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We previously identified talin rod domain-containing protein 1 (TLNRD1) as a potent actin-bundling protein in vitro. Here, we report that TLNRD1 is expressed in the vasculature in vivo. Its depletion leads to vascular abnormalities in vivo and modulation of endothelial cell monolayer integrity in vitro. We demonstrate that TLNRD1 is a component of the cerebral cavernous malformations (CCM) complex through its direct interaction with CCM2, which is mediated by a hydrophobic C-terminal helix in CCM2 that attaches to a hydrophobic groove on the four-helix domain of TLNRD1. Disruption of this binding interface leads to CCM2 and TLNRD1 accumulation in the nucleus and actin fibers. Our findings indicate that CCM2 controls TLNRD1 localization to the cytoplasm and inhibits its actin-bundling activity and that the CCM2-TLNRD1 interaction impacts endothelial actin stress fiber and focal adhesion formation. Based on these results, we propose a new pathway by which the CCM complex modulates the actin cytoskeleton and vascular integrity., (© 2024 Ball et al.)

Here, we report the generation of a transgenic Lifeact-EGFP quail line for the investigation of actin organization and dynamics during morphogenesis in vivo. This transgenic avian line allows for the high-resolution visualization of actin structures within the living embryo, from the subcellular filaments that guide cell shape to the supracellular assemblies that coordinate movements across tissues. The unique suitability of avian embryos to live imaging facilitates the investigation of previously intractable processes during embryogenesis. Using high-resolution live imaging approaches, we present the dynamic behaviors and morphologies of cellular protrusions in different tissue contexts. Furthermore, through the integration of live imaging with computational segmentation, we visualize cells undergoing apical constriction and large-scale actin structures such as multicellular rosettes within the neuroepithelium. These findings not only enhance our understanding of tissue morphogenesis but also demonstrate the utility of the Lifeact-EGFP transgenic quail as a new model system for live in vivo investigations of the actin cytoskeleton., (© 2024 Alvarez et al.)

Cell processes require precise regulation of actin polymerization that is mediated by plus-end regulatory proteins. Detailed mechanisms that explain plus-end dynamics involve regulators with opposing roles, including factors that enhance assembly, e.g., the formin mDia1, and others that stop growth (capping protein, CP). We explore IQGAP1's roles in regulating actin filament plus-ends and the consequences of perturbing its activity in cells. We confirm that IQGAP1 pauses elongation and interacts with plus ends through two residues (C756 and C781). We directly visualize the dynamic interplay between IQGAP1 and mDia1, revealing that IQGAP1 displaces the formin to influence actin assembly. Using four-color TIRF, we show that IQGAP1's displacement activity extends to formin-CP "decision complexes," promoting end-binding protein turnover at plus-ends. Loss of IQGAP1 or its plus-end activities disrupts morphology and migration, emphasizing its essential role. These results reveal a new role for IQGAP1 in promoting protein turnover on filament ends and provide new insights into how plus-end actin assembly is regulated in cells., (© 2024 Pimm et al.)

The GTPase Cdc42 regulates polarized growth in most eukaryotes. In the bipolar yeast Schizosaccharomyces pombe, Cdc42 activation cycles periodically at sites of polarized growth. These periodic cycles are caused by alternating positive feedback and time-delayed negative feedback loops. At each polarized end, negative feedback is established when active Cdc42 recruits the Pak1 kinase to prevent further Cdc42 activation. It is unclear how Cdc42 activation returns to each end after Pak1-dependent negative feedback. We find that disrupting branched actin-mediated endocytosis disables Cdc42 reactivation at the cell ends. Using experimental and mathematical approaches, we show that endocytosis-dependent Pak1 removal from the cell ends allows the Cdc42 activator Scd1 to return to that end to enable reactivation of Cdc42. Moreover, we show that Pak1 elicits its own removal via activation of endocytosis. These findings provide a deeper insight into the self-organization of Cdc42 regulation and reveal previously unknown feedback with endocytosis in the establishment of cell polarity., (© 2024 Harrell et al.)

miR-31 is a highly conserved microRNA that plays crucial roles in cell proliferation, migration and differentiation. We discovered that miR-31 and some of its validated targets are enriched on the mitotic spindle of the dividing sea urchin embryo and mammalian cells. Using the sea urchin embryo, we found that miR-31 inhibition led to developmental delay correlated with increased cytoskeletal and chromosomal defects. We identified miR-31 to directly suppress several actin remodeling transcripts, including β-actin, Gelsolin, Rab35 and Fascin. De novo translation of Fascin occurs at the mitotic spindle of sea urchin embryos and mammalian cells. Importantly, miR-31 inhibition leads to a significant a increase of newly translated Fascin at the spindle of dividing sea urchin embryos. Forced ectopic localization of Fascin transcripts to the cell membrane and translation led to significant developmental and chromosomal segregation defects, highlighting the importance of the regulation of local translation by miR-31 at the mitotic spindle to ensure proper cell division. Furthermore, miR-31-mediated post-transcriptional regulation at the mitotic spindle may be an evolutionarily conserved regulatory paradigm of mitosis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The translation elongation factor eEF1α (eukaryotic elongation factor 1α) mediates mRNA translation by delivering aminoacyl-tRNAs to ribosomes. eEF1α also has other reported roles, including the regulation of actin dynamics. However, these distinct roles of eEF1α are often challenging to uncouple and remain poorly understood in aging metazoan tissues. The genomes of mammals and Drosophila encode two eEF1α paralogs, with eEF1α1 expressed ubiquitously and eEF1α2 expression more limited to neurons and muscle cells. Here, we report that eEF1α2 plays a unique role in maintaining myofibril homeostasis during aging in Drosophila. Specifically, we generated an eEF1α2 null allele, which was viable and showed two distinct muscle phenotypes. In young flies, the mutants had thinner myofibrils in indirect flight muscles that could be rescued by expressing eEF1α1. With aging, the muscles of the mutant flies began showing abnormal distribution of actin and myosin in muscles, but without a change in actin and myosin protein levels. This age-related phenotype could not be rescued by eEF1α1 overexpression. These findings support an unconventional role of Drosophila eEF1α2 in age-related homeostasis of muscle myofibers., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: This study aimed to investigate cell degeneration, apoptosis, and ultrastructural differences in refractive lenticules (RL) obtained using small incision lenticule extraction (SMILE) compared with spherical equivalence (SE) refraction values., Methods: This study included 84 eyes from 42 patients. Patients were divided into two groups according to the SE values: those with values below 4 diopters (D) (Group 1) and above 4 diopters (D) (Group 2). Patients who did not belong to the same SE group were excluded from the study. One RL obtained from each patient was separated for light microscopy and immunohistochemical examinations, and another for transmission electron microscopy (TEM) examinations. Caspase-3 for apoptosis and alpha-smooth muscle actin (α-SMA) for cell degeneration were evaluated using immunohistochemical examinations., Results: Histological analyses showed that the density of collagen fibres was greater in Group 1 than in Group 2. Glycoaminoglycan and glycoprotein staining intensities were also higher in Group 1. TEM observations showed that Group 1 had intact cell and nuclear membranes, peripheral heterochromatin, and large nuclei, while Group 2 showed heterochromatin condensation and fragmentation, increased intracellular vacuoles, and loss of cytoplasm. Immunohistochemical analyses revealed that α-SMA and caspase-3 were significantly higher in Group 2 than in Group 1 (p < 0.001 and p < 0.001, respectively)., Conclusions: Cell degeneration and apoptosis were significantly more common in the RLs with high SE values after SMILE surgery. The tissue response induced by surgery was more severe in the RLs with high SE values. This should be considered when reusing RLs., (© 2024 The Author(s). Clinical & Experimental Ophthalmology published by John Wiley & Sons Australia, Ltd on behalf of Royal Australian and New Zealand College of Ophthalmologists.)

