RESUMO
Despite recent advances in targeted and immune-based therapies, advanced stage melanoma remains a clinical challenge with a poor prognosis. Understanding the genes and cellular processes that drive progression and metastasis is critical for identifying new therapeutic strategies. Here, we found that the GTPase RAB27A was overexpressed in a subset of melanomas, which correlated with poor patient survival. Loss of RAB27A expression in melanoma cell lines inhibited 3D spheroid invasion and cell motility in vitro, and spontaneous metastasis in vivo. The reduced invasion phenotype was rescued by RAB27A-replete exosomes, but not RAB27A-knockdown exosomes, indicating that RAB27A is responsible for the generation of pro-invasive exosomes. Furthermore, while RAB27A loss did not alter the number of exosomes secreted, it did change exosome size and altered the composition and abundance of exosomal proteins, some of which are known to regulate cancer cell movement. Our data suggest that RAB27A promotes the biogenesis of a distinct pro-invasive exosome population. These findings support RAB27A as a key cancer regulator, as well as a potential prognostic marker and therapeutic target in melanoma.
Assuntos
Exossomos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Proteínas rab27 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Meios de Cultivo Condicionados , Exossomos/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Melanoma/genética , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanossomas/genética , Melanossomas/metabolismo , Camundongos , Invasividade Neoplásica , Nevo/genética , Nevo/metabolismo , Proteômica , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Esferoides Celulares , Proteínas rab27 de Ligação ao GTP/biossíntese , Proteínas rab27 de Ligação ao GTP/genéticaRESUMO
Inflammasomes mediate inflammatory and cell death responses to pathogens and cellular stress signals via activation of procaspases-1 and -8. During inflammasome assembly, activated receptors of the NLR or PYHIN family recruit the adaptor protein ASC and initiate polymerization of its pyrin domain (PYD) into filaments. We show that ASC filaments in turn nucleate procaspase-8 death effector domain (DED) filaments in vitro and in vivo. Interaction between ASC PYD and procaspase-8 tandem DEDs optimally required both DEDs and represents an unusual heterotypic interaction between domains of the death fold superfamily. Analysis of ASC PYD mutants showed that interaction surfaces that mediate procaspase-8 interaction overlap with those required for ASC self-association and interaction with the PYDs of inflammasome initiators. Our data indicate that multiple types of death fold domain filaments form at inflammasomes and that PYD/DED and homotypic PYD interaction modes are similar. Interestingly, we observed condensation of procaspase-8 filaments containing the catalytic domain, suggesting that procaspase-8 interactions within and/or between filaments may be involved in caspase-8 activation. Procaspase-8 filaments may also be relevant to apoptosis induced by death receptors.
Assuntos
Caspase 8/metabolismo , Proteínas do Citoesqueleto/metabolismo , Inflamassomos/metabolismo , Apoptose , Proteínas Adaptadoras de Sinalização CARD , Caspase 1/metabolismo , Domínio Catalítico , Morte Celular , Células HEK293 , Humanos , Inflamação , Microscopia de Fluorescência , Mutação , Ligação Proteica , Transdução de SinaisRESUMO
Upon infection, Legionella pneumophila uses the Dot/Icm type IV secretion system to translocate effector proteins from the Legionella-containing vacuole (LCV) into the host cell cytoplasm. The effectors target a wide array of host cellular processes that aid LCV biogenesis, including the manipulation of membrane trafficking. In this study, we used a hidden Markov model screen to identify two novel, non-eukaryotic soluble NSF attachment protein receptor (SNARE) homologs: the bacterial Legionellaâ SNARE effector A (LseA) and viral SNARE homolog A proteins. We characterized LseA as a Dot/Icm effector of L. pneumophila, which has close homology to the Qc-SNARE subfamily. The lseA gene was present in multiple sequenced L. pneumophila strains including Corby and was well distributed among L. pneumophila clinical and environmental isolates. Employing a variety of biochemical, cell biological and microbiological techniques, we found that farnesylated LseA localized to membranes associated with the Golgi complex in mammalian cells and LseA interacted with a subset of Qa-, Qb- and R-SNAREs in host cells. Our results suggested that LseA acts as a SNARE protein and has the potential to regulate or mediate membrane fusion events in Golgi-associated pathways.
Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Legionella pneumophila/fisiologia , Mimetismo Molecular , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Fatores de Virulência/metabolismo , Animais , Linhagem Celular , Células Epiteliais/microbiologia , Humanos , Macrófagos/microbiologia , Camundongos , Homologia de Sequência de AminoácidosRESUMO
3-phosphorylated phosphoinositides (3-PtdIns) orchestrate endocytic trafficking pathways exploited by intracellular pathogens such as Salmonella to gain entry into the cell. To infect the host, Salmonellae subvert its normal macropinocytic activity, manipulating the process to generate an intracellular replicative niche. Disruption of the PtdIns(5) kinase, PIKfyve, be it by interfering mutant, siRNA-mediated knockdown or pharmacological means, inhibits the intracellular replication of Salmonella enterica serovar typhimurium in epithelial cells. Monitoring the dynamics of macropinocytosis by time-lapse 3D (4D) videomicroscopy revealed a new and essential role for PI(3,5)P(2) in macropinosome-late endosome/lysosome fusion, which is distinct from that of the small GTPase Rab7. This PI(3,5)P(2)-dependent step is required for the proper maturation of the Salmonella-containing vacuole (SCV) through the formation of Salmonella-induced filaments (SIFs) and for the engagement of the Salmonella pathogenicity island 2-encoded type 3 secretion system (SPI2-T3SS). Finally, although inhibition of PIKfyve in macrophages did inhibit Salmonella replication, it also appears to disrupt the macrophage's bactericidal response.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Salmonella typhimurium/patogenicidade , Aminopiridinas/farmacologia , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular , Endocitose , Endossomos/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Lisossomos/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/genética , Pinocitose , Interferência de RNA , Salmonella typhimurium/crescimento & desenvolvimento , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Plasmodium spp. parasites cause malaria in 300 to 500 million individuals each year. Disease occurs during the blood-stage of the parasite's life cycle, where the parasite is thought to replicate exclusively within erythrocytes. Infected individuals can also suffer relapses after several years, from Plasmodium vivax and Plasmodium ovale surviving in hepatocytes. Plasmodium falciparum and Plasmodium malariae can also persist after the original bout of infection has apparently cleared in the blood, suggesting that host cells other than erythrocytes (but not hepatocytes) may harbor these blood-stage parasites, thereby assisting their escape from host immunity. Using blood stage transgenic Plasmodium berghei-expressing GFP (PbGFP) to track parasites in host cells, we found that the parasite had a tropism for CD317(+) dendritic cells. Other studies using confocal microscopy, in vitro cultures, and cell transfer studies showed that blood-stage parasites could infect, survive, and replicate within CD317(+) dendritic cells, and that small numbers of these cells released parasites infectious for erythrocytes in vivo. These data have identified a unique survival strategy for blood-stage Plasmodium, which has significant implications for understanding the escape of Plasmodium spp. from immune-surveillance and for vaccine development.
Assuntos
Células Dendríticas/parasitologia , Malária/parasitologia , Plasmodium/crescimento & desenvolvimento , Plasmodium/patogenicidade , Animais , Animais Geneticamente Modificados , Antígenos CD/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/ultraestrutura , Eritrócitos/parasitologia , Feminino , Proteínas de Fluorescência Verde/genética , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Plasmodium/imunologia , Plasmodium berghei/genética , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/patogenicidade , Plasmodium chabaudi/patogenicidade , Plasmodium yoelii/patogenicidade , Proteínas Recombinantes/genética , VirulênciaRESUMO
Rab GTPases including Rab27a, Rab38 and Rab32 function in melanosome maturation or trafficking in melanocytes. A screen to identify additional Rabs involved in these processes revealed the localization of GFP-Rab17 on recycling endosomes (REs) and melanosomes in melanocytic cells. Rab17 mRNA expression is regulated by microphthalmia transcription factor (MITF), a characteristic of known pigmentation genes. Rab17 siRNA knockdown in melanoma cells quantitatively increased melanosome concentration at the cell periphery. Rab17 knockdown did not inhibit melanosome maturation nor movement, but it caused accumulation of melanin inside cells. Double knockdown of Rab17 and Rab27a indicated that Rab17 acts on melanosomes downstream of Rab27a. Filopodia are known to play a role in melanosome transfer, and in Rab17 knockdown cells filopodia formation was inhibited. Furthermore, we show that stimulation of melanoma cells with α-melanocyte-stimulating hormone induces filopodia formation, supporting a role for filopodia in melanosome release. Cell stimulation also caused redistribution of REs to the periphery, and knockdown of additional RE-associated Rabs 11a and 11b produced a similar accumulation of melanosomes and melanin to that seen after loss of Rab17. Our findings reveal new functions for RE and Rab17 in pigmentation through a distal step in the process of melanosome release via filopodia.
