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Ototoxicity is a common side effect of cisplatin cancer treatment, potentially leading to hearing loss. This study demonstrated the significant protective activity of Acanthopanax sessiliflorus ( A. sessiliflorus ) leaves against cisplatin-induced ototoxicity (CIO), investigated the active compounds, and elucidated their mechanisms in countering CIO. UPLC-Q/TOF-MS analysis identified 79 compounds. Network pharmacology and activity screening determined that chiisanoside (CSS) plays a crucial role in combating CIO. Transcriptomics combined with network pharmacology analysis and experiments revealed that CSS activates the Dock1/PIP5K1A pathway to suppress the actin-severing protein gelsolin, protecting hair cells from cisplatin-induced cytoskeleton damage. CSS also activates the SLC7A11/GPX4 pathway via TGFBR2, reducing lipid peroxidation and intracellular iron accumulation to suppress cisplatin-induced ferroptosis. This study discovers that the major component CSS in A. sessiliflorus leaves reverses CIO by regulating actin homeostasis via Dock1 and inhibiting ferroptosis through TGFBR2, providing a theoretical basis for expanding CIO treatment targets and related drug development.

Multiple myeloma (MM) is an incurable plasma cell malignancy that has prompted investigations into new potential therapeutic avenues. Epigallocatechin-3-gallate (EGCG), a major component of green tea, confers antioxidant, anti-inflammatory, and anti-tumor properties. Previous studies have shown that EGCG inhibits proliferation and induces apoptosis of multiple myeloma cells, however its underlying molecular mechanisms are largely unknown. In this study, we accordingly sought to examine the therapeutic effects and underlying mechanisms of EGCG on MM. Initially, using CCK8 (Cell Counting Kit-8) assays and Annexin V-FITC/PI staining, we demonstrated that EGCG dose-dependently reduced cell viability and induced apoptosis in the MM cell lines MM.1S and RPMI 8226. Subsequently, mRNA sequencing of EGCG-treated MM.1S cells revealed a significant upregulation of genes associated with endoplasmic reticulum stress (ERS), including P-eIF2α (phosphorylation-eukaryotic translation initiation factor 2 alpha), ATF4 (activating transcription factor 4), CHOP (C/EBP homologous protein, DDIT3), and PUMA (p53 upregulated modulator of apoptosis, BBC3), which were confirmed at the protein level by western blotting. Furthermore, treatment with the eIF2α inhibitor ISRIB reduced the rates of EGCG-induced apoptosis and promoted increases in the protein expression of all four ER stress-related molecules in MM cells. Additionally, mRNA-seq data revealed a downregulation of α-Tubulin 1b (TUBA1B) expression in EGCG-treated MM cells, which was confirmed by western blotting and immunofluorescence analyses. Moreover, we utilized a mouse model to show that EGCG inhibited myeloma tumor growth, which was inhibited by ISRIB. In summary, the findings of this novel study indicated that EGCG promotes apoptosis of MM cells, both via activation of the ER stress pathway and disruption of cytoskeletal integrity. These findings highlight the multi-faceted anti-tumor effects of EGCG and its potential clinical application in MM treatment., 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.)

One of the most dangerous aspects of cancers is their ability to metastasize, which is the leading cause of death. Hence, it holds significance to develop therapies targeting the eradication of cancer cells in parallel, inhibiting metastases in cells surviving the applied therapy. Here, we focused on two melanoma cell lines─WM35 and WM266-4─representing the less and more invasive melanomas. We investigated the mechanisms of cellular processes regulating the activation of actomyosin as an effect of colchicine treatment. Additionally, we investigated the biophysical aspects of supplement therapy using Rho-associated protein kinase (ROCK) inhibitor (Y-27632) and myosin II inhibitor ((-)-blebbistatin), focusing on the microtubules and actin filaments. We analyzed their effect on the proliferation, migration, and invasiveness of melanoma cells, supported by studies on cytoskeletal architecture using confocal fluorescence microscopy and nanomechanics using atomic force microscopy (AFM) and microconstriction channels. Our results showed that colchicine inhibits the migration of most melanoma cells, while for a small cell population, it paradoxically increases their migration and invasiveness. These changes are also accompanied by the formation of stress fibers, compensating for the loss of microtubules. Simultaneous administration of selected agents led to the inhibition of this compensatory effect. Collectively, our results highlighted that colchicine led to actomyosin activation and increased the level of cancer cell invasiveness. We emphasized that a cellular pathway of Rho-ROCK-dependent actomyosin contraction is responsible for the increased invasive potential of melanoma cells in tubulin-targeted therapy.

The legume-rhizobium symbiosis represents the most important system for terrestrial biological nitrogen fixation on land. Efficient nitrogen fixation during this symbiosis depends on successful rhizobial infection and complete endosymbiosis, which are achieved by complex cellular events including cell-wall remodeling, cytoskeletal reorganizations, and extensive membrane expansion and trafficking. In this review, we explore the dynamic remodeling of the plant-specific cell wall-membrane system-cytoskeleton (WMC) continuum during symbiotic nitrogen fixation. We focus on key processes linked to efficient nitrogen fixation, including rhizobial uptake, infection thread formation and elongation, rhizobial droplet release, cytoplasmic bridge formation, and rhizobial endosymbiosis. Additionally, we discuss the advanced techniques for investigating the cellular basis of root-nodule symbiosis and provide insights into the unsolved mysteries of robust symbiotic nitrogen fixation., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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

Plasma membranes (PMs) are highly dynamic structures where lipids and proteins can theoretically diffuse freely. However, reports indicate that PM proteins do not freely diffuse within their planes but are constrained by cytoskeleton networks, though the mechanisms for how the cytoskeleton restricts lateral diffusion of plant PM proteins are unclear. Through single-molecule tracking, we investigated the dynamics of 6 Arabidopsis (Arabidopsis thaliana) PM proteins with diverse structures and found distinctions in sizes and dynamics among these proteins. Moreover, we showed that the cytoskeleton, particularly microtubules, limits the diffusion of PM proteins, including transmembrane and membrane-anchoring proteins. Interestingly, the microfilament skeleton regulates intracellular transport of endocytic cargo. Therefore, these findings indicate that the cytoskeleton controls signal transduction by limiting diffusion of PM proteins in specific membrane compartments and participating in transport of internalized cargo vesicles, thus actively regulating plant signal transduction., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Red blood cells respond to fluctuations in blood plasma pH by changing the rate of biochemical and physical processes that affect the specific functions of individual cells. This study aimed to analyze the effect of pH changes on red blood cell morphology and structure. The findings revealed that an increase or decrease in pH above or below the physiological level of pH 7.4 results in the transformation of discocytes into echinocytes and causes significant alterations in the membrane, including its roughness, cytoskeleton structure, and the cell's elastic modulus. Furthermore, the study shown a strong connection between critical acidosis and alkalosis with increased intracellular reactive oxygen species production.

