RESUMEN

Gastric cancer is one of the most malignant digestive tract tumors worldwide and its progression is associated with gene expression and metabolic alteration. We revealed that the gastric cancer patients with lower expression level of TOB1 exhibited poorer overall survivals according to the data in Kaplan-Meier Plotter. The unphosphorylated TOB1 protein which is effective expressed lower in gastric cancer cells. The gastric cancer cells with TOB1 gene depletion performed higher abilities of proliferation, migration and invasion and lower ability of apoptosis in vitro. The TOB1 gene depletion also promoted the tumorigenesis of gastric cancer cells in vivo. The gastric cancer cells with TOB1 gene overexpression had the converse behaviors. The transcriptional and metabolic sequencing was performed. The analyzation results showed that genes correlate-expressed with TOB1 gene were enriched in the pathways related to ERK pathway, including focal adhesion pathway, which was verified using real-time quantitative PCR. After inhibiting ERK pathway, the proliferation, colony formation and migration abilities were reduced in gastric cancer cells with low phosphorylated TOB1 protein expression level. Moreover, Pearson correlation analysis was adopted to further analyze the correlation of enriched metabolic products and differentially expressed genes. The expression of Choline, UDP-N-acetylglucosamine, Adenosine and GMP were related to the function of TOB1. This study demonstrates the genes and metabolites related to focal adhesion pathway and ERK pathway are the potential diagnosis and therapeutic targets to gastric cancer with TOB1 depletion.

Asunto(s)

Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Neoplasias Gástricas , Proteínas Supresoras de Tumor , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidad , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Movimiento Celular/genética , Línea Celular Tumoral , Animales , Ratones , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Adhesiones Focales/genética , Adhesiones Focales/metabolismo , Apoptosis/genética

RESUMEN

Integrins are fundamental for cell adhesion and the formation of focal adhesions (FA). Accordingly, these receptors guide embryonic development, tissue maintenance, and haemostasis but are also involved in cancer invasion and metastasis. A detailed understanding of the molecular interactions that drive integrin activation, FA assembly, and downstream signalling cascades is critical. Here, we reveal a direct association of paxillin, a marker protein of FA sites, with the cytoplasmic tails of the integrin ß1 and ß3 subunits. The binding interface resides in paxillin's LIM3 domain, where based on the NMR structure and functional analyses, a flexible, 7-amino acid loop engages the unstructured part of the integrin cytoplasmic tail. Genetic manipulation of the involved residues in either paxillin or integrin ß3 compromises cell adhesion and motility of murine fibroblasts. This direct interaction between paxillin and the integrin cytoplasmic domain identifies an alternative, kindlin-independent mode of integrin outside-in signalling particularly important for integrin ß3 function.

Asunto(s)

Paxillin , Unión Proteica , Paxillin/metabolismo , Animales , Ratones , Dominios Proteicos , Adhesión Celular/fisiología , Adhesiones Focales/metabolismo , Humanos , Movimiento Celular , Integrina beta3/metabolismo , Integrina beta3/genética , Integrina beta3/química , Fibroblastos/metabolismo , Cadenas beta de Integrinas/metabolismo , Cadenas beta de Integrinas/química , Cadenas beta de Integrinas/genética , Integrina beta1/metabolismo , Transducción de Señal

RESUMEN

Tendons enable locomotion by transmitting high tensile mechanical forces between muscle and bone via their dense extracellular matrix (ECM). The application of extrinsic mechanical stimuli via muscle contraction is necessary to regulate healthy tendon function. Specifically, applied physiological levels of mechanical loading elicit an anabolic tendon cell response, while decreased mechanical loading evokes a degradative tendon state. Although the tendon response to mechanical stimuli has implications in disease pathogenesis and clinical treatment strategies, the cell signaling mechanisms by which tendon cells sense and respond to mechanical stimuli within the native tendon ECM remain largely unknown. Therefore, we explored the role of cell-ECM adhesions in regulating tendon cell mechanotransduction by perturbing the genetic expression and signaling activity of focal adhesion kinase (FAK) through both in vitro and in vivo approaches. We determined that FAK regulates tendon cell spreading behavior and focal adhesion morphology, nuclear deformation in response to applied mechanical strain, and mechanosensitive gene expression. In addition, our data reveal that FAK signaling plays an essential role in in vivo tendon development and postnatal growth, as FAK-knockout mouse tendons demonstrated reduced tendon size, altered mechanical properties, differences in cellular composition, and reduced maturity of the deposited ECM. These data provide a foundational understanding of the role of FAK signaling as a critical regulator of in situ tendon cell mechanotransduction. Importantly, an increased understanding of tendon cell mechanotransductive mechanisms may inform clinical practice as well as lead to the discovery of diagnostic and/or therapeutic molecular targets.

