates of ADP-P i filament depolymerization by CAP and cofilin together remain approximately 20-40 times lower than for ADP filaments. Further, we find that the promotion of ADP-P i pointed-end depolymerization is conserved for all three mammalian cofilin isoforms. Taken together, the mechanisms presented here open the possibility of newly-assembled actin filaments being directly disassembled from their pointed-ends, thus bypassing the slow step of P i release in the aging process., Competing Interests: Declaration of Competing Interest None, (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

In the development of chronic liver disease, the hepatic stellate cell (HSC) plays a pivotal role in increasing intrahepatic vascular resistance (IHVR) and inducing portal hypertension (PH) in cirrhosis. Our research demonstrated that HSC contraction, prompted by angiotensin II (Ang II), significantly contributed to the elevation of type I collagen (COL1A1) expression. This increase was intimately associated with enhanced cell tension and YAP nuclear translocation, mediated through α-smooth muscle actin (α-SMA) expression, microfilaments (MF) polymerization, and stress fibers (SF) assembly. Further investigation revealed that the Rho/ROCK signaling pathway regulated MF polymerization and SF assembly by facilitating the phosphorylation of cofilin and MLC, while Ca 2+ chiefly governed SF assembly via MLC. Inhibiting α-SMA-MF-SF assembly changed Ang II-induced cell contraction, YAP nuclear translocation, and COL1A1 expression, findings corroborated in cirrhotic mice models. Overall, our study offers insights into mitigating IHVR and PH through cell mechanics, heralding potential breakthroughs., 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., (Published by Elsevier GmbH.)

While it is well-established that F-actin networks with specific organizations and dynamics are tightly regulated by distinct sets of associated actin-binding proteins (ABPs), how ABPs self-sort to particular F-actin networks remains largely unclear. We report that actin assembly factors Arp2/3 complex and formin Cdc12 tune the association of ABPs fimbrin Fim1 and tropomyosin Cdc8 to different F-actin networks in fission yeast. Genetic and pharmacological disruption of F-actin networks revealed that Fim1 is preferentially directed to Arp2/3-complex mediated actin patches, whereas Cdc8 is preferentially targeted to formin Cdc12-mediated filaments in the contractile ring. To investigate the role of Arp2/3 complex- and formin Cdc12-mediated actin assembly, we used four-color TIRF microscopy to observe the in vitro reconstitution of ABP sorting with purified proteins. Fim1 or Cdc8 alone bind similarly well to filaments assembled by either assembly factor. However, in 'competition' reactions containing both actin assembly factors and both ABPs, ∼2.0-fold more Fim1 and ∼3.5-fold more Cdc8 accumulates on Arp2/3 complex branch points and formin Cdc12-assembled actin filaments, respectively. These findings indicate that F-actin assembly factors Arp2/3 complex and formin Cdc12 help facilitate the recruitment of specific ABPs, thereby tuning ABP sorting and subsequently establishing the identity of F-actin networks in fission yeast., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Cell shape and motility are determined by the cytoskeleton, an interpenetrating network of actin filaments, microtubules, and intermediate filaments. The biophysical properties of each filament type individually have been studied extensively by cell-free reconstitution. By contrast, the interactions between the three cytoskeletal networks are relatively unexplored. They are coupled via crosslinkers of the plakin family such as plectin. These are challenging proteins for reconstitution because of their giant size and multidomain structure. Here we engineer a recombinant actin-vimentin crosslinker protein called 'ACTIF' that provides a minimal model system for plectin, recapitulating its modular design with actin-binding and intermediate filament-binding domains separated by a coiled-coil linker for dimerisation. We show by fluorescence and electron microscopy that ACTIF has a high binding affinity for vimentin and actin and creates mixed actin-vimentin bundles. Rheology measurements show that ACTIF-mediated crosslinking strongly stiffens actin-vimentin composites. Finally, we demonstrate the modularity of this approach by creating an ACTIF variant with the intermediate filament binding domain of Adenomatous Polyposis Coli. Our protein engineering approach provides a new cell-free system for the biophysical characterization of intermediate filament-binding crosslinkers and for understanding the mechanical synergy between actin and vimentin in mesenchymal cells., 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 GmbH.. All rights reserved.)