The keratin cytoskeleton and associated desmosomes contribute to the mechanical stability of epithelial tissues, but their organization in native bladder umbrella cells and their responses to bladder filling are poorly understood. Using whole rat bladders in conjunction with confocal microscopy, super-resolution image processing, three-dimensional image reconstruction, and platinum replica electron microscopy, we identified a cortical cytoskeleton network in umbrella cells that was organized as a dense tile-like mesh comprised of tesserae bordered by cortical actin filaments, filled with keratin filaments, and cross-linked by plectin. Below these tesserae, keratin formed a subapical meshwork and at the cell periphery a band of keratin was linked via plectin to the junction-associated actin ring. Disruption of plectin led to focal keratin network dissolution, loss of the junction-associated keratin, and defects in cell-cell adhesion. During bladder filling, a junction-localized necklace of desmosomes expanded, and a subjacent girded layer formed linking the keratin network to desmosomes, including those at the umbrella cell-intermediate cell interface. Our studies reveal a novel tile- and mesh-like organization of the umbrella cell keratin network that is dependent on plectin, that reorganizes in response to bladder filling, and that likely serves to maintain umbrella cell continuity in the face of mechanical distension., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

The actin cytoskeleton is a key cellular structure subverted by pathogens to infect and survive in or on host cells. Several pathogenic strains of Escherichia coli, such as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), developed a unique mechanism to remodel the actin cytoskeleton that involves the assembly of actin filament-rich pedestals beneath the bacterial attachment sites. Actin pedestal assembly is driven by bacterial effectors injected into the host cells, and this structure is important for EPEC and EHEC colonization. While the interplay between bacterial effectors and the actin polymerization machinery of host cells is well-understood, how other mechanisms of actin filament remodelling regulate pedestal assembly and bacterial attachment are poorly investigated. This review discusses the gaps in our understanding of the complexity of the actin cytoskeletal remodelling during EPEC and EHEC infection. We describe possible roles of actin depolymerizing, crosslinking and motor proteins in pedestal dynamics, and bacterial interactions with the host cells. We also discuss the biological significance of pedestal assembly for bacterial infection., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

In plants, robust defense depends on the efficient and resilient trafficking supply chains to the site of pathogen attack. Though the importance of intracellular trafficking in plant immunity has been well established, a lack of clarity remains regarding the contribution of the various trafficking pathways in transporting immune-related proteins. We have recently identified a trans-Golgi network protein, TGN-ASSOCIATED PROTEIN 1 (TGNap1), which functionally links post-Golgi vesicles with the cytoskeleton to transport immunity-related proteins in the model plant species Arabidopsis thaliana. We propose new hypotheses on the various functional implications of TGNap1 and then elaborate on the surprising heterogeneity of TGN vesicles during immunity revealed by the discovery of TGNap1 and other TGN-associated proteins in recent years., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

Background: Finding effective pharmacological interventions to address the complex array of neurodevelopmental disorders is currently an urgent imperative within the scientific community as these conditions present significant challenges for patients and their families, often impacting cognitive, emotional, and social development. In this study, we aimed to explore non-invasive method to diagnose autism spectrum disorders (ASD) within Pakistan children population and to identify clinical drugs for its treatment., Aims: The current report outlines a comprehensive bidirectional investigation showcasing the successful utilization of saliva samples to quantify the expression patterns of profilins (PFN1, 2, and 3); and ERM (ezrin, radixin, and moesin) proteins; and additionally moesin pseudogene 1 and moesin pseudogene 1 antisense (MSNP1AS). Subsequently, these expression profiles were employed to forecast interactions between drugs and genes in children diagnosed with ASD., Methods: This study sought to delve into the intricate gene expression profiles using qualitative polymerase chain reaction of profilin isoforms (PFN1, 2, and 3) and ERM genes extracted from saliva samples obtained from children diagnosed with ASD. Through this analysis, we aimed to elucidate potential molecular mechanisms underlying ASD pathogenesis, shedding light on novel biomarkers and therapeutic targets for this complex neurological condition. (n = 22). Subsequently, we implemented a diagnostic model utilizing sparse partial least squares discriminant analysis (sPLS-DA) to predict drugs against our genes of interest. Furthermore, connectivity maps were developed to illustrate the predicted associations of 24 drugs with the genes expression., Results: Our study results showed varied expression profile of cytoskeleton linked genes. Similarly, sPLS-DA model precisely predicted drug to genes response. Sixteen of the examined drugs had significant positive correlations with the expression of the targeted genes whereas eight of the predicted drugs had shown negative correlations., Conclusion: Here we report the role of cytoskeleton linked genes (PFN and ERM) in co-relation to ASD. Furthermore, variable yet significant quantitative expression of these genes successfully predicted drug-gene interactions as shown with the help of connectivity maps that can be used to support the clinical use of these drugs to treat individuals with ASD in future studies., (© 2024 International Society for Developmental Neuroscience.)

Background Hailey-Hailey disease (HHD) is a rare, autosomal dominant, hereditary skin disorder characterised by epidermal acantholysis. The HHD-associated gene ATPase calcium-transporting type 2C member 1 (ATP2C1) encodes the protein secretory pathway Ca2+ ATPase1 (SPCA1), playing a critical role in HHD pathogenesis. Aims We aimed to investigate the effect of ATP2C1 knockdown on keratinocytes that mimicked acantholysis in HHD. Methods Immunohistochemistry (IHC) was employed to evaluate the levels of cytoskeletal and tight junction proteins such as SPCA1, P-cofilin, F-actin, claudins, occludin, and zonula occludens 1 in the skin biopsies of patients with HHD. Subsequently, the expression of these proteins in cultured ATP2C1 knockdown keratinocytes was analysed using Western blotting and immunofluorescence. Furthermore, we assessed the proliferation, apoptosis, and intracellular Ca2+ concentrations in the ATP2C1-knocked keratinocytes. Results The results showed decreased levels of these proteins (SPCA1, P-cofilin, F-actin, claudins, occluding, and zonula occludens 1) in HHD skin lesions. Moreover, their levels decreased in human keratinocytes transfected with ATP2C1 short hairpin RNA, accompanied by morphological acantholysis. Furthermore, the proliferation and apoptosis of the keratinocytes, as well as intracellular calcium concentrations in these cells, were not affected. Limitations The limitations of this study are the absence of animal experiments and the failure to explore the relationship between skeletal and tight junction proteins. Conclusion The present study indicated that ATP2C1 inhibition led to abnormal levels of the cytoskeletal and tight junction proteins in the keratinocytes. Therefore, keratinocytes can mimic HHD-like acantholysis and serve as an in vitro model, helping develop treatment strategies against HHD.

Small Heat Shock Proteins are considered as the first line of defense when proteostasis fails. Among them, αB-crystallin is expressed in striated muscles in which it interacts with desmin intermediate filaments to stabilize them, maintaining cytoskeleton's integrity and muscular functionalities. Desmin is a key actor for muscle health; its targeting by αB-crystallin is thus crucial, especially in stress conditions. αB-crystallin is phosphorylated and O-GlcNAcylated. Its phosphorylation increases consecutively to various stresses, correlated with its recruitment for cytoskeleton's safeguarding. However, phosphorylation as unique signal for cytoskeleton translocation remains controversial; indeed, O-GlcNAcylation was also proposed to be involved. Thus, there are still some gaps for a deeper comprehension of how αB-crystallin functions are finely regulated by post-translational modifications. Furthermore, desmin also bears both post-translational modifications; while desmin phosphorylation is closely linked to desmin intermediates filaments turnover, it is unclear whereas its O-GlcNAcylation could impact its proper function. In the herein paper, we aim at identifying whether phosphorylation and/or O-GlcNAcylation are involved in αB-crystallin targeting towards cytoskeleton in proteotoxic stress induced by proteasome inhibition in C2C12 myotubes. We demonstrated that proteotoxicity led to αB-crystallin's phosphorylation and O-GlcNAcylation patterns changes, both presenting a dynamic interplay depending on protein subfraction. Importantly, both post-translational modifications showed a spatio-temporal variation correlated with αB-crystallin translocation towards cytoskeleton. In contrast, we did not detect any change of desmin phosphorylation and O-GlcNAcylation. All together, these data strongly support that αB-crystallin phosphorylation/O-GlcNAcylation interplay rather than changes on desmin is a key regulator for its cytoskeleton translocation, preserving it towards stress., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Apicomplexan parasites are a group of eukaryotic protozoans with diverse biology that have affected human health like no other group of parasites. These obligate intracellular parasites rely on their cytoskeletal structures for giving them form, enabling them to replicate in unique ways and to migrate across tissue barriers. Recent progress in transgenesis and imaging tools allowed detailed insights into the components making up and regulating the actin and microtubule cytoskeleton as well as the alveolate-specific intermediate filament-like cytoskeletal network. These studies revealed interesting details that deviate from the cell biology of canonical model organisms. Here we review the latest developments in the field and point to a number of open questions covering the most experimentally tractable parasites: Plasmodium , the causative agent of malaria; Toxoplasma gondii , the causative agent of toxoplasmosis; and Cryptosporidium , a major cause of diarrhea.

