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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.)

Cell surface cortical actin is a regulatory target for viral infection. We aimed to investigate the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on host cell cortical stiffness, an indicator of cortical actin structure. The receptor-binding domain (RBD) of SARS-CoV-2 Spike (S) protein induced a reduction in cortical stiffness in ACE2-expressing cells. The interaction of RBD with ACE2 caused the inactivation of Ezrin/Radixin/Moesin (ERM) proteins. We further investigated the effects of the RBD of SARS-CoV-2 Omicron variants, BA.1 and BA.5. These RBDs influenced cortical stiffness depending on their affinity for ACE2. Our study provides the first evidence that the interaction of the SARS-CoV-2 S protein with ACE2 induces mechanobiological signals and attenuates the cortical actin., (© 2024. The Author(s) under exclusive licence to Japan Human Cell Society.)

The respiratory syncytial virus (RSV) matrix (M) protein plays an important role in infection as it can interact with viral components as well as the host cell actin microfilaments. The M-actin interaction may play a role in facilitating the transportation of virion components to the apical surface, where RSV is released. We show that M protein's association with actin is facilitated by palladin, an actin-binding protein. Cells were infected with RSV or transfected to express full-length M as a green fluorescent protein (GFP)-tagged protein, followed by removal of nuclear and cytosolic proteins to enrich for cytoskeleton and its associated proteins. M protein was present in inclusion bodies tethered to microfilaments in infected cells. In transfected cells, GFP-M was presented close to microfilaments, without association, suggesting the possible involvement of an additional protein in this interaction. As palladin can bind to proteins that also bind actin, we investigated its interaction with M. Cells were co-transfected to express GFP-M and palladin as an mCherry fluorescent-tagged protein, followed by cytoskeleton enrichment. M and palladin were observed to colocalize towards microfilaments, suggesting that palladin is involved in the M-actin interaction. In co-immunoprecipitation studies, M was found to associate with two isoforms of palladin, of 140 and 37 kDa. Interestingly, siRNA downregulation of palladin resulted in reduced titer of released RSV, while cell associated RSV titer increased, suggesting a role for palladin in virus release. Together, our data show that the M-actin interaction mediated by palladin is important for RSV budding and release.IMPORTANCERespiratory syncytial virus is responsible for severe lower respiratory tract infections in young children under 5 years old, the elderly, and the immunosuppressed. The interaction of the respiratory syncytial virus matrix protein with the host actin cytoskeleton is important in infection but has not been investigated in depth. In this study, we show that the respiratory syncytial virus matrix protein associates with actin microfilaments and the actin-binding protein palladin, suggesting a role for palladin in respiratory syncytial virus release. This study provides new insight into the role of the actin cytoskeleton in respiratory syncytial virus infection, a key host-RSV interaction in assembly. Understanding the mechanism by which the RSV M protein and actin interact will ultimately provide a basis for the development of therapeutics targeted at RSV infections., Competing Interests: The authors declare no conflict of interest.

Migration of endothelial and many other cells requires spatiotemporal regulation of protrusive and contractile cytoskeletal rearrangements that drive local cell shape changes. Unexpectedly, the small GTPase Rho, a crucial regulator of cell movement, has been reported to be active in both local cell protrusions and retractions, raising the question of how Rho activity can coordinate cell migration. Here, we show that Rho activity is absent in local protrusions and active during retractions. During retractions, Rho rapidly activated ezrin-radixin-moesin proteins (ERMs) to increase actin-membrane attachment, and, with a delay, nonmuscle myosin 2 (NM2). Rho activity was excitable, with NM2 acting as a slow negative feedback regulator. Strikingly, inhibition of SLK/LOK kinases, through which Rho activates ERMs, caused elongated cell morphologies, impaired Rho-induced cell contractions, and reverted Rho-induced blebbing. Together, our study demonstrates that Rho activity drives retractions by sequentially enhancing ERM-mediated actin-membrane attachment for force transmission and NM2-dependent contractility.

The morphology of dendritic spines, the postsynaptic compartment of most excitatory synapses, decisively modulates the function of neuronal circuits as also evident from human brain disorders associated with altered spine density or morphology. Actin filaments (F-actin) form the backbone of spines, and a number of actin-binding proteins (ABP) have been implicated in shaping the cytoskeleton in mature spines. Instead, only little is known about the mechanisms that control the reorganization from unbranched F-actin of immature spines to the complex, highly branched cytoskeleton of mature spines. Here, we demonstrate impaired spine maturation in hippocampal neurons upon genetic inactivation of cyclase-associated protein 1 (CAP1) and CAP2, but not of CAP1 or CAP2 alone. We found a similar spine maturation defect upon overactivation of inverted formin 2 (INF2), a nucleator of unbranched F-actin with hitherto unknown synaptic function. While INF2 overactivation failed in altering spine density or morphology in CAP-deficient neurons, INF2 inactivation largely rescued their spine defects. From our data we conclude that CAPs inhibit INF2 to induce spine maturation. Since we previously showed that CAPs promote cofilin1-mediated cytoskeletal remodeling in mature spines, we identified them as a molecular switch that control transition from filopodia-like to mature spines., (© 2024. The Author(s).)

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.)

Phagocytosis by macrophages is a highly polarized process to destroy large target cells. Binding to particles induces extensive cortical actin-generated forces that drive the formation of elaborate pseudopods around the target particle. Postinternalization, the resultant phagosome is driven toward the cell interior on microtubules (MTs) by cytoplasmic dynein. However, it is unclear whether dynein and cargo-adaptors contribute to the earlier steps of particle internalization and phagosome formation. Here we reveal that ninein, a MT minus-end-associated protein that localizes to the centrosome, is also present at the phagocytic cup in macrophages. Ninein depletion impairs particle internalization by delaying the early F-actin recruitment to sites of particle engagement and cup formation, with no impact on F-actin dynamics beyond this initial step. Ninein forms membrane-bound clusters on phagocytic cups that do not nucleate acentrosomal MTs but instead mediate the assembly of dynein-dynactin complex at active phagocytic membranes. Both ninein depletion and pharmacological inhibition of dynein activity reduced inward displacement of bound particles into macrophages. We found that ninein and dynein motor activity were required for timely retrograde movement of phagosomes and for phagolysosome formation. Taken together, these data show that ninein, alone and with dynein, play significant roles during phagocytosis.

The protein 4.1R is an essential component of the erythrocyte membrane skeleton, serving as a key structural element and contributing to the regulation of the membrane's physical properties, including mechanical stability and deformability, through its interaction with spectrin-actin. Recent research has uncovered additional roles of 4.1R beyond its function as a linker between the plasma membrane and the membrane skeleton. It has been found to play a crucial role in various biological processes, such as cell fate determination, cell cycle regulation, cell proliferation, and cell motility. Additionally, 4.1R has been implicated in cancer, with numerous studies demonstrating its potential as a diagnostic and prognostic biomarker for tumors. In this review, we provide an updated overview of the gene and protein structure of 4.1R, as well as its cellular functions in both physiological and pathological contexts.

