Live-cell imaging and CLEM reveal the existence of ACTN4-dependent ruffle-edge lamellipodia acting as a novel mode of cell migration.

α-Actinin-4 (ACTN4) expression levels are correlated with the invasive and metastatic potential of cancer cells; however, the underlying mechanism remains unclear. Here, we identified ACTN4-localized ruffle-edge lamellipodia using live-cell imaging and correlative light and electron microscopy (CLEM). BSC-1 cells expressing EGFP-ACTN4 showed that ACTN4 was most abundant in the leading edges of lamellipodia, although it was also present in stress fibers and focal adhesions. ACTN4 localization in lamellipodia was markedly diminished by phosphoinositide 3-kinase inhibition, whereas its localization in stress fibers and focal adhesions remained. Furthermore, overexpression of ACTN4, but not ACTN1, promoted lamellipodial formation. Live-cell analysis demonstrated that ACTN4-enriched lamellipodia are highly dynamic and associated with cell migration. CLEM revealed that ACTN4-enriched lamellipodia exhibit a characteristic morphology of multilayered ruffle-edges that differs from canonical flat lamellipodia. Similar ruffle-edge lamellipodia were observed in A549 and MDA-MB-231 invasive cancer cells. ACTN4 knockdown suppressed the formation of ruffle-edge lamellipodia and cell migration during wound healing in A549 monolayer cultures. Additionally, membrane-type 1 matrix metalloproteinase was observed in the membrane ruffles, suggesting that ruffle-edge lamellipodia have the ability to degrade the extracellular matrix and may contribute to active cell migration/invasion in certain cancer cell types.

Integrative Omics Uncovers Low Tumorous Magnesium Content as A Driver Factor of Colorectal Cancer.

Magnesium (Mg) deficiency is associated with increased risk and malignancy in colorectal cancer (CRC), yet the underlying mechanisms remain elusive. Here, we used genomic, proteomic, and phosphoproteomic data to elucidate the impact of Mg deficiency on CRC. Genomic analysis identified 160 genes with higher mutation frequencies in Low-Mg tumors, including key driver genes such as KMT2C and ERBB3. Unexpectedly, initiation driver genes of CRC, such as TP53 and APC, displayed higher mutation frequencies in High-Mg tumors. Additionally, proteomic and phosphoproteomic data indicated that low Mg content in tumors may activate epithelial-mesenchymal transition (EMT) by modulating inflammation or remodeling the phosphoproteome of cancer cells. Notably, we observed a negative correlation between the phosphorylation of DBN1 at S142 (DBN1S142p) and Mg content. A mutation in S142 to D (DBN1S142D) mimicking DBN1S142p up-regulated MMP2 and enhanced cell migration, while treatment with MgCl2 reduced DBN1S142p, thereby reversing this phenotype. Mechanistically, Mg2+ attenuated the DBN1-ACTN4 interaction by decreasing DBN1S142p, which in turn enhanced the binding of ACTN4 to F-actin and promoted F-actin polymerization, ultimately reducing MMP2 expression. These findings shed new light on the crucial role of Mg deficiency in CRC progression and suggest that Mg supplementation may be a promising preventive and therapeutic strategy for CRC.

Identification of key genes involved in collagen hydrogel-induced chondrogenic differentiation of mesenchymal stem cells through transcriptome analysis: the role of m6A modification.

