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Cationic liposomes composed of cholesteryl‐3β‐carboxyamidoethylene‐N‐hydroxyethylamine (OH‐chol) and 1,2‐dioleoyl‐sn‐glycero‐3‐phosphatidylethanolamine (DOPE) inhibit mast cell degranulation mediated via crosslinking of high‐affinity IgE receptors (FcεRI). Although the inhibitory efficiency of mast cell degranulation is altered by modifying the ratio of OH‐chol and DOPE in cationic liposomes, the manner in which physicochemical properties, such as surface charge and size, influence suppression is not clear. We observed that positive surface charge, but not the size, of liposomes plays a role in suppressing rat basophilic leukemia (RBL‐2H3) cell activation. Pretreatment with middle‐ratio OH‐chol liposomes (zeta potential, 62.2 ± 0.5 mV; diameter, 325.4 ± 7.3 nm) exhibited a larger suppression of RBL‐2H3 cell degranulation evoked by FcεRI crosslinking compared with that by low‐ratio OH‐chol liposomes (zeta potential, 48.6 ± 1.9 mV; diameter, 344.4 ± 25.0 nm), although both liposomes were similarly attached to RBL‐2H3 cells. Preparation of middle‐ratio OH‐chol liposomes, classified roughly by size using an extrusion method, revealed that the liposomal size did not affect the inhibitory efficiency of RBL‐2H3 cell activation. Mechanistically, we found that middle‐ratio OH‐chol liposomes increased the inhibition of antigen‐induced Akt phosphorylation compared to low‐ratio OH‐chol liposomes. We measured the phosphorylation of linker for activation of T cells (LAT) and paxillin, which are important proteins in FcεRI‐ and focal adhesions (FAs)‐mediated signaling, respectively. Middle ratio OH‐chol liposomes significantly suppressed antigen‐induced paxillin phosphorylation, but did not affect LAT phosphorylation, suggesting that middle‐ratio OH‐chol liposomes attached to RBL‐2H3 cells suppress the degranulation by impairing FA‐mediated Akt phosphorylation evoked by FcεRI crosslinking. [ABSTRACT FROM AUTHOR]

Meningiomas are predominantly benign tumors, but there are also malignant forms that are associated with a poor prognosis. Like almost all tumors, meningiomas metabolize glucose as part of aerobic glycolysis (Warburg effect) for energy supply, so there are attempts to influence the prognosis of tumor diseases using a glucose-reduced diet. This altered metabolism leads to so called hallmarks of cancer, such as glycation and glycosylation. In this study, we investigated the influence of low (3 mM), normal (5.5 mM) and high glucose (15 mM) on a malignant meningioma cell line (IOMM-Lee, WHO grade 3). In addition, the influence of methylglyoxal, a by-product of glycolysis and a precursor for glycation, was investigated. Impedance-based methods (ECIS and RTCA) were used to study migration and invasion, and immunoblotting was used to analyze the expression of proteins relevant to these processes, such as focal adhesion kinase (FAK), merlin or integrin ß1. We were able to show that low glucose reduced the invasive potential of the cells, which was associated with a reduced amount of sialic acid. Under high glucose, barrier function was impaired and adhesion decreased, which correlated with a decreased expression of FAK. [ABSTRACT FROM AUTHOR]

Recently, a variety of microenvironmental biophysical stimuli have been proved to play a crucial role in regulating cell functions. Among them, morpho-physical cues, like curvature, are emerging as key regulators of cellular behavior. Changes in substrate curvature have been shown to impact the arrangement of Focal Adhesions (FAs), influencing the direction and intensity of cytoskeleton generated forces and resulting in an overall alteration of cell mechanical identity. In their native environment, cells encounter varying degrees of substrate curvature, and in specific organs, they are exposed to dynamic changes of curvature due to periodic tissue deformation. However, the mechanism by which cells perceive substrate curvature remains poorly understood. To this aim, a micro-pneumatic device was designed and implemented. This device enables the controlled application of substrate curvature, both statically and dynamically. Employing a combined experimental and simulative approach, human adipose-derived stem cells were exposed to controlled curvature intensity and frequency. During this exposure, measurements were taken on FAs extension and orientation, cytoskeleton organization and cellular/nuclear alignment. The data clearly indicated a significant influence of the substrate curvature on cell adhesion processes. These findings contribute to a better understanding of the mechanisms through which cells perceive and respond to substrate curvature signals. This work is our contribution to the comprehension of substrate curvature's function as a crucial regulator of cell adhesion at the scale of focal adhesions and cell mechanical identity. In recent years, a large body of knowledge is continuously growing providing comprehension of the role of various microenvironmental biophysical stimuli in regulating cell functions. Nevertheless, little is known about the role of substrate curvature, in particular, when cells are exposed to this stimulus in a dynamic manner. To address the role of substrate curvature on cellular behavior, a micro-pneumatic device was designed and implemented. This device enables the controlled application of substrate curvature, both statically and dynamically. The experiment data made it abundantly evident that the substrate curvature had a major impact on the mechanisms involved in cell adhesion. [Display omitted] [ABSTRACT FROM AUTHOR]

Cisplatin-induced renal tubular injury largely restricts the wide-spread usage of cisplatin in the treatment of malignancies. Identifying the key signaling pathways that regulate cisplatin-induced renal tubular injury is thus clinically important. PARVB, a focal adhesion protein, plays a crucial role in tumorigenesis. However, the function of PARVB in kidney disease is largely unknown. To investigate whether and how PARVB contributes to cisplatin-induced renal tubular injury, a mouse model (PARVB cKO) was generated in which PARVB gene was specifically deleted from proximal tubular epithelial cells using the Cre-LoxP system. In this study, we found depletion of PARVB in proximal tubular epithelial cells significantly attenuates cisplatin-induced renal tubular injury, including tubular cell death and inflammation. Mechanistically, PARVB associates with transforming growth factor-β-activated kinase 1 (TAK1), a central regulator of cell survival and inflammation that is critically involved in mediating cisplatin-induced renal tubular injury. Depletion of PARVB promotes cisplatin-induced TAK1 degradation, inhibits TAK1 downstream signaling, and ultimately alleviates cisplatin-induced tubular cell damage. Restoration of PARVB or TAK1 in PARVB-deficient cells aggravates cisplatin-induced tubular cell injury. Finally, we demonstrated that PARVB regulates TAK1 protein expression through an E3 ligase ITCH-dependent pathway. PARVB prevents ITCH association with TAK1 to block its ubiquitination. Our study reveals that PARVB deficiency protects against cisplatin-induced tubular injury through regulation of TAK1 signaling and indicates targeting this pathway may provide a novel therapeutic strategy to alleviate cisplatin-induced kidney damage. [ABSTRACT FROM AUTHOR]

Ovulation is vital for successful reproduction. Following ovulation, cumulus cells and oocyte are released, while mural granulosa cells (mGCs) remain sequestered within the post‐ovulatory follicle to form the corpus luteum. However, the mechanism underlying the confinement of mGCs has been a longstanding mystery. Here, in vitro and in vivo evidence is provided demonstrating that the stiffening of mGC‐layer serves as an evolutionarily conserved mechanism that prevents mGCs from escaping the post‐ovulatory follicles. The results from spatial transcriptome analysis and experiments reveal that focal adhesion assembly, triggered by the LH (hCG)‐cAMP‐PKA‐CREB signaling cascade, is necessary for mGC‐layer stiffening. Disrupting focal adhesion assembly through RNA interference results in stiffening failure, mGC escape, and the subsequent development of an abnormal corpus luteum characterized by decreased cell density or cavities. These findings introduce a novel concept of "mGC‐layer stiffening", shedding light on the mechanism that prevents mGC escape from the post‐ovulatory follicle. [ABSTRACT FROM AUTHOR]

