Search Results (265)

The Kindlin family of scaffold proteins plays key roles in integrin-mediated processes. Kindlin-1 and -2, encoded by the FERMT1 and FERMT2 genes, respectively, are expressed in the epidermis. Kindlin-1 plays protective roles against the development of cutaneous squamous cell carcinomas (cSCCs) in epidermal keratinocytes. However, the role of Kindlin-2 in transformed epidermal keratinocytes has remained virtually unexplored. In this study, we used siRNA approaches to generate Kindlin-2-depleted cells in three isogenic transformed keratinocyte lines. PM1, MET1, and MET4 cells model, respectively, a precancerous lesion, a primary cSCC, and a metastatic lesion of the latter. MET1 cells express both Kindlin-1 and -2. However, Kindlin-1 was not detectable in PM1 and MET4 cells. FERMT2 silencing in PM1 and MET4, but not in MET1 cells, reduced proliferation and the ability to adhere to culture surfaces and spreading. Furthermore, Kindlin-2-depleted PM1 and MET4, but not MET1 cells, exhibited decreased numbers of focal adhesions, as well as an altered F-actin and microtubule cytoskeletal organization. Significantly, FERMT2 silencing reduced the directional migration in all three cell types. These findings are consistent with the concept that, in the absence of other Kindlin orthologues, Kindlin-2 plays a prominent role in the modulation of the proliferation, spreading, focal adhesion assembly, and motility of transformed keratinocytes, as exemplified by PM1 and MET4 cells. Full article

Background/Objectives: Truncating mutations in PALB2, a critical component of the BRCA1-PALB2-BRCA2 homologous recombination repair complex, are associated with increased risk and aggressiveness of breast cancer. The consequences of PALB2 truncation on the expression, localization, and functional dynamics of the scaffold protein IQGAP1 were investigated in this study based on two cases of truncated PALB2 human breast invasive ductal carcinoma (IDC), specifically, c.1240C>T (p.Arg414*) and c.2257C>T (p.Arg753*). Methods: Using confocal microscopy, we examined co-expression patterns of IQGAP1 with PALB2, PCNA, CK7, and β-tubulin in tumor tissues from both control cancer and PALB2-mutated cases. Results: In PALB2-truncated tumors, IQGAP1 exhibited enhanced peripheral and plasma membrane localization with elevated co-localization levels compared to controls, suggesting altered cytoskeletal organization. PALB2 truncation increased nuclear and cytoplasmic N-terminal PALB2 immunoreactivity, indicating the presence of truncated isoforms disrupting the homologous recombination repair system. Co-expression analyses with PCNA revealed an inverse expression pattern between IQGAP1 and proliferation markers, suggesting S-phase cell cycle-dependent heterogeneity. Furthermore, the loss of IQGAP1 dominance over CK7 and β-tubulin in mutant tumors, along with persistent intercellular spacing, implied a loss of cell–cell cohesion and the acquisition of invasive traits. Conclusions: These data support a model where PALB2 truncation triggers a reorganization of IQGAP1 that disrupts its canonical structural functions and facilitates tumor progression via enhanced motility and impaired cell–cell interaction. IQGAP1 thus serves as both a functional effector and potential biomarker in PALB2-mutated IDC, opening novel paths for diagnosis and targeted therapeutic intervention. Full article