The location of different actin-based structures is largely regulated by Rho GTPases through specific effectors. We use the apical aspect of epithelial cells as a model system to investigate how RhoA is locally regulated to contribute to two distinct adjacent actin-based structures. Assembly of the non-muscle myosin-2 filaments in the terminal web is dependent on RhoA activity, and assembly of the microvilli also requires active RhoA for phosphorylation and activation of ezrin. We show that the RhoGAP, ARHGAP18, is localized by binding active microvillar ezrin, and this interaction enhances ARHGAP18's RhoGAP activity. We present a model where ezrin-ARHGAP18 acts as a negative autoregulatory module to locally reduce RhoA activity in microvilli. Consistent with this model, loss of ARHGAP18 results in disruption of the distinction between microvilli and the terminal web including aberrant assembly of myosin-2 filaments forming inside microvilli. Thus, ARHGAP18, through its recruitment and activation by ezrin, fine-tunes the local level of RhoA to allow for the appropriate distribution of actin-based structures between the microvilli and terminal web. As RhoGAPs vastly outnumber Rho GTPases, this may represent a general mechanism whereby individual Rho effectors drive specific actin-based structures., Competing Interests: AL, CM, RZ, DM, AB No competing interests declared, (© 2024, Lombardo et al.)

Human enterocytes are primary targets of infection by invasive bacterium Salmonella Typhimurium, and studies using nonintestinal epithelial cells established that S. Typhimurium activates Rho family GTPases, primarily CDC42, to modulate the actin cytoskeletal network for invasion. The host intracellular protein network that engages CDC42 and influences the pathogen's invasive capacity are relatively unclear. Here, proteomic analyses of canonical and variant CDC42 interactomes identified a poorly characterized CDC42 interacting protein, CDC42EP1, whose intracellular localization is rapidly redistributed and aggregated around the invading bacteria. CDC42EP1 associates with SEPTIN-7 and Villin, and its relocalization and bacterial engagement depend on host CDC42 and S. Typhimurium's capability of activating CDC42. Unlike CDC42, CDC42EP1 is not required for S. Typhimurium's initial cellular entry but is found to associate with Salmonella-containing vacuoles after long-term infections, indicating a contribution to the pathogen's intracellular growth and replication. These results uncover a new host regulator of enteric Salmonella infections, which may be targeted to restrict bacterial load at the primary site of infection to prevent systemic spread., (© 2023 Wiley Periodicals LLC.)

Cyclase-associated protein (CAP) has emerged as a central player in cellular actin turnover, but its molecular mechanisms of action are not yet fully understood. Recent studies revealed that the N terminus of CAP interacts with the pointed ends of actin filaments to accelerate depolymerization in conjunction with cofilin. Here, we use in vitro microfluidics-assisted TIRF microscopy to show that the C terminus of CAP promotes depolymerization at the opposite (barbed) ends of actin filaments. In the absence of actin monomers, full-length mouse CAP1 and C-terminal halves of CAP1 (C-CAP1) and CAP2 (C-CAP2) accelerate barbed end depolymerization. Using mutagenesis and structural modeling, we show that these activities are mediated by the WH2 and CARP domains of CAP. In addition, we observe that CAP collaborates with profilin to accelerate barbed end depolymerization and that these effects depend on their direct interaction, providing the first known example of CAP-profilin collaborative effects in regulating actin. In the presence of actin monomers, CAP1 attenuates barbed end growth and promotes formin dissociation. Overall, these findings demonstrate that CAP uses distinct domains and mechanisms to interact with opposite ends of actin filaments and drive turnover. Further, they contribute to the emerging view of actin barbed ends as sites of dynamic molecular regulation, where numerous proteins compete and cooperate with each other to tune polymer dynamics, similar to the rich complexity seen at microtubule ends., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Kindlin-3 (K3) is critical for the activation of integrin adhesion receptors in hematopoietic cells. In humans and mice, K3 deficiency is associated with impaired immunity and bone development, bleeding, and aberrant erythrocyte shape. To delineate how K3 deficiency (K3KO) contributes to anemia and misshaped erythrocytes, mice deficient in erythroid (K3KO∖EpoR-cre) or myeloid cell K3 (K3KO∖Lyz2cre), knockin mice expressing mutant K3 (Q597W598 to AA) with reduced integrin-activation function (K3KI), and control wild-type (WT) K3 mice were studied. Both K3-deficient strains and K3KI mice showed anemia at baseline, reduced response to erythropoietin stimulation, and compromised recovery after phenylhydrazine (PHZ)-induced hemolytic anemia as compared with K3WT. Erythroid K3KO and K3 (Q597W598 to AA) showed arrested erythroid differentiation at proerythroblast stage, whereas macrophage K3KO showed decreased erythroblast numbers at all developmental stages of terminal erythroid differentiation because of reduced erythroblastic island (EBI) formation attributable to decreased expression and activation of erythroblast integrin α4β1 and macrophage αVβ3. Peripheral blood smears of K3KO∖EpoR-cre mice, but not of the other mouse strains, showed numerous aberrant tear drop-shaped erythrocytes. K3 deficiency in these erythrocytes led to disorganized actin cytoskeleton, reduced deformability, and increased osmotic fragility. Mechanistically, K3 directly interacted with F-actin through an actin-binding site K3-LK48. Taken together, these findings document that erythroid and macrophage K3 are critical contributors to erythropoiesis in an integrin-dependent manner, whereas F-actin binding to K3 maintains the membrane cytoskeletal integrity and erythrocyte biconcave shape. The dual function of K3 in erythrocytes and in EBIs establish an important functional role for K3 in normal erythroid function., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Palladin is an actin binding protein that is specifically upregulated in metastatic cancer cells but also colocalizes with actin stress fibers in normal cells and is critical for embryonic development as well as wound healing. Of nine isoforms present in humans, only the 90 kDa isoform of palladin, comprising three immunoglobulin (Ig) domains and one proline-rich region, is ubiquitously expressed. Previous work has established that the Ig3 domain of palladin is the minimal binding site for F-actin. In this work, we compare functions of the 90 kDa isoform of palladin to the isolated actin binding domain. To understand the mechanism of action for how palladin can influence actin assembly, we monitored F-actin binding and bundling as well as actin polymerization, depolymerization, and copolymerization. Together, these results demonstrate that there are key differences between the Ig3 domain and full-length palladin in actin binding stoichiometry, polymerization, and interactions with G-actin. Understanding the role of palladin in regulating the actin cytoskeleton may help us develop means to prevent cancer cells from reaching the metastatic stage of cancer progression., (© 2023 The Protein Society.)