Collagen hydrogel has been shown promise as an inducer for chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), contributing to the repair of cartilage defects. However, the precise molecular mechanism underlying this phenomenon remains poorly elucidated. Here, we induced chondrogenic differentiation of BMSCs using collagen hydrogel and identified 4451 differentially expressed genes (DEGs) through transcriptomic sequencing. Our analysis revealed that DEGs were enriched in the focal adhesion pathway, with a notable decrease in expression levels in the collagen hydrogel group compared to the control group. Protein-protein interaction network analysis suggested that actinin alpha 1 (ACTN1) and actinin alpha 4 (ACTN4), two proteins also involved in cytoskeletal recombination, may be crucial in collagen hydrogel-induced chondrogenic differentiation of BMSCs. Additionally, we found that N6-methyladenosine RNA methylation (m6A) modification was involved in collagen hydrogel-mediated chondrogenic differentiation, with fat mass and obesity-associated protein (FTO) implicated in regulating the expression of ACTN1 and ACTN4. These findings suggest that collagen hydrogel might regulate focal adhesion and actin cytoskeletal signaling pathways through down-regulation of ACTN1 and ACTN4 mRNA via FTO-mediated m6A modification, ultimately driving chondrogenic differentiation of BMSCs. In conclusion, our study provides valuable insights into the molecular mechanisms of collagen hydrogel-induced chondrogenic differentiation of BMSCs, which may aid in developing more effective strategies for cartilage regeneration.

Actn2 defects accelerates H9c2 hypertrophy via ERK phosphorylation under chronic stress.

BACKGROUND: In humans, ACTN2 mutations are identified as highly relevant to a range of cardiomyopathies such as DCM and HCM, while their association with sudden cardiac death has been observed in forensic cases. Although ACTN2 has been shown to regulate sarcomere Z-disc organization, a causal relationship between ACTN2 dysregulation and cardiomyopathies under chronic stress has not yet been investigated. OBJECTIVE: In this work, we explored the relationship between Actn2 dysregulation and cardiomyopathies under dexamethasone treatment. METHODS: Previous cases of ACTN2 mutations were collected and the conservative analysis was carried out by MEGA 11, the possible impact on the stability and function of ACTN2 affected by these mutations was predicted by Polyphen-2. ACTN2 was suppressed by siRNA in H9c2 cells under dexamethasone treatment to mimic the chronic stress in vitro. Then the cardiac hypertrophic molecular biomarkers were elevated, and the potential pathways were explored by transcriptome analysis. RESULTS: Actn2 suppression impaired calcium uptake and increased hypertrophy in H9c2 cells under dexamethasone treatment. Concomitantly, hypertrophic molecular biomarkers were also elevated in Actn2-suppressed cells. Further transcriptome analysis and Western blotting data suggested that Actn2 suppression led to the excessive activation of the MAPK pathway and ERK cascade. In vitro pharmaceutical intervention with ERK inhibitors could partially reverse the morphological changes and inhibit the excessive cardiac hypertrophic molecular biomarkers in H9c2 cells. CONCLUSION: Our study revealed a functional role of ACTN2 under chronic stress, loss of ACTN2 function accelerated H9c2 hypertrophy through ERK signaling. A commercial drug, Ibudilast, was identified to reverse cell hypertrophy in vitro.

The Immunosuppressive Drug LF15-0195 Acts Also on Glomerular Lesions, by a Change in Cytoskeleton Distribution in Podocyte.

INTRODUCTION: Buffalo/Mna rats spontaneously develop nephrotic syndrome (NS) which recurs after renal transplantation. The immunosuppressive drug LF15-0195 can promote regression of the initial and post-transplantation nephropathy via induction of regulatory T cells. We investigate if this drug has an additional effect on the expression and localization of podocyte specific proteins. METHODS: Buffalo/Mna kidney samples were collected before and after the occurrence of proteinuria, and after the remission of proteinuria induced by LF15-0195 treatment and compared by quantitative RT-PCR, Western blot, electron, and confocal microscopy to kidney samples of age-matched healthy rats. Cytoskeleton changes were assessed in culture by stress fibers induction by TNFα. RESULTS: We observed, by electron microscopy, a restoration of foot process architecture in the LF15-0195-treated Buff/Mna kidneys, consistent with proteinuria remission. Nephrin, podocin, CD2AP, and α-actinin-4 mRNA levels remained low during the active disease in the Buff/Mna, in comparison with healthy rats which increase, while podocalyxin and synaptopodin transcripts were elevated before the occurrence of the disease but did not differ from healthy animals after. No difference in the mRNA and protein expression between the untreated and the LF15-0195-treated proteinuric Buff/Mna were seen for these 6 proteins. No changes were observed by confocal microscopy in the protein distribution at a cellular level, but a more homogenous distribution similar to healthy rats, was observed within the glomeruli of LF15-0195-treated rats. In addition, LF15-0195 could partially restore actin cytoskeleton of endothelial cells in TNFα-induced-cell stress experiment. CONCLUSION: The effect of LF15-0195 treatment appears to be mediated by 2 mechanisms: an immunomodulatory effect via regulatory T cells induction, described in our previous work and which can act on immune cell involved in the disease pathogenesis, and an effect on the restoration of podocyte cytoskeleton, independent of expression levels of the proteins involved in the slit diaphragm and podocyte function, showed in this article.