Panton–Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus (S. aureus) and a significant virulence factor that plays a crucial role in the pathogenesis of dairy mastitis. Previous studies by our research group demonstrated that baicalin inhibits the apoptosis and hyperphosphorylation of cytoskeletal proteins induced by recombinant Panton–Valentine leukocidin (rPVL) in bovine mammary epithelial cells (BMECs). However, the effects of baicalin on the proliferation of BMECs and the underlying mechanism remain unclear. Consequently, this study aimed to explore this underlying mechanism through an LC-MS/MS analysis performed in 4D data-independent acquisition (DIA) mode. Quantitative analysis identified 757 differentially expressed phosphoproteins, among which phosphorylation levels of proteins involved in BMEC proliferation and cell cycle regulation exhibited significant alterations (p < 0.05). rPVL inhibited BMEC proliferation in a dose-dependent manner and induced G0/G1 phase arrest and dephosphorylation of the cell-cycle-related proteins BCLAF1^S285, CDK7^T170, NF2^S518, and PKM2S37. Preintervention with baicalin significantly upregulated the expression and phosphorylation of these proteins and alleviated the G0/G1 phase arrest induced by rPVL in BMECs in vitro. The establishment of the mitotic state in BMECs due to the effect of baicalin appears to be closely related to the regulation of the phosphorylation of CDK7, PKM2, BCLAF1, and NF2. Moreover, in vivo analysis revealed that S. aureus ATCC49775 and rPVL induced dramatic structural destruction and pathological impairment of mammary gland tissues in mice and that these histopathological changes were ameliorated after baicalin intervention. Quantitative immunohistochemical analysis revealed that baicalin mitigated the rPVL-induced dephosphorylation of the aforementioned cell-cycle-related proteins and increased their phosphorylation. Both in vitro and in vivo experimental evidence demonstrated that baicalin effectively reversed rPVL-induced G0/G1 phase arrest in BMECs (p < 0.01) by significantly increasing the phosphorylation levels of cell cycle regulatory proteins (p < 0.05). Additionally, baicalin alleviates pathological damage to mammary gland tissues in mouse models. These data suggest that baicalin possesses antibacterial and antitoxin effects, indicating that it is an effective preventive agent against bovine mastitis. Full article

Respiratory syncytial virus (RSV) is a major human respiratory pathogen, particularly affecting infants, the elderly, and immunocompromised individuals. RSV exists in both spherical and filamentous forms, with the filamentous morphology associated with enhanced infectivity and cell-to-cell spread. Here, we demonstrate that RhoA, a small GTPase involved in cytoskeletal regulation, is essential for filamentous RSV morphogenesis through its role in organizing lipid raft microdomains. Rhosin, a selective RhoA inhibitor developed through structure-guided screening, disrupts GEF–RhoA interactions to block RhoA activation. The pharmacological inhibition of RhoA with Rhosin significantly reduced filamentous virion formation, disrupted RSV fusion (F) protein colocalization with lipid rafts, and diminished cell-to-cell fusion, without affecting overall viral replication. Scanning electron microscopy revealed that Rhosin-treated infected HEp-2 cells exhibited fewer and shorter filamentous projections compared to the extensive filament formation seen in untreated cells. β-galactosidase-based fusion assays confirmed that reduced filamentation corresponded with decreased cell-to-cell fusion. The biophysical separation of RSV spherical and filamentous particles by sucrose gradient velocity sedimentation, coupled with fluorescence and transmission electron microscopy, showed that Rhosin treatment shifted virion morphology toward spherical forms. This suggests that RhoA activity is critical for filamentous virion assembly, which may enhance viral spread. Immunofluorescence microscopy using lipid raft-selective dyes (DiIC₁₆) and fusion protein-specific antibodies revealed the strong co-localization of RSV proteins with lipid rafts. Importantly, the pharmacological inhibition of RhoA with Rhosin disrupted F protein partitioning into raft domains, underscoring the requirement for intact lipid rafts in assembly. These findings highlight a novel role for host RhoA signaling in regulating viral assembly through raft microdomain organization, offering a potential target for host-directed antiviral intervention aimed at altering RSV structural phenotypes and limiting pathogenesis. Full article

