RESUMO
Sequential transport from early to late endosomes requires the coordinated activities of the small GTPases Rab5 and Rab7. The transition between early and late endosomes could be mediated either through transport carriers or by Rab conversion, a process in which the loss of Rab5 from an endosome occurs concomitantly to the acquisition of Rab7. We demonstrate that Rab conversion is the mechanism by which proteins pass from early to late endosomes in Caenorhabditis elegans coelomocytes. Moreover, we identified SAND-1/Mon1 as the critical switch for Rab conversion in metazoa. SAND-1 serves a dual role in this process. First, it interrupts the positive feedback loop of RAB-5 activation by displacing RABX-5 from endosomal membranes; second, it times the recruitment of RAB-7, probably through interaction with the HOPS complex to the same membranes. SAND-1/Mon1 thus acts as a switch by controlling the localization of RAB-5 and RAB-7 GEFs.
Assuntos
Caenorhabditis elegans/metabolismo , Endossomos/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
The small GTPase Arf1 plays critical roles in membrane traffic by initiating the recruitment of coat proteins and by modulating the activity of lipid-modifying enzymes. Here, we report an unexpected but evolutionarily conserved role for Arf1 and the ArfGEF GBF1 at mitochondria. Loss of function of ARF-1 or GBF-1 impaired mitochondrial morphology and activity in Caenorhabditis elegans. Similarly, mitochondrial defects were observed in mammalian and yeast cells. In Saccharomyces cerevisiae, aberrant clusters of the mitofusin Fzo1 accumulated in arf1-11 mutants and were resolved by overexpression of Cdc48, an AAA-ATPase involved in ER and mitochondria-associated degradation processes. Yeast Arf1 co-fractionated with ER and mitochondrial membranes and interacted genetically with the contact site component Gem1. Furthermore, similar mitochondrial abnormalities resulted from knockdown of either GBF-1 or contact site components in worms, suggesting that the role of Arf1 in mitochondrial functioning is linked to ER-mitochondrial contacts. Thus, Arf1 is involved in mitochondrial homeostasis and dynamics, independent of its role in vesicular traffic.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Mitocôndrias/enzimologia , Saccharomyces cerevisiae/enzimologia , Fator 1 de Ribosilação do ADP/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/genética , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In humans and rodents the adult spinal cord harbors neural stem cells located around the central canal. Their identity, precise location, and specific signaling are still ill-defined and controversial. We report here on a detailed analysis of this niche. Using microdissection and glial fibrillary acidic protein (GFAP)-green fluorescent protein (GFP) transgenic mice, we demonstrate that neural stem cells are mostly dorsally located GFAP(+) cells lying ependymally and subependymally that extend radial processes toward the pial surface. The niche also harbors doublecortin protein (Dcx)(+) Nkx6.1(+) neurons sending processes into the lumen. Cervical and lumbar spinal cord neural stem cells maintain expression of specific rostro-caudal Hox gene combinations and the niche shows high levels of signaling proteins (CD15, Jagged1, Hes1, differential screening-selected gene aberrative in neuroblastoma [DAN]). More surprisingly, the niche displays mesenchymal traits such as expression of epithelial-mesenchymal-transition zinc finger E-box-binding protein 1 (ZEB1) transcription factor and smooth muscle actin. We found ZEB1 to be essential for neural stem cell survival in vitro. Proliferation within the niche progressively ceases around 13 weeks when the spinal cord reaches its final size, suggesting an active role in postnatal development. In addition to hippocampus and subventricular zone niches, adult spinal cord constitutes a third central nervous system stem cell niche with specific signaling, cellular, and structural characteristics that could possibly be manipulated to alleviate spinal cord traumatic and degenerative diseases.