The cytoskeletal proteins tubulin and actin are the obligate substrates of TCP-1 ring complex/Chaperonin containing TCP-1 (TRiC/CCT), and their folding involves co-chaperone. Through cryo-electron microscopy analysis, we present a more complete picture of TRiC-assisted tubulin/actin folding along TRiC adenosine triphosphatase cycle, under the coordination of co-chaperone plp2. In the open S1/S2 states, plp2 and tubulin/actin engaged within opposite TRiC chambers. Notably, we captured an unprecedented TRiC-plp2-tubulin complex in the closed S3 state, engaged with a folded full-length β -tubulin and loaded with a guanosine triphosphate, and a plp2 occupying opposite rings. Another closed S4 state revealed an actin in the intermediate folding state and a plp2. Accompanying TRiC ring closure, plp2 translocation could coordinate substrate translocation on the CCT6 hemisphere, facilitating substrate stabilization and folding. Our findings reveal the folding mechanism of the major cytoskeletal proteins tubulin/actin under the coordination of the biogenesis machinery TRiC and plp2 and extend our understanding of the links between cytoskeletal proteostasis and related human diseases.

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.)

Cytoskeletal proteins not only define the shape of cells, but also have critical roles in their proliferation, migration and motility, as well as in the establishment and maintenance of tissue organization and integrity. Furthermore, these proteins influence the physiological processes of the male reproductive system and are found in the structure of some cells. This study aimed to determine differences between the pre- and post-pubertal periods for the localization and distribution of actin, desmin, vimentin and cytokeratin-18 in the testes, epididymides and ductus deferentes of Persian and Turkish Angora and Van cats, using immunohistochemistry. The study material was grouped as belonging to the pre-pubertal and post-pubertal periods. The tissue samples of both groups were subjected to routine histological processing and embedded in paraffin. Serial sections cut from the paraffin-embedded tissue blocks were immunohistochemically stained with the indirect streptavidin-biotin complex method. Immunohistochemical findings demonstrated that there was no difference between the pre- and post-pubertal periods for the staining intensity and distribution of the proteins actin, vimentin, desmin and cytokeratin-18 in Persian and Turkish Angora and Van cats. On the other hand, differences were detected between the pre- and post-pubertal periods for the cellular expression and localization of these proteins in the testes, epididymides and ductus deferentes. Thus, the study results suggest that, based on the expression of actin, desmin, vimentin and cytokeratin-18 in the testes, epididymides and ductus deferentes during both periods, these molecular factors could have a contributory role in the development of the male reproductive system and the regulation of its physiological processes., Competing Interests: Declaration of competing interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The red blood cells (RBCs) of vertebrates have evolved into two basic shapes, with nucleated nonmammalian RBCs having a biconvex ellipsoidal shape and anuclear mammalian RBCs having a biconcave disk shape. In contrast, camelid RBCs are flat ellipsoids with reduced membrane deformability, suggesting altered membrane skeletal organization. However, the mechanisms responsible for their elliptocytic shape and reduced deformability have not been determined. We here showed that in alpaca RBCs, protein 4.1R, a major component of the membrane skeleton, contains an alternatively spliced exon 14-derived cassette (e14) not observed in the highly conserved 80 kDa 4.1R of other highly deformable biconcave mammalian RBCs. The inclusion of this exon, along with the preceding unordered proline- and glutamic acid-rich peptide (PE), results in a larger and unique 90 kDa camelid 4.1R. Human 4.1R containing e14 and PE, but not PE alone, showed markedly increased ability to form a spectrin-actin-4.1R ternary complex in viscosity assays. A similar facilitated ternary complex was formed by human 4.1R possessing a duplication of the spectrin-actin-binding domain, one of the mutations known to cause human hereditary elliptocytosis. The e14- and PE-containing mutant also exhibited an increased binding affinity to β-spectrin compared with WT 4.1R. Taken together, these findings indicate that 4.1R protein with the e14 cassette results in the formation and maintenance of a hyperstable membrane skeleton, resulting in rigid red ellipsoidal cells in camelid species, and suggest that membrane structure is evolutionarily regulated by alternative splicing of exons in the 4.1R gene., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

GNE myopathy is an inherited neuromuscular disorder caused by mutations in GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase) gene catalyzing the sialic acid biosynthesis pathway. The characteristic features include muscle weakness in upper and lower extremities, skeletal muscle wasting, and rimmed vacuole formation. More than 200 GNE mutations in either epimerase or kinase domain have been reported worldwide. In Indian subcontinent, several GNE mutations have been recently identified with unknown functional correlation. Alternate role of GNE in various cellular processes such as cell adhesion, migration, apoptosis, protein aggregation, and cytoskeletal organization have been proposed in recent studies. We aim to understand and compare the effect of various GNE mutations from Indian origin on regulation of the cytoskeletal network. In particular, F-actin dynamics was determined quantitatively by determining F/G-actin ratios in immunoblots for specific proteins. The extent of F-actin polymerization was visualized by immunostaining with Phalloidin using confocal microscopy. The proteins regulating F-actin dynamics such as RhoA, cofilin, Arp2, and alpha-actinin were studied in various GNE mutants. The altered level of cytoskeletal organization network proteins affected cell migration of GNE mutant proteins as measured by wound healing assay. The functional comparison of GNE mutations will help in better understanding of the genotypic severity of the disease in the Indian population. Our study offers a potential for identification of therapeutic molecules regulating actin dynamics in GNE specific mutations., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Due to the malignancy of oral squamous cell carcinoma (OSCC), investigations of novel therapeutic targets and prognostic biomarkers are urgently needed. In our present study, significant up-regulation of Actin-like protein 8 (ACTL8) in OSCC patients was observed by bioinformatics analysis with RNA sequencing data obtained from The Cancer Genome Atlas (TCGA) database. The results of Chi-square test revealed that there was a significant correlation between ACTL8 expression and tumor status (T1 + T2/T3+T4) (P = 0.004). Survival analysis indicated a negative correlation between ACTL8 overexpression and prognosis in OSCC (P = 3.984e-02). An ACTL8 knockdown experiment was conducted to evaluate the function of ACTL8 on OSCC cell biological behaviors. The results revealed that knockdown of ACTL8 significantly inhibited the growth and mobility, arrested cell cycle and promoted apoptosis of TCA-83 and CAL27 cells. Moreover, Gene Set Enrichment Analysis (GSEA) and western blots demonstrated that activation of cell cycle signaling pathway was inhibited by knockdown of ACTL8, as we observed the down-regulation of 4 key proteins (CDK1, cyclin E1, cyclin B2 and c-Myc) in this pathway. The present investigation indicates that ACTL8 plays an oncogenic role in the pathogenesis of OSCC, suggesting that ACTL8 may be a promising therapeutic target and prognosis marker for human OSCC., (Copyright © 2021. Published by Elsevier Ltd.)