Cells sense and respond to various mechanical forces from the extracellular matrix primarily by modulating the actin cytoskeleton. Mechanical forces can be translated into biochemical signals in a process called mechanotransduction. Yes-associated protein (YAP) is an effector of Hippo signaling and a mediator of mechanotransduction, but how mechanical forces regulate Hippo signaling is still an open question. We propose that retinoic acid-induced protein 14 (RAI14) responds to mechanical forces and regulates Hippo signaling. RAI14 positively regulates the activity of YAP. RAI14 interacts with NF2, a key component of the Hippo pathway, and the interaction occurs on filamentous actin. When mechanical forces are kept low in cells, NF2 dissociates from RAI14 and filamentous actin, resulting in increased interactions with LATS1 and activation of the Hippo pathway. Clinical data show that tissue stiffness and expression of RAI14 and YAP are upregulated in tumor tissues and that RAI14 is strongly associated with adverse outcome in patients with gastric cancer. Our data suggest that RAI14 links mechanotransduction with Hippo signaling and mediates Hippo-related biological functions such as cancer progression., (© 2024. The Author(s).)

Being a bona fide actin bundler, Arabidopsis villin5 (VLN5) plays a crucial role in regulating actin stability and organization within pollen tubes. Despite its significance, the precise mechanism through which VLN5 bundles actin filaments has remained elusive. Through meticulous deletion analysis, we have unveiled that the link between gelsolin repeat 6 (G6) and the headpiece domain (VHP), rather than VHP itself, is indispensable for VLN5-mediated actin bundling. Further refinement of this region has pinpointed a critical sequence spanning from Val763 to Ser823, essential for VLN5's actin-bundling activity. Notably, the absence of Val763-Ser823 in VLN5 results in decreased filamentous decoration within pollen tubes and a diminished ability to rescue actin bundling defects in vln2vln5 mutant pollen tubes compared to intact VLN5. Moreover, our findings highlight that the Val763-Ser823 sequence harbors a binding site for F-actin, suggesting that this linker-based F-actin binding site, in conjunction with the F-actin binding site localized in G1-G6, enables a single VLN5 to concurrently bind to two adjacent actin filaments. Therefore, our study unveils a novel mechanism by which VLN5 bundles actin filaments., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Magnaporthe oryzae (M. oryzae) is a devastating hemibiotrophic pathogen. Its biotrophic invasive hyphae (IH) are enclosed in the extrainvasive hyphal membrane produced by plant cells, thus generating a front line of the battlefield between the pathogen and the host plants. In plants, defense-related complexes such as proteins, callose-rich materials and vesicles, are directionally secreted to this interface to confer defense responses, but the underlying molecular mechanism is poorly understood. In this study, we found that a Myosin gene, Myosin A1 (OsMYA1), contributed to rice defense. The OsMYA1 knockout mutant exhibited decreased resistance to M. oryzae infection. OsMYA1 localizes to the actin cytoskeleton and surrounds the IH of M. oryzae. OsMYA1 interacts with an exocyst subunit, OsExo70H1, and regulates its accumulation at the plasma membrane (PM) and pathogen-plant interface. Furthermore, OsExo70H1 interacted with the rice syntaxin of the plants121 protein (OsSyp121), and the distribution of OsSyp121 to the PM or the pathogen-plant interface was disrupted in both the OsMYA1 and OsExo70H1 mutants. Overall, these results not only reveal a new function of OsMYA1 in rice blast resistance, but also uncover a molecular mechanism by which plants regulate defense against M. oryzae by OsMYA1-initiated vesicle secretory pathway, which originates from the actin cytoskeleton to the PM., (© 2024 Institute of Botany, Chinese Academy of Sciences.)

Objectives: To explore the role of endothelial-mesenchymal transition (EndMT) mediated by the TGF-β/SMAD signalling pathway in the pathogenesis of ankylosing spondylitis (AS)., Methods: Serum levels of TGF-β1 were measured by enzyme-linked immunosorbent assay (ELISA) in 48 patients with AS and 15 healthy subjects. The expression levels of TGF-β1, SMAD7, CTGF, CD34 and EndMT-related markers (α-SMA, vimentin, FSP-1, VE-cadherin) in the sacroiliac joint (SIJ) of three AS patients were detected by immunohistochemistry, and three non-spondyloarthritis (SpA) autopsy samples were used as controls., Results: Serum TGF-β1 level of AS patients was significantly higher than that of healthy controls (22971 ± 7667 pg/ml vs. 14837±4653 pg/ml, p<0.01). Compared with the non-SpA control group, the microvascular density (MVD) at the pannus formation site of SIJ in AS patients was significantly increased, accompanied by respectively increased expressions of TGF-β1, CTGF, α-SMA, vimentin, and FSP-1 (all p<0.05), whereas respectively decreased expressions of VE-cadherin and SMAD7 (p<0.01). The expression level of FSP-1 was positively correlated with levels of TGF-β1 and MVD, and negatively correlated with SMAD7., Conclusions: Our findings show that EndMT is involved in the promotion of pannus formation by TGF-β/SMAD signalling pathway activation in AS.

