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Aims: COVID-19, caused by the SARS-CoV-2 virus, can lead to serious lung conditions, notably interstitial pulmonary fibrosis., Main Methods: Our study tracked the progression of fibrosis markers in serial bronchoalveolar lavage (BAL) measurements collected from 16 COVID-19 patients at 1, 3, and 6 months post-infection. Additionally, BAL samples from 10 healthy control subjects were included. Using RT-PCR, ELISA, and immunofluorescence, we monitored molecular markers of fibrosis and investigated the interplay between galectins-1 and -3 and key pro-fibrotic mediators., Key Findings: We found increased α-smooth muscle actin (αSMA)-positive macrophages and heightened levels of αSMA, TGFβ, and CTGF mRNA and proteins at six months compared to controls. Furthermore, galectin-1 and galectin-3 concentrations showed a time-dependent increase and correlated significantly with pro-fibrotic markers., Significance: These findings suggest that galectins contribute to fibrotic progression following COVID-19 and highlight their potential as therapeutic targets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell migration, a pivotal process in wound healing, immune response, and even cancer metastasis, manifests through intricate interplay between morphology, speed, and cytoskeletal dynamics. Mathematical modeling emerges as a powerful tool to dissect these complex interactions. This work presents a two-dimensional immersed boundary model for mammalian cell migration, incorporating both filamentous actin (F-actin) and monomeric actin (G-actin) to explicitly capture polymerization and depolymerization. This model builds upon our previous one-dimensional efforts, now enabling us to explore the impact of G-actin on not just cell velocity but also morphology. We compare predictions from both models, revealing that while the one-dimensional model captures core dynamics along the cell's axis, the two-dimensional model excels in portraying cell shape evolution and transverse variations in actin concentration and velocity. Our findings highlight the crucial role of including G-actin in shaping cell morphology. Actin velocity aligned with migration direction elongates the cell, while velocity normal to the membrane promotes spreading. Importantly, the model establishes a link between these microscopic aspects and macroscopic observables like cell shape, offering a deeper understanding of cell migration dynamics. This work not only provides a more comprehensive picture of cell migration but also paves the way for future studies exploring the interplay of actin dynamics, cell morphology, and biophysical parameters in diverse biological contexts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mitochondria are crucial for cellular metabolism and signalling. Mitochondrial activity is modulated by mitochondrial fission and fusion, which are required to properly balance metabolic functions, transfer material between mitochondria, and remove defective mitochondria. Mitochondrial fission occurs at mitochondria-endoplasmic reticulum (ER) contact sites, and requires the formation of actin filaments that drive mitochondrial constriction and the recruitment of the fission protein DRP1. The role of actin in mitochondrial fusion remains entirely unexplored. Here we show that preventing actin polymerisation on either mitochondria or the ER disrupts both fission and fusion. We show that fusion but not fission is dependent on Arp2/3, whereas both fission and fusion require INF2 formin-dependent actin polymerization. We also show that mitochondria-associated actin marks fusion sites prior to the fusion protein MFN2. Together, our work introduces a method for perturbing organelle-associated actin and demonstrates a previously unknown role for actin in mitochondrial fusion., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

To function and survive cells need to be able to sense and respond to their local environment through mechanotransduction. Crucially, mechanical and biochemical perturbations initiate cell signalling cascades, which can induce responses such as growth, apoptosis, proliferation and differentiation. At the heart of this process are actomyosin stress fibres (SFs), which form part of the cell cytoskeleton, and focal adhesions (FAs), which bind this cytoskeleton to the extra-cellular matrix (ECM). The formation and maturation of these structures (connected by a positive feedback loop) is pivotal in non-motile cells, where SFs are generally of ventral type, interconnecting FAs and producing isometric tension. In this study we formulate a one-dimensional bio-chemo-mechanical continuum model to describe the coupled formation and maturation of ventral SFs and FAs. We use a set of reaction-diffusion-advection equations to describe three sets of biochemical events: the polymerisation of actin and subsequent bundling into activated SFs; the formation and maturation of cell-substrate adhesions; and the activation of signalling proteins in response to FA and SF formation. The evolution of these key proteins is coupled to a Kelvin-Voigt viscoelastic description of the cell cytoplasm and the ECM. We employ this model to understand how cells respond to external and intracellular cues in vitro and are able to reproduce experimentally observed phenomena including non-uniform cell striation and cells forming weaker SFs and FAs on softer substrates., 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 Ltd.)

Glioblastoma (GBM) is a malignant brain tumor with diffuse infiltration. Here, we demonstrate how GBM cells usurp guidance receptor Plexin-B2 for confined migration through restricted space. Using live-cell imaging to track GBM cells negotiating microchannels, we reveal endocytic vesicle accumulation at cell front and filamentous actin assembly at cell rear in a polarized manner. These processes are interconnected and require Plexin-B2 signaling. We further show that Plexin-B2 governs membrane tension and other membrane features such as endocytosis, phospholipid composition, and inner leaflet surface charge, thus providing biophysical mechanisms by which Plexin-B2 promotes GBM invasion. Together, our studies unveil how GBM cells regulate membrane tension and mechano-electrical coupling to adapt to physical constraints and achieve polarized confined migration., Competing Interests: Competing interests: K.D. Costa discloses his role as scientific co-founder and Chief Scientific Officer of Novoheart, Medera Inc. NovoHeart did not play any role in the design, conduct, or funding of this study. The other authors declare no competing interests., (© 2024. The Author(s).)

Background: Neutrophil extracellular traps (NETs) play a significant role in the development of acute pancreatitis (AP). The actin-binding protein LASP1 regulates proteins associated with the cytoskeleton, yet its precise involvement in NETs and AP remains to be elucidated., Methods: To investigate the role of LASP1 in NETs and AP, several bioinformatics methods, such as weighted gene co-expression network analysis (WGCNA), differential analysis, and least absolute shrinkage and selection operator (LASSO) regression, were utilized to screen for feature genes based on the Gene Expression Omnibus (GEO) dataset. To further assess the impact of LASP1, both an in vitro model of 12-myristic-13-acetate phobolol (PMA)-induced NETs and a caerulein-induced AP model were employed., Results: Through WGCNA, AP-related module genes were screened, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were conducted to identify enriched pathways and functions. Six characteristic genes were identified through LASSO regression screening, with LASP1 being the most distinct. LASP1 reduces the generation of NETs induced by PMA in vitro. Mechanistically, LASP1 may increase F-actin protein levels by inhibiting the depolymerization of F-actin. Furthermore, our study utilizing a mouse AP model demonstrated that the LSAP1 recombinant protein effectively alleviated pancreatic necrosis in mice afflicted with AP., Conclusion: LASP1 inhibits the formation of NETs and may alleviate AP by increasing the level of F-actin protein., Competing Interests: Declaration of competing interest The authors declare that there is no economic conflict of interest in the described work. The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Progressive lung fibrosis is often fatal and has limited treatment options. Though the mechanisms are poorly understood, fibrosis is increasingly linked with catecholamines such as adrenaline (AD) and noradrenaline (NA) and hormones such as aldosterone (ALD). The essential functions of the adrenal glands include the production of catecholamines and numerous hormones, but the contribution of adrenal glands to lung fibrosis remains less well studied. Here, we characterized the impact of surgical adrenal ablation in the bleomycin model of lung fibrosis. Wild-type mice underwent surgical adrenalectomy or sham surgery followed by bleomycin administration. We found that although bleomycin-induced collagen overdeposition in the lung was not affected by adrenalectomy, histologic indices of lung remodeling were ameliorated. These findings were accompanied by a decrease of lymphocytes in bronchoalveolar lavage (BAL) and macrophages in lung tissues, along with concomitant reductions in alpha-smooth muscle actin (αSMA) and fibronectin. Surgical adrenalectomy completely abrogated AD, not NA, detection in all compartments. Systemic ALD levels were reduced after adrenalectomy, whereas ALD levels in lung tissues remained unaffected. Taken together, these results support the presence of a pulmonary-adrenal axis in lung fibrosis and suggest that adrenalectomy is protective in this disease. Further investigation will be needed to better understand this observation and aid in the development of novel therapeutic strategies. NEW & NOTEWORTHY The lung-adrenal axis plays a significant role in pulmonary fibrosis. Adrenalectomy provides protection against lung fibrotic ECM remodeling and lung inflammation by reducing the levels of lymphocytes in BAL and macrophages in lung of bleomycin-treated mice. Although compared with sham surgery, adrenalectomy raised collagen concentration in uninjured mice, there was no discernible difference in bleomycin-induced collagen accumulation. However, adrenalectomy significantly reversed the enhanced expression and colocalization of αSMA and fibronectin induced by bleomycin.