Chunjiao Lu,* Xuewei Wu,* Xin Meng,* Yi Liu, Ting Yang, Yan Zeng, Yang Chen, Yishan Huang, Zhou Fang, Xiaojun Yang, Juanjuan Luo Engineering Research Center of Key Technique for Biotherapy of Guangdong Province, Shantou University Medical College, Shantou, 515041, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiaojun Yang; Juanjuan Luo, Shantou University Medical College, 22 Xinling Road, Shantou, 515041, People’s Republic of China, Email yangx@stu.edu.cn; 15jjluo1@stu.edu.cnIntroduction: The potential toxic effects of wastewater discharges containing silver nanoparticles (AgNPs) and their release into aquatic ecosystems on aquatic organisms are becoming a major concern for environmental and human health. However, the potential risks of AgNPs to aquatic organisms, especially for cardiac development by Focal adhesion pathway, are still poorly understood.Methods: The cardiac development of various concentrations of AgNPs in zebrafish were examined using stereoscopic microscope. The expression levels of cardiac development-related genes were analyzed by qRT-PCR and Whole-mount in situ hybridization (WISH). In addition, Illumina high-throughput global transcriptome analysis was performed to explore the potential signaling pathway involved in the treatment of zebrafish embryos by AgNPs after 72 h.Results: We systematically investigated the cardiac developing toxicity of AgNPs on the embryos of zebrafish. The results demonstrated that 2 or 4 mg/L AgNPs exposure induces cardiac developmental malformations, such as the appearance of pericardial edema phenotype. In addition, after 72 h of exposure, the mRNA levels of cardiac development-related genes, such as myh7, myh6, tpm1, nppa, tbx5, tbx20, myl7 and cmlc1, were significantly lower in AgNPs-treated zebrafish embryos than in control zebrafish embryos. Moreover, RNA sequencing, KEGG (Kyoto Encyclopedia of Genes) and Genomes and GSEA (gene set enrichment analysis) of the DEGs (differentially expressed genes) between the AgNPs-exposed and control groups indicated that the downregulated DEGs were mainly enriched in focal adhesion pathways. Further investigations demonstrated that the mRNA levels of focal adhesion pathway-related genes, such as igf1ra, shc3, grb2b, ptk2aa, akt1, itga4, parvaa, akt3b and vcla, were significantly decreased after AgNPs treatment in zebrafish.Conclusion: Thus, our findings illustrated that AgNPs could impair cardiac development by regulating the focal adhesion pathway in zebrafish.Keywords: AgNPs, focal adhesion, cardiac development, zebrafish

Abstract Background Recycling of integrin via endosomal vesicles is critical for the migration of cancer cells, which leads to the metastasis of pancreatic cancer and devastating cancer-related death. So, new diagnostic and therapeutic molecules which target the recycling of endosomal vesicles need to be developed. Methods Public databases including TCGA, ICGC, GSE21501, GSE28735, and GENT are analyzed to derive diagnostic and therapeutic targets. To reveal biological roles and underlying mechanisms of molecular targets, various molecular biological experiments were conducted. Results First, we identified UNC13D’s overexpression in patients with pancreatic cancer (n = 824) and its prognostic significance and high hazard ratio (HR) in four independent pancreatic cancer cohorts (TCGA, n = 178, p = 0.014, HR = 3.629; ICGC, n = 91, p = 0.000, HR = 4.362; GSE21501, n = 102, p = 0.002, HR = 2.339; GSE28735, n = 45, p = 0.022, HR = 2.681). Additionally, its expression is associated with the clinicopathological progression of pancreatic cancer. Further biological studies have shown that UNC13D regulates the migration of pancreatic cancer cells by coupling the exocytosis of recycling endosomes with focal adhesion turnover via the regulation of FAK phosphorylation. Immunoprecipitation and immunocytochemistry showed the formation of the RAB11-UNC13D-FAK axis in endosomes during integrin recycling. We observed that UNC13D directly interacted with the FERM domain of FAK and regulated FAK phosphorylation in a calcium-dependent manner. Finally, we found co-expression of UNC13D and FAK showed the poorest survival (TCGA, p = 0.000; ICGC, p = 0.036; GSE28735, p = 0.006). Conclusions We highlight that UNC13D, a novel prognostic factor, promotes pancreatic cancer progression by coupling integrin recycling with focal adhesion turnover via the RAB11-UNC13D-FAK axis for the migration of pancreatic cancer cells.

Abstract Vitamin D modulates innate and adaptive immunity, the molecular mechanisms of which we aim to understand under human in vivo conditions. Therefore, we designed the study VitDHiD (NCT03537027) as a human investigation, in which 25 healthy individuals were supplemented with a single vitamin D3 bolus (80,000 IU). Transcriptome-wide differential gene expression analysis of peripheral blood mononuclear cells (PBMCs), which were isolated directly before and 24 h after supplementation, identified 452 genes significantly (FDR

Background: Recycling of integrin via endosomal vesicles is critical for the migration of cancer cells, which leads to the metastasis of pancreatic cancer and devastating cancer-related death. So, new diagnostic and therapeutic molecules which target the recycling of endosomal vesicles need to be developed. Methods: Public databases including TCGA, ICGC, GSE21501, GSE28735, and GENT are analyzed to derive diagnostic and therapeutic targets. To reveal biological roles and underlying mechanisms of molecular targets, various molecular biological experiments were conducted. Results: First, we identified UNC13D's overexpression in patients with pancreatic cancer (n = 824) and its prognostic significance and high hazard ratio (HR) in four independent pancreatic cancer cohorts (TCGA, n = 178, p = 0.014, HR = 3.629; ICGC, n = 91, p = 0.000, HR = 4.362; GSE21501, n = 102, p = 0.002, HR = 2.339; GSE28735, n = 45, p = 0.022, HR = 2.681). Additionally, its expression is associated with the clinicopathological progression of pancreatic cancer. Further biological studies have shown that UNC13D regulates the migration of pancreatic cancer cells by coupling the exocytosis of recycling endosomes with focal adhesion turnover via the regulation of FAK phosphorylation. Immunoprecipitation and immunocytochemistry showed the formation of the RAB11-UNC13D-FAK axis in endosomes during integrin recycling. We observed that UNC13D directly interacted with the FERM domain of FAK and regulated FAK phosphorylation in a calcium-dependent manner. Finally, we found co-expression of UNC13D and FAK showed the poorest survival (TCGA, p = 0.000; ICGC, p = 0.036; GSE28735, p = 0.006). Conclusions: We highlight that UNC13D, a novel prognostic factor, promotes pancreatic cancer progression by coupling integrin recycling with focal adhesion turnover via the RAB11-UNC13D-FAK axis for the migration of pancreatic cancer cells. [ABSTRACT FROM AUTHOR]

The contractile apparatus, stress fiber (SF), is connected to the cell adhesion machinery, focal adhesion (FA), at the termini of SF. The SF-FA complex is essential for various mechanical activities of cells, including cell adhesion to the extracellular matrix (ECM), ECM rigidity sensing, and cell migration. This mini-review highlights the importance of SF mechanics in these cellular activities. Actin-crosslinking proteins solidify SFs by attenuating myosindriven flows of actin and myosin filaments within the SF. In the solidified SFs, viscous slippage between actin filaments in SFs and between the filaments and the surrounding cytosol is reduced, leading to efficient transmission of myosin-generated contractile force along the SFs. Hence, SF solidification via actin crosslinking ensures exertion of a large force to FAs, enabling FA maturation, ECM rigidity sensing and cell migration. We further discuss intracellular mechanisms for tuning crosslinker-modulated SF mechanics and the potential relationship between the aberrance of SF mechanics and pathology including cancer. [ABSTRACT FROM AUTHOR]

This study investigates differences in focal adhesion (FA) morphology and Talin cleavage levels between transformed and non-transformed cell lines. Utilizing fluorescently tagged wild-type Talin and Talin mutants with calpain cleavage site mutations, FA structures were visualized. Mutations in different Talin cleavage sites showed varying impacts on FA morphology and distribution across melanoma cell lines (Meljuso, A375P, A2058) and a non-transformed cell line (HFF). Western blot analysis, ratiometric fluorescence intensity-based measurements, and FRAP experiments revealed higher Talin cleavage levels within FAs of transformed cell lines compared to non-transformed cells. Additionally, growth assays indicated that reducing calpain cleavage levels attenuated transformed cell growth. These findings suggest that Talin cleavage level is crucial for FA morphology and assembly, with higher levels observed in transformed cells, influencing their growth dynamics. [ABSTRACT FROM AUTHOR]

The role of the extracellular matrix (ECM) in Parkinson's disease (PD) is not well understood, even though it is critical for neuronal structure and signaling. This systematic review identified the top deregulated ECM-related pathways in studies that used gene set enrichment analyses (GSEA) to document transcriptomic, proteomic, or genomic alterations in PD. PubMed and Google scholar were searched for transcriptomics, proteomics, or genomics studies that employed GSEA on data from PD tissues or cells and reported ECM-related pathways among the top-10 most enriched versus controls. Twenty-seven studies were included, two of which used multiple omics analyses. Transcriptomics and proteomics studies were conducted on a variety of tissue and cell types. Of the 17 transcriptomics studies (16 data sets), 13 identified one or more adhesion pathways in the top-10 deregulated gene sets or pathways, primarily related to cell adhesion and focal adhesion. Among the 8 proteomics studies, 5 identified altered overarching ECM gene sets or pathways among the top 10. Among the 4 genomics studies, 3 identified focal adhesion pathways among the top 10. The findings summarized here suggest that ECM organization/structure and cell adhesion (particularly focal adhesion) are altered in PD and should be the focus of future studies. [ABSTRACT FROM AUTHOR]