Assuntos
Endossomos/metabolismo , Melanócitos/citologia , Melanócitos/metabolismo , Melanossomas/metabolismo , Pseudópodes/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Melaninas/metabolismo , Melanócitos/efeitos dos fármacos , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Pseudópodes/ultraestrutura , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , alfa-MSH/farmacologia , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Activated macrophages secrete an array of proinflammatory cytokines, including tumor necrosis factor-alpha (TNFalpha) and interleukin 6 (IL-6), that are temporally secreted for sequential roles in inflammation. We have previously characterized aspects of the intracellular trafficking of membrane-bound TNFalpha and its delivery to the cell surface at the site of phagocytic cups for secretion (Murray, R.Z., J.G. Kay, D.G. Sangermani, and J.L. Stow. 2005. Science. 310:1492-1495). The trafficking pathway and surface delivery of IL-6, a soluble cytokine, were studied here using approaches such as live cell imaging of fluorescently tagged IL-6 and immunoelectron microscopy. Newly synthesized IL-6 accumulates in the Golgi complex and exits in tubulovesicular carriers either as the sole labeled cargo or together with TNFalpha, utilizing specific soluble NSF attachment protein receptor (SNARE) proteins to fuse with the recycling endosome. Within recycling endosomes, we demonstrate the compartmentalization of cargo proteins, wherein IL-6 is dynamically segregated from TNFalpha and from surface recycling transferrin. Thereafter, these cytokines are independently secreted, with TNFalpha delivered to phagocytic cups but not IL-6. Therefore, the recycling endosome has a central role in orchestrating the differential secretion of cytokines during inflammation.
Assuntos
Endossomos/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , DNA Complementar/genética , Eletroporação , Endossomos/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Corantes Fluorescentes/metabolismo , Interleucina-6/análise , Macrófagos/ultraestrutura , Camundongos , Microscopia de Fluorescência , RNA Interferente Pequeno/farmacologia , Rodaminas/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/ultraestruturaRESUMO
NK cells are renowned for their ability to kill virally infected or transformed host cells by release of cytotoxic granules containing granzymes and perforin. NK cells also have important regulatory capabilities chiefly mediated by secretion of cytokines, such as IFN-gamma and TNF. The secretory pathway for the release of cytokines in NK cells is unknown. In this study, we show localization and trafficking of IFN-gamma and TNF in human NK cells in compartments and vesicles that do not overlap with perforin or other late endosome granule markers. Cytokines in post-Golgi compartments colocalized with markers of the recycling endosome (RE). REs are functionally required for cytokine release because inactivation of REs or mutation of RE-associated proteins Rab11 and vesicle-associated membrane protein-3 blocked cytokine surface delivery and release. In contrast, REs are not needed for release of perforin from preformed granules but may be involved at earlier stages of granule maturation. These findings suggest a new role for REs in orchestrating secretion in NK cells. We show that the cytokines IFN-gamma and TNF are trafficked and secreted via a different pathway than perforin. Although perforin granules are released in a polarized fashion at lytic synapses, distinct carriers transport both IFN-gamma and TNF to points all over the cell surface, including within the synapse, for nonpolarized release.
Assuntos
Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/metabolismo , Testes Imunológicos de Citotoxicidade , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Perforina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Compartimento Celular/imunologia , Membrana Celular/imunologia , Membrana Celular/metabolismo , Polaridade Celular/imunologia , Células Cultivadas , Testes Imunológicos de Citotoxicidade/métodos , Endossomos/imunologia , Endossomos/metabolismo , Humanos , Sinapses Imunológicas/metabolismo , Interferon gama/biossíntese , Células K562 , Ativação Linfocitária/imunologia , Perforina/biossíntese , Transporte Proteico/imunologia , Vesículas Secretórias/imunologia , Vesículas Secretórias/metabolismo , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.