Backgroud: Arsenic trioxide (ATO), the first-line drug in treating acute premyelogenous leukemia, has the profound side effect of inducing endothelial mesenchymal transition (EndMT) and causing cardiac fibrosis. Diosgenin (DIO), a pharmaceutical compound found in Paris polyphylla, exhibits promising potential in safeguarding cardiovascular health by mitigating EndMT., Purpose: This study aims to explore the role and mechanism of DIO in ATO-induced myocardial fibrosis to provide a novel therapeutic agent for ATO-induced cardiac fibrosis., Methods: Wistar rats were given DIO by gavage and ATO by tail vein. Cardiac function and fibrosis were evaluated by echocardiography and Masson's trichrome staining in rats. Human aortic endothelial cells (HAECs) were utilized to analyze ATO-induced EndMT in vitro. The cytoskeleton of HAECs was visualized using F-actin staining to observe cell morphology, while Dil-Ac-LDL staining was employed to assess cell functionality. EndMT-related factors (CD31 and α-SMA), glucocorticoid receptor (GR) and interleukin-6 (IL-6) were detected by immunofluorescence and Western blot in vivo and in vitro. Furthermore, GR was knocked down by si-GR, and IL-6 was blocked by IL-6 neutralizing antibody to verify their role in the effect of DIO on ATO-induced EndMT in HAECs., Results: DIO exhibited significant efficacy in ATO-induced damage to both cardiac diastolic and systolic function, along with mitigating cardiac fibrosis. Additionally, DIO alleviated the loss of cytoskeletal anisotropy and enhanced the uptake of Dil-Ac-LDL in HAECs. Furthermore, it reversed the ATO-induced downregulation of endothelial-specific markers CD31 and GR, while suppressing the upregulation of mesenchymal markers α-SMA and IL-6, both in vivo and in vitro. Notably, the protective effect of DIO was compromised upon knockdown of GR, which also led to a reversal of DIO-induced IL-6 downregulation. Furthermore, the neutralization of IL-6 with specific antibodies abolished the ATO-induced changes related to EndMT., Conclusion: In this study, we clarified the protective effect of DIO on ATO-induced myocardial fibrosis against EndMT via the GR/IL-6 axis for the first time and provided a potential therapeutic agent for preventing heart damage caused by ATO., Competing Interests: Declaration of competing interest We declare that they have no competing interests., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

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

For over 20 years, the most effective probe for live imaging of yeast actin cables has been Abp140-GFP. Here, we report that endogenously-tagged Abp140-GFP poorly decorates actin patches and cables in the bud compartment of yeast cells, while robustly decorating these structures in the mother cell. Using mutagenesis, we found that asymmetric decoration by Abp140 requires F-actin binding. By expressing integrated Bni1-Bnr1 and Bnr1-Bni1 chimeras, we demonstrate that asymmetric cable decoration by Abp140 also does not depend on which formin assembles the cables in each compartment. In contrast, the short actin-binding fragment of Abp140 (known as "Lifeact"), fused to 1x or 3xmNeonGreen and expressed from the endogenous ABP140 promoter, uniformly decorates patches and cables in both compartments. Further, this probe dramatically improves live imaging detection of cables (and patches) without altering their in vivo dynamics or cell growth. Improved detection allows us to visualize cables growing inward from the cell cortex and dynamically interacting with the vacuole. This probe also robustly decorates the cytokinetic actomyosin ring. Because Lifeact-3xmNeon expressed at relatively low levels provides intense labeling of cellular F-actin structures, this tool may improve live imaging in other organisms where higher levels of Lifeact expression are detrimental., Competing Interests: Conflict of interest: The authors declare no financial conflict of interest.

Purpose: To create a reproducible and standardized urethral stricture model in rats, evaluating both histomorphologic findings and gene expression data. In studies involving experimental animals, more standardization is needed for the creation of a urethral stricture model., Methods: Sixteen male rats were randomized into two groups. The Sham group (n:8) underwent only a penoscrotal incision, while the stricture group (n:8) had their urethras exposed through a penoscrotal incision, followed by electrocauterization to the corpus spongiosum. On the 15th day, blood and urethral tissues were harvested for histologic and molecular analyses. Histomorphologic, immunohistochemical, and reverse transcription polymerase chain reaction analyses were performed., Results: The stricture group exhibited more severe and intense spongiofibrosis, inflammation, epithelial desquamation, and congestion in vascular structures compared to the controls (p < 0.05). The urethral tissue in the stricture group showed an increased ratio of inflammation parameters, including Collagen 1A1, Collagen 3A1, elastin, Transforming growth factor β1, α Smooth muscle actin, Platelet-derived growth factor α, and Platelet-derived growth factor β. Transforming growth factor β1, Platelet-derived growth factor α, and Platelet-derived growth factor β each correlated highly with the other six parameters (r > 0.60, p < 0.05)., Conclusion: Developing electrocoagulation-induced urethral stricture in rats is a simple, reliable, inexpensive, and reproducible. Reporting histologic data with qualitative and semi-quantitative scoring will enhance data standardization, aiding reader understanding and analysis. Transforming growth factor β and Platelet-derived growth factor play key roles in fibrosis during stricture development. Incorporating these cytokines in urethral stricture animal model studies can demonstrate successful stenosis creation., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca 2+ -induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca 2+ -induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations., (© 2024 International Society for Neurochemistry.)

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

A robust and efficient cellular response to lysosomal membrane damage prevents leakage from the lysosome lumen into the cytoplasm. This response is understood to happen through either lysosomal membrane repair or lysophagy. Here we report exocytosis as a third response mechanism to lysosomal damage, which is further potentiated when membrane repair or lysosomal degradation mechanisms are impaired. We show that Connexin43 (Cx43), a protein canonically associated with gap junctions, is recruited from the plasma membrane to damaged lysosomes, promoting their secretion and accelerating cell recovery. The effects of Cx43 on lysosome exocytosis are mediated by a reorganization of the actin cytoskeleton that increases plasma membrane fluidity and decreases cell stiffness. Furthermore, we demonstrate that Cx43 interacts with the actin nucleator Arp2, the activity of which was shown to be necessary for Cx43-mediated actin rearrangement and lysosomal exocytosis following damage. These results define a novel mechanism of lysosomal quality control whereby Cx43-mediated actin remodelling potentiates the secretion of damaged lysosomes., (© 2024. The Author(s).)