SARS-CoV-2 mainly infects the respiratory tract but can also target other organs, including the central nervous system. While it was recently shown that cells of the blood-brain-barrier are permissive to SARS-CoV-2 infection in vitro, it remains debated whether neurons can be infected. In this study, we demonstrate that vesicular stomatitis virus particles pseudotyped with the spike protein of SARS-CoV-2 variants WT, Alpha, Delta and Omicron enter the neuronal model cell line SH-SY5Y. Cell biological analyses of the pseudo-virus treated cultures showed marked alterations in microtubules of SH-SY5Y cells. Because the changes in β-tubulin occurred in most cells, but only few were infected, we further asked whether interaction of the cells with spike protein might be sufficient to cause molecular and structural changes. For this, SH-SY5Y cells were incubated with trimeric spike proteins for time intervals of up to 24 h. CellProfiler™-based image analyses revealed changes in the intensities of microtubule staining in spike protein-incubated cells. Furthermore, expression of the spike protein-processing protease cathepsin L was found to be up-regulated by wild type, Alpha and Delta spike protein pseudotypes and cathepsin L was found to be secreted from spike protein-treated cells. We conclude that the mere interaction of the SARS-CoV-2 with neuronal cells can affect cellular architecture and proteolytic capacities. The molecular mechanisms underlying SARS-CoV-2 spike protein induced cytoskeletal changes in neuronal cells remain elusive and require future studies., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The aberrant expression of Stratifin (SFN) is intricately associated with the initiation and progression of numerous tumors. This study aims to investigate whether SFN regulates the metastasis of cervical cancer cells through the LIMK2/Cofilin signaling pathway. In this study, we compared the expression of SFN in normal cervical tissues and cervical carcinoma tissues. We established SFN overexpression and SFN silencing cellular models to assess the invasive and migratory capabilities of cervical cancer cells using transwell and scratch assays. YO-PRO-1/PI and EdU staining were employed to evaluate apoptotic and proliferative capacities, while Actin-Tracker Green-488 was utilized to investigate cytoskeletal remodeling. The expression levels of SFN, LIMK2, p-LIMK2, Cofilin, and p-Cofilin were examined through Western blotting and immunofluorescence. Our findings revealed elevated expression of SFN in cervical squamous cell carcinoma tissues. SFN overexpression was observed to enhance invasion and migration of cervical cancer cells, induce cytoskeletal remodeling, facilitate cell proliferation, and suppress apoptosis. Furthermore, SFN overexpression upregulated the expression levels of LIMK2, p-LIMK2, Cofilin, and p-Cofilin. Conversely, silencing SFN exerted opposite effects. SFN plays an important role in the diagnosis of cervical cancer. SFN can regulate cervical cancer cell proliferation, apoptosis, cytoskeletal remodeling and metastasis through LIMK2/Cofilin signaling., (© 2023. The Author(s).)

Background: Diabetes carries an increased risk of cardiovascular disease and thromboembolic events. Upon endothelial dysfunction, platelets bind to endothelial cells to precipitate thrombus formation; however, it is unclear which surface proteins regulate platelet-endothelium interaction. We and others have shown that peri/epicellular protein disulfide isomerase A1 (pecPDI) influences the adhesion and migration of vascular cells., Objectives: We investigated whether pecPDI regulates adhesion-related molecules on the surface of endothelial cells and platelets that influence the binding of these cells in hyperglycemia., Methods: Immunofluorescence was used to assess platelet-endothelium interaction in vitro, cytoskeleton reorganization, and focal adhesions. Hydrogen peroxide production was assessed via Amplex Red assays (ThermoFisher Scientific). Cell biophysics was assessed using atomic force microscopy. Secreted proteins of interest were identified through proteomics (secretomics), and targets were knocked down using small interfering RNA. Protein disulfide isomerase A1 (PDI) contribution was assessed using whole-cell PDI or pecPDI inhibitors or small interfering RNA., Results: Platelets of healthy donors adhered more onto hyperglycemic human umbilical vein endothelial cells (HUVECs). Endothelial, but not platelet, pecPDI regulated this effect. Hyperglycemic HUVECs showed marked cytoskeleton reorganization, increased H 2 O 2 production, and elongated focal adhesions. Indeed, hyperglycemic HUVECs were stiffer compared with normoglycemic cells. PDI and pecPDI inhibition reversed the abovementioned processes in hyperglycemic cells. A secretomics analysis revealed 8 proteins secreted in a PDI-dependent manner by hyperglycemic cells. Among these, we showed that genetic deletion of LAMC1 and SLC3A2 decreased platelet-endothelium interaction and did not potentiate the effects of PDI inhibitors., Conclusion: Endothelial pecPDI regulates platelet-endothelium interaction in hyperglycemia through adhesion-related proteins and alterations in endothelial membrane biophysics., 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.)

Previously we showed hyperosmotic solution caused TRPV1-dependent NKCC1 activation in the lens by a mechanism that involved ERK1/2 signaling. In various tissues, integrins and the cytoskeletal network play a role in responses to osmotic stress. Here, we examined the association between integrins and TRPV1-dependent activation of NKCC1 in mouse lens epithelium. Wild-type (WT) lenses exposed to the integrin agonist leukadherin-1 (LA-1) for 10 min displayed a ~33% increase in the bumetanide-sensitive rate of Rb uptake indicating NKCC activation. Paclitaxel, a microtubule stabilizing agent, abolished the Rb uptake response. In primary cultured lens epithelium LA-1 caused a robust ERK1/2 activation response that was almost fully suppressed by paclitaxel. The TRPV1 agonist capsaicin caused a similar ERK1/2 activation response. Consistent with an association between integrins and TRPV1, the TRPV1 antagonist A889425 prevented the Rb uptake response to LA-1 as did the ERK inhibitor U0126. LA-1 did not increase Rb uptake by lenses from TRPV1 knockout mice. In cells exposed to a hyperosmotic stimulus, both the ERK1/2 activation and Rb uptake responses were prevented by paclitaxel. Taken together, the findings suggest TRPV1 activation is associated with integrins and the tubulin cytoskeleton. This aligned with the observation that LA-1 elicited a robust cytoplasmic calcium rise in cells from WT lenses but failed to increase calcium in cells from TRPV1 knockout lenses. The results are consistent with the notion that integrin activation by LA-1, or a hyperosmotic stimulus, causes TRPV1 channel opening and the consequent downstream activation of the ERK1/2 and NKCC1 responses., (© 2024 Wiley Periodicals LLC.)

The Lassa virus (LASV) is a widely recognized virulent pathogen that frequently results in lethal viral hemorrhagic fever (VHF). Earlier research has indicated that macroautophagy/autophagy plays a role in LASV replication, but, the precise mechanism is unknown. In this present study, we show that LASV matrix protein (LASV-Z) is essential for blocking intracellular autophagic flux. LASV-Z hinders actin and tubulin folding by interacting with CCT2, a component of the chaperonin-containing T-complexes (TRiC). When the cytoskeleton is disrupted, lysosomal enzyme transit is hampered. In addition, cytoskeleton disruption inhibits the merge of autophagosomes with lysosomes, resulting in autophagosome accumulation that promotes the budding of LASV virus-like particles (VLPs). Inhibition of LASV-Z-induced autophagosome accumulation blocks the LASV VLP budding process. Furthermore, it is found that glutamine at position 29 and tyrosine at position 48 on LASV-Z are important in interacting with CCT2. When these two sites are mutated, LASV-mut interacts with CCT2 less efficiently and can no longer inhibit the autophagic flux. These findings demonstrate a novel strategy for LASV-Z to hijack the host autophagy machinery to accomplish effective transportation. Abbreviation: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; Baf-A1: bafilomycin A 1 ; CCT2: chaperonin containing TCP1 subunit 2; co-IP: co-immunoprecipitation; CTSD: cathepsin D; DAPI: 4',6-diamidino-2'-phenylindole; DMSO: dimethyl sulfoxide; EGFR: epidermal growth factor receptor; GFP: green fluorescent protein; hpi: hours post-infection; hpt: hours post-transfection; LAMP1: lysosomal-associated membrane protein 1; LASV: lassa virus; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry: red fluorescent protein; PM: plasma membrane; SQSTM1/p62: sequestosome 1; STX6: syntaxin 6; VLP: virus-like particle; TEM: transmission electron microscopy; TRiC: chaperonin-containing T-complex; WB: western blotting; μm: micrometer; μM: micromole.