Cellular Communication Network Factor 2, CCN2, is a profibrotic cytokine implicated in physiological and pathological processes in mammals. The expression of CCN2 is markedly increased in dystrophic muscles. Interestingly, diminishing CCN2 genetically or inhibiting its function improves the phenotypes of chronic muscular fibrosis in rodent models. Elucidating the cell-specific mechanisms behind the induction of CCN2 is a fundamental step in understanding its relevance in muscular dystrophies. Here, we show that the small lipids LPA and 2S-OMPT induce CCN2 expression in fibro/adipogenic progenitors (FAPs) through the activation of the LPA 1 receptor and, to a lower extent, by also the LPA 6 receptor. These cells show a stronger induction than myoblasts or myotubes. We show that the LPA/LPARs axis requires ROCK kinase activity and organized actin cytoskeleton upstream of YAP/TAZ signaling effectors to upregulate CCN2 levels, suggesting that mechanical signals are part of the mechanism behind this process. In conclusion, we explored the role of the LPA/LPAR axis on CCN2 expression, showing a strong cytoskeletal-dependent response in muscular FAPs., Competing Interests: Declaration of competing interest Dr. Chun has an employment relationship with Neurocrine Biosciences, Inc., a company that may potentially benefit from the research results. Dr. Chun's relationship with Neurocrine Biosciences, Inc. has been reviewed and approved by Sanford Burnham Prebys Medical Discovery Institute in accordance with its Conflict-of-Interest Policies. The other authors declare no competing or financial interests., (Copyright © 2024. Published by Elsevier B.V.)

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.)

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.)

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.)

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.)

The microtubule cytoskeleton has a well-established, instrumental role in coordinating cell migration. Decades of research has focused on understanding how microtubules couple intracellular trafficking with cortical targeting and spatial organization of signaling to facilitate locomotion. Movement in physically challenging environments requires coordination of forces generated by the actin cytoskeleton to drive cell shape changes, with microtubules acting to spatially regulate contractility. Recent work has demonstrated that the mechanical properties of microtubules are adaptive to stress, leading to a new understanding of their roles in cell migration. Herein we review new developments in how microtubules sense and adapt to changes in the physical properties of their environment during migration. We frame our discussion around our current understanding of how microtubules target cell-matrix adhesions, and their role in the spatiotemporal coordination of signaling to form mechano feedback loops. We expand on how these mechanisms may influence cell morphology in confined three-dimensional settings, and the importance of locally tuning the mechanical stability of polymers in response to mechanical cues. Finally, we discuss new roles for Golgi-derived microtubules in mechanosensing, and how preferential motor use may influence polymer stability to resist the physical constraints cells experience in confined environments., 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 Ltd.. All rights reserved.)

The actomyosin cytoskeletal network is responsible for a variety of fundamental cellular processes. Assembly and maintenance of actin networks involve an array of associated regulatory proteins for polymerization, branching, crosslinking and contractility-driven self-organization. In this study, we make the unexpected discovery in vitro that myosin VI and myosin X, motor proteins specialized in vesicle transport and filopodia formation, are capable of crosslinking and self-organizing actin into higher-order contractile structures in the absence of other actin-associated proteins. Moreover, myosin VI alone can initiate actin elongation and branching, and assemble branched force-generating networks from crosslinked actin polymers. Additional architectural control is provided by the actin crosslinking proteins α-actinin and fascin. Our data identify critical stages of tension-mediated connectivity in network development and provide a model system for further exploration of the nonequilibrium mechanics of actomyosin self-organization., 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., (Published by Elsevier Inc.)