Sitosterol (Sito) is a phytosterol with bioactive properties, including reducing atherosclerosis risk and anti-inflammatory and antitumoral effects. However, it can be oxidized by reactive oxygen species such as ozone (O₃), producing oxyphytosterols with harmful effects such as cytotoxicity, oxidative stress, and proatherogenicity. Ozone, a strong oxidant and common pollutant, can alter plant steroid compounds, raising concerns about dietary oxyphytosterol intake. Studies identify β-Secosterol (βSec) as the primary ozone-derived oxyphytosterol from Sito, exhibiting cytotoxic effects on HepG2 human liver tumor cells. This study investigated βSec’s biological effects on two rat liver cell lines: BRL-3A (immortalized) and HTC (tumoral), examining cell death, cell cycle progression, morphology, and cytoskeleton organization. While Sito influenced cell metabolic activity without affecting cell survival or morphology, βSec demonstrated significant cytotoxicity in both cell lines. It induced G0/G1 cell cycle arrest and disrupted cytoskeleton organization, with different implications: BRL-3A cells showed persistent cytoskeletal changes potentially linked to tumor induction, while HTC cells displayed chemoresistance, restoring cytoskeletal integrity and enhancing metastatic potential. These findings reveal βSec’s complex, context-dependent effects, suggesting it may promote tumor-like behavior in non-tumoral cells and resistance mechanisms in cancer cells, contributing to understanding oxyphytosterols’ implications for physiological and pathological conditions. Full article

Arthropod-borne viral infections, ranging from asymptomatic to fatal diseases, are expanding from endemic to nonendemic areas. Climate change, deforestation, and globalization favor their spread. Although arboviral manifestations mainly determine the onset of generalized symptoms, distinct clinical signs have been assessed, depending on the particular arthropod-borne virus (arbovirus) involved in the infectious process. A number of arboviruses cause neuroinvasive diseases in vertebrate hosts, with acute to chronic outcomes. Long-term neurological sequelae can include cognitive dysfunction and Parkinsonism. To increase knowledge of host interactions with arboviruses, in-depth investigations are needed to highlight how arboviruses exploit a host cell for efficient infection and clarify the molecular alterations underlying human brain diseases. This review focuses on the involvement of host cytoskeletal networks and associated signalling pathways in modulating the neurotropism of emerging arboviruses. A better understanding at the molecular level of the potential for emerging infectious diseases is fundamental for prevention and outbreak control. Full article

(1) Background: Vascular mural cells reside in the media and outer layers of the vessel wall. Their ability to proliferate and migrate or to change phenotype in response to external cues is a central feature of the vascular response to injury. Genetically engineered mice are used for loss- or gain-of-function analyses or lineage tracing in vivo, their primary cells for mechanistic studies in vitro. Whether and how cultivation conditions affect their phenotype and function is often overlooked. (2) Methods: Here, we systematically studied how the cultivation of primary mural cells isolated from the aorta of adult wild-type mice in either basal medium (DMEM) or special media formulated for the cultivation of fibroblasts or pericytes affects their phenotype and function. (3) Results: Medium composition did not alter cell viability, but the mRNA levels of differentiated smooth muscle cell markers were highest in vascular mural cells expanded in DMEM. Conversely, significantly higher numbers of proliferating and migrating cells were observed in cells expanded in Pericyte medium, and cytoskeletal rearrangements supported increased migratory capacities. Significantly reduced telomere lengths and metabolic reprogramming was observed in aortic mural cells cultured in Fibroblast medium. (4) Conclusions: Our findings underline the plasticity of primary aortic mural cells and highlight the importance of the culture media composition during their expansion, which could be exploited to interrogate their responsiveness to external stimuli or conditions observed in vivo or in patients. Full article

Zygotic genome activation (ZGA) marks the critical transition from reliance on maternal transcripts to the initiation of embryonic transcription early in development. Despite extensive characterization in model species, the regulatory framework of ZGA in sheep remains poorly defined. Here, we applied single-cell RNA sequencing (Smart-seq2) to in vivo- and in vitro-derived sheep embryos at the 8-, 16-, and 32-cell stages. Differential expression analysis revealed 114, 1628, and 1465 genes altered in the 8- vs. 16-, 16- vs. 32-, and 8- vs. 32-cell transitions, respectively, with the core pluripotency factors SOX2, NANOG, POU5F1, and KLF4 upregulated during major ZGA. To uncover coordinated regulatory modules, we constructed a weighted gene co-expression network using WGCNA, identifying the MEred module as most tightly correlated with developmental progression (r = 0.48, p = 8.6 × 10⁻¹⁴). The integration of MERed genes into the STRING v11 protein–protein interaction network furnished a high-confidence scaffold for community detection. Louvain partitioning delineated two discrete communities: Community 0 was enriched in ER–Golgi vesicle-mediated transport, transmembrane transport, and cytoskeletal dynamics, suggesting roles in membrane protein processing, secretion, and early signaling; Community 1 was enriched in G2/M cell-cycle transition and RNA splicing/processing, indicating a coordinated network for accurate post-ZGA cell division and transcript maturation. Together, these integrated analyses reveal a modular regulatory architecture underlying sheep ZGA and provide a framework for dissecting early embryonic development in this species. Full article