The leiomodin1 (LMOD1) gene, encoding a potent actin nucleator, was recently reported as a potential pathogenic gene of megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS, OMIM 619362). However, only a single patient has been reported to have LMOD1 mutations, and the underlying pathogenic mechanism remains unknown. Here, we described a male infant with LMOD1 mutations presenting typical symptoms of pediatric intestinal pseudo-obstruction (PIPO) but without megacystis and microcolon. Two compound heterozygous missense variants (c.1106C>T, p.T369M; c.1262G>A, p.R421H) were identified, both affecting highly conserved amino acid residues within the second actin-binding site (ABS2) domain of LMOD1. Expression analysis showed that both variants resulted in significantly reduced protein amounts, especially for p.T369M, which was almost undetectable. The reduction was only partially rescued by the proteasome inhibitor MG-132, indicating that there might be proteasome-independent pathways involved in the degradation of the mutant proteins. Molecular modeling showed that variant p.T369M impaired the local protein conformation of the ABS2 domain, while variant p.R421H directly impaired the intermolecular interaction between ABS2 and actin. Accordingly, both variants significantly damaged LMOD1-mediated actin nucleation. These findings provide further human genetic evidence supporting LMOD1 as a pathogenic gene underlying visceral myopathy including PIPO and MMIHS, strengthen the critical role of ABS2 domain in LMOD1-mediated actin nucleation, and moreover, reveal an unrecognized role of ABS2 in protein stability., (© 2022 Federation of American Societies for Experimental Biology.)

During Escherichia coli cell division, an intracellular complex of cell division proteins known as the Z-ring assembles at midcell during early division and serves as the site of constriction. While the predominant protein in the Z-ring is the widely conserved tubulin homolog FtsZ, the actin homolog FtsA tethers the Z-ring scaffold to the cytoplasmic membrane by binding to FtsZ. While FtsZ is known to function as a dynamic, polymerized GTPase, the assembly state of its partner, FtsA, and the role of ATP are still unclear. We report that a substitution mutation in the FtsA ATP-binding site impairs ATP hydrolysis, phospholipid vesicle remodeling in vitro, and Z-ring assembly in vivo. We demonstrate by transmission electron microscopy and Förster Resonance Energy Transfer that a truncated FtsA variant, FtsA(ΔMTS) lacking a C-terminal membrane targeting sequence, self assembles into ATP-dependent filaments. These filaments coassemble with FtsZ polymers but are destabilized by unassembled FtsZ. These findings suggest a model wherein ATP binding drives FtsA polymerization and membrane remodeling at the lipid surface, and FtsA polymerization is coregulated with FtsZ polymerization. We conclude that the coordinated assembly of FtsZ and FtsA polymers may serve as a key checkpoint in division that triggers cell wall synthesis and division progression., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Due to its extremely high prevalence and severity, non-alcoholic fatty liver disease (NALFD) is a serious health and economic concern worldwide. Developing effective methods of diagnosis and therapy demands a deeper understanding of its molecular basis. One of the strategies in such an endeavor is the analysis of alterations in the morphology of liver cells. Such alterations, widely reported in NAFLD patients and disease models, are related to the cytoskeleton. Therefore, the fate of the cytoskeleton components is useful to uncover the molecular basis of NAFLD, to further design innovative approaches for its diagnosis and therapy., Main Findings: Several cytoskeleton proteins are up-regulated in liver cells of NAFLD patients. Under pathological conditions, keratin 18 is released from hepatocytes and its detection in the blood emerges as a non-invasive diagnosis tool. α-Smooth muscle actin is up-regulated in hepatic stellate cells and its down-regulation has been widely tested as a potential NALFD therapeutic approach. Other cytoskeleton proteins, such as vimentin, are also up-regulated., Conclusions: NAFLD progression involves alterations in expression levels of proteins that build the liver cytoskeleton or associate with it. These findings provide a timely opportunity of developing novel approaches for NAFLD diagnosis and therapy., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cytoskeletal proteins are classified as a group that is defined functionally, whose members are capable of polymerizing into higher order structures, either dynamically or statically, to perform structural roles during a variety of cellular processes. In eukaryotes, the most well-studied cytoskeletal proteins are actin, tubulin, and intermediate filaments, and are essential for cell shape and movement, chromosome segregation, and intracellular cargo transport. Prokaryotes often harbor homologs of these proteins, but in bacterial cells, these homologs are usually not employed in roles that can be strictly defined as 'cytoskeletal'. However, several bacteria encode other proteins capable of polymerizing which, although they do not appear to have a eukaryotic counterpart, nonetheless appear to perform a more traditional 'cytoskeletal' function. In this review, we discuss recent reports that cover the structures and functions of prokaryotic proteins that are broadly termed as cytoskeletal, either by sequence homology or by function, to highlight how the enzymatic properties of traditionally studied cytoskeletal proteins may be used for other types of cellular functions; and to demonstrate how truly 'cytoskeletal' functions may be performed by uniquely bacterial proteins that do not display homology to eukaryotic proteins., (© 2022 IOP Publishing Ltd.)

Focal polarization is necessary for finely arranged cell-cell interactions. The yeast mating projection, with its punctate polarisome, is a good model system for this process. We explored the critical role of the polarisome scaffold protein Spa2 during yeast mating with a hypothesis motivated by mathematical modeling and tested by in vivo experiments. Our simulations predicted that two positive feedback loops generate focal polarization, including a novel feedback pathway involving the N-terminal domain of Spa2. We characterized the latter using loss-of-function and gain-of-function mutants. The N-terminal region contains a Spa2 Homology Domain (SHD) which is conserved from yeast to humans, and when mutated largely reproduced the spa2Δ phenotype. Our work together with published data show that the SHD domain recruits Msb3/4 that stimulates Sec4-mediated transport of Bud6 to the polarisome. There, Bud6 activates Bni1-catalyzed actin cable formation, recruiting more Spa2 and completing the positive feedback loop. We demonstrate that disrupting this loop at any point results in morphological defects. Gain-of-function perturbations partially restored focal polarization in a spa2 loss-of-function mutant without restoring localization of upstream components, thus supporting the pathway order. Thus, we have collected data consistent with a novel positive feedback loop that contributes to focal polarization during pheromone-induced polarization in yeast., Competing Interests: The authors have declared that no competing interests exist.

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.)

The brain capillary endothelium is highly regulatory, maintaining the chemical stability of the brain's microenvironment. The role of cytoskeletal proteins in tethering nanotubules (TENTs) during barrier-genesis was investigated using the established immortalized mouse brain endothelial cell line (bEnd5) as an in vitro blood-brain barrier (BBB) model. The morphology of bEnd5 cells was evaluated using both high-resolution scanning electron microscopy and immunofluorescence to evaluate treatment with depolymerizing agents Cytochalasin D for F-actin filaments and Nocodazole for α-tubulin microtubules. The effects of the depolymerizing agents were investigated on bEnd5 monolayer permeability by measuring the transendothelial electrical resistance (TEER). The data endorsed that during barrier-genesis, F-actin and α-tubulin play a cytoarchitectural role in providing both cell shape dynamics and cytoskeletal structure to TENTs forming across the paracellular space to provide cell-cell engagement. Western blot analysis of the treatments suggested a reduced expression of both proteins, coinciding with a reduction in the rates of cellular proliferation and decreased TEER. The findings endorsed that TENTs provide alignment of the paracellular (PC) spaces and tight junction (TJ) zones to occlude bEnd5 PC spaces. The identification of specific cytoskeletal structures in TENTs endorsed the postulate of their indispensable role in barrier-genesis and the maintenance of regulatory permeability across the BBB.

Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells., Competing Interests: The authors have declared that no competing interests exist.