To form blood vessels, endothelial cells rearrange their cytoskeleton, generate traction stresses, migrate, and proliferate, all of which require energy. Despite these energetic costs, stiffening of the extracellular matrix promotes tumor angiogenesis and increases cell contractility. However, the interplay between extracellular matrix, cell contractility, and cellular energetics remains mechanistically unclear. Here, we utilized polyacrylamide substrates with various stiffnesses, a real-time biosensor of ATP, and traction force microscopy to show that endothelial cells exhibit increasing traction forces and energy usage trend as substrate stiffness increases. Inhibition of cytoskeleton reorganization via ROCK inhibition resulted in decreased cellular energy efficiency, and an opposite trend was found when cells were treated with manganese to promote integrin affinity. Altogether, our data reveal a link between matrix stiffness, cell contractility, and cell energetics, suggesting that endothelial cells on stiffer substrates can better convert intracellular energy into cellular traction forces. Given the critical role of cellular metabolism in cell function, our study also suggests that not only energy production but also the efficiency of its use plays a vital role in regulating cell behaviors and may help explain how increased matrix stiffness promotes angiogenesis., Competing Interests: Conflict of interest The authors have no conflicts to disclose., (Copyright © 2024. Published by Elsevier B.V.)

Arabidopsis LIM proteins are named after the initials of three proteins Lin-11, Isl-1, and MEC-3, which belong to a class of transcription factors that play an important role in the developmental regulation of eukaryotes and are also involved in a variety of life processes, including gene transcription, the construction of the cytoskeleton, signal transduction, and metabolic regulation. Plant LIM proteins have been shown to regulate actin bundling in different cells, but their role in immunity remains elusive. Mitogen-activated protein kinases (MAPKs) are a family of conserved serine/threonine protein kinases that link upstream receptors to their downstream targets. Pathogens produce pathogen-associated molecular patterns (PAMPs) that trigger the activation of MAPK cascades in plants. Recently, we conducted a large-scale phosphoproteomic analysis of PAMP-induced Arabidopsis plants to identify putative MAPK targets. One of the identified phospho-proteins was WLIM2A, an Arabidopsis LIM protein. In this study, we investigated the role of WLIM2A in plant immunity. We employed a reverse-genetics approach and generated wlim2a knockout lines using CRISPR-Cas9 technology. We also generated complementation and phosphosite-mutated WLIM2A expression lines in the wlim2a background. The wlim2a lines were compromised in their response to Pseudomonas syringae Pst DC3000 but showed enhanced resistance to the necrotrophic fungus Botrytis cinereae . Transcriptome analyses of wlim2a mutants revealed the deregulation of immune hormone biosynthesis and signaling of salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways. The wlim2a mutants also exhibited altered stomatal phenotypes. Analysis of plants expressing WLIM2A variants of the phospho-dead or phospho-mimicking MAPK phosphorylation site showed opposing stomatal behavior and resistance phenotypes in response to Pst DC3000 infection, proving that phosphorylation of WLIM2A plays a crucial role in plant immunity. Overall, these data demonstrate that phosphorylation of WLIM2A by MAPKs regulates Arabidopsis responses to plant pathogens.

Vascular endothelial cytoskeletal disruption leads to increased vascular permeability and is involved in the pathogenesis and progression of various diseases. Oxidative stress can increase vascular permeability by weakening endothelial cell-to-cell junctions and decrease intracellular nicotinamide adenine dinucleotide (NAD + ) levels. However, it remains unclear how intracellular NAD + variations caused by oxidative stress alter the vascular endothelial cytoskeletal organization. In this study, we demonstrated that oxidative stress activates poly (ADP-ribose [ADPr]) polymerase (PARP), which consume large amounts of intracellular NAD + , leading to cytoskeletal disruption in vascular endothelial cells. We found that hydrogen peroxide (H 2 O 2 ) could transiently disrupt the cytoskeleton and reduce intracellular total NAD levels in human umbilical vein endothelial cells (HUVECs). H 2 O 2 stimulation led to rapid increase in ADPr protein levels in HUVECs. Pharmaceutical PARP inhibition counteracted H 2 O 2 -induced total NAD depletion and cytoskeletal disruption, suggesting that NAD + consumption by PARP induced cytoskeletal disruption. Additionally, supplementation with nicotinamide mononucleotide (NMN), the NAD + precursor, prevented both intracellular total NAD depletion and cytoskeletal disruption induced by H 2 O 2 in HUVECs. Inhibition of the NAD + salvage pathway by FK866, a nicotinamide phosphoribosyltransferase inhibitor, maintained H 2 O 2 -induced cytoskeletal disruption, suggesting that intracellular NAD + plays a crucial role in recovery from cytoskeletal disruption. Our findings provide further insights into the potential application of PARP inhibition and NMN supplementation for the treatment and prevention of diseases involving vascular hyperpermeability., Competing Interests: Declaration of competing interest The authors state no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The linker of nucleoskeleton and cytoskeleton (LINC) complex, which spans the nuclear envelope, physically connects nuclear components to the cytoskeleton and plays a pivotal role in various cellular processes, including nuclear positioning, cell migration, and chromosomal configuration. Studies have revealed that the LINC complex is essential for different aspects of the nervous system, particularly during development. The significance of the LINC complex in neural lineage cells is further corroborated by the fact that mutations in genes associated with the LINC complex have been implicated in several neurological diseases, including neurodegenerative and psychiatric disorders. In this review, we aimed to summarize the expanding knowledge of LINC complex-related neuronal functions and associated neurological diseases.

Rufy4, a protein belonging to the RUN and FYVE domain-containing protein family, participates in various cellular processes such as autophagy and intracellular trafficking. However, its role in osteoclast-mediated bone resorption remains uncertain. In this study, we investigated the expression and role of the Rufy4 gene in osteoclasts using small interfering RNA (siRNA) transfection and gene overexpression systems. Our findings revealed a significant increase in Rufy4 expression during osteoclast differentiation. Silencing Rufy4 enhanced osteoclast differentiation, intracellular cathepsin K levels, and formation of axial protrusive structures but suppressed bone resorption. Conversely, overexpressing wild-type Rufy4 in osteoclasts hindered differentiation while promoting podosome formation and bone resorption. Similarly, overexpression of a Rufy4 variant lacking the RUN domain mimics the effects of Rufy4 knockdown, significantly increasing intracellular cathepsin K levels, promoting osteoclastogenesis, and elongated axial protrusions formation, yet inhibiting bone resorption. These findings indicate that Rufy4 plays a critical role in osteoclast differentiation and bone resorption by regulating the cytoskeletal organization through its RUN domain. Our study provides new insights into the molecular mechanisms governing osteoclast activity and underscores Rufy4's potential as a novel therapeutic target for bone disorders characterized by excessive bone resorption.

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.