Background: Unresolved inflammation is a major predictor of heart failure following myocardial infarction. Exogenous ubiquitin (eUB) is shown to decrease inflammatory response and confer cardioprotection in mice 3 days post-ischemia/reperfusion (I/R) injury. Here, we hypothesized that eUB differentially modulates the phenotype and function of M1 and M2 macrophages. Methods and Results: Peritoneal macrophages, pretreated with UB for 30 min, were exposed to IFN-γ (M1 polarization) or IL-4 (M2 polarization) for 72 h. Cytokine/chemokine levels were measured in conditioned media, while cells were used for functional and biochemical assays. eUB reduced TNF-α secretion in M1, and TNF-α and IL-10 secretion in M2 macrophages. eUB induced cytoskeletal reorganization and reduced surface area in M1 macrophages. eUB enhanced M1 migration; however, it decreased M2 macrophage migration and efferocytosis. It decreased STAT1 and FAK phosphorylation in M1, while increasing STAT6 and FAK phosphorylation in M2 macrophages. Total protein ubiquitination remained unchanged. In non-activated macrophages, eUB altered morphology, suppressed IL-1β, IL-2, and IL-5 secretion, and enhanced efferocytosis. Conclusion: eUB modulates macrophage polarization, reduces pro-inflammatory cytokine secretion, and alters functional parameters and intracellular signaling. These effects may contribute to the cardioprotective potential of eUB 3 days post-I/R injury. Full article

Chronic obstructive pulmonary disease (COPD), whose main risk factor is cigarette smoking, is among the most prevalent diseases worldwide. Previous studies have shown that cigarette smoke extract (CSE) can directly affect pulmonary artery function independently of hypoxia resulting from the airway obstruction. In addition, CSE also affects bronchial smooth muscle, leading to airway hyper-responsiveness. However, its specific impact on the contractile machinery of this compartment remains unclear. In this study, using in vitro experiments with human bronchial smooth muscle cells (hBSMCs), we found that CSE exposure disrupted calcium homeostasis, increased ROS and lipid peroxidation, and reduced cell antioxidant defenses. Furthermore, CSE exposure altered the cell contractile apparatus by decreasing key cytoskeletal proteins and impairing actin dynamics, potentially contributing to the dysregulated contractile response of cells. Notably, these effects were significantly attenuated by antioxidant drugs such as mitoTEMPO and N-acetylcysteine, as well as by the inhibition of the endoplasmic reticulum (ER) calcium channels with 2-aminoethoxydiphenyl borate (2-APB). More importantly, mitoTEMPO partially restored the contractile response of bronchus upon CSE challenge. Collectively, our findings give evidence that CSE-mediated increase in ROS and intracellular calcium contribute to cytoskeletal disruption and functional impairment in airway smooth muscle. Moreover, these results also point to potential therapeutical approaches for mitigating the harmful effects of cigarette smoke in the lung. Full article

