RESUMEN
Glioblastoma (GBM) is a malignant brain tumor with uncontrolled invasive growth. Here, we demonstrate how GBM cells usurp guidance receptor Plexin-B2 to gain biomechanical plasticity for polarized migration through confined space. Using live-cell imaging to track GBM cells negotiating microchannels, we reveal active endocytosis at cell front and filamentous actin assembly at rear to propel GBM cells through constrictions. These two processes are interconnected and governed by Plexin-B2 that orchestrates cortical actin and membrane tension, shown by biomechanical assays. Molecular dynamics simulations predict that balanced membrane and actin tension are required for optimal migratory velocity and consistency. Furthermore, Plexin-B2 mechanosensitive function requires a bendable extracellular ring structure and affects membrane internalization, permeability, phospholipid composition, as well as inner membrane surface charge. Together, our studies unveil a key element of membrane tension and mechanoelectrical coupling via Plexin-B2 that enables GBM cells to adapt to physical constraints and achieve polarized confined migration.
RESUMEN
During morphogenesis, molecular mechanisms that orchestrate biomechanical dynamics across cells remain unclear. Here, we show a role of guidance receptor Plexin-B2 in organizing actomyosin network and adhesion complexes during multicellular development of human embryonic stem cells and neuroprogenitor cells. Plexin-B2 manipulations affect actomyosin contractility, leading to changes in cell stiffness and cytoskeletal tension, as well as cell-cell and cell-matrix adhesion. We have delineated the functional domains of Plexin-B2, RAP1/2 effectors, and the signaling association with ERK1/2, calcium activation, and YAP mechanosensor, thus providing a mechanistic link between Plexin-B2-mediated cytoskeletal tension and stem cell physiology. Plexin-B2-deficient stem cells exhibit premature lineage commitment, and a balanced level of Plexin-B2 activity is critical for maintaining cytoarchitectural integrity of the developing neuroepithelium, as modeled in cerebral organoids. Our studies thus establish a significant function of Plexin-B2 in orchestrating cytoskeletal tension and cell-cell/cell-matrix adhesion, therefore solidifying the importance of collective cell mechanics in governing stem cell physiology and tissue morphogenesis.
Asunto(s)
Actomiosina/metabolismo , Adhesión Celular/fisiología , Citoesqueleto/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células Madre/metabolismo , Actinas , Sistemas CRISPR-Cas , Diferenciación Celular , Uniones Célula-Matriz/metabolismo , Células Madre Embrionarias , Edición Génica , Expresión Génica , Humanos , Mecanotransducción Celular , Morfogénesis , Células-Madre Neurales , Semaforinas , Transducción de SeñalRESUMEN
Emerging evidence of crosstalk between glomerular cells in pathological settings provides opportunities for novel therapeutic discovery. Here we investigated underlying mechanisms of early events leading to filtration barrier defects of podocyte and glomerular endothelial cell crosstalk in the mouse models of primary podocytopathy (podocyte specific transforming growth factor-ß receptor 1 signaling activation) or Adriamycin nephropathy. We found that glomerular endothelial surface layer degradation and albuminuria preceded podocyte foot process effacement. These abnormalities were prevented by endothelin receptor-A antagonism and mitochondrial reactive oxygen species scavenging. Additional studies confirmed increased heparanase and hyaluronoglucosaminidase gene expression in glomerular endothelial cells in response to podocyte-released factors and to endothelin-1. Atomic force microscopy measurements showed a significant reduction in the endothelial surface layer by endothelin-1 and podocyte-released factors, which could be prevented by endothelin receptor-A but not endothelin receptor-B antagonism. Thus, our studies provide evidence of early crosstalk between activated podocytes and glomerular endothelial cells resulting in loss of endothelial surface layer, glomerular endothelial cell injury and albuminuria. Hence, activation of endothelin-1-endothelin receptor-A and mitochondrial reactive oxygen species contribute to the pathogenesis of primary podocytopathies in experimental focal segmental glomerulosclerosis.
Asunto(s)
Albuminuria/patología , Comunicación Celular/efectos de los fármacos , Células Endoteliales/patología , Glomérulos Renales/patología , Receptor de Endotelina A/metabolismo , Albuminuria/inducido químicamente , Albuminuria/tratamiento farmacológico , Albuminuria/genética , Animales , Capilares/citología , Capilares/efectos de los fármacos , Capilares/patología , Capilares/ultraestructura , Modelos Animales de Enfermedad , Doxorrubicina/toxicidad , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Antagonistas de los Receptores de la Endotelina A/administración & dosificación , Antagonistas de los Receptores de la Endotelina B/administración & dosificación , Endotelina-1/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Endotelio Vascular/ultraestructura , Humanos , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/citología , Glomérulos Renales/efectos de los fármacos , Ratones , Ratones Transgénicos , Microscopía Electrónica de Rastreo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Podocitos/citología , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Podocitos/patología , Especies Reactivas de Oxígeno/metabolismo , Receptor de Endotelina B/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Nephrotoxicity is a critical adverse event that leads to discontinuation of kinase inhibitor (KI) treatment. Here we show, through meta-analyses of FDA Adverse Event Reporting System, that dasatinib is associated with high risk for glomerular toxicity that is uncoupled from hypertension, suggesting a direct link between dasatinib and podocytes. We further investigate the cellular effects of dasatinib and other comparable KIs with varying risks of nephrotoxicity. Dasatinib treated podocytes show significant changes in focal adhesions, actin cytoskeleton, and morphology that are not observed with other KIs. We use phosphoproteomics and kinome profiling to identify the molecular mechanisms of dasatinib-induced injury to the actin cytoskeleton, and atomic force microscopy to quantify impairment to cellular biomechanics. Furthermore, chronic administration of dasatinib in mice causes reversible glomerular dysfunction, loss of stress fibers, and foot process effacement. We conclude that dasatinib induces nephrotoxicity through altered podocyte actin cytoskeleton, leading to injurious cellular biomechanics.