Stress generation by the actin cytoskeleton shapes cells and tissues. Despite impressive progress in live imaging and quantitative physical descriptions of cytoskeletal network dynamics, the connection between processes at molecular scales and spatiotemporal patterns at the cellular scale is still unclear. Here, we review studies reporting actomyosin clusters of micrometre size and with lifetimes of several minutes in a large number of organisms, ranging from fission yeast to humans. Such structures have also been found in reconstituted systems in vitro and in theoretical analyses of cytoskeletal dynamics. We propose that tracking these clusters could provide a simple readout for characterising living matter. Spatiotemporal patterns of clusters could serve as determinants of morphogenetic processes that have similar roles in diverse organisms., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The cytoskeleton is essential for spatial and temporal organisation of a wide range of cellular and tissue-level processes, such as proliferation, signalling, cargo transport, migration, morphogenesis, and neuronal development. Cytoskeleton research aims to study these processes by imaging, or by locally manipulating, the dynamics and organisation of cytoskeletal proteins with high spatiotemporal resolution: which matches the capabilities of optical methods. To date, no photoresponsive microtubule-stabilising tool has united all the features needed for a practical high-precision reagent: a low potency and biochemically stable non-illuminated state; then an efficient, rapid, and clean photoresponse that generates a high potency illuminated state; plus good solubility at suitable working concentrations; and efficient synthetic access. We now present CouEpo, a photocaged epothilone microtubule-stabilising reagent that combines these needs. Its potency increases approximately 100-fold upon irradiation by violet/blue light to reach low-nanomolar values, allowing efficient photocontrol of microtubule dynamics in live cells, and even the generation of cellular asymmetries in microtubule architecture and cell dynamics. CouEpo is thus a high-performance tool compound that can support high-precision research into many microtubule-associated processes, from biophysics to transport, cell motility, and neuronal physiology., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Phagocytosis of Mycobacterium tuberculosis ( Mtb ) followed by its integration into the matured lysosome is critical in the host defense against tuberculosis. How Mtb escapes this immune attack remains elusive. In this study, we unveiled a novel regulatory mechanism by which SIRT7 regulates cytoskeletal remodeling by modulating RAC1 activation. We discovered that SIRT7 expression was significantly reduced in CD14 + monocytes of TB patients. Mtb infection diminished SIRT7 expression by macrophages at both the mRNA and protein levels. SIRT7 deficiency impaired actin cytoskeleton-dependent macrophage phagocytosis, LC3II expression, and bactericidal activity. In a murine tuberculosis model, SIRT7 deficiency detrimentally impacted host resistance to Mtb , while Sirt7 overexpression significantly increased the host defense against Mtb , as determined by bacterial burden and inflammatory-histopathological damage in the lung. Mechanistically, we demonstrated that SIRT7 limits Mtb infection by directly interacting with and activating RAC1, through which cytoskeletal remodeling is modulated. Therefore, we concluded that SIRT7, in its role regulating cytoskeletal remodeling through RAC1, is critical for host responses during Mtb infection and proposes a potential target for tuberculosis treatment.IMPORTANCETuberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ), remains a significant global health issue. Critical to macrophages' defense against Mtb is phagocytosis, governed by the actin cytoskeleton. Previous research has revealed that Mtb manipulates and disrupts the host's actin network, though the specific mechanisms have been elusive. Our study identifies a pivotal role for SIRT7 in this context: Mtb infection leads to reduced SIRT7 expression, which, in turn, diminishes RAC1 activation and consequently impairs actin-dependent phagocytosis. The significance of our research is that SIRT7 directly engages with and activates Rac Family Small GTPase 1 (RAC1), thus promoting effective phagocytosis and the elimination of Mtb . This insight into the dynamic between host and pathogen in TB not only broadens our understanding but also opens new avenues for therapeutic development., Competing Interests: The authors declare no conflict of interest.

Studies utilizing electron microscopy and live fluorescence microscopy have significantly enhanced our understanding of the molecular mechanisms that regulate junctional dynamics during homeostasis, development and disease. To fully grasp the enormous complexity of cell-cell adhesions, it is crucial to study the nanoscale architectures of tight junctions, adherens junctions and desmosomes. It is important to integrate these junctional architectures with the membrane morphology and cellular topography in which the junctions are embedded. In this Review, we explore new insights from studies using super-resolution and volume electron microscopy into the nanoscale organization of these junctional complexes as well as the roles of the junction-associated cytoskeleton, neighboring organelles and the plasma membrane. Furthermore, we provide an overview of junction- and cytoskeletal-related biosensors and optogenetic probes that have contributed to these advances and discuss how these microscopy tools enhance our understanding of junctional dynamics across cellular environments., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Ever since Robert Hooke's 17th century discovery of the cell using a humble compound microscope, light-matter interactions have continuously redefined our understanding of cell biology. Fluorescence microscopy has been particularly transformative and remains an indispensable tool for many cell biologists. The subcellular localization of biomolecules is now routinely visualized simply by manipulating the wavelength of light. Fluorescence polarization microscopy (FPM) extends these capabilities by exploiting another optical property - polarization - allowing researchers to measure not only the location of molecules, but also their organization or alignment within larger cellular structures. With only minor modifications to an existing fluorescence microscope, FPM can reveal the nanoscale architecture, orientational dynamics, conformational changes and interactions of fluorescently labeled molecules in their native cellular environments. Importantly, FPM excels at imaging systems that are challenging to study through traditional structural approaches, such as membranes, membrane proteins, cytoskeletal networks and large macromolecular complexes. In this Review, we discuss key discoveries enabled by FPM, compare and contrast the most common optical setups for FPM, and provide a theoretical and practical framework for researchers to apply this technique to their own research questions., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Cytoskeleton remodeling which generates force and orchestrates signaling and trafficking to govern cell migration remains poorly understood, partly due to a lack of an investigation tool with high system flexibility, spatiotemporal resolution, and computational sensitivity. Herein, we developed a multimodal superresolution imaging system-based architecture-driven quantitative (ADQ) framework in spatiotemporal-angular hyperspace to enable both identification of the optimal imaging mode with well-balanced fidelity and phototoxicity and accurate postcharacterization of microtubule remodeling. In the ADQ framework, a pixel/voxel-wise metric reflecting heterogeneous intertubule alignment was proposed with improved sensitivity over previous efforts and further incorporated with temporal features to map dynamic microtubule rearrangements. The ADQ framework was verified by assessing microtubule remodeling in drug-induced (de)polymerization, lysosome transport, and migration. Different remodeling patterns from two migration modes were successfully revealed by the ADQ framework, with a front-rear polarization for individual directed migration and a contact site-centered polarization for cell-cell interaction-induced migration in an immune response model. Meanwhile, these migration modes were found to have consistent orientation changes, which exhibited the potential of predicting migration trajectory., Competing Interests: Competing interests statement:The authors declare no competing interest.