Mechanical stiffness is closely related to cell adhesion and rounding in some cells. In leukocytes, dephosphorylation of ezrin/radixin/moesin (ERM) proteins is linked to cell adhesion events. To elucidate the relationship between surface stiffness, cell adhesion, and ERM dephosphorylation in leukocytes, we examined the relationship in the myelogenous leukemia line, KG-1, by treatment with modulation drugs. KG-1 cells have ring-shaped cortical actin with microvilli as the only F-actin cytoskeleton, and the actin structure constructs the mechanical stiffness of the cells. Phorbol 12-myristate 13-acetate and staurosporine, which induced cell adhesion to fibronectin surface and ERM dephosphorylation, caused a decrease in surface stiffness in KG-1 cells. Calyculin A, which inhibited ERM dephosphorylation and had no effect on cell adhesion, did not affect surface stiffness. To clarify whether decreasing cell surface stiffness and inducing cell adhesion are equivalent, we examined KG-1 cell adhesion by treatment with actin-attenuated cell softening reagents. Cytochalasin D clearly diminished cell adhesion, and high concentrations of Y27632 slightly induced cell adhesion. Only Y27632 slightly decreased ERM phosphorylation in KG-1 cells. Thus, decreasing cell surface stiffness and inducing cell adhesion are not equivalent, but these phenomena are coordinately regulated by ERM dephosphorylation in KG-1 cells., (© 2021. Japan Human Cell Society.)

Ethnopharmacological Relevance: Huangqi Guizhi Wuwu Decoction(HQGZWWD) is a Traditional Chinese Medicine formula from Synopsis of Golden Chamber used to treat blood arthralgia. According to the principle that the same treatment can be used for different diseases, HQGZWWD has proven effective for IgA nephropathy (IgAN) associated with spleen and kidney yang deficiency., Aim of the Study: In this study, we investigated the mechanism by which HQGZWWD alleviates proteinuria and protects renal function in rats with IgAN by regulating the AT1R/Nephrin/c-Abl pathway., Methods: Rats were randomly divided into six groups: control, IgAN model, IgAN model treated with low-dose HQGZWWD, IgAN model treated with medium-dose HQGZWWD, IgAN model treated with high-dose HQGZWWD, and IgAN model treated with valsartan. IgAN was induced using bovine γ-globulin. We evaluated the mediating effects of HQGZWWD on podocyte cytoskeletal proteins, the AT1R/Nephrin/c-Abl pathway, upstream tumor necrosis factor-α (TNF-α), and TNF-α receptor-1 (TNFR1)., Results: The IgAN rats displayed proteinuria, IgA deposition in the mesangial region, and podocyte cytoskeletal protein damage. The expression of TNF-α, TNFR1, AT1R, and c-Abl was increased in the IgAN rat kidney, whereas the expression of nephrin, podocin, ACTN4, and phosphorylated nephrin (p-nephrin) was reduced. HQGZWWD treatment significantly alleviated podocyte cytoskeletal protein damage in the IgAN rats, upregulated the expression of nephrin, podocin, and ACTN4, and the colocalized expression of F-actin and nephrin. This study demonstrates that HQGZWWD attenuates podocyte cytoskeletal protein damage by regulating the AT1R-nephrin- c-Abl pathway, upregulating the expression of p-nephrin, and downregulating the expression of AT1R and c-Abl., Conclusions: These results indicate that HQGZWWD attenuates podocyte cytoskeletal protein damage in IgAN rats by regulating the AT1R/Nephrin/c-Abl pathway, providing a potential therapeutic approach for IgAN., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Mechanical forces are integral to many cellular processes, including clathrin-mediated endocytosis, a principal membrane trafficking route into the cell. During endocytosis, forces provided by endocytic proteins and the polymerizing actin cytoskeleton reshape the plasma membrane into a vesicle. Assessing force requirements of endocytic membrane remodeling is essential for understanding endocytosis. Here, we determined actin-generated force applied during endocytosis using FRET-based tension sensors inserted into the major force-transmitting protein Sla2 in yeast. We measured at least 8 pN force transmitted over Sla2 molecule, hence possibly more than 300-880 pN applied during endocytic vesicle formation. Importantly, decreasing cell turgor pressure and plasma membrane tension reduced force transmitted over the Sla2. The measurements in hypotonic conditions and mutants lacking BAR-domain membrane scaffolds then showed the limits of the endocytic force-transmitting machinery. Our study provides force values and force profiles critical for understanding the mechanics of endocytosis and potentially other key cellular membrane-remodeling processes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: Scleral remodeling plays a key role in axial elongation in myopia. The aim of the present study was to identify the proteomics changes and specific signaling networks to gain insight into the molecular basis of scleral remodeling in myopic eyes., Methods: Guinea pig form-deprivation myopia was induced with a translucent diffuser on a random eye for 4 weeks, while the other eye served as the contralateral control group. The axial length and refraction were measured at the beginning and end of the treatment. The proteins were extracted from the sclerae of each group and prepared for quantitative isobaric tags for relative and absolute quantification (iTRAQ) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The coexpression networks and protein functions were analyzed using Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA). Quantitative real-time PCR (qRT-PCR) and western blotting were performed to confirm the authenticity and accuracy of the iTRAQ results., Results: After 4 weeks, the form-deprivation eyes developed significant degrees of myopia, and the axial length increased statistically significantly (p<0.05). A total of 2,579 unique proteins with <1% false discovery rate (FDR) were identified. Furthermore, 56 proteins were found to be upregulated, and 84 proteins were found to be downregulated, with a threshold of a 1.2-fold change and p<0.05 in the myopia group, when compared to the control group. Further bioinformatics analysis indicated that 44 of 140 differentially expressed proteins were involved in cellular movement and cellular assembly and organization. The qRT-PCR or western blotting results confirmed that myosin IIB, ACTIN3, and cellular cytoskeletons were downregulated, while RhoA and RAP1A were upregulated in the sclera in myopic eyes. These results were consistent with the proteomics results., Conclusions: Proteomics and bioinformatics results can be helpful for identifying proteins and providing new insights for better understanding of the molecular mechanism underlying scleral remodeling. These results revealed that the proteins associated with cellular movement and cellular assembly and organization are altered during the development of myopia. Furthermore, RhoA plays a key role in the pathways involved in cellular movement and cellular assembly and organization., (Copyright © 2021 Molecular Vision.)

The actin cytoskeleton is a key regulator of mechanical processes in cells. The family of LIM domain proteins have recently emerged as important mechanoresponsive cytoskeletal elements capable of sensing strain in the actin cytoskeleton. The mechanisms regulating this mechanosensitive behavior, however, remain poorly understood. Here we show that the LIM domain protein testin is peculiar in that despite the full-length protein primarily appearing diffuse in the cytoplasm, the C-terminal LIM domains alone recognize focal adhesions and strained actin, while the N-terminal domains alone recognize stress fibers. Phosphorylation mutations in the dimerization regions of testin, however, reveal its mechanosensitivity and cause it to relocate to focal adhesions and sites of strain in the actin cytoskeleton. Finally, we demonstrate that activated RhoA causes testin to adorn stress fibers and become mechanosensitive. Together, our data show that testin's mechanoresponse is regulated in cells and provide new insights into LIM domain protein recognition of the actin cytoskeleton's mechanical state.