Assuntos
Endossomos , Pinocitose , Membrana Celular , Microscopia Eletrônica , VacúolosRESUMO
Stinging trees from Australasia produce remarkably persistent and painful stings upon contact of their stiff epidermal hairs, called trichomes, with mammalian skin. Dendrocnide-induced acute pain typically lasts for several hours, and intermittent painful flares can persist for days and weeks. Pharmacological activity has been attributed to small-molecule neurotransmitters and inflammatory mediators, but these compounds alone cannot explain the observed sensory effects. We show here that the venoms of Australian Dendrocnide species contain heretofore unknown pain-inducing peptides that potently activate mouse sensory neurons and delay inactivation of voltage-gated sodium channels. These neurotoxins localize specifically to the stinging hairs and are miniproteins of 4 kDa, whose 3D structure is stabilized in an inhibitory cystine knot motif, a characteristic shared with neurotoxins found in spider and cone snail venoms. Our results provide an intriguing example of inter-kingdom convergent evolution of animal and plant venoms with shared modes of delivery, molecular structure, and pharmacology.
RESUMO
Cerebellar ataxias are severe neurodegenerative disorders with an early onset and progressive and inexorable course of the disease. Here, we report a single point mutation in the gene encoding Elongator complex subunit 6 causing Purkinje neuron degeneration and an ataxia-like phenotype in the mutant wobbly mouse. This mutation destabilizes the complex and compromises its function in translation regulation, leading to protein misfolding, proteotoxic stress, and eventual neuronal death. In addition, we show that substantial microgliosis is triggered by the NLRP3 inflammasome pathway in the cerebellum and that blocking NLRP3 function in vivo significantly delays neuronal degeneration and the onset of ataxia in mutant animals. Our data provide a mechanistic insight into the pathophysiology of a cerebellar ataxia caused by an Elongator mutation, substantiating the increasing body of evidence that alterations of this complex are broadly implicated in the onset of a number of diverse neurological disorders.
Assuntos
Ataxia/genética , Comportamento Animal , Histona Acetiltransferases/genética , Mutação/genética , Degeneração Neural/genética , Animais , Ataxia/complicações , Sequência de Bases , Caspase 1/metabolismo , Feminino , Furanos , Gliose/patologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Histona Acetiltransferases/metabolismo , Indenos , Inflamassomos/metabolismo , Inflamação/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/patologia , Modelos Moleculares , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Degeneração Neural/complicações , Fenótipo , Agregados Proteicos/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Células de Purkinje/patologia , Sulfonamidas , Sulfonas/farmacologia , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
IL-1ß requires processing by caspase-1 to generate the active, pro-inflammatory cytokine. Acute IL-1ß secretion from inflammasome-activated macrophages requires caspase-1-dependent GSDMD cleavage, which also induces pyroptosis. Mechanisms of IL-1ß secretion by pyroptotic and non-pyroptotic cells, and the precise functions of caspase-1 and GSDMD therein, are unresolved. Here, we show that, while efficient early secretion of endogenous IL-1ß from primary non-pyroptotic myeloid cells in vitro requires GSDMD, later IL-1ß release in vitro and in vivo proceeds independently of GSDMD. IL-1ß maturation is sufficient for slow, caspase-1/GSDMD-independent secretion of ectopic IL-1ß from resting, non-pyroptotic macrophages, but the speed of IL-1ß release is boosted by inflammasome activation, via caspase-1 and GSDMD. IL-1ß cleavage induces IL-1ß enrichment at PIP2-enriched plasma membrane ruffles, and this is a prerequisite for IL-1ß secretion and is mediated by a polybasic motif within the cytokine. We thus reveal a mechanism in which maturation-induced IL-1ß trafficking facilitates its unconventional secretion.
Assuntos
Membrana Celular/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Humanos , TransfecçãoRESUMO
The cyclotides are macrocyclic knotted proteins characterized by a compact topology and exceptional stability. Accordingly it has been hypothesized that they may be useful as protein engineering frameworks for the stabilization and delivery of bioactive peptide sequences. This study examined the internalization of cyclotides into mammalian cells, a vital step for the delivery of bioactive peptide sequences to intracellular targets. Although the entry of various linear peptides into cells has been reported previously, this is the first report of internalization of a macrocyclic peptide. Cell uptake was examined for representatives of two cyclotide subfamilies; the first was MCoTI-II, a member of the trypsin inhibitor subfamily, which was internalized by a macrophage and breast cancer cell line and the second, the prototypic cyclotide kalata B1 from the Möbius subfamily, which remained extracellular. Biotin labeled MCoTI-II entered macrophages by macropinocytosis, resulting in vesicular encapsulation without trafficking to lysosomes for degradation. The ready uptake, coupled with low cytotoxicity, indicates that MCoTI-II has the potential to transport grafted bioactivities to intracellular targets, making it a potentially valuable framework in drug design applications.