Synaptojanin proteins are evolutionarily conserved regulators of vesicle transport and membrane homeostasis. Disruption of synaptojanin function has been implicated in a wide range of neurological disorders. Synaptojanins act as dual-functional lipid phosphatases capable of hydrolyzing a variety of phosphoinositides (PIPs) through autonomous SAC1-like PIP 4-phosphatase and PIP 2 5-phosphatase domains. The rarity of an evolutionary configuration of tethering two distinct enzyme activities in a single protein prompted us to investigate their individual and combined roles in budding yeast. Both PIP and PIP 2 phosphatase activities are encoded by multiple gene products and are independently essential for cell viability. In contrast, we observed that the synaptojanin proteins utilized both lipid-phosphatase activities to properly coordinate polarized distribution of actin during the cell cycle. Expression of each activity untethered (in trans) failed to properly reconstitute the basal actin regulatory activity; whereas tethering (in cis), even through synthetic linkers, was sufficient to complement these defects. Studies of chimeric proteins harboring PIP and PIP 2 phosphatase domains from a variety of gene products indicate synaptojanin proteins have highly specialized activities and that the length of the linker between the lipid-phosphatase domains is critical for actin regulatory activity. Our data are consistent with synaptojanin possessing a strict requirement for both two-domain configuration for some but not all functions and provide mechanistic insights into a coordinated role of tethering distinct lipid-phosphatase activities., Competing Interests: Declaration of competing interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. AI was not used in any form for the writing of the manuscript or any experimentation (design or performed). We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from john.york@vanderbilt.edu., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Using CHO-K1/A5 cells, a clonal cell line that robustly expresses adult muscle-type nicotinic acetylcholine receptor (nAChR), we explored whether insulin resistance in these mammalian cells affects cell-surface expression of the nAChR, its endocytic internalization, and actin cytoskeleton integrity. Acute nanomolar insulin stimulation resulted in a slow increase in nAChR cell-surface levels, reaching maximum levels at ∼1 h. Long periods of insulin incubation caused CHO-K1/A5 cells to become insulin resistant, as previously observed with several other cell types. Furthermore, long-term insulin treatment abolished the effects of short-term insulin exposure on cell-surface nAChR levels, suggestive of a desensitization phenomenon. It also affected the kinetics of ligand-induced nAChR internalization. Since the integrity of the cortical actin cytoskeleton affects nAChR endocytosis, we also studied the effects of long-term insulin treatment on this meshwork. We found that it significantly affected the cortical actin morphology of CHO-K1/A5 cells and the response of the actin cytoskeleton to a subsequent short-term insulin stimulus. Overall, the present results show for the first time the effects of insulin signaling on cell-surface nAChR expression and actin cytoskeleton-associated internalization., 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 Elsevier Inc. All rights reserved.)

Background: Associations between actin filaments (AFs) and intermediate filaments (IFs) are frequently observed in living cells. The crosstalk between these cytoskeletal components underpins cellular organization and dynamics; however, the molecular basis of filamentous interactions is not fully understood. Here, we describe the mode of interaction between AFs and desmin IFs (DIFs) in a reconstituted in vitro system., Methods: AFs (rabbit skeletal muscle) and DIFs (chicken gizzard) were labeled with fluorescent dyes. DIFs were immobilized on a heavy meromyosin (HMM)-coated collodion surface. HMM-driven AFs with ATP hydrolysis was assessed in the presence of DIFs. Images of single filaments were obtained using fluorescence microscopy. Vector changes in the trajectories of single AFs were calculated from microscopy images., Results: AF speed transiently decreased upon contact with DIF. The difference between the incoming and outgoing angles of a moving AF broadened upon contact with a DIF. A smaller incoming angle tended to result in a smaller outgoing angle in a nematic manner. The percentage of moving AFs decreased with an increasing DIF density, but the speed of the moving AFs was similar to that in the no-desmin control. An abundance of DIFs tended to exclude AFs from the HMM-coated surfaces., Conclusions: DIFs agitate the movement of AFs with the orientation. DIFs can bind to HMMs and weaken actin-myosin interactions., General Significance: The study indicates that apart from the binding strength, the accumulation of weak interactions characteristic of filamentous structures may affect the dynamic organization of cell architecture., 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 Elsevier B.V. All rights reserved.)

How cells utilize complex mixtures of actin binding proteins to assemble and maintain functionally diverse actin filament networks with distinct architectures and dynamics within a common cytoplasm is a longstanding question in cell biology. A compelling example of complex and specialized actin structures in cells are filopodia which sense extracellular chemical and mechanical signals to help steer motile cells. Filopodia have distinct actin architecture, composed of long, parallel actin filaments bundled by fascin, which form finger-like membrane protrusions. Elongation of the parallel actin filaments in filopodia can be mediated by two processive actin filament elongation factors, formin and Ena/VASP, which localize to the tips of filopodia. There remains debate as to how the architecture of filopodia are generated, with one hypothesis proposing that filopodia are generated from the lamellipodia, which consists of densely packed, branched actin filaments nucleated by Arp2/3 complex and kept short by capping protein. It remains unclear if different actin filament elongation factors are necessary and sufficient to facilitate the emergence of filopodia with diverse characteristics from a highly dense network of short-branched capped filaments. To address this question, we combined bead motility and micropatterning biomimetic assays with multi-color Total Internal Reflection Fluorescence microscopy imaging, to successfully reconstitute the formation of filopodia-like networks (FLN) from densely-branched lamellipodia-like networks (LLN) with eight purified proteins (actin, profilin, Arp2/3 complex, Wasp pWA, fascin, capping protein, VASP and formin mDia2). Saturating capping protein concentrations inhibit FLN assembly, but the addition of either formin or Ena/VASP differentially rescues the formation of FLN from LLN. Specifically, we found that formin/mDia2-generated FLNs are relatively long and lack capping protein, whereas VASP-generated FLNs are comparatively short and contain capping protein, indicating that the actin elongation factor can affect the architecture and composition of FLN emerging from LLN. Our biomimetic reconstitution systems reveal that formin or VASP are necessary and sufficient to induce the transition from a LLN to a FLN, and establish robust in vitro platforms to investigate FLN assembly 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.)