Asunto(s)

Mecanotransducción Celular , Ratones Noqueados , Tendones , Animales , Masculino , Ratones , Células Cultivadas , Matriz Extracelular/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Adhesiones Focales/metabolismo , Mecanotransducción Celular/fisiología , Ratones Endogámicos C57BL , Transducción de Señal/fisiología , Tendones/metabolismo , Tendones/fisiología , Tendones/citología , Femenino

RESUMEN

Talin2 is localized to large focal adhesions and is indispensable for traction force generation, invadopodium formation, cell invasion as well as metastasis. Talin2 has a higher affinity toward ß-integrin tails than talin1. Moreover, disruption of the talin2-ß-integrin interaction inhibits traction force generation, invadopodium formation and cell invasion, indicating that a strong talin2-ß-integrin interaction is required for talin2 to fulfill these functions. Nevertheless, the role of talin2 in mediation of these processes remains unknown. Here we show that talin2 binds to the N-terminus of non-muscle myosin IIA (NMIIA) through its F3 subdomain. Moreover, talin2 co-localizes with NMIIA at cell edges as well as at some cytoplasmic spots. Talin2 also co-localizes with cortactin, an invadopodium marker. Furthermore, overexpression of NMIIA promoted the talin2 head binding to the ß1-integrin tail, whereas knockdown of NMIIA reduced fibronectin and matrix metalloproteinase secretion as well as inhibited cell attachment on fibronectin-coated substrates. These results suggest that talin2 binds to NMIIA to control the secretion of extracellular matrix proteins and this interaction modulates cell adhesion.

Asunto(s)

Adhesión Celular , Fibronectinas , Miosina Tipo IIA no Muscular , Unión Proteica , Talina , Animales , Humanos , Cortactina/metabolismo , Fibronectinas/metabolismo , Adhesiones Focales/metabolismo , Integrina beta1/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Podosomas/metabolismo , Talina/metabolismo , Ratones

RESUMEN

Biophysical cues have the ability to enhance cellular signaling response to Bone Morphogenetic Proteins, an essential growth factor during bone development and regeneration. Yet, therapeutic application of Bone Morphogenetic Protein 2 (BMP2) is restricted due to uncontrolled side effects. An understanding of the temporal characteristics of mechanically regulated signaling events and underlying mechanism is lacking. Using a 3D bioreactor system in combination with a soft macroporous biomaterial substrate, we mimic the in vivo environment that BMP2 is acting in. We show that the intensity and duration of BMP2 signaling increases with increasing loading frequency in synchrony with the number and size of focal adhesions. Long-term mechanical stimulation increases the expression of BMP receptor type 1B, specific integrin subtypes and integrin clustering. Together, this triggered a short-lived mechanical echo that enhanced BMP2 signaling even when BMP2 is administered directly after mechanical stimulation, but not when it is applied after a resting period of ≥30 min. Interfering with cytoskeletal remodeling hinders focal adhesion remodeling verifying its critical role in shifting cells into a state of high BMP2 responsiveness. The design of biomaterials that exploit this potential locally at the site of injury will help to overcome current limitations of clinical growth factor treatment.

Asunto(s)

Proteína Morfogenética Ósea 2 , Citoesqueleto , Adhesiones Focales , Transducción de Señal , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 2/genética , Adhesiones Focales/metabolismo , Humanos , Citoesqueleto/metabolismo , Mecanotransducción Celular , Animales

RESUMEN

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. STATEMENT OF SIGNIFICANCE: 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.

Asunto(s)

Adhesión Celular , Adhesiones Focales , Humanos , Adhesiones Focales/metabolismo , Citoesqueleto/metabolismo , Células Madre/citología , Células Madre/metabolismo , Tejido Adiposo/citología

RESUMEN

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.