A key step in regulation of Hippo pathway signaling in response to mechanical tension is recruitment of the LIM domain proteins TRIP6 and LIMD1 to adherens junctions. Mechanical tension also triggers TRIP6 and LIMD1 to bind and inhibit the Hippo pathway kinase LATS1. How TRIP6 and LIMD1 are recruited to adherens junctions in response to tension is not clear, but previous studies suggested that they could be regulated by the known mechanosensory proteins α-catenin and vinculin at adherens junctions. We found that the three LIM domains of TRIP6 and LIMD1 are necessary and sufficient for tension-dependent localization to adherens junctions. The LIM domains of TRIP6, LIMD1, and certain other LIM domain proteins have been shown to bind to actin networks under strain/tension. Consistent with this, we show that TRIP6 and LIMD1 colocalize with the ends of actin fibers at adherens junctions. Point mutations in a key conserved residue in each LIM domain that are predicted to impair binding to f-actin under strain inhibits TRIP6 and LIMD1 localization to adherens junctions and their ability to bind to and recruit LATS1 to adherens junctions. Together these results show that the ability of TRIP6 and LIMD1 to bind to strained actin underlies their ability to localize to adherens junctions and regulate LATS1 in response to mechanical tension., (© 2024 Wiley Periodicals LLC.)

Extensive research has been conducted on the role of CXCR3 in immune responses and inflammation. However, the role of CXCR3 in the reproductive system, particularly in oocyte development, remains unknown. In this study, we present findings on the involvement of CXCR3 in the meiotic division process of mouse oocytes. We found CXCR3 was expressed consistently throughout the entire maturation process of mouse oocyte. Inhibition of CXCR3 impaired the asymmetric division of oocyte, while the injection of Cxcr3 mRNA was capable of restoring these defects. Further study showed that inhibition of CXCR3 perturbed spindle migration by affecting LIMK/cofilin pathway-mediated actin remodeling. Knockout of CXCR3 led to an upregulation of actin-binding protein and an increased ATP level in GV-stage oocytes, while maintaining normal actin dynamics during the process of meiosis. Additionally, we noticed the expression level of DYNLT1 is markedly elevated in CXCR3-null oocytes. DYNLT1 bound with the Arp2/3 complex, and knockdown of DYNLT1 in CXCR3-null oocytes impaired the organization of cytoplasmic actin, suggesting the regulatory role of DYNLT1 in actin organization, and the compensatory expression of DYNLT1 may contribute to maintain normal actin dynamics in CXCR3-knockout oocytes. In summary, our findings provide insights into the intricate network of actin dynamics associated with CXCR3 during oocyte meiosis., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Placental Derived Nucleoproteins (PDNs) is commonly associated with the process of angiogenesis, and doesn't affect the healthy vasculature. PDNs are clinically estimated for the treatment of cancer cases and severe hepatic injuries. Thus, the pathophysiological effects of PDNs targeting liver fibrosis is a concern., Objectives: To assess the molecular, histopathological, and chemical impact of PDNs on liver regeneration in Diethylnitrosamine (DEN)-induced mice liver fibrosis., Methods: Normal untreated reference group of ten mice and two groups of induced liver cirrhosis using the recommended weekly dose of Diethylnitrosamine in total of eleven doses, initially 20 mg/kg body weight, and then 30 mg/kg in the third week, followed by 50 mg/kg for the last eight weeks, one of them combined treatment aligned with injection with total dose of extracted PDNs 25 mg/kg, in comparison to PDNs only treated group. An autopsy was performed after 22 weeks of the initial dose of DEN in each group. Molecular characterization of Alpha smooth muscle actin, TGFβ and NF-κB biomarkers for liver then liver function panel were analyzed and finally hepatopathological changes were observed using H&E stain and Sirius red stain., Results: Liver enzymes, total bilirubin and total proteins in tissue in PDNs-DEN treated models were controlled in the direction of normal group and 50 % reduction of fibrosis in comparing to DEN-treated models. The cellular arrangement of fibrosis in the DEN entire groups were differentiated with high significant impact on the survival of mice. Increased levels of the biochemical markers in liver homogenate, loss of tissue architecture, and proliferation were observed in induced groups and down regulation of alpha smooth muscle actin, TGFβ and NF-κB., Conclusion: This finding demonstrates an improvement of Liver tissue induced fibrosis using DEN combined with PDNs. This strategy is to generate an animal model with a lower occurrence of fibrosis in a short time treatment regarding liver regeneration., 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 Masson SAS.. All rights reserved.)

Microtubule and actin are the two major cytoskeletal polymers that form organized functional structures in the interior of eukaryotic cells. Although the structural mechanics of the cytoskeleton has been extensively studied by direct manipulations in in vitro reconstitution systems, such unambiguous characterizations inside the living cell are sparse. Here, we report a comprehensive analysis of how the microtubule and actin cytoskeletons structurally respond to direct intracellular load. Ferrofluid-based intracellular magnetic tweezers reveal rheological properties of the microtubule complex primarily determined by filamentous actin. The strain fields of the microtubule complex and actin meshwork follow the same scaling, suggesting that the two cytoskeletal systems behave as an integrated elastic body. The structural responses of single microtubules to contact and remote forces further evidence that the individual microtubules are enclosed by the elastic medium of actin. These results, directly characterizing the microtubule and actin cytoskeletons as an interacting continuum throughout the cytoplasm, serve as a cornerstone for the physical understanding of intracellular organization., (© 2024 Orii and Tanimoto.)

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.)

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.)