URINARY EXCRETION OF ALPHA-ACTININ-4 AND TIGHT JUNCTION PROTEIN 1 IN PATIENTS WITH TYPE 2 DIABETES AND DIFFERENT PATTERNS OF CHRONIC KIDNEY DISEASE.

Abnormalities of the cytoskeleton and the slit diaphragm of podocytes have been attributed to diabetic nephropathy. In this study, we assessed urinary excretion of alpha-actinin-4 (ACTN-4), a cytoskeleton protein and a component of the slit diaphragm, and tight junction protein 1 (TJP-1, or ZO-1), a peripheral membrane protein that forms molecular complexes with actin filaments, in patients with type 2 diabetes (T2D) and albuminuric or non-albuminuric chronic kidney disease (CKD). The study included 140 patients with long-term T2D (≥10 years) and 20 healthy subjects as control. Patterns of CKD were identified based on the estimated glomerular filtration rate (eGFR) and urinary albumin-to-creatinine ratio (UACR). Urinary ACTN-4 and TJP-1 were assessed by ELISA. Patients with T2D had increased urinary excretion of ACTN-4 (p=0.03) and TJP-1 (p=0.006). In logistic regression models, both ACTN-4 and TJP-1 demonstrated associations with albuminuric CKD (UACR ≥3.0 mg/mmol and eGFR <60 mL/min×1.73 m2) after adjusting to age, sex, diabetes duration, HbA1c, and smoking. In ROC-analysis, TJP-1 excretion ≥70 pg/mmol was associated with albuminuric CKD (OR 5.45, 95% CI 1.96-15.18, p=0.001). The results demonstrate that elevated urinary ACTN-4 and TJP-1 are associated specifically with albuminuric CKD, but not with non-albuminuric CKD, in T2D patients.

Acute responses of strength-related gene expressions to maximum strength and force sense acuity.

Background/aim: Although high muscle strength worsens the sense of force, it is unknown whether there is a relationship between this deterioration and the underlying molecular mechanisms. This study examined the relationship between decreased force sense (FS) acuity and strength-related gene expressions. Materials and methods: Maximal voluntary isometric contraction (MVIC) and FS (50% MVIC) tests were performed on the knee joints of twenty-two subjects. The expression analyses were evaluated by qRT-PCR in blood samples taken before, after MVIC, after 50% MVIC, and 15 min after the test. Results: MVIC and FS error values were significantly correlated with each other (r = .659, p = .001). The qRT-PCR analyses demonstrated that the expressed mRNAs of the interleukin 6 (IL-6), alpha-actinin 3 (ACTN3), angiotensin-converting enzyme (ACE), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor receptor (CNTFR) genes dramatically increased until 50% MVIC and subsequently decreased 15 min after the exercise (p < .05). The muscle-specific creatine kinase (CKMM), myosin light chain kinase (MLCK), and G-protein ß3 subunit (GNB3) genes reached their peak expression levels 30 min after MVIC (p < .05). ACE and ACTN3 gene expression increased significantly in parallel with the increased FS error (p < .05). These gene expression fluctuations observed at 50% MVIC and after the rest could be related to changes in cellular metabolism leading to fatigue. Conclusion: The time points of gene expression levels during exercise need to be considered. The force acuity of those whose maximal force develops too much may deteriorate.

Actin Cytoskeleton Remodeling Accompanied by Redistribution of Adhesion Proteins Drives Migration of Cells in Different EMT States.