Asunto(s)

Movimiento Celular , Adhesiones Focales , Integrinas , Neoplasias Pancreáticas , Proteínas de Unión al GTP rab , Humanos , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Proteínas de Unión al GTP rab/metabolismo , Línea Celular Tumoral , Adhesiones Focales/metabolismo , Integrinas/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Femenino , Masculino , Transducción de Señal , Persona de Mediana Edad , Pronóstico , Regulación Neoplásica de la Expresión Génica , Endosomas/metabolismo , Progresión de la Enfermedad

RESUMEN

Integrin adaptor proteins, like tensin-2, are crucial for cell adhesion and signaling. However, the function of tensin-2 beyond localizing to focal adhesions remain poorly understood. We utilized proximity-dependent biotinylation and Strep-tag affinity proteomics to identify interaction partners of tensin-2 in Flp-In 293 T-REx cells. Interactomics linked tensin-2 to known focal adhesion proteins and the dystrophin glycoprotein complex, while also uncovering novel interaction with the glycolytic enzyme GAPDH. We demonstrated that Y483-phosphorylation of tensin-2 regulates the glycolytic rate in Flp-In 293 T-REx and MEF cells and found that pY483 tensin-2 is enriched in adhesions in MEF cells. Our study unveils novel interaction partners for tensin-2 and further solidifies its speculated role in cell energy metabolism. These findings shed fresh insight on the functions of tensin-2, highlighting its potential as a therapeutic target for diseases associated with impaired cell adhesion and metabolism.

Asunto(s)

Glucólisis , Tensinas , Humanos , Tensinas/metabolismo , Fosforilación , Adhesión Celular , Células HEK293 , Unión Proteica , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Adhesiones Focales/metabolismo , Proteómica/métodos , Animales , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo

RESUMEN

Although the E3 ligase Mdm2 and its homologue and binding partner MdmX are the major regulators of the p53 tumor suppressor protein, it is now evident that Mdm2 and MdmX have multiple functions that do not involve p53. As one example, it is known that Mdm2 can regulate cell migration, although mechanistic insight into this function is still lacking. Here we show in cells lacking p53 expression that knockdown of Mdm2 or MdmX, as well as pharmacological inhibition of the Mdm2/MdmX complex, not only reduces cell migration and invasion, but also impairs cell spreading and focal adhesion formation. In addition, Mdm2 knockdown decreases metastasis in vivo. Interestingly, Mdm2 downregulates the expression of Sprouty4, which is required for the Mdm2 mediated effects on cell migration, focal adhesion formation and metastasis. Further, our findings indicate that Mdm2 dampening of Sprouty4 is a prerequisite for maintaining RhoA levels in the cancer cells that we have studied. Taken together we describe a molecular mechanism whereby the Mdm2/MdmX complex through Sprouty4 regulates cellular processes leading to increase metastatic capability independently of p53.

Asunto(s)

Movimiento Celular , Adhesiones Focales , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas c-mdm2 , Proteína p53 Supresora de Tumor , Proteína de Unión al GTP rhoA , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Adhesiones Focales/metabolismo , Adhesiones Focales/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Humanos , Animales , Movimiento Celular/genética , Línea Celular Tumoral , Ratones , Proteína de Unión al GTP rhoA/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Regulación Neoplásica de la Expresión Génica

RESUMEN

The functioning of the human cornea heavily relies on the maintenance of its extracellular matrix (ECM) mechanical properties. Within this context, corneal stromal fibroblasts (CSFs) are essential, as they are responsible for remodeling the corneal ECM. In this study, we used a decellularized human amniotic membrane (dHAM) and a custom fibrillar collagen film (FCF) to explore the effects of fibrillar materials on human CSFs. Our findings indicate that substrates like FCF can enhance the early development of focal adhesions (FAs), leading to the activation and propagation of mechanotransduction signals. This is primarily achieved through FAK autophosphorylation and YAP1 nuclear translocation pathways. Remarkably, inhibiting FAK autophosphorylation negated the observed changes. Proteome analysis further confirmed the central role of FAs in mechanotransduction propagation in CSFs cultured on FCF. This analysis also highlighted complex signaling pathways, including chromatin epigenetic modifications, in response to fibrillar substrates. Overall, our research highlights the potential pathways through which CSFs undergo behavioral changes when exposed to fibrillar substrates, identifying FAs as essential mechanotransducers.