The complex regulation of traction forces (TF) produced during cellular migration remains poorly understood. We have previously found that calpain 4 (Capn4), the small non-catalytic subunit of the calpain 1 and 2 proteases, regulates the production of TF independent of the proteolytic activity of the larger subunits. Capn4 was later found to facilitate tyrosine phosphorylation and secretion of the lectin-binding protein galectin-3 (Gal3). In this study, recombinant Gal3 (rGal3) was added to the media-enhanced TF generated by capn4−/− mouse embryonic fibroblasts (MEFs). Extracellular Gal3 also rescued defects in the distribution, morphology, and adhesive strength of focal adhesions present in capn4−/− MEF cells. Surprisingly, extracellular Gal3 does not influence mechanosensing. c-Abl kinase was found to affect Gal3 secretion and the production of TF through phosphorylation of Y107 on Gal3. Our study also suggests that Gal3-mediated regulation of TF occurs through signaling pathways triggered by β1 integrin but not by focal adhesion kinase (FAK) Y397 autophosphorylation. Our findings provide insights into the signaling mechanism by which Capn4 and secreted Gal3 regulate cell migration through the modulation of TF distinctly independent from a mechanosensing mechanism. [ABSTRACT FROM AUTHOR]

ABSTRACT In the aquaculture sector, one of the challenges includes disease outbreaks such as bacterial infections, particularly from Aeromonas hydrophila (Ah), impacting both wild and farmed fish. In this study, we conducted a proteomic analysis of the intestinal tissue in Labeo rohita following Ah infection to elucidate the protein alterations and its implications for immune response. Our findings indicate significant dysregulation in extracellular matrix (ECM)-associated proteins during Ah infection, with increased abundance of elastin and collagen alpha-3(VI). Pathway and enrichment analysis of differentially expressed proteins highlights the involvement of ECM-related pathways, including focal adhesions, integrin cell surface interactions, and actin cytoskeleton organization. Focal adhesions, crucial for connecting intracellular actin bundles to the ECM, play a pivotal role in immune response during infections. Increased abundance of integrin alpha 1, integrin beta 1, and tetraspanin suggests their involvement in the host’s response to Ah infection. Proteins associated with actin cytoskeleton reorganization, such as myosin, tropomyosin, and phosphoglucomutase, exhibit increased abundance, influencing changes in cell behavior. Additionally, upregulated proteins like LTBP1 and fibrillin-2 contribute to TGF-β signaling and focal adhesion, indicating their potential role in immune regulation. The study also identifies elevated levels of laminin, galectin 3, and tenascin-C, which interact with integrins and other ECM components, potentially influencing immune cell migration and function. These proteins, along with decorin and lumican, may act as immunomodulators, coordinating pro- and anti-inflammatory responses. ECM fragments released during pathogen invasion could serve as “danger signals,” initiating pathogen clearance and tissue repair through Toll-like receptor signaling.IMPORTANCEThe study underscores the critical role of the extracellular matrix (ECM) and its associated proteins in the immune response of aquatic organisms during bacterial infections like Aeromonas hydrophila. Understanding the intricate interplay between ECM alterations and immune response pathways provides crucial insights for developing effective disease control strategies in aquaculture. By identifying key proteins and pathways involved in host defense mechanisms, this research lays the groundwork for targeted interventions to mitigate the impact of bacterial infections on fish health and aquaculture production.

Abstract Ovulation is vital for successful reproduction. Following ovulation, cumulus cells and oocyte are released, while mural granulosa cells (mGCs) remain sequestered within the post‐ovulatory follicle to form the corpus luteum. However, the mechanism underlying the confinement of mGCs has been a longstanding mystery. Here, in vitro and in vivo evidence is provided demonstrating that the stiffening of mGC‐layer serves as an evolutionarily conserved mechanism that prevents mGCs from escaping the post‐ovulatory follicles. The results from spatial transcriptome analysis and experiments reveal that focal adhesion assembly, triggered by the LH (hCG)‐cAMP‐PKA‐CREB signaling cascade, is necessary for mGC‐layer stiffening. Disrupting focal adhesion assembly through RNA interference results in stiffening failure, mGC escape, and the subsequent development of an abnormal corpus luteum characterized by decreased cell density or cavities. These findings introduce a novel concept of “mGC‐layer stiffening”, shedding light on the mechanism that prevents mGC escape from the post‐ovulatory follicle.

The editorial "Editors' showcase 2023: insights in cell adhesion and migration" published in Frontiers in Cell & Developmental Biology discusses the importance of cell adhesion for migration, focusing on both extracellular matrix and cell-to-cell adhesion. The research topic includes reviews and original research papers on various aspects of cell movement, such as cytoskeleton remodeling, molecular motor activity, and membrane trafficking. The editorial highlights the role of mechanotransduction, extracellular matrix, and molecular motor proteins in cell migration, providing insights into the complex mechanisms involved in this essential biological process. [Extracted from the article]

Biomolecular condensates are nonmembranous structures that are mainly formed through liquid-liquid phase separation. Tensins are focal adhesion (FA) proteins linking the actin cytoskeleton to integrin receptors. Here, we report that GFP-tagged tensin-1 (TNS1) proteins phase-separate to form biomolecular condensates in cells. Live-cell imaging showed that new TNS1 condensates are budding from the disassembling ends of FAs, and the presence of these condensates is cell cycle dependent. TNS1 condensates dissolve immediately prior to mitosis and rapidly reappear while postmitotic daughter cells establish new FAs. TNS1 condensates contain selected FA proteins and signaling molecules such as pT308Akt but not pS473Akt, suggesting previously unknown roles of TNS1 condensates in disassembling FAs, as the storage of core FA components and the signaling intermediates.

Abstract Background Renal fibrosis is a prevalent manifestation of chronic kidney disease (CKD), and effective treatments for this disease are currently lacking. Myofibroblasts, which originate from interstitial fibroblasts, aggregate in the renal interstitium, leading to significant accumulation of extracellular matrix and impairment of renal function. The nonreceptor tyrosine kinase c-Abl (encoded by the Abl1 gene) has been implicated in the development of renal fibrosis. However, the precise role of c-Abl in this process and its involvement in fibroblast-myofibroblast transition (FMT) remain poorly understood. Methods To investigate the effect of c-Abl in FMT during renal fibrosis, we investigated the expression of c-Abl in fibrotic renal tissues of patients with CKD and mouse models. We studied the phenotypic changes in fibroblast or myofibroblast-specific c-Abl conditional knockout mice. We explored the potential targets of c-Abl in NRK-49F fibroblasts. Results In this study, fibrotic mouse and cell models demonstrated that c-Abl deficiency in fibroblasts mitigated fibrosis by suppressing fibroblast activation, fibroblast-myofibroblast transition, and extracellular matrix deposition. Mechanistically, c-Abl maintains the stability of the RACK1 protein, which serves as a scaffold for proteins such as c-Abl and focal adhesion kinase at focal adhesions, driving fibroblast activation and differentiation during renal fibrosis. Moreover, specifically targeting c-Abl deletion in renal myofibroblasts could prove beneficial in established kidney fibrosis by reducing RACK1 expression and diminishing the extent of fibrosis. Conclusions Our findings suggest that c-Abl plays a pathogenic role in interstitial fibrosis through the regulation of RACK1 protein stabilization and myofibroblast differentiation, suggesting a promising strategy for the treatment of CKD.