Epithelial-mesenchymal transition (EMT) is a process during which epithelial cells lose epithelial characteristics and gain mesenchymal features. Here, we used several cell models to study migratory activity and redistribution of cell-cell adhesion proteins in cells in different EMT states: EGF-induced EMT of epithelial IAR-20 cells; IAR-6-1 cells with a hybrid epithelial-mesenchymal phenotype; and their more mesenchymal derivatives, IAR-6-1-DNE cells lacking adherens junctions. In migrating cells, the cell-cell adhesion protein α-catenin accumulated at the leading edges along with ArpC2/p34 and α-actinin. Suppression of α-catenin shifted cell morphology from fibroblast-like to discoid and attenuated cell migration. Expression of exogenous α-catenin in MDA-MB-468 cells devoid of α-catenin drastically increased their migratory capabilities. The Y654 phosphorylated form of ß-catenin was detected at integrin adhesion complexes (IACs). Co-immunoprecipitation studies indicated that α-catenin and pY654-ß-catenin were associated with IAC proteins: vinculin, zyxin, and α-actinin. Taken together, these data suggest that in cells undergoing EMT, catenins not participating in assembly of adherens junctions may affect cell migration.

AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells.

BACKGROUND: Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis. METHODS: To identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs. RESULTS: Proteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs. CONCLUSION: This study elucidates AGR2's pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.

KLF5 regulates actin remodeling to enhance the metastasis of nasopharyngeal carcinoma.

Transcription factors (TFs) engage in various cellular essential processes including differentiation, growth and migration. However, the master TF involved in distant metastasis of nasopharyngeal carcinoma (NPC) remains largely unclear. Here we show that KLF5 regulates actin remodeling to enhance NPC metastasis. We analyzed the msVIPER algorithm-generated transcriptional regulatory networks and identified KLF5 as a master TF of metastatic NPC linked to poor clinical outcomes. KLF5 regulates actin remodeling and lamellipodia formation to promote the metastasis of NPC cells in vitro and in vivo. Mechanistically, KLF5 preferentially occupies distal enhancer regions of ACTN4 to activate its transcription, whereby decoding the informative DNA sequences. ACTN4, extensively localized within actin cytoskeleton, facilitates dense and branched actin networks and lamellipodia formation at the cell leading edge, empowering cells to migrate faster. Collectively, our findings reveal that KLF5 controls robust transcription program of ACTN4 to modulate actin remodeling and augment cell motility which enhances NPC metastasis, and provide new potential biomarkers and therapeutic interventions for NPC.

miR-129-5p inhibits anchorage-independent growth through silencing of ACTN1 and the ELK4/c-FOS axis in HPV-transformed keratinocytes.

A persistent infection with human papillomavirus (HPV) can induce precancerous lesions of the cervix that may ultimately develop into cancer. Cervical cancer development has been linked to altered microRNA (miRNA) expression, with miRNAs regulating anchorage-independent growth being particularly important for the progression of precancerous lesions to cancer. In this study, we set out to identify and validate targets of miR-129-5p, a previously identified tumor suppressive miRNA involved in anchorage-independent growth and HPV-induced carcinogenesis. We predicted 26 potential miR-129-5p targets using online databases, followed by KEGG pathway enrichment analysis. RT-qPCR and luciferase assays confirmed that 3'UTR regions of six genes (ACTN1, BMPR2, CAMK4, ELK4, EP300, and GNAQ) were targeted by miR-129-5p. Expressions of ACTN1, CAMK4, and ELK4 were inversely correlated to miR-129-5p expression in HPV-transformed keratinocytes, and their silencing reduced anchorage-independent growth. Concordantly, miR-129-5p overexpression decreased protein levels of ACTN1, BMPR2, CAMK4 and ELK4 in anchorage-independent conditions. Additionally, c-FOS, a downstream target of ELK4, was downregulated upon miR-129-5p overexpression, suggesting regulation through the ELK4/c-FOS axis. ACTN1 and ELK4 expression was also upregulated in high-grade precancerous lesions and cervical cancers, supporting their clinical relevance. In conclusion, we identified six targets of miR-129-5p involved in the regulation of anchorage-independent growth, with ACTN1, BMPR2, ELK4, EP300, and GNAQ representing novel targets for miR-129-5p. For both ACTN1 and ELK4 functional and clinical relevance was confirmed, indicating that miR-129-5p-regulated ACTN1 and ELK4 expression contributes to HPV-induced carcinogenesis.

