RESUMO
Chemotactic and angiogenic factors secreted within the tumor microenvironment eventually facilitate the metastatic dissemination of cancer cells. Calcium-sensing receptor (CaSR) activates secretory pathways in breast cancer cells via a mechanism driven by vesicular trafficking of this receptor. However, it remains to be elucidated how endosomal proteins in secretory vesicles are controlled by CaSR. In the present study, we demonstrate that CaSR promotes expression of Rab27B and activates this secretory small GTPase via PI3K, PKA, mTOR and MADD, a guanine nucleotide exchange factor, also known as DENN/Rab3GEP. Active Rab27B leads secretion of various cytokines and chemokines, including IL-6, IL-1ß, IL-8, IP-10 and RANTES. Expression of Rab27B is stimulated by CaSR in MDA-MB-231 and MCF-7 breast epithelial cancer cells, but not in non-cancerous MCF-10A cells. This regulatory mechanism also occurs in HeLa and PC3 cells. Our findings provide insightful information regarding how CaSR activates a Rab27B-dependent mechanism to control secretion of factors known to intervene in paracrine communication circuits within the tumor microenvironment.
Assuntos
Neoplasias da Mama/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Quimiotaxia , Proteínas Quinases Dependentes de AMP Cíclico , Citocinas/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Feminino , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Fosfatidilinositol 3-Quinase , Receptores de Detecção de Cálcio/fisiologia , Via Secretória/fisiologia , Serina-Treonina Quinases TOR , Microambiente Tumoral , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
Neuronal cells are characterized by the presence of two confined domains, which are different in their cellular properties, biochemical functions and molecular identity. The generation of asymmetric domains in neurons should logically require specialized membrane trafficking to both promote neurite outgrowth and differential distribution of components. Members of the Rab family of small GTPases are key regulators of membrane trafficking involved in transport, tethering and docking of vesicles through their effectors. RabGTPases activity is coupled to the activity of guanine nucleotide exchange factors or GEFs, and GTPase-activating proteins known as GAPs. Since the overall spatiotemporal distribution of GEFs, GAPs and Rabs governs trafficking through the secretory and endocytic pathways, affecting exocytosis, endocytosis and endosome recycling, it is likely that RabGTPases could have a major role in neurite outgrowth, elongation and polarization. In this review we summarize the evidence linking the functions of several RabGTPases to axonal and dendritic development in primary neurons, as well as neurite formation in neuronal cell lines. We focused on the role of RabGTPases from the trans-Golgi network, early/late and recycling endosomes, as well as the function of some Rab effectors in neuritogenesis. Finally, we also discuss the participation of the ADP-ribosylation factor 6, a member of the ArfGTPase family, in neurite formation since it seems to have an important cross-talk with RabGTPases.
Assuntos
Neuritos/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia , Fator 6 de Ribosilação do ADP , Animais , Endossomos/fisiologia , Humanos , Transdução de Sinais/fisiologia , Rede trans-Golgi/fisiologiaRESUMO
Rab24 is an atypical member of the Rab GTPase family whose distribution in interphase cells has been characterized; however, its function remains largely unknown. In this study, we have analyzed the distribution of Rab24 throughout cell division. We have observed that Rab24 was located at the mitotic spindle in metaphase, at the midbody during telophase and in the furrow during cytokinesis. We have also observed partial co-localization of Rab24 and tubulin and demonstrated its association to microtubules. Interestingly, more than 90% of transiently transfected HeLa cells with Rab24 presented abnormal nuclear connections (i.e., chromatin bridges). Furthermore, in CHO cells stably transfected with GFP-Rab24wt, we observed a large percentage of binucleated and multinucleated cells. In addition, these cells presented an extremely large size and multiple failures in mitosis, as aberrant spindle formation (metaphase), delayed chromosomes (telophase) and multiple cytokinesis. A marked increase in binucleated, multinucleated and multilobulated nucleus formation was observed in HeLa cells depleted of Rab24. We also present evidence that a fraction of Rab24 associates with microtubules. In addition, Rab24 knock down resulted in misalignment of chromosomes and abnormal spindle formation in metaphase leading to the appearance of delayed chromosomes during late telophase and failures in cytokinesis. Our findings suggest that an adequate level of Rab24 is necessary for normal cell division. In summary, Rab24 modulates several mitotic events, including chromosome segregation and cytokinesis, perhaps through the interaction with microtubules.