Assuntos
Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Nicho de Células-Tronco/citologia , Nicho de Células-Tronco/metabolismo , Células-Tronco/citologia , Actinas/metabolismo , Animais , Proliferação de Células , Proteína Duplacortina , Regulação da Expressão Gênica no Desenvolvimento , Proteína Glial Fibrilar Ácida/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Neurônios/metabolismo , Células-Tronco/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Endoplasmic reticulum (ER)-mitochondrial contact sites play a pivotal role in exchange of lipids and ions between the two organelles. How size and function of these contact sites are regulated remains elusive. Here we report a previously unanticipated, but conserved role of the small GTPase Sar1 in the regulation of ER-mitochondrial contact site size. Activated Sar1 introduces membrane curvature through its N-terminal amphiphatic helix at the ER-mitochondria interphase and thereby reducing contact size. Conversely, the S. cerevisiae N3-Sar1 mutant, in which curvature induction is decreased, caused an increase in ER-mitochondrial contacts. As a consequence, ER tubules are no longer able to mark the prospective scission site on mitochondria, thereby impairing mitochondrial dynamics. Consistently, blocking mitochondrial fusion partially rescued, whereas deletion of the dynamin-like protein enhanced the phenotype in the sar1D32G mutant. We conclude that Sar1 regulates the size of ER-mitochondria contact sites through its effects on membrane curvature.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/genética , Mitocôndrias/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Substituição de Aminoácidos , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , Dinaminas/genética , Dinaminas/metabolismo , Retículo Endoplasmático/química , Retículo Endoplasmático/ultraestrutura , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Transporte de Íons , Metabolismo dos Lipídeos , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial , Proteínas Monoméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteínas de Transporte Vesicular/metabolismoRESUMO
Small GTPases of the Sar/Arf family are essential to generate transport containers that mediate communication between organelles of the secretory pathway. Guanine nucleotide exchange factor (GEFs) activate the small GTPases and help their anchorage in the membrane. Thus, GEFs in a way temporally and spatially control Sar1/Arf1 GTPase activation. We investigated the role of the ArfGEF GBF-1 in C. elegans oocytes and intestinal epithelial cells. GBF-1 localizes to the cis-Golgi and is part of the t-ER-Golgi elements. GBF-1 is required for secretion and Golgi integrity. In addition, gbf-1(RNAi) causes the ER reticular structure to become dispersed, without destroying ER exit sites (ERES) because the ERES protein SEC-16 was still localized in distinct punctae at t-ER-Golgi units. Moreover, GBF-1 plays a role in receptor-mediated endocytosis in oocytes, without affecting recycling pathways. We find that both the yolk receptor RME-2 and the recycling endosome-associated RAB-11 localize similarly in control and gbf-1(RNAi) oocytes. While RAB5-positive early endosomes appear to be less prominent and the RAB-5 levels are reduced by gbf-1(RNAi) in the intestine, RAB-7-positive late endosomes were more abundant and formed aggregates and tubular structures. Our data suggest a role for GBF-1 in ER structure and endosomal traffic.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Caenorhabditis elegans/genética , Células Cultivadas , Endocitose , Retículo Endoplasmático/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/citologia , Oócitos/metabolismo , Interferência de RNARESUMO
Mesenchymal stem cells (MSC) are multipotent cells found as part of the stromal compartment of the bone marrow and in many other organs. They can be identified in vitro as CFU-F (colony forming unit-fibroblast) based on their ability to form adherent colonies of fibroblast-like cells in culture. MSC expanded in vitro retain characteristics appropriate to their tissue of origin. This is reflected in their propensity for differentiating towards specific lineages, and their capacity to generate, upon retransplantation in vivo, a stroma supporting typical lineages of hematopoietic cells. Hox genes encode master regulators of regional specification and organ development in the embryo and are widely expressed in the adult. We investigated whether they could be involved in determining tissue-specific properties of MSC. Hox gene expression profiles of individual CFU-F colonies derived from various organs and anatomical locations were generated, and the relatedness between these profiles was determined using hierarchical cluster analysis. This revealed that CFU-F have characteristic Hox expression signatures that are heterogeneous but highly specific for their anatomical origin. The topographic specificity of these Hox codes is maintained during differentiation, suggesting that they are an intrinsic property of MSC. Analysis of Hox codes of CFU-F from vertebral bone marrow suggests that MSC originate over a large part of the anterioposterior axis, but may not originate from prevertebral mesenchyme. These data are consistent with a role for Hox proteins in specifying cellular identity of MSC.