Cell migration, shape maintenance, and intracellular signaling are closely linked to dynamic changes in cell morphology and the cytoskeleton. These processes involve the reorganization of the cytoskeleton within the cytoplasm, affecting all its key components: intermediate filaments, microtubules, and microfilaments. A promising strategy for remotely controlling cellular functions is the use of magnetic nanoparticles, which can influence cellular physiology. This approach, known as magnetogenetics, has been applied in various areas of cell and molecular biology. Applying a magnetic field allows for the non-invasive modulation of biochemical processes, cell migration, and morphological changes in cells containing magnetic nanoparticles. In our study, magnetic nanoparticles were conjugated with antibodies targeting cytoskeletal components, enabling the magnetically induced manipulation and deformation of the cell cytoskeleton. Our research introduces a novel approach to manipulating specific cytoskeletal components and altering cell polarity with spatial precision in vitro using magnetic nanoparticles associated with the cytoskeleton. Full article

Methylglyoxal is a reactive dicarbonyl intermediate in the advanced glycation end-product (AGE) pathway, and alterations in its levels have been detected in the plasma, cerebrospinal fluid, and brain parenchyma in various pathologies, particularly in diabetes. In this study, we investigate the effects of methylglyoxal (MGO) on murine brain microvascular endothelial cells at both physiological and pathological concentrations. We evaluate molecular parameters, including reactive oxygen species (ROS) production, cytosolic calcium signaling, and ATP synthesis, as well as cellular responses such as cytoskeletal remodeling, cell migration, adhesion, and permeability, across a concentration range of 0–1000 μM. At low concentrations (below ~250 μM), MGO does not induce oxidative stress; instead, it leads to an increase in cytosolic calcium levels and ATP production. At higher concentrations, however, MGO induces significant oxidative stress, which is accompanied by a marked decrease in cell viability, particularly at concentrations exceeding 500 μM. The modulation of key functional processes, including purinergic calcium signaling, actin filament synthesis, cell migration, and adhesion, reveals a threshold concentration beyond which cellular function is impaired due to oxidative stress. Below this threshold, the observed effects appear to be mediated primarily by non-oxidative mechanisms, likely involving protein glycation. In conclusion, our results suggest a dual action of methylglyoxal on brain endothelial cells, with distinct molecular mechanisms underlying its effects at physiological versus pathological concentrations. Full article

Hypoxia is a critical factor affecting tissue homeostasis that dramatically alters the tumor microenvironment (TME) through genetic, metabolic, and structural changes, promoting tumor survival and proliferation. Hypoxia-inducible factor-1α (HIF-1α) plays a central role in this process by regulating hundreds of genes involved in the processes of tumorigenesis, angiogenesis, metabolic reprogramming, and immune evasion. This review provides a comprehensive examination of the role of HIF-1α in hypoxia and how hypoxia weakens intercellular junctions—including gap junctions, adherens junctions, tight junctions, and desmosomes. The disruption of gap junctions decreases intercellular communication, which alters signal transduction cascades and tumor suppressive properties. Adherens junctions are comprised of proteins that characterize the tissues and link cells to the actin cytoskeleton, whereby their disruption promotes the epithelial-to-mesenchymal transition (EMT). Under hypoxic conditions, the tight junction proteins are dysregulated, altering paracellular transport and cell polarity. In addition, desmosomes provide linkage to intermediate filaments, and hypoxia compromises tissue integrity. Collectively, the influence of hypoxia on cellular junctions promotes tumorigenesis through reducing cell communication, cytoskeletal interactions, and altering signaling pathways. Activation of matrix metalloproteinases (MMPs) further degrades the extracellular matrix and enhances tumor invasion and metastasis. This process also involves hypoxia-induced angiogenesis, regulated by HIF-1α. A comprehensive understanding of the mechanisms of hypoxia-driven tumor adaptation is essential for developing effective therapeutic strategies. Furthermore, this review examines current treatments aimed at targeting HIF-1α and explores future directions to enhance treatment efficacy and improve patient outcomes. Full article