A bioluminescence-based RNA monitoring method in living cells was developed using a split NanoLuc (NLuc) reconstitution technique. For specific recognition of a target RNA sequence, a mutant PUM-HD (mPUM) was used. The method was applied to β-actin mRNA in various cells, including primary cultures of rat hippocampal neurons. It allowed for continuous observation of intracellular localization distribution of the target mRNA in living cells, providing insights into various biological phenomena involving intracellular RNA localization and dynamics., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The LIM domain kinases (LIMKs) are important actin cytoskeleton regulators. These proteins, LIMK1 and LIMK2, are nodes downstream of Rho GTPases and are the key enzymes that phosphorylate cofilin/actin depolymerization factors to regulate filament severing. They therefore perform an essential role in cascades that control actin depolymerization. Signaling of the LIMKs is carefully regulated by numerous inter- and intra-molecular mechanisms. In this review, we discuss recent findings that improve the understanding of LIM domain kinase regulation mechanisms. We also provide an up-to-date review of the role of the LIM domain kinases, their architectural features, how activity is impacted by other proteins, and the implications of these findings for human health and disease., (© 2024 Wiley Periodicals LLC.)

While apoptosis dismantles the cell to enforce immunological silence, pyroptotic cell death provokes inflammation. Little is known of the structural architecture of cells undergoing pyroptosis, and whether pyroptotic corpses are immunogenic. Here we report that inflammasomes trigger the Gasdermin-D- and calcium-dependent eruption of filopodia from the plasma membrane minutes before pyroptotic cell rupture, to crown the resultant corpse with filopodia. As a rich store of F-actin, pyroptotic filopodia are recognized by dendritic cells through the F-actin receptor, CLEC9A (DNGR1). We propose that cells assemble filopodia before cell rupture to serve as a posthumous mark for a cell that has died by gasdermin-induced pyroptosis, or MLKL-induced necroptosis, for recognition by dendritic cells. This study reveals the spectacular morphology of pyroptosis and identifies a mechanism by which inflammasomes induce pyroptotic cells to construct a de novo alarmin that activates dendritic cells via CLEC9A, which coordinates the transition from innate to adaptive immunity 1,2 ., Competing Interests: Competing interests: K.S., M.H.L. and J.C.K. declare the following competing interests: K.S. is a coinventor on patent applications for NLRP3 inhibitors that have been licensed to Inflazome Ltd. K.S. served on the Scientific Advisory Board of Inflazome (2016–2017) and Quench Bio, USA (2018–2021) and serves on a Scientific Advisory Board for Novartis, Switzerland (since 2020). M.H.L. is an inventor on patents relating to CLEC9A antibodies. J.C.K. consults and holds equity in Corner Therapeutics, Larkspur Biosciences, MindImmune Therapeutics and Neumora Therapeutics; none of these relationships affected this study. The other authors declare no competing interests., (© 2024. The Author(s).)

To determine the impact of increased glucose concentration on gene expression of primary healthy human limbal fibroblasts (LFCs) and congenital aniridia human limbal fibroblasts (AN-LFCs), in vitro. LFCs (n = 8) and AN-LFCs (n = 8) were isolated and cultured in serum containing DMEM, including either normal glucose (17.5 mM) or increased glucose (70 mM) concentration for 48h or 72h, respectively. mRNA and protein expression of transforming growth factor beta 1 (TGF-β1), alpha-smooth muscle actin (ACTA)2A1, SMAD 2/3, hypoxia markers such as nuclear factor kappa B (NFκB), inducible nitric oxide synthase (iNOS), hypoxia-inducible factor 1-alpha (HIF-1ɑ), oxidative stress markers such as nuclear factor erythroid 2-related factor 2 (Nrf2) and Catalase (CAT) were analyzed using qPCR and Western blot. In 70 mM glucose concentration medium for 48 h, TGF-β1 mRNA expression was significantly lower (p = 0.001, p < 0.001), Nrf2 (p = 0.001, p = 0.001) and CAT (p = 0.001, p = 0.001) mRNA expression was significantly higher in LFCs and AN-LFCs, than using 17.5 mM glucose concentration medium. In addition, in 70 mM glucose concentration medium for 48 h, SMAD 2, SMAD 3, NFκB, HIF-1ɑ mRNA expression was significantly lower in AN-LFCs, than in 17.5 mM glucose concentration medium (p = 0.003, p = 0.002, p = 0.008, p = 0.020). At this time-point in 70 mM glucose concentration medium, at protein level, TGF-β1, SMAD2/3 and NFκB were significantly lower in AN-LFCs, than in 17.5 mM glucose concentration medium (p = 0.041, p = 0.002, p = 0.012). In 70 mM glucose concentration medium for 72h, TGF-β1 was significantly higher (p < 0.001, p < 0.001) and Nrf2 (p = 0.001, p = 0.001) and CAT (p < 0.001, p < 0.001) mRNA were significantly lower in LFCs and AN-LFCs, than in 17.5 mM glucose concentration medium. At this time-point, in 70 mM glucose concentration medium, NFκB mRNA was significantly higher (p < 0.001) in LFCs, than in 17.5 mM glucose concentration DMEM medium. In 70 mM glucose concentration medium for 72 h, TGF-β1 and NFκB protein were significantly lower in AN-LFCs, than in 17.5 mM glucose concentration medium (p < 0.001, p < 0.001). Our study confirmed that high glucose concentration has an impact on TGF-β1 and NFκB signaling both in AN-LFCs and LFCs. These findings highlight that prolonged exposure to high glucose levels may contribute to cellular stress and dysfunction in LFCs and AN-LFCs., (Copyright © 2024. Published by Elsevier Ltd.)

Actin is one of the most widespread and most conserved proteins. At the same time, six actin isoforms are known, encoded by different genes. These isoforms differ slightly in amino acid sequence and have similar structures, but differ in localization and functioning. During functioning, actin interacts with a large number of proteins, which are combined according to this feature into a pool of so-called actin-binding proteins. The question arises whether and how the proteins interacting with different actin isoforms differ. Since the pool of actin-binding proteins includes hundreds of proteins, it was logical to use bioinformatics analysis to solve the questions. In this work, it is shown that the functionality of the α-, β-, and γ-actin interactomes differ significantly, but their structural characteristics are close., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The force produced by a muscle depends on its contractile history, yet human movement simulations typically employ muscle models that define the force-length relationship from measurements of fiber force during isometric contractions. In these muscle models, the total force-length curve can have a negative slope at fiber lengths greater than the fiber length at which peak isometric force is produced. This region of negative stiffness can cause numerical instability in simulations. Experiments have found that the steady-state force in a muscle fiber following active stretching is greater than the force produced during a purely isometric contraction. This behavior is called residual force enhancement. We present a constitutive model that exhibits force enhancement, implemented as a hyperelastic material in the febio finite element software. There is no consensus on the mechanisms responsible for force enhancement; we adopt the assumption that the passive fiber force depends on the sarcomere length at the instant that the muscle is activated above a threshold. We demonstrate the numerical stability of our model using an eigenvalue analysis and by simulating a muscle whose fibers are of different lengths. We then use a three-dimensional muscle geometry to verify the effect of force enhancement on the development of stress and the distribution of fiber lengths. Our proposed muscle material model is one of the few models available that exhibits force enhancement and is suitable for simulations of active lengthening. We provide our implementation in febio so that others can reproduce and extend our results., (Copyright © 2025 by ASME.)