Multimeric cytoskeletal protein complexes orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed, differential detergent fractionation to simultaneously detect protein complexes in hundreds of individual cells. Fractionation occurs by 60 s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Proteins are measured with a ~5-hour immunoassay. Co-detection of cytoskeletal protein complexes in U2OS cells treated with filamentous actin (F-actin) destabilizing Latrunculin A detects a unique subpopulation (~2%) exhibiting downregulated F-actin, but upregulated microtubules. Thus, some cells may upregulate other cytoskeletal complexes to counteract the stress of Latrunculin A treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin. We find heat shock may dysregulate filamentous and globular actin correlation. In this work, our assay overcomes selectivity limitations to biochemically quantify single-cell protein complexes perturbed with diverse stimuli., (© 2021. The Author(s).)

Divisions of the genetic material and cytoplasm are coordinated spatially and temporally to ensure genome integrity. This coordination is mediated in part by the major cell cycle regulator cyclin-dependent kinase (Cdk1). Cdk1 activity peaks during mitosis, but during mitotic exit/cytokinesis Cdk1 activity is reduced, and phosphorylation of its substrates is reversed by various phosphatases including Cdc14, PP1, PP2A, and PP2B. Cdk1 is known to phosphorylate several components of the actin- and myosin-based cytokinetic ring (CR) that mediates division of yeast and animal cells. Here we show that Cdk1 also phosphorylates the Schizosaccharomyces pombe CR component paxillin Pxl1. We determined that both the Cdc14 phosphatase Clp1 and the PP1 phosphatase Dis2 contribute to Pxl1 dephosphorylation at mitotic exit, but PP2B/calcineurin does not. Preventing Pxl1 phosphorylation by Cdk1 results in increased Pxl1 levels, precocious Pxl1 recruitment to the division site, and increased duration of CR constriction. In vitro Cdk1-mediated phosphorylation of Pxl1 inhibits its interaction with the F-BAR domain of the cytokinetic scaffold Cdc15, thereby disrupting a major mechanism of Pxl1 recruitment. Thus, Pxl1 is a novel substrate through which S. pombe Cdk1 and opposing phosphatases coordinate mitosis and cytokinesis.

This review extensively reports data from the literature concerning the complex relationships between the stress-induced c-Jun N-terminal kinases (JNKs) and the four main cytoskeleton elements, which are actin filaments, microtubules, intermediate filaments, and septins. To a lesser extent, we also focused on the two membrane-associated cytoskeletons spectrin and ESCRT-III. We gather the mechanisms controlling cytoskeleton-associated JNK activation and the known cytoskeleton-related substrates directly phosphorylated by JNK. We also point out specific locations of the JNK upstream regulators at cytoskeletal components. We finally compile available techniques and tools that could allow a better characterization of the interplay between the different types of cytoskeleton filaments upon JNK-mediated stress and during development. This overview may bring new important information for applied medical research.

The mechanism that mediates the interaction between the contractile ring and the plasma membrane during cytokinesis remains elusive. We previously found that ERM (Ezrin/Radixin/Moesin) proteins, which usually mediate cellular pole contraction, become over-accumulated at the cell equator and support furrow ingression upon the loss of other actin-membrane associated proteins, anillin and supervillin. In this study, we addressed the molecular basis of the exchangeability between ezrin and other actin-membrane associated proteins in mediating cortical contraction during cytokinesis. We found that depletion of anillin and supervillin caused over-accumulation of the membrane-associated FERM domain and actin-binding C-terminal domain (C-term) of ezrin at the cleavage furrow, respectively. This finding suggests that ezrin differentially shares its binding sites with these proteins on the actin cytoskeleton or inner membrane surface. Using chimeric mutants, we found that ezrin C-term, but not the FERM domain, can substitute for the corresponding anillin domains in cytokinesis and cell proliferation. On the other hand, either the membrane-associated or the actin/myosin-binding domains of anillin could not substitute for the corresponding ezrin domains in controlling cortical blebbing at the cell poles. Our results highlight specific designs of actin- or membrane-associated moieties of different actin-membrane associated proteins with limited exchangeability, which enables them to support diverse cortical activities on the shared actin-membrane interface during cytokinesis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Vascular smooth muscle cells (VSMC) switch to macrophage-like cells after cholesterol loading, and this change may play an important role in atherogenesis. Muscleblind-like splicing regulator 1 (MBNL1) is a well-known splicing factor that has been implicated in many cellular processes. However, the role of MBNL1 in VSMC macrophage-like transdifferentiation is largely unknown. In this study, we aim to characterize the role of MBNL1-induced gene splicing during atherogenesis., Methods: The expression of MBNL1 and Abelson interactor 1 (Abi1) splice variants (Abi1-e10 and Abi1-Δe10) was compared between artery tissues from healthy donors and atherosclerosis patients. Regulatory mechanisms of MBNL1-induced Abi1 gene splicing were studied, and the signal pathways mediated by Abi1 splice variants were investigated in VSMC., Results: Loss of MBNL1 was found in the macrophage-like VSMC (VSMC-M) in artery wall from atherosclerosis patients. In vitro and in vivo evidence confirmed that Abi1 is one of the MBNL1 target genes. Loss of MBNL1 significantly induces the Abi1-Δe10 isoform expression. Compared to the known actin organization activities of the Abi1 gene, we discovered a novel action of Abi1-Δe10, whereby Abi1-Δe10 activates Rac1 independent of upstream stimulation and triggers the Rac1-NOX1-ROS pathway, which results in increased expression of transcription factor Kruppel-like factor 4 (KLF4). While Abi1-Δe10 inhibits contractile VSMC biomarkers expression and cell contraction, it stimulates VSMC proliferation, migration and macrophage-like transdifferentiation., Conclusion: Loss-of-function of MBNL1 activates VSMC-M transdifferentiation to promote atherogenesis through regulating Abi1 RNA splicing., (© 2021 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Lung cancer is one of the most intractable diseases with high incidence and mortality worldwide. Adenylate cyclase-associated protein 1 (CAP1), a well-known actin depolymerization factor, is recently reported to be an oncogene accelerating cancer cell proliferation. However, the physiological significance of CAP1 in lung cancer is incompletely understood and the novel functions of CAP1 in transcriptional regulation remain unknown. Here we found that CAP1 was highly expressed in lung cancer tissues and cells, which was also negatively associated with prognosis in lung cancer patients. Moreover, CAP1 promoted A549 cells proliferation by promoting protein synthesis to accelerate cell cycle progression. Mechanistically, we revealed that CAP1 facilitated cyclin-dependent kinase 9 (CDK9)-mediated RNA polymerases (Pol) II-Ser2 phosphorylation and subsequent transcription elongation, and CAP1 performed its function in this progress depending on its actin-depolymerization activity in nucleoplasm. Furthermore, our in vivo findings confirmed that CAP1-promoted A549 xenograft tumor growth was associated with CDK9-mediated Pol II-Ser2 phosphorylation. Our study elucidates a novel role of CAP1 in modulating transcription by promoting polymerase II phosphorylation and suggests that CAP1 is a newly identified biomarker for lung cancer treatment and prognosis prediction.