Together with actin filaments and microtubules, intermediate filaments (IFs) constitute the eukaryotic cytoskeleton and each of the three filament types contributes very distinct mechanical properties to this intracellular biopolymer network. IFs assemble hierarchically, rather than polymerizing from nuclei of a small number of monomers or dimers, as is the case with actin filaments and microtubules, respectively. This pathway leads to a molecular architecture specific to IFs and intriguing mechanical and dynamic properties: they are the most flexible cytoskeletal filaments and extremely extensible. Moreover, IFs are very stable against disassembly. Thus, they contribute important properties to cell mechanics, which recently have been investigated with state-of-the-art experimental and computational methods., 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 Elsevier Ltd. All rights reserved.)

Podosomes have been known for several decades as micron-sized, F-actin-rich structures that play a pivotal role in cell migration and invasion, as they are able to mediate both cell-matrix attachment as well as extracellular matrix degradation. Particularly in monocytic cells, podosomes have been shown to fulfill a variety of additional functions such as sensing of substrate rigidity and topography, or cell-cell fusion. Increasing evidence now points to the involvement of podosome-like structures also during phagocytosis by immune cells such as macrophages, dendritic cells, and neutrophils. Here, we compare the different cell models and experimental set ups where "phagocytic podosomes" have been described. We also discuss the composition and architecture of these structures, their potential involvement in mechanosensing and particle disruption, as well as the pros and cons for addressing them as bona fide podosomes., Competing Interests: Declaration of Competing Interest The authors declare no conflicting interests., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Cells rely on their cytoskeleton for key processes including division and directed motility. Actin filaments are a primary constituent of the cytoskeleton. Although actin filaments can create a variety of network architectures linked to distinct cell functions, the microscale molecular interactions that give rise to these macroscale structures are not well understood. In this work, we investigate the microscale mechanisms that produce different branched actin network structures using an iterative classification approach. First, we employ a simple yet comprehensive agent-based model that produces synthetic actin networks with precise control over the microscale dynamics. Then we apply machine learning techniques to classify actin networks based on measurable network density and geometry, identifying key mechanistic processes that lead to particular branched actin network architectures. Extensive computational experiments reveal that the most accurate method uses a combination of supervised learning based on network density and unsupervised learning based on network symmetry. This framework can potentially serve as a powerful tool to discover the molecular interactions that produce the wide variety of actin network configurations associated with normal development as well as pathological conditions such as cancer., 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., (Published by Elsevier Ltd.)

Cellular physiology has been mainly studied by using two-dimensional cell culture substrates which lack in vivo-mimicking extracellular environment and interactions. Thus, there is a growing need for more complex model systems in life sciences. Micro-engineered scaffolds have been proven to be a promising tool in understanding the role of physical cues in the co-regulation of cellular functions. These tools allow, for example, probing cell morphology and migration in response to changes in chemo-physical properties of their microenvironment. In order to understand how microtopographical features, what cells encounter in vivo, affect cytoskeletal organization and nuclear mechanics, we used direct laser writing via two-photon polymerization (TPP) to fabricate substrates which contain different surface microtopographies. By combining with advanced high-resolution spectral imaging, we describe how the constructed grid and vertical line microtopographies influence cellular alignment, nuclear morphology and mechanics. Specifically, we found that growing cells on grids larger than 10 × 20 μm 2 and on vertical lines increased 3D actin cytoskeleton orientation along the walls of microtopographies and abolished basal actin stress fibers. In concert, the nuclei of these cells were also more aligned, elongated, deformed and less flattened, indicating changes in nuclear force transduction. Importantly, by using fluorescence lifetime imaging microscopy for measuring Förster resonance energy transfer for a genetically encoded nesprin-2 molecular tension sensor, we show that growing cells on these microtopographic substrates induce lower mechanical tension at the nuclear envelope. To conclude, here used substrate microtopographies modulated the cellular mechanics, and affected actin organization and nuclear force transduction., 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. Published by Elsevier Ltd.)