Assuntos
Divisão Celular , Cromossomos/ultraestrutura , Microtúbulos/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Células CHO , Núcleo Celular/metabolismo , Cricetinae , Cricetulus , Citocinese , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Mitose , RNA Interferente Pequeno/metabolismo , Fuso Acromático , Telófase , Transfecção , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
At the phagosome level, Mycobacterium spp. alters activation and recruitment of several "Ras gene from rat brain" proteins, commonly known as Rab. Mycobacterial phagosomes have a greater and sustained expression of Rab5, Rab11, Rab14 and Rab22a, and lowered or no expression of Rab7, Rab9 and Rab6. This correlates with increased fusion of the phagosomes with early and recycling endosomes acquiring some features of early phagosomes, allowing the bacteria to gain access to nutrients and preventing the activation of anti-mycobacterial mechanisms. The expression of constitutively active mutants of Rab from the early stage endosomes prevents the maturation of phagosomes containing latex beads or heat-inactivated mycobacteria. Silencing of these mutants by interference RNA or dominant negative forms induces the maturation of mycobacterial phagosomes. The mechanisms have not been established by which mycobacteria alter the expression of these GTPases and thereby shift the phagolysosomal maturation. The problem can be explained by alterations in the recruitment of proteins that interact with Rab, such as phosphoinositide 3-kinases and early endosomal antigen 1. Identifying the mechanisms used by Mycobacterium spp. to disrupt the cycle of Rab activation will be essential to understand the pathophysiology of mycobacterial infections and usefully to potential drug targets.
Assuntos
Infecções por Mycobacterium/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Humanos , Fagossomos/fisiologiaRESUMO
A fundamental feature of eukaryotic cells is the presence of distinct membrane-bound compartments having unique protein and lipid composition. These compartments are interconnected by active trafficking mechanisms that must direct macromolecules to defined locations, and at the same time maintain the protein and lipid composition of each organelle. It is well accepted that Rab proteins play a central role in intracellular transport regulating the recognition, fusion and fission of organelles. However, how the transport is achieved is not completely understood. We propose a model whereby a soluble component in the luminal compartment is transported along different Rab-containing organelles that interact according to the following simple principles: (i) only organelles with the same or compatible Rab membrane domains can fuse; (ii) after fusion, an asymmetric fission occurs producing a tubule and a round-shaped vesicle; and (iii) Rab membrane domains distribute asymmetrically between the two resulting organelles. When this model was tested in a simulation, efficient unidirectional transport was observed, while the compartment identity was preserved. All three principles were absolutely necessary for transport. The model is compatible with Rab association/dissociation dynamics and with Rab conversion. In simulations mimicking a simplified endocytic pathway, soluble and membrane-associated markers were efficiently transported preserving the identity of the interacting compartments.
Assuntos
Membranas Intracelulares/metabolismo , Fusão de Membrana/fisiologia , Modelos Biológicos , Células Eucarióticas/metabolismo , Complexo de Golgi/metabolismo , Organelas/metabolismo , Transporte Proteico/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
Venezuelan equine encephalitis virus (VEEV) is a New World alphavirus that can cause fatal encephalitis in humans. It remains a naturally emerging disease as well as a highly developed biological weapon. VEEV is transmitted to humans in nature by mosquito vectors. Little is known about VEEV entry, especially in mosquito cells. Here, a novel luciferase-based virus entry assay is used to show that the entry of VEEV into mosquito cells requires acidification. Furthermore, mosquito homologs of key human proteins (Rab5 and Rab7) involved in endocytosis were isolated and characterized. Rab5 is found on early endosomes and Rab7 on late endosomes and both are important for VEEV entry in mammalian cells. Each was shown to have analogous function in mosquito cells to that seen in mammalian cells. The wild-type, dominant negative and constitutively active mutants were then used to demonstrate that VEEV requires passage through early and late endosomes before infection can take place. This work indicates that the infection mechanism in mosquitoes and mammals is through a common and ancient evolutionarily conserved pathway.