Main Conclusion: Our studies reveal the involvement of SPI in cytoskeleton-associated trichome morphogenesis, expanding the roles of SPI in regulating plant epidermal cell development. Acquisition of distinct shapes is crucial for cells to perform their biological functions in multicellular organisms. Trichomes are specialized epidermal cells of plant aerial parts, offering an excellent paradigm for dissecting the underlying regulatory mechanism of plant cell shape development at the single-cell level. SPIRRIG (SPI) that encodes a BEACH domain-containing protein was initially identified to regulate trichome branch extension, but the possible pathway(s) through which SPI regulates trichome morphogenesis remain unclear. Here, we report that SPI facilitates microtubule-associated regulation on trichome branching in Arabidopsis. Functional loss of SPI results in trichome morphogenesis hyper-sensitive to the microtubule-disrupting drug oryzalin, implying SPI may mediate microtubule stability during trichome development. Accordingly, spi mutant has less-branched trichomes. Detailed live-cell imaging showed that the spatio-temporal microtubule organization during trichome morphogenesis is aberrant in spi mutants. Further genetic investigation indicated that SPI may cooperate with ZWICHEL (ZWI) to modulate microtubule dynamics during trichome morphogenesis. ZWI encodes a kinesin-like calmodulin-binding protein (KCBP), whose distribution is necessary for the proper microtubule organization in trichomes, and zwi mutants produce less-branched trichomes as well. Trichome branching is further inhibited in spi-3 zwi-101 double mutants compared to either of the single mutant. Moreover, we found SPI could co-localize with the MYTH4 domain of ZWI. Taken together, our results expand the role of SPI in regulating trichome morphogenesis and also reveal a molecular and genetic pathway in plant cell shape formation control., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We propose a novel quantitative method to explore the forces affecting mitochondria within living cells in an almost non-invasive fashion. This new tool enables the detection of localized mechanical impulses on these organelles that occur amidst the stationary fluctuations caused by the thermal jittering in the cytoplasm. Recent experimental evidence shows that the action of mechanical forces has important effects on the dynamics, morphology and distribution of mitochondria in cells. In particular, their crosstalk with the cytoskeleton has been found to alter these organelles function; however, the mechanisms underlying this phenomenon are largely unknown. Our results highlight the different functions that cytoskeletal networks play in shaping mitochondrial dynamics. This work presents a novel technique to extend our knowledge of how the impact of mechanical cues can be quantified at the single organelle level. Moreover, this approach can be expanded to the study of other organelles or biopolymers., (© 2024. The Author(s).)

Endothelial cells are key players in the cardiovascular system. Among other things, they are responsible for sprouting angiogenesis, the process of new blood vessel formation essential for both health and disease. Endothelial cells are strongly regulated by the juxtacrine signaling pathway Notch. Recent studies have shown that both Notch and angiogenesis are influenced by extracellular matrix stiffness; however, the underlying mechanisms are poorly understood. Here, we addressed this challenge by combining computational models of Notch signaling and YAP/TAZ, stiffness- and cytoskeleton-regulated mechanotransducers whose activity inhibits both Dll4 (Notch ligand) and LFng (Notch-Dll4 binding modulator). Our simulations successfully mimicked previous experiments, indicating that this YAP/TAZ-Notch crosstalk elucidates the Notch and angiogenesis mechanoresponse to stiffness. Additional simulations also identified possible strategies to control Notch activity and sprouting angiogenesis via cytoskeletal manipulations or spatial patterns of alternating stiffnesses. Our study thus inspires new experimental avenues and provides a promising modeling framework for further investigations into the role of Notch, YAP/TAZ, and mechanics in determining endothelial cell behavior during angiogenesis and similar processes., (© 2024. The Author(s).)

SLMAP3 is a tail-anchored membrane protein that targets subcellular organelles and is believed to regulate Hippo signaling. The global loss of SLMAP3 causes late embryonic lethality in mice, with some embryos exhibiting neural tube defects such as craniorachischisis. We show here that SLMAP3 -/- embryos display reduced length and increased width of neural plates, signifying arrested convergent extension. The expression of planar cell polarity (PCP) components Dvl2/3 and the activity of the downstream targets ROCK2, cofilin, and JNK1/2 were dysregulated in SLMAP3 -/- E12.5 brains. Furthermore, the cytoskeletal proteins (γ-tubulin, actin, and nestin) and apical components (PKCζ and ZO-1) were mislocalized in neural tubes of SLMAP3 -/- embryos, with a subsequent decrease in colocalization of PCP proteins (Fzd6 and pDvl2). However, no changes in PCP or cytoskeleton proteins were found in cultured neuroepithelial cells depleted of SLMAP3, suggesting an essential requirement for SLMAP3 for these processes in vivo for neurulation. The loss of SLMAP3 had no impact on Hippo signaling in SLMAP3 -/- embryos, brains, and neural tubes. Proteomic analysis revealed SLMAP3 in an interactome with cytoskeletal components, including nestin, tropomyosin 4, intermediate filaments, plectin, the PCP protein SCRIB, and STRIPAK members in embryonic brains. These results reveal a crucial role of SLMAP3 in neural tube development by regulating the cytoskeleton organization and PCP pathway., (© 2024 Rehmani et al.)

Convergent extension of epithelial tissue is a key motif of animal morphogenesis. On a coarse scale, cell motion resembles laminar fluid flow; yet in contrast to a fluid, epithelial cells adhere to each other and maintain the tissue layer under actively generated internal tension. To resolve this apparent paradox, we formulate a model in which tissue flow in the tension-dominated regime occurs through adiabatic remodeling of force balance in the network of adherens junctions. We propose that the slow dynamics within the manifold of force-balanced configurations is driven by positive feedback on myosin-generated cytoskeletal tension. Shifting force balance within a tension network causes active cell rearrangements (T1 transitions) resulting in net tissue deformation oriented by initial tension anisotropy. Strikingly, we find that the total extent of tissue deformation depends on the initial cellular packing order. T1s degrade this order so that tissue flow is self-limiting. We explain these findings by showing that coordination of T1s depends on coherence in local tension configurations, quantified by a geometric order parameter in tension space. Our model reproduces the salient tissue- and cell-scale features of germ band elongation during Drosophila gastrulation, in particular the slowdown of tissue flow after approximately twofold elongation concomitant with a loss of order in tension configurations. This suggests local cell geometry contains morphogenetic information and yields experimentally testable predictions. Defining biologically controlled active tension dynamics on the manifold of force-balanced states may provide a general approach to the description of morphogenetic flow., Competing Interests: Competing interests statement:The authors declare no competing interest.

Bactofilins are rigid, nonpolar bacterial cytoskeletal filaments that link cellular processes to specific curvatures of the cytoplasmic membrane. Although homologs of bactofilins have been identified in archaea and eukaryotes, functional studies have remained confined to bacterial systems. Here, we characterize representatives of two families of archaeal bactofilins from the pleomorphic archaeon Haloferax volcanii , halofilin A (HalA) and halofilin B (HalB). HalA and HalB polymerize in vitro, assembling into straight bundles. HalA polymers are highly dynamic and accumulate at positive membrane curvatures in vivo, whereas HalB forms more static foci that localize in areas of local negative curvatures on the outer cell surface. Gene deletions and live-cell imaging show that halofilins are critical in maintaining morphological integrity during shape transition from disk (sessile) to rod (motile). Morphological defects in Δ halA result in accumulation of highly positive curvatures in rods but not in disks. Conversely, disk-shaped cells are exclusively affected by halB deletion, resulting in flatter cells. Furthermore, while Δ halA and Δ halB cells imprecisely determine the future division plane, defects arise predominantly during the disk-to-rod shape remodeling. The deletion of halA in the haloarchaeon Halobacterium salinarum , whose cells are consistently rod-shaped, impacted morphogenesis but not cell division. Increased levels of halofilins enforced drastic deformations in cells devoid of the S-layer, suggesting that HalB polymers are more stable at defective S-layer lattice regions. Our results suggest that halofilins might play a significant mechanical scaffolding role in addition to possibly directing envelope synthesis., Competing Interests: Competing interests statement:The authors declare no competing interest.