The non-cholinergic molecular targets of organophosphate (OP) compounds have recently been investigated to explain their role in the generation of non-neurological diseases, such as immunotoxicity and cancer. Here, we evaluated the effects of malathion and its dialkylphosphate (DAP) metabolites on the cytoskeleton components and organization of RAW264.7 murine macrophages as non-cholinergic targets of OP and DAPs toxicity. All OP compounds affected actin and tubulin polymerization. Malathion, dimethyldithiophosphate (DMDTP) dimethylthiophosphate (DMTP), and dimethylphosphate (DMP) induced elongated morphologies and the formation of pseudopods rich in microtubule structures, and increased filopodia formation and general actin disorganization in RAW264.7 cells and slightly reduced stress fibers in the human fibroblasts GM03440, without significantly disrupting the tubulin or vimentin cytoskeleton. Exposure to DMTP and DMP increased cell migration in the wound healing assay but did not affect phagocytosis, indicating a very specific modification in the organization of the cytoskeleton. The induction of actin cytoskeleton rearrangement and cell migration suggested the activation of cytoskeletal regulators such as small GTPases. We found that DMP slightly reduced Ras homolog family member A activity but increased the activities of Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) from 5 min to 2 h of exposure. Chemical inhibition of Rac1 with NSC23766 reduced cell polarization and treatment with DMP enhanced cell migration, but Cdc42 inhibition by ML-141 completely inhibited the effects of DMP. These results suggest that methylated OP compounds, especially DMP, can modify macrophage cytoskeleton function and configuration via activation of Cdc42, which may represent a potential non-cholinergic molecular target for OP compounds., 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 Elsevier B.V. All rights reserved.)

Asymmetric cell divisions, where cells divide with respect to a polarized axis and give rise to daughter cells with different fates, are critically important for development. In many such divisions, the conserved PAR polarity proteins accumulate in distinct cortical domains in response to a symmetry breaking cue. The one-cell C. elegans embryo is a paradigm for understanding mechanisms of PAR polarization, but much less is known about polarity in subsequent divisions. Here, we investigate the polarization of the P 1 cell of the two-cell embryo. A posterior PAR-2 domain forms in the first 4 ​min, and polarization becomes stronger over time. Initial polarization depends on the PAR-1 and PKC-3 kinases, and the downstream polarity regulators MEX-5 and PLK-1. However, par-1 and plk-1 mutants exhibit delayed polarization. This late polarization correlates with the time of centrosome maturation and actomyosin flow, and loss of centrosome maturation or myosin in par-1 mutant embryos causes an even stronger polarity phenotype. Based on these and other results, we propose that PAR polarity in the P 1 cell is generated by at least two redundant mechanisms: There is a novel early pathway dependent on PAR-1, PKC-3 and cytoplasmic polarity, and a late pathway that resembles symmetry breaking in the one-cell embryo and requires PKC-3, centrosome associated AIR-1 and myosin flow., (Copyright © 2023. Published by Elsevier Inc.)

Dedicator of cytokinesis (DOCK) proteins play a central role in actin cytoskeleton regulation. This is highlighted by the DOCK2 and DOCK8 deficiencies leading to actinopathies and immune deficiencies. DOCK8 and DOCK11 activate CDC42, a Rho-guanosine triphosphate hydrolases involved in actin cytoskeleton dynamics, among many cellular functions. The role of DOCK11 in human immune disease has been long suspected but, to the best of our knowledge, has never been described to date. We studied 8 male patients, from 7 unrelated families, with hemizygous DOCK11 missense variants leading to reduced DOCK11 expression. The patients were presenting with early-onset autoimmunity, including cytopenia, systemic lupus erythematosus, skin, and digestive manifestations. Patients' platelets exhibited abnormal ultrastructural morphology and spreading as well as impaired CDC42 activity. In vitro activated T cells and B-lymphoblastoid cell lines from patients exhibited aberrant protrusions and abnormal migration speed in confined channels concomitant with altered actin polymerization during migration. Knock down of DOCK11 recapitulated these abnormal cellular phenotypes in monocytes-derived dendritic cells and primary activated T cells from healthy controls. Lastly, in line with the patients' autoimmune manifestations, we also observed abnormal regulatory T-cell (Treg) phenotype with profoundly reduced FOXP3 and IKZF2 expression. Moreover, we found reduced T-cell proliferation and impaired STAT5B phosphorylation upon interleukin-2 stimulation of the patients' lymphocytes. In conclusion, DOCK11 deficiency is a new X-linked immune-related actinopathy leading to impaired CDC42 activity and STAT5 activation, and is associated with abnormal actin cytoskeleton remodeling as well as Treg phenotype, culminating in immune dysregulation and severe early-onset autoimmunity., (© 2023 by The American Society of Hematology.)