The epiblast is a pluripotent cell population formed in the late blastocyst stage of preimplantation embryos. During the process of epiblast formation from the inner cell mass (ICM) of the early blastocyst, activation of the Hippo pathway transcription factor TEAD by the nuclear translocation of the coactivator protein YAP is required for the robust expression of pluripotency factors. However, the mechanisms that alter YAP localization during epiblast formation remain unknown. Here, we reveal two such mechanisms. Expansion of the blastocoel promotes nuclear YAP localization by increasing cytoplasmic F-actin and reducing YAP phosphorylation. Additionally, cell differentiation regulates YAP. Expression of the junctional Hippo component, AMOT, gradually decreases during epiblast formation through a tankyrase-mediated degradation. SOX2 expression in the ICM is necessary for the reduction of AMOT and YAP phosphorylation. These two mechanisms function in parallel. Thus, the blastocoel-F-actin and SOX2-AMOT axes cooperatively suppress YAP phosphorylation and promote YAP nuclear localization during epiblast formation. The cooperation of these two distinct mechanisms likely contributes to the robustness of epiblast cell differentiation., Competing Interests: Declarations of interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

SIL1 is a nucleotide exchange factor for the molecular chaperone protein Bip in the endoplasmic reticulum that plays a crucial role in protein folding. The Sil1 gene is currently the only known causative gene of Marinesco-Sjögren syndrome (MSS). Intellectual developmental disability is the main symptom of MSS, and its mechanism has not been fully elucidated. Studies have shown that mutations in the Sil1 gene can delay neuronal migration during cortical development, but the underlying molecular mechanisms remain unclear. To further identify potential molecules involved in the regulation of central nervous system development by SIL1, we established a cortical neuron model with SIL1 protein deficiency and used proteomic analysis to screen for differentially expressed proteins after Sil1 silencing, followed by GO functional enrichment and protein‒protein interaction (PPI) network analysis. We identified 68 upregulated and 137 downregulated proteins in total, and among them, 10 upregulated and 3 downregulated proteins were mainly related to actin cytoskeleton dynamics. We further validated the differential changes in actin-related molecules using qRT‒PCR and Western blotting of a Sil1 gene knockout (Sil1 -/- ) mouse model. The results showed that the protein levels of ACTN1 and VIM decreased, while their mRNA levels increased as a compensatory response to protein deficiency. The mRNA and protein levels of IQGAP1 both showed a secondary increase. In conclusion, we identified ACTN1 and VIM as the key molecules regulated by SIL1 that are involved in neuronal migration during cortical development., Competing Interests: Declarations. Ethics Approval: All procedures involving animals were reviewed and approved by the Animal Care and Use Committee of Kunming Medical University (license number: SCXK (Dian) k2015-0002 and kmmu20230085). Consent to Participate: All authors have read the journal’s authorship agreement and conflict of interest policy. Consent for Publication: Not applicable. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

We present protocols for using an optogenetic tool called LILAC for actin imaging. LILAC is a light-controlled version of Lifeact that uses the Avena sativa LOV2 (AsLOV2) domain. By significantly reducing Lifeact's affinity for the cytoskeleton in the dark, LILAC reduces concentration-dependent negative side effects while enabling new image processing methods. We discuss the considerations for using this probe of live-cell actin dynamics, including fluorescent protein selection, cell maintenance, microscopy protocols, and image processing. Our work highlights the potential of AsLOV2-based optogenetics for novel imaging and control tools in cell biology., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The endothelial glycocalyx, lining the apical surface of the endothelium, is involved in a host of vascular processes. The glycocalyx is comprised of a network of membrane-bound proteoglycans and glycoproteins along with associated plasma proteins. One such glycoprotein is endomucin (EMCN), which our lab has revealed is a modulator of VEGFR2 function. Intravitreal injection of siEMCN into the eyes of P5 mice impairs vascular development. In vitro silencing of EMCN suppresses VEGF-induced proliferation and migration. Signaling pathways that drive cell migration converge on cytoskeletal remodeling. By coupling co-immunoprecipitation with liquid chromatography/mass spectrometry, we identified interactions between EMCN and proteins associated with actin cytoskeleton organization. The aim of the study was to investigate the influence of EMCN on cytoskeleton dynamics in angiogenesis. EMCN depletion resulted in reduction of F-actin levels, whereas overexpression of EMCN induced increased membrane protrusions in cells that were rich in stress fibers. The reorganization of the actin filaments did not depend on VEGFR2 signaling, suggesting that EMCN connects the cytoskeleton and the glycocalyx., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