Asunto(s)

Sustancia Propia , Fibroblastos , Adhesiones Focales , Mecanotransducción Celular , Humanos , Adhesiones Focales/metabolismo , Fibroblastos/metabolismo , Sustancia Propia/citología , Sustancia Propia/metabolismo , Fosforilación , Matriz Extracelular/metabolismo , Células Cultivadas , Proteínas Señalizadoras YAP/metabolismo , Colágenos Fibrilares/metabolismo , Amnios/citología , Amnios/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo

RESUMEN

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

Asunto(s)

Hemangioma Cavernoso del Sistema Nervioso Central , Células Endoteliales de la Vena Umbilical Humana , Humanos , Animales , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales/metabolismo , Adhesiones Focales/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Fibras de Estrés/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Unión Proteica , Ratones , Núcleo Celular/metabolismo , Talina

RESUMEN

Ectodermal organs, such as hair follicles, originate from simple epithelial and mesenchymal sheets through a complex developmental process driven by interactions between these cell types. This process involves dermal condensation, placode formation, bud morphogenesis, and organogenesis, and all of these processes require intricate interactions among various tissues. Recent research has emphasized the crucial role of reciprocal and dynamic interactions between cells and the extracellular matrix (ECM), referred to as the "dynamic duo", in the development of ectodermal organs. These interactions provide spatially and temporally changing biophysical and biochemical cues within tissues. Using the hair follicle as an example, this review highlights two types of cell-ECM adhesion units-focal adhesion-type and hemidesmosome-type adhesion units-that facilitate communication between epithelial and mesenchymal cells. This review further explores how these adhesion units, along with other cell-ECM interactions, evolve during hair follicle development and regeneration, underscoring their importance in guiding both developmental and regenerative processes.

Asunto(s)

Matriz Extracelular , Folículo Piloso , Regeneración , Folículo Piloso/metabolismo , Folículo Piloso/fisiología , Folículo Piloso/citología , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiología , Animales , Regeneración/fisiología , Humanos , Adhesión Celular/fisiología , Adhesiones Focales/metabolismo , Adhesiones Focales/fisiología , Comunicación Celular/fisiología , Hemidesmosomas/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/fisiología

RESUMEN

This study investigates how dynamic fluctuations in matrix stiffness affect the behavior of cardiac fibroblasts (CFs) within a three-dimensional (3D) hydrogel environment. Using hybrid hydrogels with tunable stiffness, we created an in vitro model to mimic the varying stiffness of the cardiac microenvironment. By manipulating hydrogel stiffness, we examined CF responses, particularly the expression of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation. Our findings reveal that increased matrix stiffness promotes the differentiation of CFs into myofibroblasts, while matrix softening reverses this process. Additionally, we identified the role of focal adhesions and integrin ß1 in mediating stiffness-induced phenotypic switching. This study provides significant insights into the mechanobiology of cardiac fibrosis and suggests that modulating matrix stiffness could be a potential therapeutic strategy for treating cardiovascular diseases.

Asunto(s)

Diferenciación Celular , Matriz Extracelular , Fibroblastos , Hidrogeles , Miofibroblastos , Fenotipo , Hidrogeles/química , Matriz Extracelular/metabolismo , Animales , Fibroblastos/metabolismo , Fibroblastos/citología , Miofibroblastos/metabolismo , Miofibroblastos/citología , Integrina beta1/metabolismo , Adhesiones Focales/metabolismo , Miocardio/citología , Miocardio/metabolismo , Células Cultivadas , Ratas , Actinas/metabolismo

RESUMEN

Purpose: Bee pollen possesses favorable anticancer activities. As a medicinal plant source, Schisandra chinensis bee pollen (SCBP) possesses potential pharmacological properties, such as reducing cisplatin-induced liver injury, but its anti-liver cancer effect is still rarely reported. This paper aims to investigate the effect and mechanism of SCBP extract (SCBPE) on hepatocellular carcinoma HepG2 cells. Methods: The effect of SCBPE on cell proliferation and migration of HepG2 cells was evaluated based on MTT assay, morphology observation, or scratching assay. Furthermore, tandem mass tag-based quantitative proteomics was used to study the effect mechanisms. The mRNA expression levels of identified proteins were verified by RT-qPCR. Results: Tandem mass tag-based quantitative proteomics showed that 61 differentially expressed proteins were obtained in the SCBPE group compared with the negative-control group: 18 significantly downregulated and 43 significantly upregulated proteins. Bioinformatic analysis showed the significantly enriched KEGG pathways were predominantly ferroptosis-, Wnt-, and hepatocellular carcinoma-signaling ones. Protein-protein interaction network analysis and RT-qPCR validation revealed SCBPE also downregulated the focal adhesion-signaling pathway, which is abrogated by PF-562271, a well-known inhibitor of FAK. Conclusion: This study confirmed SCBPE suppressed the cell proliferation and migration of hepatocellular carcinoma HepG2 cells, mainly through modulation of ferroptosis-, Wnt-, hepatocellular carcinoma-, and focal adhesion-signaling pathways, providing scientific data supporting adjuvant treatment of hepatocellular carcinoma using SCBP.