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.)

Contact guidance, the widely-known phenomenon of cell alignment, is an essential step in the organization of adherent cells. This guidance is known to occur by, amongst other things, anisotropic features in the environment including elastic heterogeneity. To understand the origins of this guidance we employed a novel statistical thermodynamics framework, which recognises the non-thermal fluctuations in the cellular response, for modelling the response of the cells seeded on substrates with alternating soft and stiff stripes. Consistent with observations, the modelling framework predicts the existence of three regimes of cell guidance: (i) in regime I for stripe widths much larger than the cell size guidance is primarily entropic; (ii) for stripe widths on the order of the cell size in regime II guidance is biochemically mediated and accompanied by changes to the cell morphology while (iii) in regime III for stripe widths much less than the cell size there is no guidance as cells cannot sense the substrate heterogeneity. Guidance in regimes I and II is due to "molli-avoidance" with cells primarily residing on the stiff stripes. While the molli-avoidance tendency is not lost with decreasing density of collagen coating the substrate, the reduced focal adhesion formation with decreasing collagen density tends to inhibit contact guidance. Our results provide clear physical insights into the interplay between cell mechano-sensitivity and substrate elastic heterogeneity that ultimately leads to the contact guidance of cells in heterogeneous tissues. STATEMENT OF SIGNIFICANCE: Cellular morphology and organization play a crucial role in the micro-architecture of tissues and dictates their biological and mechanical functioning. Despite the importance of cellular organization in all facets of tissue biology, the fundamental question of how a cell organizes itself in an anisotropic environment is still poorly understood. We employ a novel statistical thermodynamics framework which recognises the non-thermal fluctuations in the cellular response to investigate cell guidance on substrates with alternating soft and stiff stripes. The propensity of cells to primarily reside on stiff stripes results in strong guidance when the period of the stripes is larger than the cell size. For smaller stripe periods, cells sense a homogeneous substrate and guidance is lost., 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 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The actin cytoskeleton impacts practically every function of a eukaryotic cell. Historically, the best-characterized cytoskeletal activities are in cell morphogenesis, motility, and division. The structural and dynamic properties of the actin cytoskeleton are also crucial for establishing, maintaining, and changing the organization of membrane-bound organelles and other intracellular structures. Such activities are important in nearly all animal cells and tissues, although distinct anatomical regions and physiological systems rely on different regulatory factors. Recent work indicates that the Arp2/3 complex, a broadly expressed actin nucleator, drives actin assembly during several intracellular stress response pathways. These newly described Arp2/3-mediated cytoskeletal rearrangements are coordinated by members of the Wiskott-Aldrich Syndrome Protein (WASP) family of actin nucleation-promoting factors. Thus, the Arp2/3 complex and WASP-family proteins are emerging as crucial players in cytoplasmic and nuclear activities including autophagy, apoptosis, chromatin dynamics, and DNA repair. Characterizations of the functions of the actin assembly machinery in such stress response mechanisms are advancing our understanding of both normal and pathogenic processes, and hold great promise for providing insights into organismal development and interventions for disease., 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.)

Metallothionein 3 (MT-3) is a cysteine-rich metal-binding protein that is expressed in the mammalian central nervous system and kidney. Various reports have posited a role for MT-3 in regulating the actin cytoskeleton by promoting the assembly of actin filaments. We generated purified, recombinant mouse MT-3 of known metal compositions, either with zinc (Zn), lead (Pb), or copper/zinc (Cu/Zn) bound. None of these forms of MT-3 accelerated actin filament polymerization in vitro, either with or without the actin binding protein profilin. Furthermore, using a co-sedimentation assay, we did not observe Zn-bound MT-3 in complex with actin filaments. Cu 2+ ions on their own induced rapid actin polymerization, an effect that we attribute to filament fragmentation. This effect of Cu 2+ is reversed by adding either EGTA or Zn-bound MT-3, indicating that either molecule can chelate Cu 2+ from actin. Altogether, our data indicate that purified recombinant MT-3 does not directly bind actin but it does attenuate the Cu-induced fragmentation of actin filaments., 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 Elsevier Inc. All rights reserved.)