To understand the mechanics and kinetic properties of cytoskeletal molecular motors such as myosin, typically the motor of interest needs to be expressed and purified and then analyzed using a range of in vitro-based assays. In this chapter, we describe how to express and purify myosin using the insect cell system, how to characterize the purified protein by mass photometry and negative-stain EM to assess its quality, and how to perform in vitro assays in which fluorescently labeled myosin walks along actin tracks, including a brief description of adapting these assays for MINFLUX imaging., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mechanosensation allows cells to generate intracellular signals in response to mechanical cues from their environment. Previous research has demonstrated that mechanical stress can alter the mechanical properties of the nucleus, affecting gene transcription, chromatin methylation, and nuclear mechanoprotection during mechanical loading. PIEZO1, a mechanically gated Ca 2+ ion channel, has been shown to be important in sensing mechanical stress, however its signal transduction pathway is not thoroughly understood. In this study, we used primary porcine chondrocytes to determine whether PIEZO1 activation and subsequent Ca 2+ influx altered nuclear mechanical properties, and whether these effects involved the actin cytoskeleton. We discovered that activating PIEZO1 with Yoda1, a specific small-molecule agonist, induces transient nuclear softening-a previously identified mechanoprotective response. This PIEZO1-mediated nuclear softening is abolished by inhibiting actin cytoskeleton remodeling with Latrunculin A or by removing extracellular Ca 2+ . Notably, PIEZO1-mediated nuclear softening did not lead to significant changes in gene expression or heterochromatin methylation. Our findings demonstrate that actin cytoskeleton remodeling following Ca 2+ influx facilitates PIEZO1 signal transduction to the nucleus but does not induce lasting gene expression changes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background/aim: Silicosis, the most severe type of occupational pneumoconiosis, leads to diffuse pulmonary fibrosis without specific therapy. Ferroptosis is triggered by reactive oxygen species (ROS) and Fe 2+ overload-induced lipid peroxidation, which is involved in the progression of pulmonary fibrosis. As an important coenzyme in the process of aerobic respiration, Coenzyme Q10 (CoQ10) can enhance mitochondrial function and energy supply and reduce malondialdehyde (MDA) to limit the risk of fibrosis. We aimed to clarify whether ferroptosis is involved in the process of coenzyme CoQ10-treated silicosis fibrosis., Materials and Methods: C57BL/6J mice were divided in 3 groups (n=6 in each group). In the normal group, mice underwent sham operation; in the silicosis group, mice were tracheally instilled with SiO 2 suspension; in CoQ10 group, mice with silicosis were treated with CoQ10 solution. Histological analyses were performed to assess the lung injury level. Iron content was measured by colorimetry in lung tissue. The levels of MDA in lung tissue were characterized by immunofluorescence staining. The level of alpha smooth muscle actin (α-SMA), Collagen I, GPX4, p53 expression was analyzed by qRT-PCR and western blotting., Results: CoQ10 significantly reduced the mRNA and protein expression levels of α-SMA and collagen I in silicosis lung tissues. It is worth noting that CoQ10 significantly inhibited the accumulation of lipid peroxidation and Fe 2+ level by increasing the expression of ferroptosis regulatory core enzyme GPX4 and reducing its upstream regulator p53 in silicosis lung tissues., Conclusion: CoQ10 alleviated silicosis fibrosis via inhibiting ferroptosis in mice. This finding is a new perspective for exploring the pathogenesis and treatment for silicosis., (Copyright © 2025, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of glaucoma, the leading cause of irreversible vision loss and blindness. An overall increase in resistance to aqueous humor outflow causes sustained elevation in IOP. Glaucomatous insults in the aqueous humor outflow pathway, including the trabecular meshwork (TM), precede such chronic physiological changes in IOP. These insults include ultrastructural changes with excessive extracellular matrix deposition and actin cytoskeletal reorganization that leads to pathological stiffening of the ocular tissues. One of the most common cytoskeletal changes associated with TM tissue stiffness in glaucoma is the increased prevalence of cross-linked actin networks (CLANs) in cells of the trabecular meshwork (TM) and lamina cribrosa (LC). In glaucomatous cells, rearrangement of linear actin stress fibers leads to formation of polygonal arrays within the cytoplasm, resembling a geodesic dome-like structure, that we identified as CLANs. In addition to increased amounts of CLANs in POAG TM cells and tissues, we also discovered that glucocorticoid (GC) and TGFβ2 signaling pathways associated with the development of ocular hypertension (OHT) and glaucoma also induced CLANs in the TM. Despite a clear association, we are yet to completely understand the mechanisms involved in CLAN formation and their direct relevance to disease pathology. In this chapter, we will describe methods to identify and characterize CLANs using fluorescent microscopy in primary TM cell cultures, ex vivo perfusion cultured human anterior segments, and in situ in human donor eyes., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

An emerging instigator of endothelial dysfunction in type 2 diabetes (T2D) is the stiffening of the cell. Previous reports suggest that polymerization of filamentous actin (F-actin) is a potential mediator of endothelial stiffening. Actin polymerization is limited by active cofilin, an F-actin-severing protein that can be oxidized, leading to its inactivation and loss of severing capability. Yet, whether these mechanisms are implicated in endothelial stiffening in T2D remains unknown. Herein, we report that endothelial cells exposed to plasma from male and female subjects with T2D, and the aortic endothelium of diabetic male mice ( db/db ), exhibit evidence of increased oxidative stress, F-actin, and stiffness. Furthermore, we show reactive oxygen species, including H 2 O 2 , are increased in the endothelium of mesenteric arteries isolated from db/db male mice, and that exposure of endothelial cells to H 2 O 2 induces F-actin formation. We also demonstrate, in vitro, that cofilin-1 can be oxidized by H 2 O 2 , leading to reduced F-actin severing activity. Finally, we provide evidence that genetic silencing or pharmacological inhibition of LIM kinase 1, an enzyme that phosphorylates and thus inactivates cofilin, reduces F-actin and cell stiffness. In aggregate, this work supports the inactivation of cofilin as a potential novel mechanism underlying endothelial stiffening in T2D. NEW & NOTEWORTHY Cell stiffening is an emerging contributor to endothelial dysfunction, a classic feature of type 2 diabetes (T2D). However, the mechanisms underlying endothelial stiffening remain largely unknown. This work provides evidence that oxidative stress-induced inactivation of cofilin, a key F-actin severing protein, may be implicated in increasing endothelial F-actin and cell stiffness in T2D.

Damage-accumulation failure models are broadly used to examine tissue property changes caused by mechanical loading. However, damage accumulation models are purely phenomenological. The underlying justification in using this type of model is often that damage occurs to the extracellular fibers and/or cells which changes the fundamental mechanical behavior of the system. In this work, we seek to align damage accumulation models with microstructural models to predict alterations in the mechanical behavior of biological materials that arise from structural heterogeneity associated with nonuniform remodeling of tissues. Further, we seek to extend this multiscale model toward assessing catastrophic failure events such as cerebral aneurysm rupture. First, we demonstrate that a model made up of linear elastin and actin and nonlinear collagen fibers can replicate bot the pre-failure and failure tissue-scale mechanics of uniaxially-stretched cerebral aneurysms. Next, we investigate how mechanical heterogeneities, like those observed in cerebral aneurysms, influence fiber and tissue failure. Notably, we find that failure occurs and the interface between regions of high and low material stiffness, suggesting that spatial mechanical heterogeneity influences aneurysm failure behavior. This model system is a step toward linking structural changes in growth and remodeling to failure properties., 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.)

Purpose: To examine microscopically the distribution of the lymphatic channels in comparison with the blood vessels in the upper eyelid., Methods: Central sagittal sections of 13 upper eyelids (8 right, 5 left) from 11 Japanese cadavers, aged 36 to 87 years old (average age: 66.6 years), were histologically examined. The specimens were fixed in 10% formalin. Staining with D2-40 for lymphatic channels and with the antibody for α-smooth muscle actin for blood vessels was utilized., Results: The lymphatic channels were mainly distributed superficially, just under the skin, but the blood vessels were situated deeper, and diffusely spread throughout the upper eyelid., Conclusion: The positional predispositions of the lymphatic channels and the blood vessels were different. The former is mainly superficial and the latter is deeper and diffuse., Competing Interests: The authors have no financial or conflicts of interest to disclose., (Copyright © 2024 The American Society of Ophthalmic Plastic and Reconstructive Surgery, Inc.)