Assuntos
Ciclotídeos/metabolismo , Macrófagos/metabolismo , Pinocitose/fisiologia , Sequência de Aminoácidos , Animais , Biotinilação , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ciclotídeos/química , Ciclotídeos/farmacologia , Dextranos/metabolismo , Endossomos/química , Endossomos/metabolismo , Humanos , Proteínas de Membrana Lisossomal/análise , Lisossomos/química , Lisossomos/metabolismo , Macrófagos/citologia , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/análise , Microscopia de Fluorescência , Dados de Sequência Molecular , Momordica/química , Rodaminas/metabolismo , Sementes/química , Homologia de Sequência de Aminoácidos , Temperatura , Proteínas de Transporte Vesicular/análiseRESUMO
A growing body of evidence suggests that the Golgi complex contains an actin-based filament system. We have previously reported that one or more isoforms from the tropomyosin gene Tm5NM (also known as gamma-Tm), but not from either the alpha- or beta-Tm genes, are associated with Golgi-derived vesicles (Heimann et al., (1999). J. Biol. Chem. 274, 10743-10750). We now show that Tm5NM-2 is sorted specifically to the Golgi complex, whereas Tm5NM-1, which differs by a single alternatively spliced internal exon, is incorporated into stress fibers. Tm5NM-2 is localized to the Golgi complex consistently throughout the G1 phase of the cell cycle and it associates with Golgi membranes in a brefeldin A-sensitive and cytochalasin D-resistant manner. An actin antibody, which preferentially reacts with the ends of microfilaments, newly reveals a population of short actin filaments associated with the Golgi complex and particularly with Golgi-derived vesicles. Tm5NM-2 is also found on these short microfilaments. We conclude that an alternative splice choice can restrict the sorting of a tropomyosin isoform to short actin filaments associated with Golgi-derived vesicles. Our evidence points to a role for these Golgi-associated microfilaments in vesicle budding at the level of the Golgi complex.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo de Golgi/metabolismo , Fibras de Estresse/metabolismo , Tropomiosina/metabolismo , Actinas/metabolismo , Processamento Alternativo/genética , Animais , Brefeldina A/farmacologia , Citocalasina D/farmacologia , Vesículas Citoplasmáticas/metabolismo , Fase G1 , Camundongos , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Células NIH 3T3 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico/fisiologia , Tropomiosina/genéticaRESUMO
For the past 30 years, oocytes from Xenopus laevis have been extensively used to express and characterise ion channels in an easily controlled environment. Here we report the first use of oocytes from the closely related species Xenopus borealis as an alternative expression system for neuronal ion channels. Using the two-electrode voltage-clamp technique, we show that a wide variety of voltage- and ligand-gated ion channels have the same channel properties and pharmacological profiles when expressed in either X. laevis or X. borealis oocytes. Potential advantages of the X. borealis oocytes include a smaller endogenous chloride current and the ability to produce more intense fluorescence signals when studied with voltage-clamp fluorometry. Scanning electron microscopy revealed a difference in vitelline membrane structure between the two species, which may be related to the discrepancy in fluorescence signals observed. We demonstrate that X. borealis oocytes are a viable heterologous system for expression of neuronal ion channels with some potential advantages over X. laevis oocytes for certain applications.
Assuntos
Canais Iônicos/metabolismo , Neurônios/fisiologia , Oócitos/fisiologia , Xenopus , Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Canais Iônicos Sensíveis a Ácido/metabolismo , Animais , Feminino , Microscopia Eletrônica de Varredura , Neurônios/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Técnicas de Patch-Clamp , Membrana Vitelina/ultraestrutura , Xenopus laevisRESUMO
We have compared the melanogenic activities of cultured melanocytes carrying two common TYR alleles as homozygous 192S-402R wild-type, heterozygous and homozygous variant. This includes assays of TYR protein, DOPAoxidase activity, glycosylation and temperature sensitivity of protein and DOPAoxidase levels. Homozygous wild-type strains on average had higher levels of TYR protein and enzyme activity than other genotypes. Homozygous 402Q/Q melanocytes produced significantly less TYR protein, displayed altered trafficking and glycosylation, with reduced DOPAoxidase. However, near wild-type TYR activity levels could be recovered at lower growth temperature. In a sample population from Southeast Queensland, these two polymorphisms were present on four TYR haplotypes, designated as WT 192S-402R, 192Y-402R and 192S-402Q with a double-variant 192Y-402Q of low frequency at 1.9%. Based on cell culture findings and haplotype associations, we have used an additive model to assess the penetrance of the ten possible TYR genotypes derived from the combination of these haplotypes.