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 endoplasmic reticulum (ER) network is highly complex and highly dynamic in its geometry, and undergoes extensive remodeling and bulk flow. It is known that the ER dynamics are driven by actin-myosin dependent processes. ER motion through the cytoplasm will cause forces on the cytoplasm that will induce flow. However, ER will also clearly be passively transported by the bulk cytoplasmic streaming. We take the complex ER network structure into account and propose a positive-feedback mechanism among myosin-like motors, actin alignment, ER network dynamics for the emergence of ER flow. Using this model, we demonstrate that ER streaming may be an emergent feature of this three-way interaction and that the persistent-point density may be a key driver of the emergence of ER streaming., Competing Interests: Declaration of competing interest No conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Transglutaminase 2 (TG2) is a multifunctional enzyme involved in fibrosis by promoting transforming-growth-factor-β1 and crosslinking of extracellular matrix proteins. These functions are dependent on the open conformation, while the closed state of TG2 can induce vasodilation. We explored the putative protective role of TG2 in its closed state on development of renal fibrosis and blood pressure (BP) regulation., Methods: We studied the unilateral ureteral obstruction (UUO) mouse model treated with LDN27219, which promotes the closed conformation of TG2. Mice were subjected to 7 days UUO or sham operation and treated with vehicle (n = 10), LDN27219 (15 mg/kg/12 h, n = 9) or candesartan (5 mg/kg/day, n = 10) as a clinically comparator. Renal expression of TG2 and pro-fibrotic mediators were evaluated by Western blotting, qPCR and histology, and BP by tail-cuff measurements., Results: Obstructed kidneys showed increased mRNA and protein expression of fibronectin, collagen 3α1 (Col3α1), α-smooth muscle actin and collagen staining. Despite increased renal TG2 mRNA, protein expression was reduced in all UUO groups, but with increased transamidase activity in the vehicle and candesartan groups. LDN27219 reduced mRNA expression of fibronectin and Col3α1, but their protein expression remained unchanged. In contrast to LDN27219, candesartan lowered BP without affecting expression of pro-fibrotic biomarkers., Conclusion: Renal TG2 mRNA and protein expression levels seem dissociated, with transamidase activity being increased. LDN27219 influences kidney pro-fibrotic markers at the mRNA level and attenuates transamidase activity but without affecting collagen content or BP. Our findings suggest that TG2 in its closed conformation has anti-fibrotic effects at the molecular level., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Estefano Pinilla reports financial support was provided by Erik Hørslev and Birgit Hørslev Fund. Niels Henrik Buus reports financial support was provided by Aarhus University Research Foundation. Aarhus University has patent #EP3607948A1 licensed to Estéfano Pinilla. Aarhus University has patent #EP3607948A1 licensed to Ulf Simonsen. If there are other authors, they 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 B.V. All rights reserved.)

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

Metastasis initiates when cancer cells escape from the primary tumor, which requires changes to intercellular junctions. Claudins are transmembrane proteins that form the tight junction, and their expression is reduced in aggressive breast tumors. However, claudins' roles during breast cancer metastasis remain unclear. We used gain- and loss-of-function genetics in organoids isolated from murine breast cancer models to establish that Cldn7 suppresses invasion and metastasis. Transcriptomic analysis revealed that Cldn7 knockdown induced smooth muscle actin (SMA)-related genes and a broader mesenchymal phenotype. We validated our results in human cell lines, fresh human tumor tissue, bulk RNA-seq, and public single-cell RNA-seq data. We consistently observed an inverse relationship between Cldn7 expression and expression of SMA-related genes. Furthermore, knockdown and overexpression of SMA-related genes demonstrated that they promote breast cancer invasion. Our data reveal that Cldn7 suppresses breast cancer invasion and metastasis through negative regulation of SMA-related and mesenchymal gene expression., (© 2024 West et al.)

We repurposed micropillar arrays to quantify spatiotemporal inter-adhesion communication. Following the observation that integrin adhesions formed around pillar tops we relied on the precise repetitive spatial control of the pillars to reliably monitor F-actin dynamics in mouse embryonic fibroblasts as a model for spatiotemporal adhesion-related intracellular signaling. Using correlation-based analyses, we revealed localized information flows propagating between adjacent pillars that were integrated over space and time to synchronize the adhesion dynamics within the entire cell. Probing the mechanical regulation, we discovered that stiffer pillars or partial actomyosin contractility inhibition enhances inter-adhesion F-actin synchronization, and that inhibition of Arp2/3, but not formin, reduces synchronization. Our results suggest that adhesions can communicate and highlight the potential of using micropillar arrays as a tool to measure spatiotemporal intracellular signaling., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

The organization of cytoskeletal elements is pivotal for coordinating intracellular transport in eukaryotic cells. Several quantitative measures based on image analysis have been proposed to characterize morphometric features of fluorescently labeled actin networks. While helpful in detecting differences in actin organization between treatments or genotypes, the accuracy of these measures could not be rigorously assessed due to a lack of ground-truth data to which they could be compared. To overcome this limitation, we utilized coarse-grained computer simulations of actin filaments and cross-linkers to generate synthetic actin networks with varying levels of bundling. We converted the simulated networks into pseudofluorescence images similar to images obtained using confocal microscopy. Using both published and novel analysis procedures, we extracted a series of morphometric parameters and benchmarked them against analogous measures based on the ground-truth actin configurations. Our analysis revealed a set of parameters that reliably reports on actin network density, orientation, ordering, and bundling. Application of these morphometric parameters to root epidermal cells of Arabidopsis thaliana revealed subtle changes in network organization between wild-type and mutant cells. This work provides robust measures that can be used to quantify features of actin networks and characterize changes in actin organization for different experimental conditions., Competing Interests: Conflicts of interests: The authors declare no financial conflict of interest.

Upon activation by vascular injury or extracellular agonists, platelets undergo rapid change shape, a process regulated by the actin cytoskeleton and accessory proteins. Platelet shape change is accompanied by the secretion of hemostatic factors and immunomodulatory cytokines from their intracellular granules, as well as the release of microvesicles (MVs) containing pro-inflammatory cytokines and procoagulant phosphatidylserine (PS). However, the role of actin dynamics in MV generation remains unclear. In this study, we found that blocking actin polymerization with cytochalasin D attenuated the release of PS-positive MVs in human platelets stimulated by thrombin or the calcium ionophore A23187. The actin-severing protein gelsolin (Gsn) facilitates normal actin filament turnover in activated platelets. Platelets from Gsn-deficient (Gsn -/- ) mice showed reduced MV release compared to platelets from control mice. These findings indicate that the proper dynamics of the actin cytoskeleton are essential for MV generation in platelets, which has implications for their pro-inflammatory and procoagulant functions., 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.)

Viruses are dependent on the host factors for their replication and survival. Therefore, identification of host factors that druggable for antiviral development is crucial. The actin cytoskeleton plays an important role in the virus infection. The dynamics change of actin and its function are regulated by multiple actin-associated proteins (AAPs). However, the role and mechanism of various AAPs in the life cycle of virus are still enigmatic. In this study, we analyzed the roles of actin and AAPs in the replication of pseudorabies virus (PRV). Using a library of compounds targeting AAPs, our data found that multiple AAPs, such as Rho-GTPases, Rock, Myosin and Formin were involved in PRV infection. Besides, our result demonstrated that the actin-binding protein Drebrin was also participated in PRV infection. Further studies are necessary to elucidate the molecular mechanism of AAPs in the virus life cycle, in the hope of mining host factors for antiviral developments., 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.)