Aims: Aortic aneurysms (AA) frequently involve dysregulation of transforming growth factor β (TGF-β)-signalling in the aorta. Here, FURIN was tested as aneurysm predisposition gene given its role as proprotein convertase in pro-TGF-β maturation., Methods and Results: Rare FURIN variants were detected by whole-exome sequencing of 781 unrelated aortic aneurysm patients and affected relatives. Thirteen rare heterozygous FURIN variants occurred in 3.7% (29) unrelated index AA patients, of which 72% had multiple aneurysms or a dissection. FURIN maturation and activity of these variants were decreased in vitro. Patient-derived fibroblasts showed decreased pro-TGF-β processing, phosphorylation of downstream effector SMAD2 and kinases ERK1/2, and steady-state mRNA levels of the TGF-β-responsive ACTA2 gene. In aortic tissue, collagen and fibrillin fibres were affected. One variant (R745Q), observed in 10 unrelated cases, affected TGF-β signalling variably, indicating effect modification by individual genetic backgrounds., Conclusion: FURIN is a novel, frequent genetic predisposition for abdominal-, thoracic-, and multiple aortic or middle sized artery aneurysms in older patients, by affecting intracellular TGF-β signalling, depending on individual genetic backgrounds., Competing Interests: Conflict of interest The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Th2 inflammation and epithelial-mesenchymal transition (EMT) play crucial roles in the pathophysiology of chronic rhinosinusitis with nasal polyps (CRSwNP). This study aimed to investigate the hypothesis that MMP-12, produced by M2 macrophages, induces EMT in nasal epithelial cells, thereby contributing to airway inflammation and remodeling in CRSwNP. The expression levels of MMP-12 were measured by RT-PCR in CRS nasal mucosa and THP-1 cells. mRNA and protein levels of E-cadherin, vimentin, α-SMA, and fibronectin were determined using RT-PCR, western blotting, and immunofluorescence staining in primary nasal epithelial cells and air-liquid interface culture. The expression of MMP-12 was significantly increased in CRSwNP and M2-like THP-1 cells. In co-culture with primary nasal epithelial cells and M2-like THP-1 cells, E-cadherin expression was inhibited, and fibronectin, vimentin, and α-SMA expression were increased. MMP-12 decreased E-cadherin but induced fibronectin, vimentin, and α-SMA mRNA and protein expression in primary nasal epithelial cells and air-liquid interface culture. MMP408, an MMP-12 inhibitor, inhibited EMT-related factors. These findings suggest that MMP-12 expression in M2 macrophages induces EMT in nasal epithelial cells and may contribute to the pathogenesis of CRSwNP., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Park et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In addition to the well-known monomeric and polymeric forms of actin there is another unique thermodynamically stable state of this protein, called "inactivated actin" (I-actin). I-actin is formed at moderate concentration of a denaturant, release of Ca 2+ ions and/or ATP, or after heating. This state is a monodisperse associate and it has the same spectral characteristics regardless of the method of preparation. The interest in I-actin arises from the discovery of similar-sized short oligomers of actin in the cell nucleus, which structurally differ from polymeric actin. In this work, we investigated the intramolecular mobility of I-actin using the time-resolved anisotropy method. Our findings indicate that its tryptophan residues participate in structural oscillations, although their correlation time is significantly longer than that of native actin. Using the dynamic light scattering, we demonstrated that I-actin obtained by heating possesses the same dimensions as I-actin in 1.8 M GdnHCl. Using the fluorescent probe ANS, it was shown that I-actin has a unique structure with hydrophobic pockets on the surface and tryptophan residues in the polar internal regions of the structure., 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.)

In this paper, we present a refined version of the previously proposed suspension contractile model based on catch muscle proteins of the Gray's mussel (Crenomytilus grayanus). The objective of this model was to test the current hypotheses about the catch state, a unique phenomenon observed in the adductor muscle of bivalve molluscs. This state allows the muscle to maintain the force developed by contraction for a long time with minimum energy expenditure. Our study has resolved the issue of constructing a catch muscle model capable of controlled contraction and relaxation in an ATP-resistant manner. As a result, we have gained insight into the functional role of myorod, a unique protein of the catch muscle, and have tested the modified myorod-links hypothesis of the catch state. Myorod was shown capable of forming strong and functional cross-links between actin and synthetic thick filaments. This finding removes the last shortcoming of the myorod-links hypothesis of the catch state. There is now no doubt that myorod plays the main role of the catch-link we have been seeking all this time., 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.)

Idiopathic pulmonary fibrosis (IPF) is a fatal disease defined by a progressive decline in lung function due to scarring and accumulation of extracellular matrix (ECM) proteins. The SOCS (Suppressor Of Cytokine Signaling) domain is a 40 amino acid conserved domain known to form a functional ubiquitin ligase complex targeting the Von Hippel Lindau (VHL) protein for proteasomal degradation. Here we show that the SOCS conserved domain operates as a molecular tool, to disrupt collagen and fibronectin fibrils in the ECM associated with fibrotic lung myofibroblasts. Our results demonstrate that fibroblasts differentiated using TGFβ, followed by transduction with the SOCS domain, exhibit significantly reduced levels of the contractile myofibroblast-marker, α-SMA. Furthermore, in support of its role to retard differentiation, we find that lung fibroblasts expressing the SOCS domain present with significantly reduced levels of α-SMA and fibrillar fibronectin after differentiation with TGFβ. We show that adenoviral delivery of the SOCS domain in the fibrotic phase of experimental lung fibrosis in mice, significantly reduces collagen accumulation in disease lungs. These data underscore a novel function for the SOCS domain and its potential in ameliorating pathologic matrix deposition in lung fibroblasts and experimental lung fibrosis., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The actomyosin cytoskeleton, a protein assembly comprising actin fibers and the myosin molecular motor, drives various cellular dynamics through contractile force generation at high densities. However, the relationship between the density dependence of the actomyosin cytoskeleton and force-controlled ordered structure remains poorly understood. In this study, we measured contraction-driven flow generation by varying the concentration of cell extracts containing the actomyosin cytoskeleton and associated nucleation factors. We observed continuous actin flow toward the center at a critical actomyosin density in cell-sized droplets. The actin flow exhibited an emergent oscillation in which the tracer advection in the bulk solution periodically changed in a stop-and-go fashion. In the vicinity of the actomyosin density where oscillatory dynamics occur, the velocity of tracer particle motion decreases with actomyosin density but exhibits superdiffusive motion. Furthermore, the increase or decrease in myosin activity causes the oscillatory flow generation to become irregular, indicating that the density-dependent flow generation of actomyosin is driven by an interplay between actin density and myosin force generation., Competing Interests: Declarations. Competing interests: There are no conflicts of interest to declare., (© 2024. The Author(s).)

Background: Understanding drug addiction as a disorder of maladaptive learning, where drug-associated or environmental cues trigger drug cravings and seeking, is crucial for developing effective treatments. Actin polymerization, a biochemical process, plays a crucial role in drug-related memory formation, particularly evident in conditioned place preference paradigms involving drugs like morphine and methamphetamine. However, the role of actin polymerization in the reconsolidation of heroin-associated memories remains understudied., Methods: This study employed a rodent model of self-administered heroin to investigate the involvement of actin polymerization in the reconsolidation of heroin-associated memories. Rats underwent ten days of intravenous heroin self-administration paired with conditioned cues. Subsequently, a 10-day extinction phase aimed to reduce heroin-seeking behaviors. Following this, rats participated in a 15-minute retrieval trial with or without cues. Immediately post-retrieval, rats received bilateral injections of the actin polymerization inhibitor Latrunculin A (Lat A) into the nucleus accumbens core (NACc), a critical brain region for memory reconsolidation., Results: Immediate administration of Lat A into the NACc post-retrieval significantly reduced cue-induced and heroin-primed reinstatement of heroin-seeking behavior for at least 28 days. However, administering Lat A 6-hour post-retrieval or without a retrieval trial, as well as administering Jasplakionlide prior to memory reactivation did not affect heroin-seeking behaviors., Conclusions: Inhibiting actin polymerization during the reconsolidation window disrupts heroin-associated memory reconsolidation, leading to decreased heroin-seeking behavior and prevention of relapse. These effects are contingent upon the presence of a retrieval trial and exhibit temporal specificity, shedding light on addiction mechanisms and potential therapeutic interventions., (© The Author(s) 2024. Published by Oxford University Press on behalf of CINP.)