Background: Colonic diverticulosis is a common condition, particularly in the elderly population. While dietary habits, obesity, smoking, and physical inactivity contribute to its pathogenesis, emerging evidence highlights a genetic predisposition affecting extracellular matrix (ECM) remodeling, inflammation, and connective tissue integrity. The aim of this systematic review was to summarize genetic determinants of colonic diverticulosis. Methods: The PubMed^® database was searched for original studies in humans. The inclusion criteria were named genetic factor and confirmed diverticulosis. Patients with diverticulitis and diverticular diseases were excluded from this review. Results: Out of 137 publications, 10 articles met the inclusion criteria: six large association studies (GWAS) and four cross-sectional studies. The genes regulating ECM turnover, including TIMP1, MMP3, and MMP9, are involved in diverticulosis development. The TIMP1 (rs4898) T allele has been associated with increased susceptibility, potentially due to its role in ECM remodeling. Similarly, MMP3 (rs3025058) and MMP9 (rs3918242) polymorphisms contribute to altered collagen degradation. The COL3A1 (rs3134646) variant coding modified collagen type III may promote diverticular formation. Other genes, such as ARHGAP15 (rs4662344, rs6736741), affect cytoskeletal dynamics. Identified in GWAS studies, gene candidates may be grouped into blood group and immune system-related genes (ABO, HLA-DQA1, HLA-H, OAS1, TNFSF13, FADD), extracellular matrix and connective tissue genes (COL6A1, COLQ, EFEMP1, ELN, HAS2, TIMP2), signaling and cell communication (BMPR1B, WNT4, RHOU, PHGR1, PCSK5), nervous system and neurodevelopment (BDNF, CACNB2, GPR158, SIRT1, SCAPER, TRPS1), metabolism and transporters (SLC25A28, SLC35F3, RBKS, PPP1R14A, PPP1R16B), lipids and cholesterol (LDAH, LYPLAL1, STARD13), transcription and gene regulation (ZBTB4, UBTF, TNRC6B), apoptosis (FADD, PIAS1), and poorly characterized genes (C1TNF7, ENSG00000224849, ENSG00000251283, LINC01082, DISP2, SNX24, THEM4, UBL4B, UNC50, WDR70, SREK1IP1). Conclusions: There are a number of gene variants that probably predispose to colonic diverticulosis. Detailed characterization of the multigene background of diverticulosis will enable appropriate therapeutic or preventive interventions in the future. Full article

Type 1 diabetes (T1D) is a chronic metabolic disease defined by sustained hyperglycemia, leading to oxidative stress (OS) and systemic complications, including male subfertility. This study investigates the potential impact of T1D-induced OS on microtubule (MTs) dynamics and microtubule-associated proteins (MAPs) in the testis and spermatozoa (SPZ). Using a streptozotocin-induced T1D rat model, we examined the expression and localization of key MAPs, including Microtubule Affinity-Regulating Kinase 4 (MARK4), Microtubule-Associated Protein 1A (MAP1A), Dynein Light Chain LC8-Type 1 (DYNLL1), Prolyl Endopeptidase (PREP), and Radial Spoke Head 6 Homolog A (RSPH6A), alongside sperm functional parameters. Our findings showed that T1D significantly impaired the expression and distribution of these proteins, which may affect MTs organization and be associated with cytoskeletal disorganization, and impaired germ cell differentiation. Moreover, T1D rats exhibited reduced sperm count, viability, and motility, accompanied by increased DNA fragmentation and chromatin defects. Elevated levels of 4-hydroxy-2-nonenal (4-HNE), a marker of OS, were detected in SPZ, particularly in the acrosome and flagellum, correlating with mitochondrial dysfunction and ATP depletion. Additionally, decreased intracellular Ca²⁺ levels, downregulation of Cation Channel of Sperm (CATSPER) and Voltage-Dependent Anion Channel 3 (VDAC3), and altered tubulin acetylation, possibly due to imbalanced Alpha-Tubulin N-Acetyltransferase 1 (ATAT1) and Histone Deacetylase 6 (HDAC6) expression, were also associated with impaired sperm motility. The combined data suggest that T1D-induced OS is linked to disrupted MTs dynamics, which may contribute to testicular dysfunction and reduced sperm quality, potentially affecting male fertility. A better understanding of these associations may support the development of therapeutic strategies to mitigate the reproductive consequences of T1D and improve male fertility outcomes. Full article