Oxidative stress causes damage to cancer cells and plays an important role in cancer therapy. Antagonizing oxidative stress is crucial for cancer cells to survive during the oxidation-based therapy. In this study, we defined the role of nuclear receptor co-activator 7 (NCOA7) in anti-oxidation in lung cancer cells and found that NCOA7 protects lung cancer A549 cells from the oxidative damage caused by hydrogen peroxide. Knockdown of NCOA7 in A549 cells significantly enhanced the hydrogen peroxide-caused inhibition of cell proliferation and migration, and markedly increased the damage effect of hydrogen peroxide on F-actin and focal adhesion structure, suggesting that NCOA7 protects F-actin and focal adhesion structure, thus the cell proliferation and migration, from oxidation-caused damage. Mechanistically, the anti-oxidation effect of NCOA7 is mediated by its nuclear receptor binding domain, the ERbd domain, suggesting that the anti-oxidation function of NCOA7 is dependent on its nuclear receptor co-activator activity. Our studies identified NCOA7 as an anti-oxidative protein through its nuclear receptor co-activator function and revealed the mechanism underlying the anti-oxidative effect of NCOA7 on cancer cell proliferation and migration., 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.)

This study aimed to evaluate the effects of ursolic acid (UA) on Angiotensin II (Ang II)-treated neonatal rat cardiac fibroblasts (rCFs) as an in vitro model of cardiac fibrosis. The rCFs were isolated from two-day-old neonatal rats. An in vitro model of cardiac fibrosis was established using 500 nm Ang II treatment for 48 h. The cells were then treated with 5 and 10 μM of UA for 24 and 48 h. Masson's trichrome staining, hydroxyproline content assay, scratch assay, apoptosis assay, measurements of superoxide dismutase (SOD) and malondialdehyde (MDA) levels, real-time PCR, immunocytology and western blotting, were employed to assess the impact of UA. Ang II induced fibrosis in rCFs, as evidenced by the examination of various fibrotic markers. Upon treatment with 5 and 10 μM of UA, the amount of fibrosis in Ang II-treated rCFs was significantly decreased, so that the hydroxyproline concentration was reduced to 0.3 and 0.7 times, respectively. The RNA expression of the Col1a1, Col3a1, Tgfb1, Acta2 and Mmp2 genes had a decrease as well as Nrf2 and HO-1 had an increase after UA treatment. UA could lessen the harmful effects of cardiac fibrosis in a dose- and time-dependent manner, due to its antiapoptotic, antioxidant and cardioprotective properties. This suggests the potential of UA for treatment of cardiac fibrosis., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Key Message: ARID-HMG DNA binding protein, AtHMGB15, regulates pollen development and pollen germination in Arabidopsis. Previous studies have shown that ARID-HMG DNA binding protein, AtHMGB15 regulate pollen development and pollen germination in Arabidopsis. Here, we performed transcriptome and cytological studies to understand the role of AtHMGB15 in regulating pollen wall morphology and the pollen tube germination rate. Our result showed abnormal vacuolization in the tapetal cells during anther maturation and prolonged PCD in AtHMGB15 loss-of-function mutant. The tapetum has the ability to perform both secretory and biosynthetic activities critical for pollen maturation and pollen viability. Interestingly, expression of PCD executer genes CEP1, MC9 and RNS3 were significant down-regulation of in athmgb15-4. The growth of pollen tubes is regulated by the actin cytoskeleton dynamics. To address the defect in pollen tube growth of athmgb15, we monitored the actin network in growing pollen tubes of wildtype and athmgb15-4 using Rhodamine-phalloidin fluorescence. Our results indicate a highly fragmented actin distribution in athmgb15-4 pollen tubes with a lesser number of long actin fibers and significantly low f-actin concentration at the apex. q-RTPCR further indicates significant downy-regulation of actin regulatory proteins VLN2 and PRF4. Collectively, our results suggest that AtHMGB15 being a nuclear architectural protein orchestrates high-order chromatin organization to promote the transcription of genes responsible for pollen development and pollen germination., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The view on nuclear filaments formed by non-skeletal β-actin has significantly changed over the decades. Initially, filamentous actin was observed in amphibian oocyte nuclei and only under specific cell stress conditions in mammalian cell nuclei. Improved labeling and imaging technologies have permitted insights into a transient but microscopically apparent filament network that is relevant for chromatin organization, biomechanics of the mammalian cell nucleus, gene expression, and DNA damage repair. Here, we will provide a historical perspective on the developing insight into nuclear actin filaments.

Objectives:  Astaxanthin (ATX) is a strong antioxidant drug. This study aimed to investigate the effects of ATX on podocytes in diabetic nephropathy and the underlying renal protective mechanism of ATX, which leads to pathological crosstalk with mesangial cells. Methods:  In this study, diabetic rats treated with ATX exhibited reduced 24-h urinary protein excretion and decreased blood glucose and lipid levels compared to vehicle-treated rats. Glomerular mesangial matrix expansion and renal tubular epithelial cell injury were also attenuated in ATX-treated diabetic rats compared to control rats. Results:  ATX treatment markedly reduced the α-SMA and collagen IV levels in the kidneys of diabetic rats. Additionally, ATX downregulated autophagy levels. In vitro , compared with normal glucose, high glucose inhibited LC3-II expression and increased p62 expression, whereas ATX treatment reversed these changes. ATX treatment also inhibited α-SMA and collagen IV expression in cultured podocytes. Secreted factors (vascular endothelial growth factor B and transforming growth factor-β) generated by high glucose-induced podocytes downregulated autophagy in human mesangial cells (HMCs); however, this downregulation was upregulated when podocytes were treated with ATX. Conclusions:  The current study revealed that ATX attenuates diabetes-induced kidney injury likely through the upregulation of autophagic activity in podocytes and its antifibrotic effects. Crosstalk between podocytes and HMCs can cause renal injury in diabetes, but ATX treatment reversed this phenomenon.

Cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), lead to inflammation and severe hepatic damage with limited therapeutic options. This study assessed the efficacy of the inverse RORγt agonist, GSK805, both in vitro using the hepatic stellate cell-line LX-2 and in vivo using male bile duct-ligated BALB/c mice. In vitro, 0.3 μM GSK805 reduced alpha-smooth muscle actin expression in LX-2 cells. In vivo, GSK805 significantly decreased IL-23R, TNF-α, and IFN-γ expression in cholestatic liver. Despite high concentrations of GSK805 in the liver, no significant reduction in fibrosis was noticed. GSK805 significantly increased aspartate aminotransferase and alanine aminotransferase activity in the blood, while levels of glutamate dehydrogenase, alkaline phosphatase, and bilirubin were not substantially increased. Importantly, GSK805 did neither increase an animal distress score nor substantially reduce body weight, burrowing activity, or nesting behavior. These results suggest that a high liver concentration of GSK805 is achieved by daily oral administration and that this drug modulates inflammation in cholestatic mice without impairing animal well-being., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Purpose: The abnormal growth factors-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells was known as a vital pathogenesis of proliferative vitreoretinopathy (PVR). This study aims to explore how survivin inhibition affects EMT induced by epidermal growth factor (EGF) in RPE cells., Methods: Human primary RPE cells were identified in vitro. EMT in RPE cells was induced by EGF. Inhibition of survivin in RPE cells was accomplished through the use of a survivin inhibitor (YM155) and survivin siRNA. The viability, proliferation and migration of RPE cells was detected by methylthiazol tetrazolium assay, bromodeoxyuridine labeling assay, and wound healing assay, respectively. The EGF receptor /mitogen-activated protein kinase (EGFR/MAPK) proteins and EMT-related proteins were measured by western blot and immunofluorescence assay., Results: EGF induced significant EMT in RPE cells, activated the phosphorylation of EGFR/MAPK signaling proteins, and caused changes to EMT-related proteins. YM155 suppressed RPE cells' viability, proliferation, and migration; induced the phosphorylation of EGFR, JNK, and P38MAPK; and down regulated EGFR and phosphorylated ERK. YM155 also increased expression of E-cadherin and ZO-1 proteins and reduced expression of N-cadherin, Vimentin, and α-SMA proteins. The EGF-induced increase of RPE cell proliferation and migration was constrained by survivin inhibition. Moreover, survivin inhibition in RPE cells suppressed the EGF-caused phosphorylation of EGFR/MAPK proteins and attenuated the EGF-induced reduction of E-cadherin and ZO-1 proteins and increase of N-cadherin, Vimentin, and α-SMA proteins., Conclusions: Survivin inhibition attenuates EGF-induced EMT of RPE cells by affecting the EGFR/MAPK signaling pathway. Survivin might be a promising target for preventing PVR., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhu 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.)

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, a leading cause of cancer-related deaths globally. Initial lesions of PDAC develop within the exocrine pancreas' functional units, with tumor progression driven by interactions between PDAC and stromal cells. Effective therapies require anatomically and functionally relevant in vitro human models of the pancreatic cancer microenvironment. We employed tomographic volumetric bioprinting, a novel biofabrication method, to create human fibroblast-laden constructs mimicking the tubuloacinar structures of the exocrine pancreas. Human pancreatic ductal epithelial (HPDE) cells overexpressing the KRAS oncogene (HPDE-KRAS) were seeded in the multiacinar cavity to replicate pathological tissue. HPDE cell growth and organization within the structure were assessed, demonstrating the formation of a thin epithelium covering the acini inner surfaces. Immunofluorescence assays showed significantly higher alpha smooth muscle actin ( α -SMA) vs. F-actin expression in fibroblasts co-cultured with cancerous versus wild-type HPDE cells. Additionally, α -SMA expression increased over time and was higher in fibroblasts closer to HPDE cells. Elevated interleukin (IL)-6 levels were quantified in supernatants from co-cultures of stromal and HPDE-KRAS cells. These findings align with inflamed tumor-associated myofibroblast behavior, serving as relevant biomarkers to monitor early disease progression and target drug efficacy. To our knowledge, this is the first demonstration of a 3D bioprinted model of exocrine pancreas that recapitulates its true 3-dimensional microanatomy and shows tumor triggered inflammation., (Creative Commons Attribution license.)

The migration of breast cancer cells is the main cause of death and significantly regulated by physical factors of the extracellular matrix (ECM). To be specific, the curvature and stiffness of the ECM were discovered to effectively guide cell migration in velocity and direction. However, it is not clear what the extent of effect is when these dual-physical factors regulate cell migration. Moreover, the mechanobiology mechanism of breast cancer cell migration in the molecular level and analysis of cell traction force (CTF) are also important, but there is a lack of systematic investigation. Therefore, we employed a microfluidic platform to construct hydrogel microspheres with an independently adjustable curvature and stiffness as a three-dimensional substrate for breast cancer cell migration. We found that the cell migration velocity was negatively correlated to curvature and positively correlated to stiffness. In addition, curvature was investigated to influence the focal adhesion expression as well as the assignment of F-actin at the molecular level. Further, with the help of a motor-clutch mathematical model and hydrogel microsphere stress sensors, it was concluded that cells perceived physical factors (curvature and stiffness) to cause changes in CTF, which ultimately regulated cell motility. In summary, we employed a theoretical model (motor-clutch) and experimental strategy (stress sensors) to understand the mechanism of curvature and stiffness regulating breast cancer cell motility. These results provide evidence of force driven cancer cell migration by ECM physical factors and explain the mechanism from the perspective of mechanobiology.

Macropinocytosis is a broadly conserved endocytic process discovered nearly 100 years ago, yet still poorly understood. It is prominent in cancer cell feeding, immune surveillance, uptake of RNA vaccines and as an invasion route for pathogens. Macropinocytic cells extend large cups or flaps from their plasma membrane to engulf droplets of medium and trap them in micron-sized vesicles. Here they are digested and the products absorbed. A major problem - discussed here - is to understand how cups are shaped and closed. Recently, lattice light-sheet microscopy has given a detailed description of this process in Dictyostelium amoebae, leading to the 'stalled-wave' model for cup formation and closure. This is based on membrane domains of PIP3 and active Ras and Rac that occupy the inner face of macropinocytic cups and are readily visible with suitable reporters. These domains attract activators of dendritic actin polymerization to their periphery, creating a ring of protrusive F-actin around themselves, thus shaping the walls of the cup. As domains grow, they drive a wave of actin polymerization across the plasma membrane that expands the cup. When domains stall, continued actin polymerization under the membrane, combined with increasing membrane tension in the cup, drives closure at lip or base. Modelling supports the feasibility of this scheme. No specialist coat proteins or contractile activities are required to shape and close cups: rings of actin polymerization formed around PIP3 domains that expand and stall seem sufficient. This scheme may be widely applicable and begs many biochemical questions., (© 2024 The Author(s).)