Pterygium is an ocular disease in which the conjunctival tissue invades the cornea. When the pterygium tissue reaches the pupillary region, the visual function of the patient is affected. Currently, surgical removal is the only effective treatment. However, the recurrence rate of pterygium after surgery can be high. Pterygium is also a health disparity issue since it is more prevalent in the Hispanic and Latino American population. In this study, we determined if the Rho kinase inhibitor can be used to prevent pterygium recurrence since its anti-fibrosis effects have been reported in other cell and tissue types. We cultured primary pterygium cells from pterygium tissues from Hispanic and Latino American, African American, Caucasian, and Asian donors, and used those cells for viability assays, scratch assays, migration assays, and immunostaining of F-actin, fibronectin, collagen I and α smooth muscle actin. We found that the Rho kinase inhibitor Y27632 decreased cell viability, wound healing, cell migration, as well as the expression of extracellular matrix and myofibroblast markers in cultured pterygium cells. We believe that Rho kinase inhibitors are a potential post-surgical treatment to prevent pterygium recurrence., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The Phenotypic states of vascular smooth muscle cells (SMCs) are essential to understanding vascular pathophysiology. SMCs in vessels generally express a specific set of contractile proteins, but decreased contractile protein expression, indicating a phenotypic shift, is a hallmark of vascular diseases. Recent studies have suggested the relation of abnormally high wall shear stress (WSS) of approximately 20 Pa with the aortic disease pathogenesis. However, due to the lack of appropriate experimental models to assess SMC phenotypic states, the details of the phenotypic shift under high WSS conditions remain unclear. In this study, we developed a coculture model where vascular endothelial cells (ECs) were cocultured with SMCs expressing calponin 1, a contractile protein involved in the phenotypic shift of SMCs. We investigated the effects of a pathologically high WSS condition on the phenotypic states of SMCs. Increased calponin 1 expression was found upon exposure to 20 Pa WSS compared with a physiological 2 Pa condition, whereas the expression of another contractile protein, α-smooth muscle actin (αSMA) remained unchanged. Furthermore, the inhibition of EC-derived nitric oxide (NO), which is associated with endothelial dysfunction in vascular diseases, resulted in a trend of decreasing αSMA and Calponin 1 expression under 20 Pa WSS conditions compared with 2 Pa. Our findings suggest that EC-derived NO under pathologically high WSS conditions may impact the expression of contractile proteins implicated in aortic pathophysiology., 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.)

Filopodia, microspikes and cytonemes are implicated in sensing the environment and in dissemination of morphogens, organelles and pathogens across tissues. Their major structural component is parallel bundles of actin filaments that assemble from the cell membrane. Whilst the length of filopodia is central to their function, it is not known how their lengths are determined by actin bundle dynamics. Here, we identified a set of monoclonal antibodies that lengthen filopodia-like structures formed in a cell-free reconstitution system, and used them to uncover a key molecular switch governing length regulation. Using immunolabelling, enzyme-linked immunosorbent assays, immunoprecipitation and immunoblock experiments, we identified four antibodies that lengthen actin bundles by selectively binding the open DNase 1-binding loop (D-loop) of actin filaments. The antibodies inhibit actin disassembly and their effects can be alleviated by providing additional actin or cofilin. This work indicates that maintaining an open state of the actin filament D-loop is a mechanism of generating long filopodia-like actin bundles., (© 2024 The Author(s).)

Objectives: To investigate the inhibitory effect of Danshen Injection on endothelial-mesenchymal transition (EndMT) induced by peritoneal dialysis fluid in HMrSV5 cells and the role of the TGF‑β/Smad signaling pathway in mediating this effect., Methods: HMrSV5 cells cultured in 40% peritoneal dialysis solution for 72 h to induce EndMT were treated with 0.05%, 0.1% and 0.5% Danshen Injection. CCK-8 assay was used to assess the changes in viability of the treated cells, and the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF) in the cell supernatant were detected using ELISA; Western blotting was performed to detect the protein expressions of E-cadherin, α-smooth muscle actin (α-SMA), p-Smad 2/3, and Smad 7 in the cells., Results: Culture in 40% peritoneal dialysis fluid for 72 induced significant EndMT in HMrSV5 cells, which exhibited obviously lowered cell viability. Danshen Injection within the concentration range of 0.025%-1.5% did not significantly affect the viability of the cells. Exposure of HMrSV5 cells to peritoneal dialysis fluid for 72 h significantly increased the production of IL-6, TNF‑α, TGF‑β and VEGF, upregulated the protein expressions of α‑SMA and p-Smad 2/3, and lowered the expressions of E-cadherin and Smad7 proteins. Treatment of the exposed cells with Danshen injection significantly increased cell viability and cellular expressions of E-cadherin and Smad 7 proteins and reduced the production of IL-6, TNF-α, TGF-β and VEGF and the protein expressions of α‑SMA and p-Smad 2/3., Conclusions: Danshen Injection can suppress peritoneal dialysis fluid-induced EndMT in HMrSV5 cells possibly by regulating the TGF-β/Smad signaling pathway.

A growing body of evidence suggests that actin plays a role in nuclear architecture, genome organisation, and regulation. Our study of human lung adenocarcinoma cells demonstrates that the equilibrium between actin isoforms affects the composition of the nuclear lamina, which in turn influences nuclear stiffness and cellular behaviour. The downregulation of β-actin resulted in an increase in nuclear area, accompanied by a decrease in A-type lamins and an enhancement in lamin B2. In contrast, the suppression of γ-actin led to upregulation of the lamin A/B ratio through an increase in A-type lamins. Histone H3 post-translational modifications display distinct patterns in response to decreased actin isoform expression. The level of dimethylated H3K9me2 declined while acetylated H3K9ac increased in β-actin-depleted A549 cells. In contrast, the inhibition of γ-actin expression resulted in a reduction in H3K9ac. Based on our observations, we propose that β-actin plays a role in chromatin compaction and deactivation, and is involved in the elevation of nuclear stiffness through the control of the lamins ratio. The non-muscle γ-actin is presumably responsible for chromatin decondensation and activation. The identification of novel functions for actin isoforms offers insights into the mechanisms through which they influence cell fate during development and cancer progression.

Background: Oral cancer; categorised under head and neck cancers (HNC) predominantly originates from squamous cells and is referred as oral squamous cell carcinoma (OSCC). Various factors (internal and external) causes OSCC. PI3K/AKT/mTOR pathways are known to be primarily mutated in HNC. mTOR remains as a key regulator for various physiological and developmental processes in normal and cancer cells. Cancer cells are surrounded by tumor microenvironment, majorly composed of immune cells. Cancer associated fibroblasts, macrophages and regulatory T cells controlled by mTOR, plays an important role in the progression of cancer., Methods: Two hundred sixty retrospective patient samples were collected along with their demographical and clinic-pathological data. Here, we have analysed expression of mTOR, α-SMA, CD163 and FOXp3 using immunohistochemistry and their survival outcomes were calculated using Kaplan-Meier statistical method., Results: Overexpression of CD163 and α-SMA was detected in samples of patients compared to mTOR and FOXp3. Their expression was compared with clinico-oncological parameters. We also observed two and three combinations of markers and its association with the prognosis of the cancer. The results suggest, higher the expression of all the four markers in combination correlated to poor prognosis of patients and vice-versa., Conclusion: The study reveals that over expression of CD163 and α-SMA is strongly associated with disease outcome. The combinations of all the four marker expression profile will be emerging strategy towards prognosis and also to determine survival outcomes in patients. This is a pioneering observation of these combinations of markers in OSCC despite certain limitations., Competing Interests: Declarations. Ethics approval and consent to participate: The study was approved by Institutional Review Board known as Institutional Ethics Committee of Sri Ramachandra Institute of Higher Education and Research, bearing reference number IEC-NI/21/APR/78/53 dated 19.03.2021. The present study utilised samples (tissue blocks) from Department of Oral Pathology, Sri Ramachandra Dental College and Hospital and Department of Pathology, Sri Ramachandra Medical College and Research Institute, SRIHER. Written informed consent from patients towards sample collection have been obtained as part of intervention/procedure. Consent for publication: Not Applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Cortactin (CTTN) is an actin-binding protein regulating actin polymerization and stabilization, which are vital processes for maintaining skeletal muscle homeostasis. Despite the established function of CTTN in actin cytoskeletal dynamics, its role in the myogenic differentiation of progenitor cells remains largely unexplored. In this study, we investigated the role of CTTN in the myogenic differentiation of C2C12 myoblasts by analyzing its effects on actin cytoskeletal remodeling, myocardin-related transcription factor A (MRTFA) nuclear translocation, serum response factor (SRF) activation, expression of myogenic transcription factors, and myotube formation. CTTN expression declined during myogenic differentiation, paralleling the reduction in MyoD, suggesting a potential role in the early stages of myogenesis. We also found that CTTN knockdown in C2C12 myoblasts reduced filamentous actin, enhanced globular actin levels, and inhibited the nuclear translocation of MRTFA, resulting in suppressed SRF activity. This led to the subsequent downregulation of myogenic regulatory factors, such as MyoD and MyoG. Furthermore, CTTN knockdown reduced the nuclear localization of YAP1, a mechanosensitive transcription factor, further supporting its regulatory roles in cell cycle and proliferation. Consequently, CTTN depletion impeded proliferation, differentiation, and myotube formation in C2C12 myoblasts, highlighting its dual role in the coordination of cell cycle regulation and myogenic differentiation of progenitor cells during myogenesis. This study identifies CTTN as an essential regulator of myogenic differentiation via affecting the actin remodeling-MRTFA-SRF signaling axis and cell proliferation.