The cytoophidium is a novel type of membraneless organelle, first observed in the ovaries of Drosophila using fluorescence microscopy. In vitro, purified Drosophila melanogaster CTPS (dmCTPS) can form metabolic filaments under the presence of either substrates or products, and their structures that have been analyzed using cryo-electron microscopy (cryo-EM). These dmCTPS filaments are considered the fundamental units of cytoophidia. However, due to the resolution gap between light and electron microscopy, the precise assembly pattern of cytoophidia remains unclear. In this study, we find that dmCTPS filaments can spontaneously assemble in vitro, forming network structures that reach micron-scale dimensions. Using cryo-electron tomography (cryo-ET), we reconstruct the network structures formed by dmCTPS filaments under substrate or product binding conditions and elucidate their assembly process. The dmCTPS filaments initially form structural bundles, which then further assemble into larger networks. By identifying, tracking, and statistically analyzing the filaments, we observed distinct characteristics of the structural bundles formed under different conditions. This study provides the first systematic analysis of dmCTPS filament networks, offering new insights into the relationship between cytoophidia and metabolic filaments., Competing Interests: Declaration of competing interest The authors declare that no interest exists., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Morphological complexity and plasticity are hallmarks of polyextremotolerant fungi. Septins are conserved cytoskeletal proteins and key contributors to cell polarity and morphogenesis. They sense membrane curvature, coordinate cell division, and influence diffusion at the plasma membrane. Four septin homologues are conserved from yeasts to humans, the systems in which septins have been most studied. But there is also a fifth family of opisthokont septins that remain biochemically mysterious. Members of this family, Group 5 septins, appear in the genomes of filamentous fungi, but are understudied due to their absence from ascomycete yeasts. Knufia petricola is an emerging model polyextremotolerant black fungus that can also serve as a model system for Group 5 septins. We have recombinantly expressed and biochemically characterized Kp AspE, a Group 5 septin from K. petricola . This septin--by itself in vitro--recapitulates many functions of canonical septin hetero-octamers. Kp AspE is an active GTPase that forms diverse homo-oligomers, binds shallow membrane curvatures, and interacts with the terminal subunit of canonical septin hetero-octamers. These findings raise the possibility that Group 5 septins govern the higher-order structures formed by canonical septins, which in K. petricola cells form extended filaments, and provide insight into how septin hetero-oligomers evolved from ancient homomers., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

Background: Plasma concentration of PAI-1 (plasminogen activator inhibitor-1) correlates with arterial stiffness. Vascular smooth muscle cells (SMCs) express PAI-1, and the intrinsic stiffness of SMCs is a major determinant of total arterial stiffness. We hypothesized that PAI-1 promotes SMC stiffness by regulating the cytoskeleton and that pharmacological inhibition of PAI-1 decreases SMC and aortic stiffness., Methods: PAI-039, a specific inhibitor of PAI-1, and small interfering RNA were used to inhibit PAI-1 expression in cultured human SMCs. Effects of PAI-1 inhibition on SMC stiffness, F-actin (filamentous actin) content, and cytoskeleton-modulating enzymes were assessed. WT (wild-type) and PAI-1-deficient murine SMCs were used to determine PAI-039 specificity. RNA sequencing was performed to determine the effects of PAI-039 on SMC gene expression. In vivo effects of PAI-039 were assessed by aortic pulse wave velocity., Results: PAI-039 significantly reduced intrinsic stiffness of human SMCs, which was accompanied by a significant decrease in cytoplasmic F-actin content. PAI-1 gene knockdown also decreased cytoplasmic F-actin. PAI-1 inhibition significantly increased the activity of cofilin, an F-actin depolymerase, in WT murine SMCs, but not in PAI-1-deficient SMCs. RNA-sequencing analysis suggested that PAI-039 upregulates AMPK (AMP-activated protein kinase) signaling in SMCs, which was confirmed by Western blotting. Inhibition of AMPK prevented activation of cofilin by PAI-039. In mice, PAI-039 significantly decreased aortic stiffness and tunica media F-actin content without altering the elastin or collagen content., Conclusions: PAI-039 decreases intrinsic SMC stiffness and cytoplasmic stress fiber content. These effects are mediated by AMPK-dependent activation of cofilin. PAI-039 also decreases aortic stiffness in vivo. These findings suggest that PAI-1 is an important regulator of the SMC cytoskeleton and that pharmacological inhibition of PAI-1 has the potential to prevent and treat cardiovascular diseases involving arterial stiffening., Competing Interests: None.

The actin cytoskeleton is involved in a large number of cellular signaling events in addition to providing structural integrity to the cell. Actin polymerization is a key event during cellular signaling. Although the role of actin cytoskeleton in cellular processes such as trafficking and motility has been extensively studied, the reorganization of the actin cytoskeleton upon signaling has been rarely explored due to lack of suitable assays. Keeping in mind this lacuna, we developed a confocal microscopy based approach that relies on high magnification imaging of cellular F-actin, followed by image reconstruction using commercially available software. In this review, we discuss the context and relevance of actin quantitation, followed by a detailed hands-on approach of the methodology involved with specific points on troubleshooting and useful precautions. In the latter part of the review, we elucidate the method by discussing applications of actin quantitation from our work in several important problems in contemporary membrane biology ranging from pathogen entry into host cells, to GPCR signaling and membrane-cytoskeleton interaction. We envision that future discovery of cell-permeable novel fluorescent probes, in combination with genetically encoded actin-binding reporters, would allow real-time visualization of actin cytoskeleton dynamics to gain deeper insights into active cellular processes in health and 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 Elsevier Inc. All rights reserved.)

Eukaryotic cell rheology has important consequences for vital processes such as adhesion, migration, and differentiation. Experiments indicate that cell cytoplasm can exhibit both elastic and viscous characteristics in different regimes, while the transport of fluid (cytosol) through the cross-linked filamentous scaffold (cytoskeleton) is reminiscent of mass transfer by diffusion through a porous medium. To gain insights into this complex rheological behaviour, we construct a computational model for the cell cytoplasm as a poroviscoelastic material formulated on the principles of nonlinear continuum mechanics, where we model the cytoplasm as a porous viscoelastic scaffold with an embedded viscous fluid flowing between the pores to model the cytosol. Baseline simulations (neglecting the viscosity of the cytosol) indicate that the system exhibits seven different regimes across the parameter space spanned by the viscoelastic relaxation timescale of the cytoskeleton and the poroelastic diffusion timescale; these regimes agree qualitatively with experimental measurements. Furthermore, the theoretical model also allows us to elucidate the additional role of pore fluid viscosity, which enters the system as a distinct viscous timescale. We show that increasing this viscous timescale hinders the passage of the pore fluid (reducing the poroelastic diffusion) and makes the cytoplasm rheology increasingly incompressible, shifting the phase boundaries between the regimes., (© 2024. The Author(s).)