Sprint and Anaerobic Power with the Soccer-Specific ACTN3 Gene: A Distintive Example / Sprint y Potencia Anaeróbica con el Gen ACTN3 Específico del Fútbol: Un Ejemplo Distintivo

SUMMARY: The aim of this study is twofold: (1) to identify differences in certain anaerobic parameters (10m sprint, 30m sprint, anaerobic power, and Illinois agility tests) between professional and amateur soccer players, and (2) to determine whether there is a difference in the ACTN3 gene polymorphism between professional and amateur soccer players. Ultimately, the goal is to reveal which parameters contribute to the differentiation in these two aspects. A total of 133 volunteer soccer players, including 71 professionals and 62 amateurs, participated in the research. DNA extraction from buccal epithelial cells was performed using a commercial kit to determine the genetic background of the athletes, and Real-Time PCR was conducted for genotyping. Statistical analysis of the findings obtained from the test results was performed using the SPSS 23 (SPSS Inc., Chicago, IL, USA) package program. The homogeneity of variance of the data was assessed using the Levene Test, and normal distribution analyses were conducted using the Shapiro-Wilk Test. Chi-square and Mann-Whitney U tests were employed for parameter analysis. The significance level was set at p0.05). However, there is a statistically significant difference in anaerobic parameters (10m sprint, 30m sprint, and anaerobic power) except for the Illinois test (p<0.05). In conclusion, our study found that gene polymorphism is not a differentiating factor between professional and amateur soccer players, but speed (10m and 30m) and anaerobic power parameters are differentiating factors.

Los objetivos de este estudio fueron: 1º identificar diferencias en ciertos parámetros anaeróbicos (sprint de 10 m, sprint de 30 m, potencia anaeróbica y pruebas de agilidad de Illinois) entre jugadores de fútbol profesionales y amateurs, y 2º determinar si existe una diferencia en el polimorfismo del gen ACTN3 entre jugadores de fútbol profesionales y aficionados. En definitiva, el objetivo fue revelar qué parámetros contribuyen a la diferenciación en estos dos aspectos. En la investigación participaron un total de 133 jugadores de fútbol voluntarios, incluidos 71 profesionales y 62 aficionados. La extracción de ADN de las células epiteliales orales se realizó utilizando un kit comercial para determinar los antecedentes genéticos de los atletas y se realizó una PCR en tiempo real para el genotipado. El análisis estadístico de los hallazgos obtenidos a partir de los resultados de las pruebas se realizó utilizando el programa de paquete SPSS 23 (SPSS Inc., Chicago, IL, EE. UU.). La homogeneidad de la varianza de los datos se evaluó mediante la prueba de Levene y los análisis de distribución normal se realizaron mediante la prueba de Shapiro-Wilk. Para el análisis de parámetros se emplearon las pruebas de Chi-cuadrado y U de Mann-Whitney. El nivel de significancia se fijó en p0,05). Sin embargo, existe una diferencia estadísticamente significativa en los parámetros anaeróbicos (sprint de 10 m, sprint de 30 m y potencia anaeróbica) excepto para la prueba de Illinois (p<0,05). En conclusión, nuestro estudio encontró que el polimorfismo genético no es un fac- tor diferenciador entre jugadores de fútbol profesionales y amateurs, pero sí los parámetros de velocidad (10 m y 30 m) y potencia anaeróbica.

Genetic Variations in Susceptibility to Traumatic Muscle Injuries and Muscle Pain among Brazilian High-Performance Athletes.