Symmetry breaking, which is ubiquitous in biological cells, functionally enables directed cell movement and organized embryogenesis. Prior to movement, cells break symmetry to form a well-defined cell front and rear in a process called polarization. In developing and regenerating tissues, collective cell movement requires the coordination of the polarity of the migration machineries of neighboring cells. Though several works shed light on the molecular basis of polarity, fewer studies have focused on the regulation across the cell-cell junction required for collective polarization, thus limiting our ability to connect tissue-level dynamics to subcellular interactions. Here, we investigated how polarity signals are communicated from one cell to its neighbor to ensure coordinated front-to-rear symmetry breaking with the same orientation across the group. In a theoretical setting, we systematically searched a variety of intercellular interactions and identified that co-alignment arrangement of the polarity axes in groups of two and four cells can only be achieved with strong asymmetric regulation of Rho GTPases or enhanced assembly of complementary F-actin structures across the junction. Our results held if we further assumed the presence of an external stimulus, intrinsic cell-to-cell variability, or larger groups. The results underline the potential of using quantitative models to probe the molecular interactions required for macroscopic biological phenomena. Lastly, we posit that asymmetric regulation is achieved through junction proteins and predict that in the absence of cytoplasmic tails of such linker proteins, the likeliness of doublet co-polarity is greatly diminished., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Levandosky, Copos. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Aims: This study investigates the role of actin polymerization and Myh9 in mediating lipid droplet (LD) fission during rosiglitazone-induced browning of white adipocytes. The aim is to understand how LD splitting might contribute to the beige conversion of white adipose tissue, providing insights into adipocyte plasticity and metabolic regulation., Materials and Methods: C3H10 T1/2-differentiated adipocytes were used as a classical model to study white adipocyte browning. Rosiglitazone was applied to induce browning, and the interactions between LDs and actin, as well as the distribution of Myh9, were assessed using immunofluorescence and Western blotting. In vivo, we employed a microfilament inhibitor to block actin polymerization in cold-stimulated mice and evaluated changes in LD morphology and browning. Furthermore, dynamic live-cell imaging using confocal microscopy was conducted to observe the real-time behavior of LDs during the browning process and to determine whether they undergo fission., Main Findings: Our results demonstrate that rosiglitazone significantly induces LD size reduction, a process correlated with the increased contact of LDs with microfilaments. Inhibition of actin polymerization prevented both the reduction in LD size and the browning of white adipocytes, indicating that actin plays a critical role. Myh9 was enriched at the LD fission sites, forming a structure resembling a contractile ring. Overexpression of Myh9 promoted the shrinkage of LD, suggesting that it may be involved in LD fission., Significance: This study identifies actin and Myh9 as key regulators of LD fission in rosiglitazone-induced browning of white adipocytes, offering new insights into the cellular mechanisms of adipocyte plasticity. The findings propose a novel pathway by which LD dynamics contribute to the beige conversion of white fat, with potential implications for metabolic disease therapies targeting adipocyte function and energy expenditure., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tight junctions regulate epithelial barrier function and have been shown to be influenced by multiple classes of proteins. Apical integrins have been identified as potential regulators of epithelial barrier function; however, only indirect approaches have been used to measure integrin regulation of the epithelial barrier. Here, we used polymeric nanowires conjugated with anti-integrin β1 antibodies to specifically target apically localized integrins in either their closed or open conformation. Barrier regulation by apical integrins was found to be conformation specific. Nanowires targeting integrins in the closed conformation increased epithelial permeability and caused zonula occludens-1 (ZO-1, also known as TJP1) to change from a linear to a ruffled morphology. Claudin-2 and claudin-4 colocalized with ZO-1 and were also ruffled; however, claudin-1 and claudin-7 remained linear. Ruffling was dependent on myosin light chain kinases (MLCKs) and Rho kinases (ROCKs). Conversely, targeting integrins in the open conformation decreased epithelial permeability and made junctions more linearized. Anti-integrin β1 nanowires differentially affected actin and talin (analyzed using pan-talin antibodies), depending on whether they contained activating or inhibitory antibodies. Thus, apical integrins can act as a conformation-sensitive switch that regulates epithelial barrier function., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Decidualization of endometrial stromal cells is a prerequisite for successful embryo implantation and early pregnancy. Decidualization dysregulation results in implantation failure. In our previous study, we reported that PAI-1 is abnormally downregulated in the endometrial tissue samples of patients with recurrent implantation failure. This study will explore the dynamic expression changes of PAI-1 in the endometrium during the menstrual cycle and its molecular mechanism affecting endometrial decidualization. Our findings indicated that the abundance of PAI-1 increased in the mid-secretory phase and attached a peak in the decidual phase in the endometrium of women with regular menstrual cycles. In human endometrial stromal cells (HESCs), PAI-1 knockdown attenuated endometrial decidualization by upregulating VEGFR2/PI3K/AKT signaling pathway and impaired the F-actin reorganization. Furthermore, axitinib (a VEGFR2 inhibitor) was used to inhibit the VEGFR2 protein activity and the results suggested that it eliminated the effects of PAI-1 on PI3K/AKT signaling pathways and F-actin remodeling. In addition, the interaction between PAI-1 and KNG1 was confirmed by coimmunoprecipitation assay in HESCs. Altogether, PAI-1-KNG1 may enhance the decidualization of endometrium by inhibiting VEGFR2/PI3K/AKT signaling pathway-mediated F-actin reorganization in healthy females., (© 2024 Federation of American Societies for Experimental Biology.)