Simple Summary: α-Catenin mediates cell-matrix interactions while Piezo1 at focal adhesions likely mechanosenses traction force through the matrix hydrogel during cell mechanical communication. Cell-to-cell distant mechanical communication has been demonstrated using in vitro and in vivo models. However, the molecular mechanisms underlying long-range cell mechanoresponsive interactions remain to be fully elucidated. This study further examined the roles of α-Catenin and Piezo1 in traction force-induced rapid branch assembly of airway smooth muscle (ASM) cells on a Matrigel hydrogel containing type I collagen. Our findings demonstrated that siRNA-mediated downregulation of α-Catenin or Piezo1 expression or chemical inhibition of Piezo1 activity significantly reduced both directional cell movement and branch assembly. Regarding the role of N-cadherin in regulating branch assembly but not directional migration, our results further confirmed that siRNA-mediated downregulation of α-Catenin expression caused a marked reduction in focal adhesion formation, as assessed by focal Paxillin and Integrin α5 localization. These observations imply that mechanosensitive α-Catenin is involved in both cell–cell and cell-matrix adhesions. Additionally, Piezo1 partially localized in focal adhesions, which was inhibited by siRNA-mediated downregulation of α-Catenin expression. This result provides insights into the Piezo1-mediated mechanosensing of traction force on a hydrogel. Collectively, our findings highlight the significance of α-Catenin in the regulation of cell-matrix interactions and provide a possible interpretation of Piezo1-mediated mechanosensing activity at focal adhesions during cell–cell mechanical communication. [ABSTRACT FROM AUTHOR]

Talin (herein referring collectively to talin 1 and 2) couples the actomyosin cytoskeleton to integrins and transmits tension to the extracellular matrix. Talin also interacts with numerous additional proteins capable of modulating the actin-integrin linkage and thus downstream mechanosignaling cascades. Here, we demonstrate that the scaffold protein Caskin2 interacts directly with the R8 domain of talin through its C-terminal LD motif. Caskin2 also associates with the WAVE regulatory complex to promote cell migration in an Abi1-dependent manner. Furthermore, we demonstrate that the Caskin2-Abi1 interaction is regulated by growth factor-induced phosphorylation of Caskin2 on serine 878. In MCF7 and UACC893 cells, which contain an amplification of CASKIN2, Caskin2 localizes in plasma membrane-associated plaques and around focal adhesions in cortical microtubule stabilization complexes. Taken together, our results identify Caskin2 as a novel talin-binding protein that might not only connect integrin-mediated adhesion to actin polymerization but could also play a role in crosstalk between integrins and microtubules. [ABSTRACT FROM AUTHOR]

Background: Renal fibrosis is a prevalent manifestation of chronic kidney disease (CKD), and effective treatments for this disease are currently lacking. Myofibroblasts, which originate from interstitial fibroblasts, aggregate in the renal interstitium, leading to significant accumulation of extracellular matrix and impairment of renal function. The nonreceptor tyrosine kinase c-Abl (encoded by the Abl1 gene) has been implicated in the development of renal fibrosis. However, the precise role of c-Abl in this process and its involvement in fibroblast-myofibroblast transition (FMT) remain poorly understood. Methods: To investigate the effect of c-Abl in FMT during renal fibrosis, we investigated the expression of c-Abl in fibrotic renal tissues of patients with CKD and mouse models. We studied the phenotypic changes in fibroblast or myofibroblast-specific c-Abl conditional knockout mice. We explored the potential targets of c-Abl in NRK-49F fibroblasts. Results: In this study, fibrotic mouse and cell models demonstrated that c-Abl deficiency in fibroblasts mitigated fibrosis by suppressing fibroblast activation, fibroblast-myofibroblast transition, and extracellular matrix deposition. Mechanistically, c-Abl maintains the stability of the RACK1 protein, which serves as a scaffold for proteins such as c-Abl and focal adhesion kinase at focal adhesions, driving fibroblast activation and differentiation during renal fibrosis. Moreover, specifically targeting c-Abl deletion in renal myofibroblasts could prove beneficial in established kidney fibrosis by reducing RACK1 expression and diminishing the extent of fibrosis. Conclusions: Our findings suggest that c-Abl plays a pathogenic role in interstitial fibrosis through the regulation of RACK1 protein stabilization and myofibroblast differentiation, suggesting a promising strategy for the treatment of CKD. [ABSTRACT FROM AUTHOR]

The 5-year survival rate of kidney cancer drops dramatically from 93% to 15% when it is metastatic. Metastasis constitutes for 30% of kidney cancer cases, in which clear cell renal cell carcinoma (ccRCC) is the most prominent subtype. By sequencing mRNA of ccRCC patient samples, we found that apolipoprotein L1 (APOL1) was highly expressed in tumors compared to their adjacent normal tissues. This gene has been previously identified in a large body of kidney disease research and was reported as a potential prognosis marker in many types of cancers. However, the molecular function of APOL1 in ccRCC, especially in metastasis, remained unknown. In this study, we modulated the expression of APOL1 in various renal cancer cell lines and analyzed their proliferative, migratory, and invasive properties. Strikingly, APOL1 overexpression suppressed ccRCC metastasis both in vitro and in vivo. We then explored the mechanism by which APOL1 alleviated ccRCC malignant progression by investigating its downstream pathways. APOL1 overexpression diminished the activity of focal adhesive molecules, Akt signaling pathways, and EMT processes. Furthermore, in the upstream, we discovered that miR-30a-3p could inhibit APOL1 expression. In conclusion, our study revealed that APOL1 play a role as a tumor suppressor in ccRCC and inhibit metastasis, which may provide novel potential therapeutic approaches for ccRCC patients. [ABSTRACT FROM AUTHOR]

Angiogenesis is a tightly controlled dynamic process demanding a delicate equilibrium between pro-angiogenic signals and factors that promote vascular stability. The spatiotemporal activation of the transcriptional co-factors YAP (herein referring to YAP1) and TAZ (also knownWWTR1), collectively denoted YAP/TAZ, is crucial to allow for efficient collective endothelial migration in angiogenesis. The focal adhesion protein deleted-in-liver-cancer-1 (DLC1) was recently described as a transcriptional downstream target of YAP/TAZ in endothelial cells. In this study, we uncover a negative feedback loop between DLC1 expression and YAP activity during collective migration and sprouting angiogenesis. In particular, our study demonstrates that signaling via theRhoGAP domain of DLC1 reduces nuclear localization of YAP and its transcriptional activity. Moreover, the RhoGAP activity of DLC1 is essential for YAP-mediated cellular processes, including the regulation of focal adhesion turnover, traction forces, and sprouting angiogenesis. We show that DLC1 restricts intracellular cytoskeletal tension by inhibiting Rho signaling at the basal adhesion plane, consequently reducing nuclear YAP localization. Collectively, these findings underscore the significance of DLC1 expression levels and its function in mitigating intracellular tension as a pivotal mechanotransductive feedback mechanism that finely tunes YAP activity throughout the process of sprouting angiogenesis. [ABSTRACT FROM AUTHOR]

Integrin‐based focal adhesion is one of the major mechanosensory in osteocytes. The aim of this study was to mine the hub genes associated with focal adhesion and investigate their roles in osteoporosis based on the data of single‐cell RNA sequencing and RNA‐sequencing. Two hub genes (FAM129A and RNF24) with the same expression trend and AUC values greater than 0.7 in both GSE56815 and GSE56116 cohorts were uncovered. The nomogram was created to predict the risk of OP based on two hub genes. Subsequently, the competing endogenous RNA network was established based on two hub genes, 14 microRNAs and five long noncoding RNAs. Meanwhile, transcription factors‐hub gene network was established based on two hub genes and 14 TFs. Finally, 73 drugs were predicted, of which there were 13 drugs targeting FAM129A and 66 drugs targeting RNF24. In both mouse and human blood samples, FAM129A expression was decreased in granulocytes and RNF24 expression was increased in monocytes. In the mouse experiment, FAM129A and anti‐RNF24 were found to partially alleviate the progression of osteoporosis. In conclusion, two hub genes related to focal adhesion were identified by combined scRNA‐seq and RNA‐seq analyses, which might supply a new insight for the treatment and evaluation of OP. [ABSTRACT FROM AUTHOR]

The function of kidney podocytes is closely associated with actin cytoskeleton regulated by Rho small GTPases. Loss of actin-driven cell adhesions and processes is connected to podocyte dysfunction, proteinuria, and kidney diseases. FilGAP, a GTPase-activating protein for Rho small GTPase Rac1, is abundantly expressed in kidney podocytes, and its gene is linked to diseases in a family with focal segmental glomerulosclerosis. In this study, we have studied the role of FilGAP in podocytes in vitro. Depletion of FilGAP in cultured podocytes induced loss of actin stress fibers and increased Rac1 activity. Conversely, forced expression of FilGAP increased stress fiber formation whereas Rac1 activation significantly reduced its formation. FilGAP localizes at the focal adhesion (FA), an integrin-based protein complex closely associated with stress fibers, that mediates cell-extracellular matrix (ECM) adhesion, and FilGAP depletion decreased FA formation and impaired attachment to the ECM. Moreover, in unique podocyte cell cultures capable of inducing the formation of highly organized processes including major processes and foot process-like projections, FilGAP depletion or Rac1 activation decreased the formation of these processes. The reduction of FAs and process formations in FilGAP-depleted podocyte cells was rescued by inhibition of Rac1 or P21-activated kinase 1 (PAK1), a downstream effector of Rac1, and PAK1 activation inhibited their formations. Thus, FilGAP contributes to both cell-ECM adhesion and process formation of podocytes by suppressing Rac1/PAK1 signaling. [ABSTRACT FROM AUTHOR]

