RESUMO
The topography of biological membranes is critical for formation of protein and lipid microdomains. One prominent example in the yeast plasma membrane (PM) are BAR domain-induced PM furrows. Here we report a novel function for the Sur7 family of tetraspanner proteins in the regulation of local PM topography. Combining TIRF imaging, STED nanoscopy, freeze-fracture EM and membrane simulations we find that Sur7 tetraspanners form multimeric strands at the edges of PM furrows, where they modulate forces exerted by BAR domain proteins at the furrow base. Loss of Sur7 tetraspanners or Sur7 displacement due to altered PIP2 homeostasis leads to increased PM invagination and a distinct form of membrane tubulation. Physiological defects associated with PM tubulation are rescued by synthetic anchoring of Sur7 to furrows. Our findings suggest a key role for tetraspanner proteins in sculpting local membrane domains. The maintenance of stable PM furrows depends on a balance between negative curvature at the base which is generated by BAR domains and positive curvature at the furrows' edges which is stabilized by strands of Sur7 tetraspanners.
Assuntos
Proteínas , Membrana Celular/metabolismo , Proteínas/metabolismoRESUMO
BACKGROUND: Monogenic diseases provide favorable opportunities to elucidate the molecular mechanisms of disease progression and improve medical diagnostics. However, the complex interplay between genetic and environmental factors in disease etiologies makes it difficult to discern the mechanistic links between different alleles of a single locus and their associated pathophysiologies. Inverted formin 2 (INF2), an actin regulator, mediates a stress response-calcium mediated actin reset, or CaAR-that reorganizes the actin cytoskeleton of mammalian cells in response to calcium influx. It has been linked to the podocytic kidney disease focal segemental glomerulosclerosis (FSGS), as well as to cases of the neurologic disorder Charcot-Marie-Tooth disease that are accompanied by nephropathy, mostly FSGS. METHODS: We used a combination of quantitative live cell imaging and validation in primary patient cells and Drosophila nephrocytes to systematically characterize a large panel of >50 autosomal dominant INF2 mutants that have been reported to cause either FSGS alone or with Charcot-Marie-Tooth disease. RESULTS: We found that INF2 mutations lead to deregulated activation of formin and a constitutive stress response in cultured cells, primary patient cells, and Drosophila nephrocytes. We were able to clearly distinguish between INF2 mutations that were linked exclusively to FSGS from those that caused a combination of FSGS and Charcot-Marie-Tooth disease. Furthermore, we were able to identify distinct subsets of INF2 variants that exhibit varying degrees of activation. CONCLUSIONS: Our results suggest that CaAR can be used as a sensitive assay for INF2 function and for robust evaluation of diseased-linked variants of formin. More broadly, these findings indicate that cellular profiling of disease-associated mutations has potential to contribute substantially to sequence-based phenotype predictions.
Assuntos
Doença de Charcot-Marie-Tooth/etiologia , Forminas/genética , Glomerulosclerose Segmentar e Focal/etiologia , Mutação , Animais , Cálcio/metabolismo , Drosophila , Feminino , Forminas/fisiologia , Células HeLa , Humanos , Masculino , Camundongos , Estresse FisiológicoRESUMO
Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress.
Assuntos
Actinas/metabolismo , Cálcio/metabolismo , Fenômenos Fisiológicos Celulares , Citoesqueleto de Actina/metabolismo , Adaptação Fisiológica , Animais , Linhagem Celular , HumanosRESUMO
Myeloid-related protein 8 (MRP8) and MRP14, S100 proteins secreted by activated phagocytes, bind specifically to endothelial cells. The endothelial response to MRP8/MRP14, however, is unknown. Using oligonucleotide microarray analysis, we show for the first time that MRP8/MRP14 induce a thrombogenic, inflammatory response in human microvascular endothelial cells by increasing the transcription of proinflammatory chemokines and adhesion molecules and by decreasing the expression of cell junction proteins and molecules involved in monolayer integrity. All changes on the gene expression level could be confirmed using biochemical and functional assays. We demonstrated that the expression of MRP8/MRP14 closely correlated with the inflammatory activity in systemic vasculitis, confirming the important role of these proteins for distinct inflammatory reactions in endothelia. MRP8/MRP14 may represent novel targets for anti-inflammatory strategies.
Assuntos
Calgranulina A/imunologia , Calgranulina B/imunologia , Células Endoteliais/imunologia , Vasculite/imunologia , Vasculite/fisiopatologia , Calgranulina A/genética , Calgranulina B/genética , Calgranulina B/farmacologia , Capilares/citologia , Capilares/imunologia , Permeabilidade Capilar/imunologia , Células Cultivadas , Células Endoteliais/citologia , Expressão Gênica/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/genética , Junções Intercelulares/imunologia , Interleucina-8/genética , Análise de Sequência com Séries de Oligonucleotídeos , Trombose/imunologiaRESUMO
Endothelial cell-cell contacts control the vascular permeability, thereby regulating the flow of solutes, macromolecules, and leukocytes between blood vessels and interstitial space. Because of specific needs, the endothelial permeability differs significantly between the tight blood-brain barrier endothelium and the more permeable endothelial lining of the non-brain microvasculature. Most likely, such differences are due to a differing architecture of the respective interendothelial cell contacts. However, while the molecules and junctional complexes of macrovascular endothelial cells and the blood-brain barrier endothelium are fairly well characterized, much less is known about the organization of intercellular contacts of microvascular endothelium. Toward this end, we developed a combined cross-linking and immunoprecipitation protocol which enabled us to map nearest neighbor interactions of junctional proteins in the human dermal microvascular endothelial cell line HMEC-1. We show that proteins typically located in tight or adherens junctions of epithelial cells are in the proximity in HMEC-1 cells. This contrasts with the separation of the different types of junctions observed in polarized epithelial cells and "tight" endothelial layers of the blood-brain barrier and argues for a need of the specific junctional contacts in microvascular endothelium possibly required to support an efficient transendothelial migration of leukocytes.
Assuntos
Junções Aderentes/química , Moléculas de Adesão Celular/metabolismo , Comunicação Celular/fisiologia , Endotélio Vascular/química , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Pele/química , Junções Íntimas/química , Junções Aderentes/metabolismo , Animais , Antígenos CD , Encéfalo/citologia , Encéfalo/metabolismo , Caderinas , Linhagem Celular , Reagentes de Ligações Cruzadas/metabolismo , Cães , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Microcirculação/química , Microcirculação/citologia , Microcirculação/metabolismo , Testes de Precipitina , Pele/irrigação sanguínea , Pele/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1RESUMO
Bacterial toxins affecting Rho activity in microvascular endothelial cells were employed to elucidate whether endothelial Rho participates in regulating the migration of monocytes across monolayers of cultured endothelial cells. Inactivation of Rho by the Clostridium C3 exoenzyme resulted in an increased adhesion of peripheral blood monocytes to the endothelium and a decreased rate of transendothelial monocyte migration. Cytotoxic necrotizing factor 1-mediated activation of endothelial Rho also reduced the rate of monocyte transmigration, but did not affect monocyte-endothelium adhesion. Thus, efficient leukocyte extravasation requires Rho signaling not only within the migrating leukocytes but also within the endothelial lining of the vessel wall.