Traumatic muscle injuries (TMIs) and muscle pain (MP) negatively impact athletes' performance and quality of life. Both conditions have a complex pathophysiology involving the interplay between genetic and environmental factors. Yet, the existing data are scarce and controversial. To provide more insights, this study aimed to investigate the association of single-nucleotide polymorphisms (SNPs) previously linked to athletic status with TMI and MP after exercise among Brazilian high-performance athletes from different sports modalities (N = 345). The impact of important environmental determinants was also assessed. From the six evaluated SNPs (ACTN3 rs1815739, FAAH rs324420, PPARGC1A rs8192678, ADRB2 rs1042713, NOS3 rs1799983, and VDR rs731236), none was significantly associated with TMI. Regarding MP after exercise, ACTN3 rs1815739 (CC/CT vs. TT; adjusted odds ratio (aOR) = 1.90; 95% confidence interval (95%Cl), 1.01-3.57) and FAAH rs324420 (AA vs. AC/CC; aOR = 2.30; 95%Cl, 1.08-4.91) were independent predictors according to multivariate binomial analyses adjusted for age (≥23 vs. <23 years), sex (male vs. female), and tobacco consumption (yes vs. no). External validation is warranted to assess the predictive value of ACTN3 rs1815739 and FAAH rs324420. This could have implications for prophylactic interventions to improve athletes' quality of life.

Actinin-4 controls survival signaling in B cells by limiting the lateral mobility of B-cell antigen receptors.

The structure and dynamics of F-actin networks in the cortical area of B cells control the signal efficiency of B-cell antigen receptors (BCRs). Although antigen-induced signaling has been studied extensively, the role of cortical F-actin in antigen-independent tonic BCR signaling is less well understood. Because these signals are essential for the survival of B cells and are consequently exploited by several B-cell lymphomas, we assessed how the cortical F-actin structure influences tonic BCR signal transduction. We employed genetic variants of a primary cell-like B-cell line that can be rendered quiescent to show that cross-linking of actin filaments by α-actinin-4 (ACTN4), but not ACTN1, is required to preserve the dense architecture of F-actin in the cortical area of B cells. The reduced cortical F-actin density in the absence of ACTN4 resulted in increased lateral BCR diffusion. Surprisingly, this was associated with reduced tonic activation of BCR-proximal effector proteins, extracellular signal-regulated kinase, and pro-survival pathways. Accordingly, ACTN4-deficient B-cell lines and primary human B cells exhibit augmented apoptosis. Hence, our findings reveal that cortical F-actin architecture regulates antigen-independent tonic BCR survival signals in human B cells.

Cofilin-mediated actin filament network flexibility facilitates 2D to 3D actomyosin shape change.

The organization of actin filaments (F-actin) into crosslinked networks determines the transmission of mechanical stresses within the cytoskeleton and subsequent changes in cell and tissue shape. Principally mediated by proteins such as α-actinin, F-actin crosslinking increases both network connectivity and rigidity, thereby facilitating stress transmission at low crosslinking yet attenuating transmission at high crosslinker concentration. Here, we engineer a two-dimensional model of the actomyosin cytoskeleton, in which myosin-induced mechanical stresses are controlled by light. We alter the extent of F-actin crosslinking by the introduction of oligomerized cofilin. At pH 6.5, F-actin severing by cofilin is weak, but cofilin bundles and crosslinks filaments. Given its effect of lowering the F-actin bending stiffness, cofilin- crosslinked networks are significantly more flexible and softer in bending than networks crosslinked by α-actinin. Thus, upon local activation of myosin-induced contractile stress, the network bends out-of-plane in contrast to the in-plane compression as observed with networks crosslinked by α-actinin. Here, we demonstrate that local effects on filament mechanics by cofilin introduces novel large-scale network material properties that enable the sculpting of complex shapes in the cell cytoskeleton.