Aortic valve disease (AVD) represents a global public health challenge. Research indicates a higher prevalence of diabetes in AVD patients, accelerating disease advancement. Although the specific mechanisms linking diabetes to valve dysfunction remain unclear, alterations of valvular endothelial cells (VECs) homeostasis due to high glucose (HG) or their crosstalk with monocytes play pivotal roles. The aim of this study was to determine the molecular signatures of VECs in HG and upon their interaction with monocytes in normal (NG) or high glucose conditions and to propose novel mechanisms underlying valvular dysfunction in diabetes. VECs and THP-1 monocytes cultured in NG/HG conditions were used. The RNAseq analysis revealed transcriptomic changes in VECs, in processes related to cytoskeleton regulation, focal adhesions, cellular junctions, and cell adhesion. Key molecules were validated by qPCR, Western blot, and immunofluorescence assays. The alterations in cytoskeleton and intercellular junctions impacted VEC function, leading to changes in VECs adherence to extracellular matrix, endothelial permeability, monocyte adhesion, and transmigration. The findings uncover new molecular mechanisms of VEC dysfunction in HG conditions and upon their interaction with monocytes in NG/HG conditions and may help to understand mechanisms of valvular dysfunction in diabetes and to develop novel therapeutic strategies in AVD. [ABSTRACT FROM AUTHOR]

The pericellular matrix (PCM) is a specialized extracellular matrix that surrounds cells. Interactions with the PCM enable the cells to sense and respond to mechanical signals, triggering a proper adaptive response. Collagen VI is a component of muscle and tendon PCM. Mutations in collagen VI genes cause a distinctive group of inherited skeletal muscle diseases, and Ullrich congenital muscular dystrophy (UCMD) is the most severe form. In addition to muscle weakness, UCMD patients show structural and functional changes of the tendon PCM. In this study, we investigated whether PCM alterations due to collagen VI mutations affect the response of tendon fibroblasts to mechanical stimulation. By taking advantage of human tendon cultures obtained from unaffected donors and from UCMD patients, we analyzed the morphological and functional properties of cellular mechanosensors. We found that the length of the primary cilia of UCMD cells was longer than that of controls. Unlike controls, in UCMD cells, both cilia prevalence and length were not recovered after mechanical stimulation. Accordingly, under the same experimental conditions, the activation of the Hedgehog signaling pathway, which is related to cilia activity, was impaired in UCMD cells. Finally, UCMD tendon cells exposed to mechanical stimuli showed altered focal adhesions, as well as impaired activation of Akt, ERK1/2, p38MAPK, and mechanoresponsive genes downstream of YAP. By exploring the response to mechanical stimulation, for the first time, our findings uncover novel unreported mechanistic aspects of the physiopathology of UCMD-derived tendon fibroblasts and point at a role for collagen VI in the modulation of mechanotransduction in tendons. [ABSTRACT FROM AUTHOR]

Abstract Background The R-RAS2 is a small GTPase highly similar to classical RAS proteins at the regulatory and signaling levels. The high evolutionary conservation of R-RAS2, its links to basic cellular processes and its role in cancer, make R-RAS2 an interesting research topic. To elucidate the evolutionary history of R-RAS proteins, we investigated and compared structural and functional properties of ancestral type R-RAS protein with human R-RAS2. Methods Bioinformatics analysis were used to elucidate the evolution of R-RAS proteins. Intrinsic GTPase activity of purified human and sponge proteins was analyzed with GTPase-GloTM Assay kit. The cell model consisted of human breast cancer cell lines MCF-7 and MDA-MB-231 transiently transfected with EsuRRAS2-like or HsaRRAS2. Biological characterization of R-RAS2 proteins was performed by Western blot on whole cell lysates or cell adhesion protein isolates, immunofluorescence and confocal microscopy, MTT test, colony formation assay, wound healing and Boyden chamber migration assays. Results We found that the single sponge R-RAS2-like gene/protein probably reflects the properties of the ancestral R-RAS protein that existed prior to duplications during the transition to Bilateria, and to Vertebrata. Biochemical characterization of sponge and human R-RAS2 showed that they have the same intrinsic GTPase activity and RNA binding properties. By testing cell proliferation, migration and colony forming efficiency in MDA-MB-231 human breast cancer cells, we showed that the ancestral type of the R-RAS protein, sponge R-RAS2-like, enhances their oncogenic potential, similar to human R-RAS2. In addition, sponge and human R-RAS2 were not found in focal adhesions, but both homologs play a role in their regulation by increasing talin1 and vinculin. Conclusions This study suggests that the ancestor of all animals possessed an R-RAS2-like protein with oncogenic properties similar to evolutionarily more recent versions of the protein, even before the appearance of true tissue and the origin of tumors. Therefore, we have unraveled the evolutionary history of R-RAS2 in metazoans and improved our knowledge of R-RAS2 properties, including its structure, regulation and function.

Abstract Background Metastasis is one of the leading cause contributes to treatment failure and poor prognosis of hepatocellular carcinoma (HCC) patients. The underlying mechanism of HCC metastasis remains to be determined. Although several RNA binding proteins (RBPs) have been found to participate in tumorigenesis and progression of liver cancer, the role of RBPs in HCC patients with extrahepatic metastases is poorly understood. Methods By performing RNA-seq of primary HCC tissues (including HCC with extrahepatic metastasis and those did not develop metastasis), we identified a set of HCC metastasis-associated RBPs candidates. Among which, ribosomal protein S7 (RPS7) was found to be remarkably increased in HCC tissues and be strongly related to HCC poor survival. Overexpression or CRISPR-Cas9–mediated knockout were applied to investigate the role of RPS7 on the metastasis-associated phenotypes of HCC cells. RNA sequencing, RIP, RNA-pull down, dual luciferase reporter assay, nascent RNA capture assay, and RNA decay and so on, were applied to reveal the underlying mechanism of RPS7 induced HCC metastasis. Results Gain- and loss- of function analyses revealed that RPS7 promoted HCC cells adhesion, migration and invasion capabilities, as well as lung metastasis. Mechanistically, we uncovered that lysyl oxidase-like 2 (LOXL2) was a critical downstream target of RPS7. RPS7 could stabilize LOXL2 mRNA by binding to AUUUA motifs in the 3155–3375 region of the 3’UTR of LOXL2 mRNA, thus increased LOXL2 expression via elevating LOXL2 mRNA abundance. Further research revealed that LOXL2 could accelerate focal adhesion formation through maintaining the protein stability of ITGB1 and activating ITGB1-mediated FAK/SRC signaling pathway, and thereby contribute to the pro-metastasis effect of RPS7. Conclusions Taken together, our data reveal a novel function of RPS7 in HCC metastasis, also reveal the critical roles of the RPS7/LOXL2/ITGB1 axis in HCC metastasis and shed new light on the exploration of molecular drugs against HCC.

Abstract Background Understanding the molecular mechanism of early lymph node metastasis among lung adenocarcinoma (LUAD) is essential for developing novel therapeutic agents. Proteomic studies helped generate molecular landscape for LUAD. However, the molecular basis of early lymph node metastases remains unknown in patients with LUAD. Methods Surgically resected LUAD tissues and paired normal tissues were selected from 25 patients with stage pT1bN2M0 (group of metastases [GM]) and 27 patients with stage T2b‐3N0M0 (group of large primary focus, GL) who had not undergone any anti‐tumor treatment. 4D‐Label‐free proteomics sequencing was performed among these tissues. The clinicopathological information was retrieved from the electronic medical record system in Guangdong Provincial People's Hospital. Results Compared with GL tumor tissue, 89 upregulated and 155 downregulated proteins were identified in GM tumor tissue. Upregulated proteins of GM were enriched in the ECM‐receptor interaction and PI3K‐AKT pathway under Kyoto Encyclopedia of Genes and Genomes enrichment analysis. And then, the median disease‐free survival (DFS) of GM phenotype patients was used as the cut‐off value, and GM was divided into two groups with significantly different survival outcomes (DFS good vs. DFS Poor: DFS: p