Assuntos
Culicidae/virologia , Vírus da Encefalite Equina Venezuelana/crescimento & desenvolvimento , Endossomos/virologia , Internalização do Vírus , Proteínas rab de Ligação ao GTP/fisiologia , Proteínas rab5 de Ligação ao GTP/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Culicidae/citologia , Endossomos/química , Genes Reporter , Humanos , Luciferases/biossíntese , Luciferases/genética , Microscopia Confocal , Dados de Sequência Molecular , Alinhamento de Sequência , Proteínas rab de Ligação ao GTP/análise , Proteínas rab de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/análise , Proteínas rab5 de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
During reticulocyte maturation, hematopoietic progenitors undergo numerous changes to reach the final functional stage which concludes with the release of reticulocytes and erythrocytes into circulation. During this process some proteins, which are not required in the mature stage, are sequestered in the internal vesicles present in multivesicular bodies (MVBs). These small vesicles are known as exosomes because they are released into the extracellular medium by fusion of the MVB with the plasma membrane. Interestingly, during this maturation process some organelles, such as mitochondria and endoplasmic reticulum, are wrapped in double membrane vacuoles and degraded via autophagy. We have demonstrated in human leukemic K562 cells a role for calcium and Rab11 in the biogenesis of MVBs and exosome release. Here we discuss evidence indicating that K562 cells present a high basal level of autophagy, and that there is an association between MVBs and autophagosomes, suggesting a role for the autophagic pathway in the maturation process of this cell type.
Assuntos
Autofagia/fisiologia , Eritrócitos/fisiologia , Eritropoese/fisiologia , Organelas/fisiologia , Animais , Células CHO , Cálcio/fisiologia , Membrana Celular/fisiologia , Cricetinae , Cricetulus , Retículo Endoplasmático/fisiologia , Endossomos/fisiologia , Humanos , Células K562 , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/fisiologia , Reticulócitos/fisiologia , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
During reticulocyte maturation, some membrane proteins and organelles that are not required in the mature red cell are lost. These proteins are released into the extracellular medium associated with vesicles present in multivesicular bodies (MVBs). Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles, termed exosomes. By studying MVBs fusion and exosome release in K562 cells, a human erythroleukemic cell line, we have determined the functional significance of Rab11 and calcium in these events. Additionally, in the transformation process that occurs during erythrocyte maturation, intracellular organelles are likely removed as a consequence of autophagic sequestration and degradation. We propose K562 cells as a useful tool to analyze, at the molecular level, the role of autophagy in the terminal differentiation of red cells.
Assuntos
Endossomos/metabolismo , Eritrócitos/citologia , Fusão de Membrana , Autofagia , Cálcio/fisiologia , Diferenciação Celular , Endossomos/fisiologia , Eritrócitos/ultraestrutura , Exocitose , Humanos , Células K562 , Reticulócitos/citologia , Reticulócitos/ultraestrutura , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
We analyzed the expression and location of EhRabB in clone L-6, a phagocytosis-deficient mutant of Entamoeba histolytica, in comparison with the wild-type clone A. Intriguingly, trophozoites of clone L-6 express more EhRabB than those of clone A. However, the majority of EhRabB-containing vesicles remained in the cytoplasm of clone L-6 during phagocytosis. To investigate molecular alterations in EhRabB of clone L-6 we compared the EhrabB gene sequences from clones L-6 and A. We also isolated, sequenced and compared the RabB protein of Entamoeba dispar. Results showed that EhrabB gene of clone L-6 is 98.2 and 94.1% identical to rabB genes of E. dispar and clone A, respectively. The rabB genes from clone A and E. dispar have 92.2% identity. Four out of five amino acids changes in RabB proteins of clone L-6 and E. dispar are shared. These changes may alter the binding of effector proteins and the specific subcellular location of EhRabB.
Assuntos
Entamoeba histolytica/química , Fagocitose/fisiologia , Proteínas rab de Ligação ao GTP/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , DNA Complementar/química , DNA de Protozoário/química , Densitometria , Eletroforese em Gel de Poliacrilamida , Entamoeba/química , Entamoeba/genética , Entamoeba/imunologia , Entamoeba histolytica/genética , Entamoeba histolytica/imunologia , Dados de Sequência Molecular , Mutação , Fagocitose/genética , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
During invasion of nonphagocytic cells by Trypanosoma cruzi (T. cruzi), host cell lysosomes are recruited to the plasma membrane attachment site followed by lysosomal enzyme secretion. The membrane trafficking events involved in invasion have not been delineated. We demonstrate here that T. cruzi invasion of nonphagocytic cells was completely abolished by overexpression of a dominant negative mutant of dynamin. Likewise, overexpression of a dominant negative mutant of Rab5, the rate-limiting GTPase for endocytosis, resulted in reduced infection rates compared with cells expressing Rab5 wild-type. Moreover, cells expressing the activated mutant of Rab5 experienced higher infection rates. A similar pattern was also observed when Rab7-transfected cells were examined. Confocal microscopy experiments showed that parasites colocalized with green fluorescent protein-Rab5-positive early endosomes after 5 min of invasion. These data clearly indicate that newly forming T. cruzi phagosomes first interact with an early endosomal compartment and subsequently with other late component markers before lysosomal interaction occurs.