Asunto(s)

Carcinoma Hepatocelular , Movimiento Celular , Proliferación Celular , Ferroptosis , Neoplasias Hepáticas , Polen , Schisandra , Humanos , Proliferación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Células Hep G2 , Animales , Schisandra/química , Polen/química , Ferroptosis/efectos de los fármacos , Abejas/química , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Antineoplásicos/farmacología , Antineoplásicos/química , Transducción de Señal/efectos de los fármacos , Productos Biológicos , Polifenoles

RESUMEN

Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.

Asunto(s)

Matriz Extracelular , Adhesiones Focales , Familia-src Quinasas , Adhesiones Focales/metabolismo , Matriz Extracelular/metabolismo , Humanos , Familia-src Quinasas/metabolismo , Familia-src Quinasas/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Animales , Proteína Tirosina Quinasa CSK/metabolismo , Transducción de Señal , Células Endoteliales/metabolismo , Células Endoteliales/patología , Metaloproteinasas de la Matriz/metabolismo

RESUMEN

Kinases are known to have kinase activity independent functions. To gain further insights into potential kinase-independent functions of SLK/STK2, we have developed a kinase-dead allele, SLKK63R using in vivo CRISPR/Cas technology. Our studies show that blastocysts homozygote for SLKK63R do not develop into viable mice. However, heterozygotes are viable and fertile with no overt phenotypes. Analyses of mouse embryonic fibroblasts show that expression of SLKK63R results in a 50% decrease in kinase activity in heterozygotes. In contrast to previous studies, our data show that SLK does not form homodimers and that the kinase defective allele does not act in a dominant negative fashion. Expression of SLKK63R leads to altered Rac1 and RhoA activity, increased stress fiber formation and delayed focal adhesion turnover. Our data support a previously observed role for SLK in cell migration and suggest that at least 50% kinase activity is sufficient for embryonic development.

Asunto(s)

Movimiento Celular , Fibroblastos , Proteínas Serina-Treonina Quinasas , Proteína de Unión al GTP rac1 , Animales , Femenino , Ratones , Movimiento Celular/genética , Sistemas CRISPR-Cas , Pérdida del Embrión/genética , Pérdida del Embrión/patología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Fibroblastos/citología , Adhesiones Focales/metabolismo , Adhesiones Focales/genética , Neuropéptidos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/genética

RESUMEN

Vinculin is a cytoskeletal linker strengthening cell adhesion. The Shigella IpaA invasion effector binds to vinculin to promote vinculin supra-activation associated with head-domain-mediated oligomerization. Our study investigates the impact of mutations of vinculin D1D2 subdomains' residues predicted to interact with IpaA VBS3. These mutations affected the rate of D1D2 trimer formation with distinct effects on monomer disappearance, consistent with structural modeling of a closed and open D1D2 conformer induced by IpaA. Notably, mutations targeting the closed D1D2 conformer significantly reduced Shigella invasion of host cells as opposed to mutations targeting the open D1D2 conformer and later stages of vinculin head-domain oligomerization. In contrast, all mutations affected the formation of focal adhesions (FAs), supporting the involvement of vinculin supra-activation in this process. Our findings suggest that IpaA-induced vinculin supra-activation primarily reinforces matrix adhesion in infected cells, rather than promoting bacterial invasion. Consistently, shear stress studies pointed to a key role for IpaA-induced vinculin supra-activation in accelerating and strengthening cell-matrix adhesion.

Asunto(s)

Adhesión Celular , Adhesiones Focales , Vinculina , Vinculina/metabolismo , Vinculina/genética , Humanos , Adhesiones Focales/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Mutación , Interacciones Huésped-Patógeno , Células HeLa , Unión Proteica , Shigella/metabolismo , Shigella/genética , Antígenos Bacterianos/metabolismo , Antígenos Bacterianos/genética , Disentería Bacilar/microbiología , Disentería Bacilar/metabolismo