Background: The R-RAS2 is a small GTPase highly similar to classical RAS proteins at the regulatory and signaling levels. The high evolutionary conservation of R-RAS2, its links to basic cellular processes and its role in cancer, make R-RAS2 an interesting research topic. To elucidate the evolutionary history of R-RAS proteins, we investigated and compared structural and functional properties of ancestral type R-RAS protein with human R-RAS2. Methods: Bioinformatics analysis were used to elucidate the evolution of R-RAS proteins. Intrinsic GTPase activity of purified human and sponge proteins was analyzed with GTPase-GloTM Assay kit. The cell model consisted of human breast cancer cell lines MCF-7 and MDA-MB-231 transiently transfected with EsuRRAS2-like or HsaRRAS2. Biological characterization of R-RAS2 proteins was performed by Western blot on whole cell lysates or cell adhesion protein isolates, immunofluorescence and confocal microscopy, MTT test, colony formation assay, wound healing and Boyden chamber migration assays. Results: We found that the single sponge R-RAS2-like gene/protein probably reflects the properties of the ancestral R-RAS protein that existed prior to duplications during the transition to Bilateria, and to Vertebrata. Biochemical characterization of sponge and human R-RAS2 showed that they have the same intrinsic GTPase activity and RNA binding properties. By testing cell proliferation, migration and colony forming efficiency in MDA-MB-231 human breast cancer cells, we showed that the ancestral type of the R-RAS protein, sponge R-RAS2-like, enhances their oncogenic potential, similar to human R-RAS2. In addition, sponge and human R-RAS2 were not found in focal adhesions, but both homologs play a role in their regulation by increasing talin1 and vinculin. Conclusions: This study suggests that the ancestor of all animals possessed an R-RAS2-like protein with oncogenic properties similar to evolutionarily more recent versions of the protein, even before the appearance of true tissue and the origin of tumors. Therefore, we have unraveled the evolutionary history of R-RAS2 in metazoans and improved our knowledge of R-RAS2 properties, including its structure, regulation and function. [ABSTRACT FROM AUTHOR]

Background: Metastasis is one of the leading cause contributes to treatment failure and poor prognosis of hepatocellular carcinoma (HCC) patients. The underlying mechanism of HCC metastasis remains to be determined. Although several RNA binding proteins (RBPs) have been found to participate in tumorigenesis and progression of liver cancer, the role of RBPs in HCC patients with extrahepatic metastases is poorly understood. Methods: By performing RNA-seq of primary HCC tissues (including HCC with extrahepatic metastasis and those did not develop metastasis), we identified a set of HCC metastasis-associated RBPs candidates. Among which, ribosomal protein S7 (RPS7) was found to be remarkably increased in HCC tissues and be strongly related to HCC poor survival. Overexpression or CRISPR-Cas9–mediated knockout were applied to investigate the role of RPS7 on the metastasis-associated phenotypes of HCC cells. RNA sequencing, RIP, RNA-pull down, dual luciferase reporter assay, nascent RNA capture assay, and RNA decay and so on, were applied to reveal the underlying mechanism of RPS7 induced HCC metastasis. Results: Gain- and loss- of function analyses revealed that RPS7 promoted HCC cells adhesion, migration and invasion capabilities, as well as lung metastasis. Mechanistically, we uncovered that lysyl oxidase-like 2 (LOXL2) was a critical downstream target of RPS7. RPS7 could stabilize LOXL2 mRNA by binding to AUUUA motifs in the 3155–3375 region of the 3'UTR of LOXL2 mRNA, thus increased LOXL2 expression via elevating LOXL2 mRNA abundance. Further research revealed that LOXL2 could accelerate focal adhesion formation through maintaining the protein stability of ITGB1 and activating ITGB1-mediated FAK/SRC signaling pathway, and thereby contribute to the pro-metastasis effect of RPS7. Conclusions: Taken together, our data reveal a novel function of RPS7 in HCC metastasis, also reveal the critical roles of the RPS7/LOXL2/ITGB1 axis in HCC metastasis and shed new light on the exploration of molecular drugs against HCC. [ABSTRACT FROM AUTHOR]

Objective To investigate the role and the mechanism of Ras-associated binding protein23 (RAB23) in the migration and invasion of esophageal squamous cell carcinoma (ESCC) cells. MethodsRAB23 mRNA levels were measured in 16 pairs of ESCC and adjacent normal tissues via real-time polymerase chain reactions. RAB23 mRNA levels in the ESCC and adjacent normal tissues of dataset GSE20347 deposited in the Gene Expression Omnibus (GEO) database were also analyzed. Immunohistochemistry (IHC) was used to detect the RAB23 protein expressions in 106 pairs of ESCC and adjacent normal tissues, as well as in the lymph glands and primary tumor tissues of 33 patients with positive lymph nodes and 10 patients with negative lymph nodes. Endogenous RAB23 expression was transiently depleted using siRNAs (si-NC, si-RAB23-1, and si-RAB23-9) or stably reduced using shRNAs (sh-NC and sh-RAB23) in ESCC KYSE30 and KYSE150 cells, and the knockdown efficiency was tested using Western blot assays. Cell counting kit-8 assays and mouse xenograft models were used to test the proliferation of ESCC cells . Transwell assays and tail vein-pulmonary metastasis models in immunocompromised mice were used to examine the migration and invasion of ESCC cells. Cell adhesion assays were used to test the adhesion of ESCC cells. RNA-seq assays were used to analyze how RAB23 knockdown influenced the expression profile of ESCC cells and the implicated signal pathways were confirmed using Western blot assays. Results The RAB23 mRNA expression in 16 cases of ESCC tissues was 0.009 7±0.008 9, which was markedly higher than that in adjacent normal tissues (0.003 2±0.003 7, P=0.006). GEO analysis on RAB23 expressions in ESCC and adjacent normal tissues showed that the RAB23 mRNA level in ESCC tissues (4.30±0.25) was remarkably increased compared with their normal counterparts (4.10±0.17, P=0.037). Among the 106 pairs of ESCC and tumor-adjacent normal tissues, 51 cases exhibited low expression of RAB23 and 55 cases showed high expression of RAB23, whereas in the paired tumor-adjacent normal tissues 82 cases were stained weakly and 24 strongly for RAB23 protein. These results indicated that RAB23 expression was markedly increased in ESCC tissues ( P<0.001). Additionally, only 1 out of 33 primary ESCC tissues with positive lymph nodes showed low RAB23 protein expression. On the other hand, 7 samples of primary ESCC tissues with negative lymph nodes were stained strongly for RAB23 while its level in the other 3 samples was weak. These results showed that RAB23 expression was remarkably increased in primary ESCC tissues with positive lymph nodes compared with those with negative lymph nodes ( P=0.024). Further tests showed that 32 out of 33 positive lymph nodes were stained strongly for RAB23, whereas no negative lymph nodes ( n=10) exhibited high expression of RAB23 ( P<0.001). Both transient and stable knockdown of endogenous RAB23 expression failed to cause detectable changes in the proliferation of KYSE30 cells in vitro and in vivo, but attenuated the migration and invasion of KYSE30 cells as well as the invasion of KYSE150 cells. RAB23 knockdown was found to significantly decrease the number of adhesive KYSE30 cells in the sh-RAB23 group (313.75±89.34) compared with control cells in the sh-NC group (1 030.75±134.29, P<0.001). RAB23 knockdown was also found to significantly decrease the number of adhesive KYSE150 cells in the sh-RAB23 group (710.5±31.74) compared with the number of control cells in the sh-NC group (1 005.75±61.09, P<0.001). RNA-seq assays demonstrated that RAB23 knockdown using two siRNAs targeting RAB23 mRNA markedly impaired focal adhesion-related signal pathways, and decreased the levels of phosphorylated FAK (p-FAK) and phosphorylated paxillin (p-paxillin) in KYSE30 and KYSE150 cells. Conclusions Significantly increased RAB23 in ESCC tissues positively correlates with lymph node metastasis. Depleted RAB23 expression attenuates focal adhesion-related signal pathways, thus impairing the invasion, metastasis, and adhesion of ESCC cells. [ABSTRACT FROM AUTHOR]

Smart electroactive materials that can dynamically regulate stem cell fate without external stimuli have fascinated increasing attention. Meanwhile, noninvasive electrical stimulation has emerged as a less restrictive approach, generating considerable interest in its potential biomedical applications. The intricate interplay between cells and materials within complex microenvironments includes encompassing substrate response, ion exchange, and membrane potential alterations. However, the mechanisms by which smart materials influence stem cells have yet to be fully elucidated. Herein, electrospinning technology is utilized to fabricate bone‐mimicking microenvironments comprising disordered and highly oriented polyvinylidene fluoride (PVDF) nanofibers. The aligned annealed PVDF (AA) and random annealed PVDF (RA) membranes present high fractions of β‐phase. By comparing the osteogenic ability, calcium activity, and F‐actin distribution of bone marrow‐derived mesenchymal stem cells (BMSCs) cultured with these PVDF nanofibers, it is proposed that the stem cells autonomously regulate their differentiation by remodeling the cytoskeleton on the electrospun membranes. Electrical stimulation, more adhesion area, and active calcium influx support the greater osteogenesis of BMSCs on RA than AA. This mechanism can provide a basic theory for the design and preparation of bone tissue engineering scaffolds and contribute to the further study of cells and microenvironments. [ABSTRACT FROM AUTHOR]