ACTN1 promotes HNSCC tumorigenesis and cisplatin resistance by enhancing MYH9-dependent degradation of GSK-3ß and integrin ß1-mediated phosphorylation of FAK.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignant tumors globally. Understanding the molecular basis of tumor progression and drug resistance can offer innovative strategies to enhance clinical outcomes for HNSCC patients. METHODS: The cytoskeletal remodeling genes associated with cisplatin resistance were screened using a PCR array. The role of alpha-actinin 1 (ACTN1) in modulating cisplatin resistance and tumorigenesis in HNSCC was evaluated both in vitro and in vivo. Co-immunoprecipitation (Co-IP), IP-mass spectrometry (MS), western blotting, dual-luciferase assay, and bioinformatics analysis were performed to elucidate the underlying mechanisms involved. RESULTS: Our study identifies ACTN1 as a crucial contributor to cisplatin resistance and tumorigenesis in HNSCC, as evidenced across cellular, animal, and patient-derived xenograft models. From a clinical perspective, overexpression of ACTN1 significantly correlates with a suboptimal response to neoadjuvant chemotherapy and reduced overall survival in HNSCC patients. Mechanistically, ACTN1 predominantly activates ß-catenin-mediated signaling by promoting the interaction between myosin heavy chain 9 (MYH9) and GSK-3ß, leading to the ubiquitin-dependent degradation of GSK-3ß. ACTN1 also interacts with integrin ß1, subsequently activating the FAK/PI3K/AKT pathway, providing an additional avenue for the activation of ß-catenin signaling. Our study also unveils that the ß-catenin/c-Myc axis transcriptionally regulates ACTN1, thereby creating a positive feedback loop promoting HNSCC tumorigenesis and drug resistance. CONCLUSIONS: These insights underscore the novel mechanisms that highlight ACTN1's pivotal role in driving HNSCC progression and resistance to chemotherapy, suggesting ACTN1 as a promising therapeutic target in HNSCC management.

Cytosolic actin isoforms form networks with different rheological properties that indicate specific biological function.

The implications of the existence of different actins expressed in epithelial cells for network mechanics and dynamics is investigated by microrheology and confocal imaging. γ-actin predominately found in the apical cortex forms stiffer networks compared to ß-actin, which is preferentially organized in stress fibers. We attribute this to selective interactions with Mg2+-ions interconnecting the filaments' N-termini. Bundling propensity of the isoforms is different in the presence of Mg2+-ions, while crosslinkers such as α-actinin, fascin, and heavy meromyosin alter the mechanical response independent of the isoform. In the presence of myosin, ß-actin networks show a large number of small contraction foci, while γ-actin displays larger but fewer foci indicative of a stronger interaction with myosin motors. We infer that subtle changes in the amino acid sequence of actin isoforms lead to alterations of the mechanical properties on the network level with potential implications for specific biological functions.

The Distinguishing Factor in Soccer Players is Aerobic Performance, not the ACTN3 Gene / El Factor Distintivo en los Jugadores de Fútbol es el Rendimiento Aeróbico y no el gen ACTN3

SUMMARY: The purpose of this study was to reveal the differences between ACTN3 genotype (RR, RX, XX) and aerobic performance [Yo-Yo IRT1 (m), VO2 max (ml/kg/min)] in professional and regional amateur league soccer players and to reveal which of these parameters was a distinctive factor in these athletes.71 professional soccer players (age: 23.66 ± 4.11 years; body height: 1.79 ± 6.99 m; body weight: 76.02 ± 6.76 kg; body fat: 11.59±3.11 %) and 62 regional amateur soccer players (age: 23.63 ±3.77 years; body height: 1.81 ± 5.77 m; body weight: 76.36 ± 7.53 kg; body fat: 15.60±4.65 %) volunteered for the study. After DNA extraction from buccal epithelial cells via a commercial kit was performed for the genetic background of the athletes, Real-Time PCR was carried out for genotyping. Furthermore, Yo-Yo IRT1 test was performed to determine the aerobic performance of the soccer players. SPSS 23 (SPSS Inc., Chicago, IL, USA) package program was used for the statistical analysis of the data obtained in the tests. Shapiro-Wilk test for normality and Levene's test for homogeneity of variance were performed. Chi-Square, Independent Sample T Test and One Way ANOVA test were used in the analysis of the parameters. Statistical significance was set as p0.05); however, there was a statistical significance in favor of professional soccer players in terms of aerobic parameters (p<0.05). Consequently, it can be said that aerobic performance is the distinguishing factor, not the ACTN3 gene, in soccer players.