During morphogenesis, epithelial sheets undergo sequential folding to form three-dimensional organ structures. The resulting folds are often irreversible, ensuring that morphogenesis progresses in one direction. However, the mechanism establishing folding irreversibility remains unclear. Here, we report a mechanical property of epithelia that determines folding irreversibility. Using a mechanical assay, we demonstrate that long-term, high-curvature folding induces plastic, irreversible deformations, while short-term or low-curvature folding results in an elastic, shape-restoring response. This elastic-plastic transition occurs in a switch-like manner, with critical thresholds in folding curvature and duration. The transition is induced by F-actin accumulating into a bracket-like structure across the fold, triggered by cells sensing deformations via mechanosensitive signaling pathways, including TRPC 3/6-mediated calcium influx and ligand-independent EGFR activation. These results demonstrate that cells control epithelial folding irreversibility by detecting folding characteristics and adaptively switching between elastic and plastic responses, providing mechanical insight into the directionality of morphogenesis., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Amoebozoa is a group of single-celled organisms that change their shape during locomotion. However, there is a taxon-specific complex of morphological characters inherent in the moving amoebae, known as locomotive forms. Actin is one of the proteins most important for amoeboid movement that, together with actin-binding proteins, construct the architecture of the cytoskeleton in the amoeboid cells. One of the actin-binding proteins is the Arp2/3 complex that provides a connection between actin filaments at an angle of 70°. In this paper, we predicted 3D models of bonded subunits Arp2 and Arp3 for 30 species from different taxa of Amoebozoa based on the publicly available transcriptomic data. Moreover, we predicted the binding free energy (ΔG) of bonded subunits Arp2 and Arp3 for 30 species and tried to link it to the morphology of the locomotive forms of amoebae. The ΔG values are the lowest in amoebae with the broad hyaline area, like Vannella spp. Amoebae that produce thin hyaline projections, like Vexillifera abyssalis , are characterized by intermediate ΔG values. Finally, the highest ΔG values are typical for the group of amoebae that have no conspicuous hyaline areas of the cytoplasm, like Pelomyxa shiedti , or have small hyaline caps, like Arcella intermedia . The presented analysis provides new insights into the molecular mechanisms of shape formation in amoeboid cells.

The living cell creates a unique internal molecular environment that is challenging to characterize. By combining single-molecule displacement/diffusivity mapping (SM d M) with physiologically active extracts prepared from Xenopus laevis eggs, we sought to elucidate molecular properties of the cytoplasm. Quantification of the diffusion coefficients of 15 diverse proteins in extract showed that, compared to in water, negatively charged proteins diffused ~50% slower, while diffusion of positively charged proteins was reduced by ~80 to 90%. Adding increasing concentrations of salt progressively alleviated the suppressed diffusion observed for positively charged proteins, signifying electrostatic interactions within a predominately negatively charged macromolecular environment. To investigate the contribution of RNA, an abundant, negatively charged component of cytoplasm, extracts were treated with ribonuclease, which resulted in low diffusivity domains indicative of aggregation, likely due to the liberation of positively charged RNA-binding proteins such as ribosomal proteins, since this effect could be mimicked by adding positively charged polypeptides. Interestingly, in extracts prepared under typical conditions that inhibit actin polymerization, negatively charged proteins of different sizes showed similar diffusivity suppression consistent with our separately measured 2.22-fold higher viscosity of extract over water. Restoring or enhancing actin polymerization progressively suppressed the diffusion of larger proteins, recapitulating behaviors observed in cells. Together, these results indicate that molecular interactions in the crowded cell are defined by an overwhelmingly negatively charged macromolecular environment containing cytoskeletal networks., Competing Interests: Competing interests statement:The authors declare no competing interest.

Cortical condensates, transient punctate-like structures rich in actin and the actin nucleation pathway member Neural Wiskott-Aldrich syndrome protein (N-WASP), form during activation of the actin cortex in the Caenorhabditis elegans oocyte. Their emergence and spontaneous dissolution is linked to a phase separation process driven by chemical kinetics. However, the mechanisms that drive the onset of cortical condensate formation near membranes remain unexplored. Here, using a reconstituted phase separation assay of cortical condensate proteins, we demonstrate that the key component, N-WASP, can collectively undergo surface condensation on supported lipid bilayers via a prewetting transition. Actin partitions into the condensates, where it polymerizes and counteracts the N-WASP prewetting transition. Taken together, the dynamics of condensate-assisted cortex formation appear to be controlled by a balance between surface-assisted condensate formation and polymer-driven condensate dissolution. This opens perspectives for understanding how the formation of complex intracellular structures is affected and controlled by phase separation., Competing Interests: Competing interests statement:A.A.H. is cofounder and member of the scientific advisory board of Dewpoint Therapeutics Inc.

The regulation of heart function is attributed to a dual filament mechanism: i) the Ca 2+ -dependent structural changes in the regulatory proteins of the thin, actin-containing filament making actin available for myosin motor attachment, and ii) the release of motors from their folded (OFF) state on the surface of the thick filament allowing them to attach and pull the actin filament. Thick filament mechanosensing is thought to control the number of motors switching ON in relation to the systolic performance, but its molecular basis is still controversial. Here, we use high spatial resolution X-ray diffraction data from electrically paced rat trabeculae and papillary muscles to provide a molecular explanation of the modulation of heart performance that calls for a revision of the mechanosensing hypothesis. We find that upon stimulation, titin-mediated structural changes in the thick filament switch motors ON throughout the filament within ~½ the maximum systolic force. These structural changes also drive Myosin Binding Protein-C (MyBP-C) to promote first motor attachments to actin from the central 1/3 of the half-thick filament. Progression of attachments toward the periphery of half-thick filament with increase in systolic force is carried on by near-neighbor cooperative thin filament activation by attached motors. The identification of the roles of MyBP-C, titin, thin and thick filaments in heart regulation enables their targeting for potential therapeutic interventions., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background/objectives: Actin plays a crucial role not only in the cytoplasm, but also in the nucleus, influencing various cellular behaviors, including cell migration and gene expression. Recent studies reveal that nuclear actin dynamics is altered by cellular stresses, such as DNA damage; however, the effect of heat shock on nuclear actin dynamics, particularly in the nucleolus, remains unclear. This study aims to elucidate the contribution of nucleolar actin to cellular responses under heat shock conditions., Methods: Nuclear actin dynamics in response to heat shock were investigated using nAC-GFP, a GFP-tagged actin chromobody, to visualize nuclear actin in HeLa cells. Bioinformatic analyses were also performed., Results: Heat shock induced the reversible assembly of nAC-GFP in the nucleolus, with disassembly occurring upon recovery in a heat shock protein (Hsp) 70-dependent manner. Because the nucleolus, formed via liquid-liquid phase separation (LLPS), sequesters misfolded proteins under heat shock to prevent irreversible aggregation, we hypothesized that nucleolar actin-binding proteins might also be sequestered in a similar manner. Using several databases, we identified 47 actin-binding proteins localized in the nucleolus and determined the proportion of intrinsically disordered regions (IDRs) known to promote LLPS. Our analysis revealed that many of these 47 proteins exhibited high levels of IDRs., Conclusions: The findings from our bioinformatics analysis and further cellular studies may help elucidate new roles for actin in the heat shock response.

Cutaneous wound healing is a complex process involving various cellular and molecular interactions, resulting in the formation of a collagen-rich scar with imperfect function and morphology. Dermal fibroblasts are crucial to successful wound healing, migrating to the wound site where they are activated to provide extracellular matrix remodeling and wound closure. Peripheral nerves have been shown to play an important role in wound healing, with loss or damage to these nerves often leading to impaired healing and the formation of chronic nonhealing wounds. Previous research has suggested that sensory nerves secrete trophic factors that can regulate wound healing, including fibroblast activation; however, the direct cell-cell interaction between nerves and fibroblasts has not been extensively studied. To address this knowledge gap, we developed an in vitro co-culture model using a device called the IFlowPlate. This model supports the long-term viability of multiple cell types while allowing for direct contact between sensory nerve cells and dermal fibroblasts. Using the IFlowPlate, we demonstrate that co-culture of dorsal root ganglia with dermal fibroblasts increases fibroblast proliferation, collagen and α-smooth muscle actin expression, and secretion of pro-wound healing factors, suggesting that nerves can promote wound healing by modulating fibroblast activation. The IFlowPlate offers a user-friendly and high-throughput platform to study the in vitro interactions between nerves and a variety of cell types that can be applied to wound healing and other important biological processes.