The generation of F-actin bundles is controlled by the action of actin-binding proteins. In Drosophila bristle development, two major actin-bundling proteins-Forked and Fascin-were identified, but still the molecular mechanism by which these actin-bundling proteins and other proteins generate bristle actin bundles is unknown. In this study, we developed a technique that allows recapitulation of bristle actin module organization using the Drosophila ovary by a combination of confocal microscopy, super-resolution structured illumination microscopy, and correlative light and electron microscope analysis. Since Forked generated a distinct ectopic network of actin bundles in the oocyte, the additive effect of two other actin-associated proteins, namely, Fascin and Javelin (Jv), was studied. We found that co-expression of Fascin and Forked demonstrated that the number of actin filaments within the actin bundles dramatically increased, and in their geometric organization, they resembled bristle-like actin bundles. On the other hand, co-expression of Jv with Forked increased the length and density of the actin bundles. When all three proteins co-expressed, the actin bundles were longer and denser, and contained a high number of actin filaments in the bundle. Thus, our results demonstrate that the Drosophila oocyte could serve as a test tube for actin bundle analysis.

Kindlin3 (K3), a FERM domain containing protein expressed in hematopoietic cells controls integrin activation and thus hemostatic and inflammatory responses. However, its role in the mechanics of plasma membrane remains unclear. Here, we show that genetic knockout of K3 in microglia and macrophages resulted in defective plasma membrane tension and membrane blebbing. Atomic force microscopy (AFM) of K3-deficient cells revealed a significant loss in membrane-to-cortex attachment (MCA), and consequently reduced membrane tension. This loss in MCA is amplified by the mislocalization of the cell cortex proteins-ezrin, radixin, and moesin (ERM)-to the plasma membrane of microglia and macrophages. Re-expression of K3 in K3-deficient macrophages rescued the defects and localization of ERMs implying a key role for K3 in MCA. Analysis of two K3 mutants, K3 int affecting integrin binding and activation, and K3 pxn/act disrupting binding to paxillin and actin but not integrin functions, demonstrated that the role of K3 in membrane mechanics is separate from integrin activation. The K3 pxn/act mutant substantially diminished both membrane tension and Yes-associated protein (YAP) translocation to the nucleus, while preserving integrin activation, cell spreading, and migration. Together, our results show that K3 coordinates membrane mechanics, ERM protein recruitment to the membrane, and YAP translocation by linking integrin at the membrane to paxillin and actin of the cytoskeleton. This novel function of K3 is distinct from its role in integrin activation.

Despite the well-established role of actin polymerization as a driving mechanism for cell protrusion, upregulated actin polymerization alone does not initiate protrusions. Using a combination of theoretical modeling and quantitative live-cell imaging experiments, we show that local depletion of actin-membrane links is needed for protrusion initiation. Specifically, we show that the actin-membrane linker ezrin is depleted prior to protrusion onset and that perturbation of ezrin's affinity for actin modulates protrusion frequency and efficiency. We also show how actin-membrane release works in concert with actin polymerization, leading to a comprehensive model for actin-driven shape changes. Actin-membrane release plays a similar role in protrusions driven by intracellular pressure. Thus, our findings suggest that protrusion initiation might be governed by a universal regulatory mechanism, whereas the mechanism of force generation determines the shape and expansion properties of the protrusion., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In the eukaryotic cell nucleus, cytoskeletal proteins are emerging as essential players in nuclear function. In particular, actin regulates chromatin as part of ATP-dependent chromatin remodeling complexes, it modulates transcription and it is incorporated into nascent ribonucleoprotein complexes, accompanying them from the site of transcription to polyribosomes. The nuclear actin pool is undistinguishable from the cytoplasmic one in terms of its ability to undergo polymerization and it has also been implicated in the dynamics of chromatin, regulating heterochromatin segregation at the nuclear lamina and maintaining heterochromatin levels in the nuclear interiors. One of the next frontiers is, therefore, to determine a possible involvement of nuclear actin in the functional architecture of the cell nucleus by regulating the hierarchical organization of chromatin and, thus, genome organization. Here, we discuss the repertoire of these potential actin functions and how they are likely to play a role in the context of cellular differentiation.

Purpose: To determine the temporal effects of dexamethasone (DEX) and glucocorticoid-induced matrix (GIM) on integrins/integrin adhesomes, caveolins, cytoskeletal-related proteins, and stiffness in human trabecular meshwork (hTM) cells., Methods: Primary hTM cells were plated on plastic dishes (TCP), treated with vehicle (Veh) or 100 nM DEX in 1% serum media for 1, 3, 5, and 7 day(s). Concurrently, hTM cells were also plated on vehicle control matrices (VehMs) and GIMs for similar time points; VehMs and GIMs had been generated from chronic cultures of Veh-/DEX-stimulated hTM cells and characterized biochemically. Subsets of cells prior to plating on TCP or VehMs / GIMs served as baseline. Protein expression of mechanoreceptors, cytoskeletal-related proteins, and elastic moduli of hTM cells were determined., Results: Compared with Veh, DEX temporally overexpressed αV, β3, and β5 integrins from day 3 to day 7, and integrin linked kinase at day 7, in hTM cells. However, DEX decreased β1 integrin at day 1 and day 7, while increasing Cavin1 at day 7, in a time-independent manner. Further, DEX temporally upregulated α-smooth muscle actin(α-SMA) and RhoA at day 7 and day 5, respectively; while temporally downregulating Cdc42 at day 3 and day 7 in hTM cells. Conversely, GIM showed increased immunostaining of fibronectin extra-domain A and B isoforms. Compared with VehM, GIM temporally increased αV integrin, Cavin1, and RhoA from day 3 to day 7, at day 3 and day 7, and at day 5, respectively, in hTM cells. Further, GIM overexpressed α-SMA at day 3 and day 7, and stiffened hTM cells from day 1 to day 7, in a time-independent fashion., Conclusions: Our data highlight crucial mechanoreceptors, integrin adhesomes, and actin-related proteins that may temporally sustain fibrotic phenotypes precipitated by DEX and/or GIM in hTM cells.

Contractile myofiber units are mainly composed of thick myosin and thin actin (F-actin) filaments. F-Actin interacts with Microtubule Associated Monooxygenase, Calponin And LIM Domain Containing 2 (MICAL2). Indeed, MICAL2 modifies actin subunits and promotes actin filament turnover by severing them and preventing repolymerization. In this study, we found that MICAL2 increases during myogenic differentiation of adult and pluripotent stem cells (PSCs) towards skeletal, smooth and cardiac muscle cells and localizes in the nucleus of acute and chronic regenerating muscle fibers. In vivo delivery of Cas9-Mical2 guide RNA complexes results in muscle actin defects and demonstrates that MICAL2 is essential for skeletal muscle homeostasis and functionality. Conversely, MICAL2 upregulation shows a positive impact on skeletal and cardiac muscle commitments. Taken together these data demonstrate that modulations of MICAL2 have an impact on muscle filament dynamics and its fine-tuned balance is essential for the regeneration of muscle tissues.