El objetivo de este estudio fue revelar las diferencias entre el genotipo ACTN3 (RR, RX, XX) y el rendimiento aeróbico [Yo-Yo IRT1 (m), VO2 max (ml/kg/min)] en jugadores de fútbol de ligas profesionales y amateurs regionales y determinar cuál de estos parámetros es un factor distintivo en estos deportistas. 71 futbolistas profesionales (edad: 23,66 ±4,11 años; altura corporal: 1,79 ± 6,99 m; peso corporal: 76,02 ± 6,76 kg; grasa corporal: 11,59±3,11 %) y 62 jugadores de fútbol amateur regionales (edad: 23,63 ± 3,77 años; altura corporal: 1,81 ± 5,77 m; peso corporal: 76,36 ± 7,53 kg; grasa corporal: 15,60 ± 4,65 %) se ofrecieron como voluntarios para el estudio. Después de realizar la extracción de ADN de las células epiteliales orales mediante un kit comercial para obtener los antecedentes genéticos de los atletas, se llevó a cabo una PCR en tiempo real para el genotipado. Además, se realizó la prueba Yo-Yo IRT1 para determinar el rendimiento aeróbico de los futbolistas. Para el análisis estadístico de los datos obtenidos en las pruebas se utilizó el programa SPSS 23 (SPSS Inc., Chicago, IL, EE. UU.). Se realizó la prueba de normalidad de Shapiro- Wilk y la prueba de homogeneidad de la varianza de Levene. En el análisis de los parámetros se utilizaron Chi-cuadrado, prueba T para muestra independiente y prueba ANOVA unidireccional. La significancia estadística se estableció en p0,05); sin embargo, hubo significación estadística a favor de los futbolistas profesionales en cuanto a los parámetros aeróbicos (p<0,05). En consecuencia, se puede decir que el rendimiento aeróbico es el factor distintivo, no el gen ACTN3, en los jugadores de fútbol.

Cardiomyocyte external mechanical unloading activates modifications of α-actinin differently from sarcomere-originated unloading.

Loss of myocardial mass in a neonatal rat cardiomyocyte culture is studied to determine whether there is a distinguishable cellular response based on the origin of mechano-signals. The approach herein compares the sarcomeric assembly and disassembly processes in heart cells by imposing mechano-signals at the interface with the extracellular matrix (extrinsic) and at the level of the myofilaments (intrinsic). Experiments compared the effects of imposed internal (inside/out) and external (outside/in) loading and unloading on modifications in neonatal rat cardiomyocytes. Unloading of the cellular substrate by myosin inhibition (1 µm mavacamten), or cessation of cyclic strain (1 Hz, 10% strain) after preconditioning, led to significant disassembly of sarcomeric α-actinin by 6 h. In myosin inhibition, this was accompanied by redistribution of intracellular poly-ubiquitin K48 to the cellular periphery relative to the poly-ubiquitin K48 reservoir at the I-band. Moreover, loading and unloading of the cellular substrate led to a three-fold increase in post-translational modifications (PTMs) when compared to the myosin-specific activation or inhibition. Specifically, phosphorylation increased with loading while ubiquitination increased with unloading, which may involve extracellular signal-regulated kinase 1/2 and focal adhesion kinase activation. The identified PTMs, including ubiquitination, acetylation, and phosphorylation, are proposed to modify internal domains in α-actinin to increase its propensity to bind F-actin. These results demonstrate a link between mechanical feedback and sarcomere protein homeostasis via PTMs of α-actinin that exemplify how cardiomyocytes exhibit differential responses to the origin of force. The implications of sarcomere regulation governed by PTMs of α-actinin are discussed with respect to cardiac atrophy and heart failure.