S100A11 is a small Ca2+-activatable protein known to localize along stress fibers (SFs). Analyzing S100A11 localization in HeLa and U2OS cells further revealed S100A11 enrichment at focal adhesions (FAs). Strikingly, S100A11 levels at FAs increased sharply, yet transiently, just before FA disassembly. Elevating intracellular Ca2+ levels with ionomycin stimulated both S100A11 recruitment and subsequent FA disassembly. However, pre-incubation with the nonmuscle myosin II (NMII) inhibitor blebbistatin or with an inhibitor of the stretch-activatable Ca2+ channel Piezo1 suppressed S100A11 recruitment, implicating S100A11 in an actomyosin-driven FA recruitment mechanism involving Piezo1-dependent Ca2+ influx. Applying external forces on peripheral FAs likewise recruited S100A11 to FAs even if NMII activity was inhibited, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ and Piezo1 function were indispensable, indicating that NMII contraction forces act upstream of Piezo1-mediated Ca2+ influx, in turn leading to S100A11 activation and FA recruitment. S100A11-knockout cells display enlarged FAs and had delayed FA disassembly during cell membrane retraction, consistent with impaired FA turnover in these cells. Our results thus demonstrate a novel function for S100A11 in promoting actomyosin contractility-driven FA disassembly. [ABSTRACT FROM AUTHOR]

Alternative splicing is one of the major cellular processes that determine the tissue-specific expression of protein variants. However, it remains challenging to identify physiologically relevant and tissue-selective proteins that are generated by alternative splicing. Hence, we investigated the target spectrum of the splicing factor Rbfox1 in the cardiac muscle context in more detail. By using a combination of in silico target prediction and in-cell validation, we identified several focal adhesion proteins as alternative splicing targets of Rbfox1. We focused on the alternative splicing patterns of vinculin (metavinculin isoform) and paxillin (extended paxillin isoform) and identified both as potential Rbfox1 targets. Minigene analyses suggested that both isoforms are promoted by Rbfox1 due to binding in the introns. Focal adhesions play an important role in the cardiac muscle context, since they mainly influence cell shape, cytoskeletal organization, and cell–matrix association. Our data confirmed that depletion of Rbfox1 changed cardiomyoblast morphology, cytoskeletal organization, and multinuclearity after differentiation, which might be due to changes in alternative splicing of focal adhesion proteins. Hence, our results indicate that Rbfox1 promotes alternative splicing of focal adhesion genes in cardiac muscle cells, which might contribute to heart disease progression, where downregulation of Rbfox1 is frequently observed. [ABSTRACT FROM AUTHOR]

DLC1 is a focal adhesion molecule that regulates cell polarity, proliferation, migration, and survival. DLC1 functions as a tumor suppressor and its expression is often down-regulated in various malignant neoplasms of epithelial origin. Recent studies have suggested that lack of DLC1 in endothelial cells may contribute to the development of angiosarcoma, and that DLC1 mutations have been identified in patients with nephrotic syndrome, a disease mainly due to leaky glomerular filtration barriers. To demonstrate whether lack of endothelial DLC1 induces angiosarcoma and/or damages glomerular capillaries leading to nephrotic syndrome, we have extended our analyses on endothelial cell-specific DLC1 knockout mice with focuses on their liver and kidney function. Mice were monitored up to 24 months of age. However, no histological or clinical difference was found between DLC1 knockout and wild type mice, indicating that lack of endothelial DLC1 alone does not compromise kidney and liver function in mice.

Focal adhesion complexes function as the mediators of cell-extracellular matrix interactions to sense and transmit the extracellular signals. Previous studies have demonstrated that cardiomyocyte focal adhesions can be modulated by surface topographic features. However, the response of focal adhesions to dynamic surface topographic changes remains underexplored. To study this dynamic responsiveness of focal adhesions, we utilized a shape memory polymer-based substrate that can produce a flat-to-wrinkle surface transition triggered by an increase of temperature. Using this dynamic culture system, we analyzed three proteins (paxillin, vinculin and zyxin) from different layers of the focal adhesion complex in response to dynamic extracellular topographic change. Hence, we quantified the dynamic profile of cardiomyocyte focal adhesion in a time-dependent manner, which provides new understanding of dynamic cardiac mechanobiology.

Abstract A variety of cell behaviors, such as cell adhesion, motility, and fate, can be controlled by substrate characteristics such as surface topology and chemistry. In particular, the surface topology of substrates strongly affects cell behaviors, and the topological spacing is a critical factor in inducing cell responses. Various works have demonstrated that cell adhesion was enhanced with decreasing topological spacing although differentiation progressed slowly. However, there are exceptions, and thus, correlations between topological spacing and cell responses are still debated. We show that a nanoporous gold substrate affected cell adhesion while it neither affected osteogenic nor adipogenic differentiation. In addition, the cell adhesion was reduced with decreasing pore size. These do not agree with previous findings. A focal adhesion (FA) is an aggregate of modules comprising specific proteins such as FA kinase, talin, and vinculin. Therefore, it is suggested that because various extracellular signals can be independently branched off from the FA modules, the unusual effects of nanoporous gold substrates are related to the multi-branching of FAs. Graphical Abstract

The complex regulation of traction forces (TF) produced during cellular migration remains poorly understood. We have previously found that calpain 4 (Capn4), the small non-catalytic subunit of the calpain 1 and 2 proteases, regulates the production of TF independent of the proteolytic activity of the larger subunits. Capn4 was later found to facilitate tyrosine phosphorylation and secretion of the lectin-binding protein galectin-3 (Gal3). In this study, recombinant Gal3 (rGal3) was added to the media-enhanced TF generated by capn4−/− mouse embryonic fibroblasts (MEFs). Extracellular Gal3 also rescued defects in the distribution, morphology, and adhesive strength of focal adhesions present in capn4−/− MEF cells. Surprisingly, extracellular Gal3 does not influence mechanosensing. c-Abl kinase was found to affect Gal3 secretion and the production of TF through phosphorylation of Y107 on Gal3. Our study also suggests that Gal3-mediated regulation of TF occurs through signaling pathways triggered by β1 integrin but not by focal adhesion kinase (FAK) Y397 autophosphorylation. Our findings provide insights into the signaling mechanism by which Capn4 and secreted Gal3 regulate cell migration through the modulation of TF distinctly independent from a mechanosensing mechanism.

Abstract Cell migration is a highly orchestrated process requiring the coordination between the cytoskeleton, cell membrane and extracellular matrix adhesions. Our previous study demonstrated that Hax1 interacts with EB2, a microtubule end-binding protein, and this interaction regulate cell migration in keratinocytes. However, little is known about the underlying regulatory mechanism. Here, we show that Hax1 links dynamic focal adhesions to regulate cell migration via interacting with IQGAP1, a multidomain scaffolding protein, which was identified by affinity purification coupled with LC–MS/MS. Biochemical characterizations revealed that C-terminal region of Hax1 and RGCT domain of IQGAP1 are the most critical binding determinants for its interaction. IQGAP1/Hax1 interaction is essential for cell migration in MCF7 cells. Knockdown of HAX1 not only stabilizes focal adhesions, but also impairs the accumulation of IQGAP in focal adhesions. Further study indicates that this interaction is critical for maintaining efficient focal adhesion turnover. Perturbation of the IQGAP1/Hax1 interaction in vivo using a membrane-permeable TAT-RGCT peptide results in impaired focal adhesion turnover, thus leading to inhibition of directional cell migration. Together, our findings unravel a novel interaction between IQGAP1 and Hax1, suggesting that IQGAP1 association with Hax1 plays a significant role in focal adhesion turnover and directional cell migration. Video Abstract