RESUMEN

Mesenchymal stem cells (MSCs), pivotal for tissue repair, utilize collagen to restore structural integrity in damaged tissue, preserving its organization through concomitant remodeling. The non-enzymatic glycation of collagen potentially compromises MSC communication, particularly upon advancing the process, underlying various pathologies such as late-stage diabetic complications and aging. However, an understanding of the impact of early-stage collagen glycation on MSC interaction is lacking. This study examines the fate of in vitro glycated rat tail collagen (RTC) upon exposure to glucose for 1 or 5 days in contact with MSCs. Utilizing human adipose tissue-derived MSCs (ADMSCs), we demonstrate their significantly altered interaction with glycated collagen, characterized morphologically by reduced cell spreading, diminished focal adhesions formation, and attenuated development of the actin cytoskeleton. The morphological findings were confirmed by ImageJ 1.54g morphometric analysis with the most significant drop in the cell spreading area (CSA), from 246.8 µm2 for the native collagen to 216.8 µm2 and 163.7 µm2 for glycated ones, for 1 day and 5 days, respectively, and a similar trend was observed for cell perimeter 112.9 µm vs. 95.1 µm and 86.2 µm, respectively. These data suggest impaired recognition of early glycated collagen by integrin receptors. Moreover, they coincide with the reduced fibril-like reorganization of adsorbed FITC-collagen (indicating impaired remodeling) and a presumed decreased sensitivity to proteases. Indeed, confirmatory assays reveal diminished FITC-collagen degradation for glycated samples at 1 day and 5 days by attached cells (22.8 and 30.4%) and reduced proteolysis upon exogenous collagenase addition (24.5 and 40.4%) in a cell-free system, respectively. The mechanisms behind these effects remain uncertain, although differential scanning calorimetry confirms subtle structural/thermodynamic changes in glycated collagen.

Asunto(s)

Colágeno , Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Humanos , Colágeno/metabolismo , Glicosilación , Animales , Ratas , Comunicación Celular , Células Cultivadas , Glucosa/metabolismo , Tejido Adiposo/metabolismo , Tejido Adiposo/citología , Adhesiones Focales/metabolismo , Adhesiones Focales/efectos de los fármacos

RESUMEN

Understanding cellular force transmission dynamics is crucial in mechanobiology. We developed the DNA-based ForceChrono probe to measure force magnitude, duration, and loading rates at the single-molecule level within living cells. The ForceChrono probe circumvents the limitations of in vitro single-molecule force spectroscopy by enabling direct measurements within the dynamic cellular environment. Our findings reveal integrin force loading rates of 0.5-2 pN/s and durations ranging from tens of seconds in nascent adhesions to approximately 100 s in mature focal adhesions. The probe's robust and reversible design allows for continuous monitoring of these dynamic changes as cells undergo morphological transformations. Additionally, by analyzing how mutations, deletions, or pharmacological interventions affect these parameters, we can deduce the functional roles of specific proteins or domains in cellular mechanotransduction. The ForceChrono probe provides detailed insights into the dynamics of mechanical forces, advancing our understanding of cellular mechanics and the molecular mechanisms of mechanotransduction.

Asunto(s)

Mecanotransducción Celular , Imagen Individual de Molécula , Animales , Humanos , Ratones , Fenómenos Biomecánicos , Adhesión Celular , ADN/química , ADN/metabolismo , Adhesiones Focales/metabolismo , Integrinas/metabolismo , Microscopía de Fuerza Atómica/métodos , Imagen Individual de Molécula/métodos , Línea Celular , Supervivencia Celular , Emparejamiento Base , Calibración

RESUMEN

Immune cells are highly dynamic and able to migrate through environments with diverse biochemical and mechanical compositions. Their migration has classically been defined as amoeboid under the assumption that it is integrin independent. Here, we show that activated primary Th1 T cells require both confinement and extracellular matrix proteins to migrate efficiently. This migration is mediated through small and dynamic focal adhesions that are composed of the same proteins associated with canonical mesenchymal cell focal adhesions, such as integrins, talin, and vinculin. These focal adhesions, furthermore, localize to sites of contractile traction stresses, enabling T cells to pull themselves through confined spaces. Finally, we show that Th1 T cells preferentially follow tracks of other T cells, suggesting that these adhesions modify the extracellular matrix to provide additional environmental guidance cues. These results demonstrate not only that the boundaries between amoeboid and mesenchymal migration modes are ambiguous, but that integrin-mediated focal adhesions play a key role in T cell motility.

Asunto(s)

Movimiento Celular , Matriz Extracelular , Adhesiones Focales , Integrinas , Talina , Adhesiones Focales/metabolismo , Animales , Integrinas/metabolismo , Talina/metabolismo , Ratones , Matriz Extracelular/metabolismo , Vinculina/metabolismo , Ratones Endogámicos C57BL , Linfocitos T/inmunología , Linfocitos T/metabolismo , Activación de Linfocitos , Adhesión Celular