Actin cytoskeleton remodelling drives cell motility, cell to cell contacts, as well as membrane and organelle dynamics. Those cellular activities operate at a particularly high pace in immune cells since these cells migrate through various tissues, interact with multiple cellular partners, ingest microorganisms and secrete effector molecules. The central and multifaceted role of actin cytoskeleton remodelling in sustaining immune cell tasks in humans is highlighted by rare inborn errors of immunity due to mutations in genes encoding proximal and distal actin regulators. In line with the specificity of some of the actin-based processes at work in immune cells, the expression of some of the affected genes, such as WAS, ARPC1B and HEM1 is restricted to the hematopoietic compartment. Exploration of these natural deficiencies highlights the fact that the molecular control of actin remodelling is tuned distinctly in the various subsets of myeloid and lymphoid immune cells and sustains different networks associated with a vast array of specialized tasks. Furthermore, defects in individual actin remodelling proteins are usually associated with partial cellular impairments highlighting the plasticity of actin cytoskeleton remodelling. This review covers the roles of disease-associated actin regulators in promoting the actin-based processes of immune cells. It focuses on the specific molecular function of those regulators across various immune cell subsets and in response to different stimuli. Given the fact that numerous immune-related actin defects have only been characterized recently, we further discuss the challenges lying ahead to decipher the underlying patho-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 © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Classical cadherins are transmembrane proteins whose extracellular domains link neighboring cells, and whose intracellular domains connect to the actin cytoskeleton via β-catenin and α-catenin. The cadherin-catenin complex transmits forces that drive tissue morphogenesis and wound healing. In addition, tension-dependent changes in αE-catenin conformation enables it to recruit the actin-binding protein vinculin to cell-cell junctions, which contributes to junctional strengthening. How and whether multiple cadherin-complexes cooperate to reinforce cell-cell junctions in response to load remains poorly understood. Here, we used single-molecule optical trap measurements to examine how multiple cadherin-catenin complexes interact with F-actin under load, and how this interaction is influenced by the presence of vinculin. We show that force oriented toward the (-) end of the actin filament results in mean lifetimes 3-fold longer than when force was applied towards the barbed (+) end. We also measured force-dependent actin binding by a quaternary complex comprising the cadherin-catenin complex and the vinculin head region, which cannot itself bind actin. Binding lifetimes of this quaternary complex increased as additional complexes bound F-actin, but only when load was oriented toward the (-) end. In contrast, the cadherin-catenin complex alone did not show this form of cooperativity. These findings reveal multi-level, force-dependent regulation that enhances the strength of the association of multiple cadherin/catenin complexes with F-actin, conferring positive feedback that may strengthen the junction and polarize F-actin to facilitate the emergence of higher-order cytoskeletal organization., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The cell motility of smooth muscle cells (SMCs) is essential for vascular and internal organ development and tissue regeneration in response to damage. Cell migration requires dynamic changes in the actin-cytoskeleton via the p-21 activated kinase (Pak)-Cofilin signaling cascade, which is the central axis of the actin filaments. We previously identified that the Inka2 gene was preferentially expressed in the central nervous system (CNS) and revealed that Inka2 directly binds Pak4 to suppress its kinase activity, thereby regulating actin de-polymerization in dendritic spine formation of the forebrain neurons. However, its physiological significance outside the CNS remains unclear. Here we determined the Inka2 expression profile in various organs using in situ hybridization analysis and lacZ staining on Inka2 flox/+ mice. Robust Inka2 expression was consistently detected in the SMCs of many peripheral organs, including the arteries, esophagus, stomach, intestine, and bladder. The scratch assay was used on primary cultured SMCs and revealed that Inka2 -/- SMC exhibits accelerated cell migration ability without a change in the cell proliferation rate. Inka2 -/- SMCs displayed Cofilin activation/phosphorylation, a downstream molecule of Pak4 signal cascade. These results suggest that Inka2 regulates SMC motility through modulating actin reorganization as the endogenous inhibitor of Pak4., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest or financial interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Branched actin networks have emerged as major force-generating structures driving the protrusions in various distinct cell types and processes, ranging from lamellipodia operating in mesenchymal and epithelial cell migration or tails pushing intracellular pathogens and vesicles to developing spine heads on neurons. Many key molecular features are conserved among all those Arp2/3 complex-containing, branched actin networks. Here, we will review recent progress in our molecular understanding of the core biochemical machinery driving branched actin nucleation, from the generation of filament primers to Arp2/3 activator recruitment, regulation and turnover. Due to the wealth of information on distinct, Arp2/3 network-containing structures, we are largely focusing-in an exemplary fashion-on canonical lamellipodia of mesenchymal cells, which are regulated by Rac GTPases, their downstream effector WAVE Regulatory Complex and its target Arp2/3 complex. Novel insight additionally confirms that WAVE and Arp2/3 complexes regulate or are themselves tuned by additional prominent actin regulatory factors, including Ena/VASP family members and heterodimeric capping protein. Finally, we are considering recent insights into effects exerted by mechanical force, both at the branched network and individual actin regulator level., Competing Interests: Conflict of interest statement Nothing declared, (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Osteogenic differentiation has been reportedly regulated by various mechanical stresses, including fluid shear stress and tensile and compressive loading. The promotion of osteoblastic differentiation by these mechanical stresses is accompanied by reorganization of the F-actin cytoskeleton, which is deeply involved in intracellular forces and the mechanical environment. However, there is limited information about the effect on the mechanical environment of the intracellular nucleus, such as the mechanical properties of the nucleus and intracellular forces exerted on the nucleus, which have recently been found to be directly involved in various cellular functions. Here, we investigated the changes in the intracellular force applied to the nucleus and the effect on nuclear morphology and mechanical properties during osteogenic differentiation in human osteoblast-like cells (Saos-2). We carried out cell morphological analyses with confocal fluorescence microscopy, nuclear indentation test with atomic force microscopy (AFM), and fluorescence recovery after photobleaching (FRAP) for intranuclear DNA. The results revealed that a significant reorganization of the F-actin cytoskeleton from the nuclear surfaces to the cell periphery occurred in the osteogenic differentiation processes, simultaneously with the reduction of compressive forces to the nucleus. Such changes also facilitated nuclear shrinkage and stiffening, and further intranuclear chromatin compaction. The results indicate that the reduction of the intracellular compressive force due to reorganization of the F-actin cytoskeleton affects the intra- and extra-mechanical environment of the nucleus, and this change may affect gene expression and DNA replication in the osteogenic differentiation process., 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 © 2022 Elsevier Ltd. All rights reserved.)