Abstract Background Focal adhesions (FAs) are integrin-containing, multi-protein structures that link intracellular actin to the extracellular matrix and trigger multiple signaling pathways that control cell proliferation, differentiation, survival and motility. Microtubules (MTs) are stabilized in the vicinity of FAs through interaction with the components of the cortical microtubule stabilizing complex (CMSC). KANK (KN motif and ankyrin repeat domains) family proteins within the CMSC, KANK1 or KANK2, bind talin within FAs and thus mediate actin-MT crosstalk. We previously identified in MDA-MB-435S cells, which preferentially use integrin αVβ5 for adhesion, KANK2 as a key molecule enabling the actin-MT crosstalk. KANK2 knockdown also resulted in increased sensitivity to MT poisons, paclitaxel (PTX) and vincristine and reduced migration. Here, we aimed to analyze whether KANK1 has a similar role and to distinguish which talin isoform binds KANK2. Methods The cell model consisted of human melanoma cell line MDA-MB-435S and stably transfected clone with decreased expression of integrin αV (3αV). For transient knockdown of talin1, talin2, KANK1 or KANK2 we used gene-specific siRNAs transfection. Using previously standardized protocol we isolated integrin adhesion complexes. SDS-PAGE and Western blot was used for protein expression analysis. The immunofluorescence analysis and live cell imaging was done using confocal microscopy. Cell migration was analyzed with Transwell Cell Culture Inserts. Statistical analysis using GraphPad Software consisted of either one-way analysis of variance (ANOVA), unpaired Student’s t-test or two-way ANOVA analysis. Results We show that KANK1 is not a part of the CMSC associated with integrin αVβ5 FAs and its knockdown did not affect the velocity of MT growth or cell sensitivity to PTX. The talin2 knockdown mimicked KANK2 knockdown i.e. led to the perturbation of actin-MT crosstalk, which is indicated by the increased velocity of MT growth and increased sensitivity to PTX and also reduced migration. Conclusion We conclude that KANK2 functionally interacts with talin2 and that the mechanism of increased sensitivity to PTX involves changes in microtubule dynamics. These data elucidate a cell-type-specific role of talin2 and KANK2 isoforms and we propose that talin2 and KANK2 are therefore potential therapeutic targets for improved cancer therapy. Graphical Abstract

BackgroundLung squamous cell carcinoma (LUSC) is a devastating and difficult-to-treat type of lung cancer, and the prognosis of LUSC is the worst. The functional roles of focal adhesion-related genes were explored in LUSC based on data from The Cancer Genome Atlas (TCGA).MethodsRNA sequencing data and clinical characteristics of LUSC patients in TCGA-LUSC were obtained from the TCGA database. Through systematic analysis, we screened the prognostic genes and determined the focal adhesion-related pathways closely associated with LUSC.ResultsWe identified 444 prognostic genes and focal adhesion-related pathways intimately associated with LUSC. According to the focal adhesion-related genes, TCGA-LUSC patients were well divided into two groups: the low-risk group (G1) and the high-risk group (G2). A differential expression analysis identified 44 differentially expressed genes (DEGs) upregulated in the low-risk G1 group and 379 DEGs upregulated in the high-risk G2 group. The upregulated DEGs in the G1 group were primarily related to tyrosine metabolism, steroid hormone biosynthesis, retinol metabolism, platinum drug resistance, pentose and glucuronate interconversions, and metabolism of xenobiotics by cytochrome P450, while the downregulated DEGs in the G1 group were primarily related to ECM-receptor interaction, focal adhesion, proteoglycans in cancer, small cell lung cancer, cytokine-cytokine receptor interaction, and TGF-beta signaling pathway. The immune activity of the G1 group was lower than that of the G2 group, and the half-maximal inhibitory concentration (IC50) of five chemotherapy drugs (i.e., gemcitabine, methotrexate, vinorelbine, paclitaxel, and cisplatin) was significantly different between the G1 and G2 groups. Furthermore, a 10-gene prognostic model was constructed to predict the prognosis for LUSC patients: ITGA3, VAV2, FLNC, FLT4, HGF, MYL2, ITGB1, PDGFRA, CCND2, and PPP1CB.ConclusionThe status of focal adhesion-related genes has a close relationship with tumor classification and immunity in LUSC patients. A novel focal adhesion-related signature had good prognostic and predictive performance for LUSC. Our findings may provide new insight into the diagnosis and treatment of LUSC.

Endothelial cells lining blood vessels are essential for maintaining vascular homeostasis and mediate several pathological and physiological processes. Mechanical stresses generated by blood flow and other biomechanical factors significantly affect endothelial cell activity. Here, we review how mechanical stresses, both in situ and in vitro, affect endothelial cells. We review the basic principles underlying the cellular response to mechanical stresses. We also consider the implications of these findings for understanding the mechanisms of mechanotransducer and mechano-signal transduction systems by cytoskeletal components. [ABSTRACT FROM AUTHOR]

Self-renewal and differentiation of mouse embryonic stem cells (mESCs) are greatly affected by the extracellular matrix (ECM) environment; the composition and stiffness of which are sensed by the cells via integrin-associated focal adhesions (FAs) which link the cells to the ECM. Although FAs have been studied extensively in differentiated cells, their composition and function in mESCs are not as well elucidated. To gain more detailed knowledge of the molecular compositions of FAs in mESCs, we adopted the proximity-dependent biotinylation (BioID) proteomics approach. Paxillin, a known FA protein (FAP), is fused to the promiscuous biotin ligase TurboID as bait. We employed both SILAC- and label-free (LF)-based quantitative proteomics to strengthen as well as complement individual approach. The mass spectrometry data derived from SILAC and LF identified 38 and 443 proteins, respectively, with 35 overlapping candidates. Fifteen of these shared proteins are known FAPs based on literature-curated adhesome and 7 others are among the reported "meta-adhesome", suggesting the components of FAs are largely conserved between mESCs and differentiated cells. Furthermore, the LF data set contained an additional 18 literature-curated FAPs. Notably, the overlapped proteomics data failed to detect LIM-domain proteins such as zyxin family proteins, which suggests that FAs in mESCs are less mature than differentiated cells. Using the LF approach, we are able to identify PDLIM7, a LIM-domain protein, as a FAP in mESCs. This study illustrates the effectiveness of TurboID in mESCs. Importantly, we found that application of both SILAC and LF methods in combination allowed us to analyze the TurboID proteomics data in an unbiased, stringent and yet comprehensive manner. • Application of SILAC and LF LC-MS/MS provides unbiased and high-confident data. • We identified key proteins present in the focal adhesions (FAs) of mESCs. • The identified FA proteins share high similarity with literature-curated adhesome. • LIM-domain proteins from the zyxin family are rarely found in the FA of mESCs. [ABSTRACT FROM AUTHOR]

A variety of cell behaviors, such as cell adhesion, motility, and fate, can be controlled by substrate characteristics such as surface topology and chemistry. In particular, the surface topology of substrates strongly affects cell behaviors, and the topological spacing is a critical factor in inducing cell responses. Various works have demonstrated that cell adhesion was enhanced with decreasing topological spacing although differentiation progressed slowly. However, there are exceptions, and thus, correlations between topological spacing and cell responses are still debated. We show that a nanoporous gold substrate affected cell adhesion while it neither affected osteogenic nor adipogenic differentiation. In addition, the cell adhesion was reduced with decreasing pore size. These do not agree with previous findings. A focal adhesion (FA) is an aggregate of modules comprising specific proteins such as FA kinase, talin, and vinculin. Therefore, it is suggested that because various extracellular signals can be independently branched off from the FA modules, the unusual effects of nanoporous gold substrates are related to the multi-branching of FAs. [ABSTRACT FROM AUTHOR]

Despite decades of research presenting insulin-like growth factor-1 receptor (IGF1R) as an attractive target for cancer therapy, IGF1R inhibitors ultimately failed in clinical trials. This was surprising due to the known cancer-promoting functions of IGF1R, including stimulation of cell invasion, proliferation, and survival. Discourse in the literature has acknowledged that a lack of patient stratification may have impacted the success of IGF1R-inhibitor trials. This argument alludes to the possibility that IGF1R function may be contingent on tumor type and cellular composition. Looking into the known roles of IGF1R, it becomes clear that this receptor interacts with a multitude of different proteins and even has tumor-suppressing functions. IGF1R is implicated in both cell–cell and cell–surface adhesion dynamics, and the effects of either IGF1R downregulation or pharmacological inhibition on cellular adhesion remain poorly understood. In turn, adhesion receptors modulate IGF1R signaling. In addition, our understanding of IGF1R function in tumor-associated immune and stromal cells is lacking, which could contribute to the overwhelming failure of IGF1R inhibitors in the clinic. In this review, we re-investigate clinical trial data to make connections between the failure of these drugs in human cancer patients and the understudied facets of IGF1R function. We describe lesser-known and potentially tumor-suppressive functions of IGF1R that include promoting cell–cell adhesion through E-cadherin, augmenting a pro-inflammatory macrophage phenotype, and stimulating B cells to produce immunoglobulins. We also highlight the important role of adhesion receptors in regulating IGF1R function, and we use this information to infer stratification criteria for selecting patients that might benefit from IGF1R inhibitors. [ABSTRACT FROM AUTHOR]