Ligand-engaged chemokine receptors trigger nucleotide exchange in heterotrimeric Gα i proteins, which stimulates cytoskeletal reorganization and cell polarity changes. To better understand the signaling events responsible for these cellular changes, we focused on early changes in F-actin dynamics after engagement of the chemokine receptor CXCR5 in murine splenic B cells. Within 10 seconds of exposure to the CXCR5 ligand CXCL13, three-dimensional lamellar-like pseudopods and F-actin-rich ridges appeared. The transient F-actin increase depended on Gα i2/3 signaling, the PI3K/AKT pathway, ERK activation, phospholipase C activity, and Rac1/2 activation mediated by Dock2 (dedicator of cytokinesis 2). Immunoblot analyses identified the kinase WNK1 (with no lysine kinase 1) as a potential early AKT effector. Treating B cells with specific WNK inhibitors disrupted F-actin dynamics and impaired B cell polarity, motility, and chemotaxis. These changes were mimicked in a murine B cell line by CRISPR-Cas9 gene editing of Wnk1 , which also suggested that WNK1 contributed to B cell proliferation. Administration of a single dose of a WNK inhibitor transiently reduced B cell motility and polarity in the lymph nodes of live mice. These results indicate that WNK1 signaling maintains B cell responsiveness to CXCL13 and suggest that pharmacological inhibition of WNK1, which is involved in cancer progression and blood pressure regulation, may affect humoral immunity.

Collective cell migration is an emergent phenomenon, with long-range cell-cell communication influenced by various factors, including transmission of forces, viscoelasticity of individual cells, substrate interactions, and mechanotransduction. We investigate how alterations in cell-substrate distance fluctuations, cell-substrate adhesion, and traction forces impact the average velocity and temporal-spatial correlation of confluent monolayers formed by either wild-type (WT) MDCKII cells or zonula occludens (ZO)-1/2-depleted MDCKII cells (double knockdown [dKD]) representing highly contractile cells. The data indicate that confluent dKD monolayers exhibit decreased average velocity compared to less contractile WT cells concomitant with increased substrate adhesion, reduced traction forces, a more compact shape, diminished cell-cell interactions, and reduced cell-substrate distance fluctuations. Depletion of basal actin and myosin further supports the notion that short-range cell-substrate interactions, particularly fluctuations driven by basal actomyosin, significantly influence the migration speed of the monolayer on a larger length scale., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nuclear actin-based movements support DNA double-strand break (DSB) repair. However, molecular determinants that promote filamentous actin (F-actin) formation on the damaged chromatin remain undefined. Here we describe the DYRK1A kinase as a nuclear activity that promotes local F-actin assembly to support DSB mobility and repair, accomplished in part by its targeting of actin nucleator spire homolog 1 (Spir1). Indeed, perturbing DYRK1A-dependent phosphorylation of S482 mis-regulated Spir1 accumulation at damaged-modified chromatin, and led to compromised DSB-associated actin polymerization and attenuated DNA repair. Our findings uncover a role of the DYRK1A-Spir1 axis in nuclear actin dynamics during early DSB responses, and highlight the intricate details of nuclear cytoskeletal network in DSB repair and genome stability maintenance., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Human cortical neurons (hCNs) exhibit high dendritic complexity and synaptic density, and the maturation process is greatly protracted. However, the molecular mechanism governing these specific features remains unclear. Here, we report that the hominoid-specific gene TBC1D3 promotes dendritic arborization and protracts the pace of synaptogenesis. Ablation of TBC1D3 in induced hCNs causes reduction of dendritic growth and precocious synaptic maturation. Forced expression of TBC1D3 in the mouse cortex protracts synaptic maturation while increasing dendritic growth. Mechanistically, TBC1D3 functions via interaction with MICAL1, a monooxygenase that mediates oxidation of actin filament. At the early stage of differentiation, the TBC1D3/MICAL1 interaction in the cytosol promotes dendritic growth via F-actin oxidation and enhanced actin dynamics. At late stages, TBC1D3 escorts MICAL1 into the nucleus and downregulates the expression of genes related with synaptic maturation through interaction with the chromatin remodeling factor ATRX. Thus, this study delineates the molecular mechanisms underlying human neuron development., Competing Interests: Declaration of interests The authors have no competing financial interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Clathrin-mediated endocytosis has characteristic features in neuronal dendrites and presynapses, but how membrane proteins are internalized along the axon shaft remains unclear. We focused on clathrin-coated structures and endocytosis along the axon initial segment (AIS) and their relationship to the periodic actin-spectrin scaffold that lines the axonal plasma membrane. A combination of super-resolution microscopy and platinum-replica electron microscopy on cultured neurons revealed that AIS clathrin-coated pits form within "clearings", circular areas devoid of actin-spectrin mesh. Actin-spectrin scaffold disorganization increased clathrin-coated pit formation. Cargo uptake and live-cell imaging showed that AIS clathrin-coated pits are particularly stable. Neuronal plasticity-inducing stimulation triggered internalization of the clathrin-coated pits through polymerization of branched actin around them. Thus, spectrin and actin regulate clathrin-coated pit formation and scission to control endocytosis at the AIS.

Depletion interactions are thought to significantly contribute to the organization of intracellular structures in the crowded cytosol. The strength of depletion interactions depends on physical parameters such as the depletant number density and the depletant size ratio. Cells are known to dynamically regulate these two parameters by varying the copy number of proteins of a wide distribution of sizes. However, mammalian cells are also known to keep the total protein mass density remarkably constant, to within 0.5% throughout the cell cycle. We thus ask how the strength of depletion interactions varies when the total depletant mass is held fixed, a.k.a. fixed-mass depletion. We answer this question via scaling arguments, as well as by studying depletion effects on networks of reconstituted semiflexible actin in silico and in vitro. We examine the maximum strength of the depletion interaction potential U∗ as a function of q, the size ratio between the depletant and the matter being depleted. We uncover a scaling relation U∗ ∼ qζ for two cases: fixed volume fraction φ and fixed mass density ρ. For fixed volume fraction, we report ζ < 0. For the fixed mass density case, we report ζ > 0, which suggests that the depletion interaction strength increases as the depletant size ratio is increased. To test this prediction, we prepared our filament networks at fixed mass concentrations with varying sizes of the depletant molecule poly(ethylene glycol) (PEG). We characterize the depletion interaction strength in our simulations via the mesh size. In experiments, we observe two distinct actin network morphologies, which we call weakly bundled and strongly bundled. We identify a mass concentration where different PEG depletant sizes lead to weakly bundled or strongly bundled morphologies. For these conditions, we find that the mesh size and intra-bundle spacing between filaments across the different morphologies do not show significant differences, while the dynamic light scattering relaxation time and storage modulus between the two states do show significant differences. Our results demonstrate the ability to tune actin network morphology and mechanics by controlling depletant size and give insights into depletion interaction mechanisms under the fixed-depletant-mass constraint relevant to living cells., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)