ERM proteins are conserved regulators of cortical membrane specialization that function as membrane-actin linkers and molecular hubs. The activity of ERM proteins requires a conformational switch from an inactive cytoplasmic form into an active membrane- and actin-bound form, which is thought to be mediated by sequential PIP 2 binding and phosphorylation of a conserved C-terminal threonine residue. Here, we use the single Caenorhabditis elegans ERM ortholog, ERM-1, to study the contribution of these regulatory events to ERM activity and tissue formation in vivo Using CRISPR/Cas9-generated erm-1 mutant alleles, we demonstrate that a PIP 2 -binding site is crucially required for ERM-1 function. By contrast, dynamic regulation of C-terminal T544 phosphorylation is not essential but modulates ERM-1 apical localization and dynamics in a tissue-specific manner, to control cortical actin organization and support lumen formation in epithelial tubes. Our work highlights the dynamic nature of ERM protein regulation during tissue morphogenesis and the importance of C-terminal phosphorylation in fine-tuning ERM activity in a tissue-specific context., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Cilia and flagella are microtubule-based cellular projections with important sensory and motility functions. Their absence or malfunction is associated with a growing number of human diseases collectively referred to as ciliopathies. However, the fundamental mechanisms underpinning cilia biogenesis and functions remain only partly understood. Here, we show that depleting LUZP1 or its interacting protein, EPLIN, increases the levels of MyosinVa at the centrosome and primary cilia formation. We further show that LUZP1 localizes to both actin filaments and the centrosome/basal body. Like EPLIN, LUZP1 is an actin-stabilizing protein that regulates actin dynamics, at least in part, by mobilizing ARP2 to the centrosomes. Both LUZP1 and EPLIN interact with known ciliogenesis and cilia-length regulators and as such represent novel players in actin-dependent centrosome to basal body conversion. Ciliogenesis deregulation caused by LUZP1 or EPLIN loss may thus contribute to the pathology of their associated disease states., (© 2020 Gonçalves et al.)

Ezrin, a membrane-cytoskeleton linker protein, plays an essential role in cell polarity establishment, cell migration, and division. Recent studies show that ezrin phosphorylation regulates breast cancer metastasis by promoting cancer cell survivor and promotes intrahepatic metastasis via cell migration. However, it was less characterized whether there are additional post-translational modifications and/or post-translational crosstalks on ezrin underlying context-dependent breast cancer cell migration and invasion. Here we show that ezrin is acetylated by p300/CBP-associated factor (PCAF) in breast cancer cells in response to CCL18 stimulation. Ezrin physically interacts with PCAF and is a cognate substrate of PCAF. The acetylation site of ezrin was mapped by mass spectrometric analyses, and dynamic acetylation of ezrin is essential for CCL18-induced breast cancer cell migration and invasion. Mechanistically, the acetylation reduced the lipid-binding activity of ezrin to ensure a robust and dynamic cycling between the plasma membrane and cytosol in response to CCL18 stimulation. Biochemical analyses show that ezrin acetylation prevents the phosphorylation of Thr567. Using atomic force microscopic measurements, our study revealed that acetylation of ezrin induced its unfolding into a dominant structure, which prevents ezrin phosphorylation at Thr567. Thus, these results present a previously undefined mechanism by which CCL18-elicited crosstalks between the acetylation and phosphorylation on ezrin control breast cancer cell migration and invasion. This suggests that targeting PCAF signaling could be a potential therapeutic strategy for combating hyperactive ezrin-driven cancer progression., (© The Author(s) (2019). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)

Smooth muscle cell migration is essential for many diverse biological processes such as pulmonary/cardiovascular development and homeostasis. Abi1 (Abelson interactor 1) is an adapter protein that has been implicated in nonmuscle cell migration. However, the role and mechanism of Abi1 in smooth muscle migration are largely unknown. Here, Abi1 knockdown by shRNA reduced human airway smooth muscle cell migration, which was restored by Abi1 rescue. Abi1 localized at the tip of lamellipodia and its protrusion coordinated with F-actin at the leading cell edge of live cells. In addition, we identified profilin-1 (Pfn-1), a G-actin transporter, as a new partner for Abi1. Abi1 knockdown reduced the recruitment of Pfn-1 to the leading cell edge. Moreover, Abi1 knockdown reduced the localization of the actin-regulatory proteins c-Abl (Abelson tyrosine kinase) and N-WASP (neuronal Wiskott-Aldrich Syndrome Protein) at the cell edge without affecting other migration-related proteins including pVASP (phosphorylated vasodilator stimulated phosphoprotein), cortactin and vinculin. Furthermore, we found that c-Abl and integrin β1 regulated the positioning of Abi1 at the leading edge. Taken together, the results suggest that Abi1 regulates cell migration by affecting Pfn-1 and N-WASP, but not pVASP, cortactin and focal adhesions. Integrin β1 and c-Abl are important for the recruitment of Abi1 to the leading edge.

Endometrial cancer (EC) is generally considered as a disease that affects older women. We attempt to explore the role of actin‑like protein 8 (ACTL8) in EC and how it achieves its function. Based on the data from The Cancer Genome Atlas (TCGA), we found that ACTL8 expression was up-regulated in EC tissues and correlated with shorter overall survival of EC patients. ACTL8 expression was significantly associated with age, clinical-stage, or grade. Cox proportional hazards model analysis revealed that ACTL8 expression, grade, and clinical-stage were promising independent prognostic factors of EC. Knockdown of ACTL8 repressed the proliferative, migrating and invading capabilities of human EC cell lines KLE and Ishikawa. Silencing ACTL8 up-regulated the negative cell cycle regulator p21 and epithelial marker E-cadherin, and down-regulated the positive cell cycle regulator Cyclin A, mesenchymal markers MMP-9 and N-cadherin in KLE cells. Collectively, these outcomes illustrated that ACTL8 might act as a tumor facilitator during EC progression.

Mitochondria adapt to cellular needs by changes in morphology through fusion and fission events, referred to as mitochondrial dynamics. Mitochondrial function and morphology are intimately connected and the dysregulation of mitochondrial dynamics is linked to several human diseases. In this work, we investigated the role of mitochondrial dynamics in wound healing in the Drosophila embryonic epidermis. Mutants for mitochondrial fusion and fission proteins fail to close their wounds, indicating that the regulation of mitochondrial dynamics is required for wound healing. By live-imaging, we found that loss of function of the mitochondrial fission protein Dynamin-related protein 1 (Drp1) compromises the increase of cytosolic and mitochondrial calcium upon wounding and leads to reduced reactive oxygen species (ROS) production and F-actin defects at the wound edge, culminating in wound healing impairment. Our results highlight a new role for mitochondrial dynamics in the regulation of calcium, ROS and F-actin during epithelial repair., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)