Cell migration is essential in animal development and co-opted during metastasis and inflammatory diseases. Some cells migrate collectively, which requires them to balance epithelial characteristics such as stable cell-cell adhesions with features of motility like rapid turnover of adhesions and dynamic cytoskeletal structures. How this is regulated is not entirely clear but important to understand. While investigating Drosophila oogenesis, we found that the putative E3 ubiquitin ligase, Mind bomb 2 (Mib2), is required to promote epithelial stability and the collective cell migration of border cells. Through biochemical analysis, we identified components of Mib2 complexes, which include E-cadherin and α- and β-catenins, as well as actin regulators. We also found that three Mib2 interacting proteins, RhoGAP19D, Supervillin, and Myosin heavy chain-like, affect border cell migration. mib2 mutant main body follicle cells have drastically reduced E-cadherin-based adhesion complexes and diminished actin filaments. We conclude that Mib2 acts to stabilize E-cadherin-based adhesion complexes and promote a robust actin cytoskeletal network, which is important for maintenance of epithelial integrity. The interaction with cadherin adhesion complexes and other cytoskeletal regulators contribute to its role in collective cell migration. Since Mib2 is well conserved, it may have similar functional significance in other organisms., Competing Interests: Declaration of competing interest No competing interests declared., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Mitochondria are highly dynamic organelles that constantly divide and fuse to maintain their proper structure and function. Cancer cells are often accompanied by an imbalance of mitochondrial fusion and fission, cancer progression is greatly affected by this imbalance. Here, we found that high-metastatic breast cancer MDA-MB-231 cells possess higher caveolin-1 (Cav-1) expression compared with low-metastatic breast cancer MCF-7 cells or normal breast epithelial MCF-10A cells. Downregulation of Cav-1 decreases the migratory and invasive abilities of MDA-MB-231 cells. Our results further demonstrated that downregulation of Cav-1 facilitated DRP1 and MFN2 to translocate to mitochondria, increasing the inhibitory phosphorylation level of DRP1 at Ser637 by protein kinase A (PKA), resulting in mitochondria elongation. We also showed that downregulation of Cav-1 significantly reduced the Rac1 activity by affecting intracellular reactive oxygen species (ROS) generation, which then inhibited F-actin formation. Based on these findings, we proposed that Cav-1 mediated mitochondrial fission-affected intracellular ROS generation and activated Rho GTPases, leading to F-actin-dependent formation of lamellipodia and promotion of breast cancer motility., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)