ABSTRACT
BACKGROUND: Endothelial cells provide a barrier between blood and tissues, which is regulated to allow molecules and cells in out of tissues. Patients with cerebral cavernous malformations (CCM) have dilated leaky blood vessels, especially in the central nervous system. A subset of these patients has loss-of-function mutations in CCM3. CCM3 is part of the STRIPAK protein complex that includes the little-characterized proteins FAM40A and FAM40B. RESULTS: We show here that FAM40A and FAM40B can interact with CCM3. Knockdown of CCM3, FAM40A or FAM40B in endothelial cells by RNAi causes an increase in stress fibers and a reduction in loop formation in an in vitro angiogenesis assay, which can be reverted by inhibiting the Rho-regulated ROCK kinases. FAM40B depletion also increases endothelial permeability. CONCLUSIONS: These results demonstrate the importance of the FAM40 proteins for endothelial cell physiology, and suggest that they act as part of the CCM3-containing STRIPAK complex.
Subject(s)
Carrier Proteins/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , rho-Associated Kinases/metabolism , Animals , Apoptosis Regulatory Proteins/metabolism , COS Cells , Cell Membrane Permeability , Chlorocebus aethiops , Cytoskeletal Proteins , Humans , Membrane Proteins/metabolism , Myosin Light Chains/metabolism , Neovascularization, Physiologic , Phosphate-Binding Proteins , Phosphorylation , Proto-Oncogene Proteins/metabolism , Signal Transduction , Stress Fibers/metabolism , rho GTP-Binding Proteins/metabolismABSTRACT
Nesprins are large multi-domain proteins that link the nuclear envelope to the cytoskeleton and nucleoskeleton. Here we show that nesprin-1 and nesprin-2 play important roles in regulating cell shape and migration in endothelial cells. Nesprin-1 or nesprin-2 depletion by RNAi increased endothelial cell spread area and the length of cellular protrusions, as well as stimulating stress fibre assembly which correlated with an increase in F-actin levels. Nuclear area was also increased by nesprin depletion, and localization of the inner nuclear membrane protein emerin to the nuclear envelope was reduced. Depletion of nesprin-1 or nesprin-2 reduced migration of endothelial cells into a cell-free area, and decreased loop formation in an in vitro angiogenesis assay. Taken together, our results indicate that nesprin-1 and nesprin-2 both regulate nuclear and cytoplasmic architecture, which we propose leads to their effects on endothelial cell migration and angiogenic loop formation.
Subject(s)
Cell Movement , Cell Shape , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Microfilament Proteins/metabolism , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Actins/metabolism , Cytoskeletal Proteins , Exons/genetics , Humans , Membrane Proteins/metabolism , Neovascularization, Physiologic , Nuclear Envelope/metabolism , Protein Transport , RNA, Small Interfering/metabolismABSTRACT
The Rho GTPases are members of the Ras superfamily of GTPases that are pivotal regulators of the actin cytoskeleton. They also contribute to other cellular processes such as gene transcription, cell polarity, microtubule dynamics, cell cycle progression and vesicle trafficking. Most Rho GTPases act as molecular switches cycling between an "active" GTP-bound form and an "inactive" GDP-bound form. Hence, to elucidate the mechanisms by which Rho GTPases regulate cellular responses, an important parameter to determine is the GTP-loading of each Rho family member in cells under different conditions. Here we describe a biochemical technique to assess this based on affinity-precipitation of the GTP-bound form from whole cell lysates.
Subject(s)
Actin Cytoskeleton/metabolism , Molecular Biology/methods , Transcription, Genetic , rho GTP-Binding Proteins/isolation & purification , Cell Cycle/genetics , Guanosine Triphosphate/metabolism , Humans , rho GTP-Binding Proteins/metabolismABSTRACT
BACKGROUND: Cell migration is essential during development and in human disease progression including cancer. Most cell migration studies concentrate on known or predicted components of migration pathways. RESULTS: Here we use data from a genome-wide RNAi morphology screen in Drosophila melanogaster cells together with bioinformatics to identify 26 new regulators of morphology and cytoskeletal organization in human cells. These include genes previously implicated in a wide range of functions, from mental retardation, Down syndrome and Huntington's disease to RNA and DNA-binding genes. We classify these genes into seven groups according to phenotype and identify those that affect cell migration. We further characterize a subset of seven genes, FAM40A, FAM40B, ARC, FMNL3, FNBP3/FBP11, LIMD1 and ZRANB1, each of which has a different effect on cell shape, actin filament distribution and cell migration. Interestingly, in several instances closely related isoforms with a single Drosophila homologue have distinct phenotypes. For example, FAM40B depletion induces cell elongation and tail retraction defects, whereas FAM40A depletion reduces cell spreading. CONCLUSIONS: Our results identify multiple regulators of cell migration and cytoskeletal signalling that are highly conserved between Drosophila and humans, and show that closely related paralogues can have very different functions in these processes.