Biologists have the capability to edit a genome at the nanometer scale and then observe whether or not the edit affects the structure of a developing organ or organism at the centimeter scale. Our understanding of the underlying mechanisms driving this emergent phenomenon from a multiscale perspective remains incomplete. This review focuses predominantly on recent experimental developments in uncovering the mechanical interplay between the chromatin and cell scale since mechanics plays a major role in determining nuclear, cellular, and tissue structure. Here, we discuss the generation and transmission of forces through the cytoskeleton, affecting chromatin diffusivity and organization. Decoding such pieces of these multiscale connections lays the groundwork for solving the genotype-to-phenotype puzzle in biology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Förster resonance energy transfer (FRET) serves as a tool for measuring protein-protein interactions using various sensor molecules. The tension sensor module relies on FRET technology. In our study, this module was inserted within the actinin molecule to measure the surface tension of the cells. Given that the decay curve of FRET efficiency correlates with surface tension increase, precise and accurate efficiency measurement becomes crucial. Among the methods of FRET measurements, FRET efficiency remains the most accurate if sample fixation is successful. However, when cells were fixed with 4% paraformaldehyde (PFA), the actinin-FRET sensor diffused across the cytoplasm; this prompted us to explore fixation method enhancements. Glyoxal fixative has been reported to improve cytoskeletal morphologies compared to PFA. However, it was not known whether glyoxal fits FRET measurements. Glyoxal necessitates an acetic acid solution for fixation; however, acidic conditions could compromise fluorescence stability. We observed that the pH working range of glyoxal fixative aligns closely with MES (methyl-ethylene sulfonic acid) Good's buffer. Initially, we switched the acidic solution for MES buffer and optimized the fixation procedure for in vitro and in vivo FRET imaging. By comparing FRET measurements on hydrogels with known stiffness to tumor nodules in mouse lung, we estimated in vivo stiffness. The estimated stiffness of cancerous tissue was harder than the reported stiffness of smooth muscle. This discovery shed lights on how cancer cells perceive environmental stiffness during metastasis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Piezo1 functions as a special transducer of mechanostress into electrochemical signals and is implicated in the pathogenesis of various diseases across different disciplines. However, whether Piezo1 contributes to the pathogenesis of lupus nephritis (LN) remains elusive. To study this, we applied an agonist and antagonist of Piezo1 to treat lupus-prone MRL/lpr mice. Additionally, a podocyte-specific Piezo1 knockout mouse model was also generated to substantiate the role of Piezo1 in podocyte injury induced by pristane, a murine model of LN. A marked upregulation of Piezo1 was found in podocytes in both human and murine LN. The Piezo1 antagonist, GsMTx4, significantly alleviated glomerulonephritis and tubulointerstitial damage, improved kidney function, decreased proteinuria, and mitigated podocyte foot process effacement in MRL/lpr mice. Moreover, podocyte-specific Piezo1 deletion showed protective effects on the progression of proteinuria and podocyte foot process effacement in the murine LN model. Mechanistically, Piezo1 expression was upregulated by inflammatory cytokines (IL-6, TNF-α and IFN-γ), soluble urokinase Plasminogen Activator Receptor and its own activation. Activation of Piezo1 elicited calcium influx, which subsequently enhanced Rac1 activity and increased active paxillin, thereby promoting cytoskeleton remodeling and decreasing podocyte motility. Thus, our work demonstrated that Piezo1 contributed to podocyte injury and proteinuria progression in LN. Hence, targeted therapy aimed at decreasing or inhibiting Piezo1 could represent a novel strategy to treat LN., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD), a multifactorial disease, is characterized by the accumulation of neurofibrillary tangles (NFTs) and amyloid beta (Aβ) plaques. AD is triggered via several factors like alteration in cytoskeletal proteins, a mutation in presenilin 1 (PSEN1), presenilin 2 (PSEN2), amyloid precursor protein (APP), and post-translational modifications (PTMs) in the cytoskeletal elements. Owing to the major structural and functional role of cytoskeletal elements, like the organization of axon initial segmentation, dendritic spines, synaptic regulation, and delivery of cargo at the synapse; modulation of these elements plays an important role in AD pathogenesis; like Tau is a microtubule-associated protein that stabilizes the microtubules, and it also causes inhibition of nucleo-cytoplasmic transportation by disrupting the integrity of nuclear pore complex. One of the major cytoskeletal elements, actin and its dynamics, regulate the dendritic spine structure and functions; impairments have been documented towards learning and memory defects. The second major constituent of these cytoskeletal elements, microtubules, are necessary for the delivery of the cargo, like ion channels and receptors at the synaptic membranes, whereas actin-binding protein, i.e., Cofilin's activation form rod-like structures, is involved in the formation of paired helical filaments (PHFs) observed in AD. Also, the glial cells rely on their cytoskeleton to maintain synaptic functionality. Thus, making cytoskeletal elements and their regulation in synaptic structure and function as an important aspect to be focused for better management and targeting AD pathology. This review advocates exploring phytochemicals and Ayurvedic plant extracts against AD by elucidating their neuroprotective mechanisms involving cytoskeletal modulation and enhancing synaptic plasticity. However, challenges include their limited bioavailability due to the poor solubility and the limited potential to cross the blood-brain barrier (BBB), emphasizing the need for targeted strategies to improve therapeutic efficacy., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In this study, we have explored the role of the KATNB1 gene, a microtubule-severing protein, in the seminiferous epithelium of the rat testis. Our data have shown that KATNB1 expressed in rat brain, testes, and Sertoli cells. KATNB1 was found to co-localize with α-tubulin showing a unique stage-specific distribution across the seminiferous epithelium. Knockdown of KATNB1 by RNAi led to significant disruption of the tight junction (TJ) permeability barrier function in primary Sertoli cells cultured in vitro with an established functional TJ-barrier, as well as perturbations in the microtubule and actin cytoskeleton organization. The disruption in these cytoskeletal structures, in turn, led to improper distribution of TJ and basal ES proteins essential for maintaining the Sertoli TJ function. More importantly, overexpression of KATNB1 in the testis in vivo was found to block cadmium-induced blood-testis barrier (BTB) disruption and testis injury. KATNB1 exerted its promoting effects on BTB and spermatogenesis through corrective spatiotemporal expression of actin- and microtubule-based regulatory proteins by maintaining the proper organization of cytoskeletons in the testis, illustrating its plausible therapeutic implication. In summary, Katanin regulatory subunit B1 (KATNB1) plays a crucial role in BTB and spermatogenesis through its effects on the actin- and microtubule-based cytoskeletons in Sertoli cells and testis, providing important insights into male reproductive biology., (© 2024 Federation of American Societies for Experimental Biology.)

Posttranslational modifications (PTMs) of tubulin, termed the "tubulin code", play important roles in regulating microtubule functions within subcellular compartments for specialized cellular activities. While numerous tubulin PTMs have been identified, a comprehensive understanding of the complete repertoire is still underway. In this study, we report that α-tubulin lactylation is catalyzed by HDAC6 by using lactate to increase microtubule dynamics in neurons. We identify lactylation on lysine 40 of α-tubulin in the soluble tubulin dimers. Notably, lactylated α-tubulin enhances microtubule dynamics and facilitates neurite outgrowth and branching in cultured hippocampal neurons. Moreover, we discover an unexpected function of HDAC6, acting as the primary lactyltransferase to catalyze α-tubulin lactylation. HDAC6-catalyzed lactylation is a reversible process, dependent on lactate concentrations. Intracellular lactate concentration triggers HDAC6 to lactylate α-tubulin, a process dependent on its deacetylase activity. Additionally, the lactyltransferase activity may be conserved in HDAC family proteins. Our study reveals the primary role of HDAC6 in regulating α-tubulin lactylation, establishing a link between cell metabolism and cytoskeleton functions., (© 2024. The Author(s).)

Microtubule (MT) dynamics is tightly regulated by microtubule-associated proteins (MAPs) and various post-translational modifications (PTMs) of tubulin. Here, we introduce OligoMT and OligoTIP as genetically encoded oligomeric MT binders designed for real-time visualization and manipulation of MT behaviors within living cells. OligoMT acts as a reliable marker to label the MT cytoskeleton, while OligoTIP allows for live monitoring of the growing MT plus-ends. These engineered MT binders have been successfully utilized to label the MT network, monitor cell division, track MT plus-ends, and assess the effect of tubulin acetylation on the MT stability at the single-cell level. Moreover, OligoMT and OligoTIP can be repurposed as biosensors for quantitative assessment of drug actions and for reporting enzymatic activity. Overall, these engineered MT binders hold promise for advancing the mechanistic dissection of MT biology and have translational applications in cell-based high-throughput drug discovery efforts.

The exon junction complex (EJC), nucleated by EIF4A3, is indispensable for mRNA fate and function throughout eukaryotes. We discover that EIF4A3 directly controls microtubules, independent of RNA, which is critical for neural wiring. While neuronal survival in the developing mouse cerebral cortex depends upon an intact EJC, axonal tract development requires only Eif4a3. Using human cortical organoids, we show that EIF4A3 disease mutations also impair neuronal growth, highlighting conserved functions relevant for neurodevelopmental pathology. Live imaging of growing neurons shows that EIF4A3 is essential for microtubule dynamics. Employing biochemistry and competition experiments, we demonstrate that EIF4A3 directly binds to microtubules, mutually exclusive of the EJC. Finally, in vitro reconstitution assays and rescue experiments demonstrate that EIF4A3 is sufficient to promote microtubule polymerization and that EIF4A3-microtubule association is a major contributor to axon growth. This reveals a fundamental mechanism by which neurons re-utilize core gene expression machinery to directly control the cytoskeleton., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)