Assuntos
Alelos , Haplótipos , Melanócitos/enzimologia , Monofenol Mono-Oxigenase , Polimorfismo Genético , Locos de Características Quantitativas , Pigmentação da Pele/genética , Humanos , Masculino , Modelos Genéticos , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Penetrância , QueenslandRESUMO
Single nucleotide polymorphisms (SNPs) within the SLC45A2/MATP, SLC24A5/NCKX5, and OCA2/P genes have been associated with natural variation of pigmentation traits in human populations. Here, we describe the characterization of human primary melanocytic cells genotyped for polymorphisms within the MATP, NCKX5, or OCA2 loci. On the basis of genotype, these cultured cells reflect the phenotypes observed by others in terms of both melanin content and tyrosinase (TYR) activity when comparing skin designated as either "White" or "Black". We found a statistically significant association of MATP-374L (darker skin) with higher TYR protein abundance that was not observed for any NCKX5-111 or OCA2 rs12913832 allele. MATP-374L/L homozygous strains displayed significantly lower MATP transcript levels compared to MATP-374F/F homozygous cells, but this did not reach statistical significance based on NCKX5 or OCA2 genotype. Similarly, we observed significantly increased levels of OCA2 mRNA in rs12913832-T (brown eye) homozygotes compared to rs12913832-C (blue eye) homozygous strains, which was not observed for MATP or NCKX5 gene transcripts. In genotype-phenotype associations performed on a collection of 226 southern European individuals using these same SNPs, we were able to show strong correlations in MATP-L374F, OCA2, and melanocortin-1 receptor with skin, eye, and hair color variation, respectively.
Assuntos
Antígenos de Neoplasias/genética , Antiporters/genética , Melanócitos/fisiologia , Proteínas de Membrana Transportadoras/genética , Pigmentação da Pele/genética , Células Cultivadas , Cor de Olho/genética , Regulação da Expressão Gênica/fisiologia , Genótipo , Cor de Cabelo/genética , Humanos , Melaninas/metabolismo , Melanócitos/citologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único/fisiologia , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , Receptor Tipo 1 de Melanocortina/genética , Pele/citologia , População Branca/genéticaRESUMO
The GRIP domain is a targeting sequence found in a family of coiled-coil peripheral Golgi proteins. Previously we demonstrated that the GRIP domain of p230/golgin245 is specifically recruited to tubulovesicular structures of the trans-Golgi network (TGN). Here we have characterized two novel Golgi proteins with functional GRIP domains, designated GCC88 and GCC185. GCC88 cDNA encodes a protein of 88 kDa, and GCC185 cDNA encodes a protein of 185 kDa. Both molecules are brefeldin A-sensitive peripheral membrane proteins and are predicted to have extensive coiled-coil regions with the GRIP domain at the C terminus. By immunofluorescence and immunoelectron microscopy GCC88 and GCC185, and the GRIP protein golgin97, are all localized to the TGN of HeLa cells. Overexpression of full-length GCC88 leads to the formation of large electron dense structures that extend from the trans-Golgi. These de novo structures contain GCC88 and co-stain for the TGN markers syntaxin 6 and TGN38 but not for alpha2,6-sialyltransferase, beta-COP, or cis-Golgi GM130. The formation of these abnormal structures requires the N-terminal domain of GCC88. TGN38, which recycles between the TGN and plasma membrane, was transported into and out of the GCC88 decorated structures. These data introduce two new GRIP domain proteins and implicate a role for GCC88 in the organization of a specific TGN subcompartment involved with membrane transport.
Assuntos
Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Compartimento Celular , Clonagem Molecular , Primers do DNA , Proteínas da Matriz do Complexo de Golgi , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans-Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Rede trans-Golgi/fisiologia , Rede trans-Golgi/ultraestrutura , Animais , Fracionamento Celular , Proteínas Ativadoras de GTPase/genética , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Vídeo , Mutagênese , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Deleção de SequênciaRESUMO
N4WBP5A (Ndfip2) belongs to an evolutionarily conserved group of Nedd4-interacting proteins with two homologues in mammalian species. We have previously shown that N4WBP5A expression in Xenopus oocytes results in increased cell-surface expression of the epithelial sodium channel. N4WBPs are characterized by one or two amino terminal PPxY motifs and three transmembrane domains. Here we show that both PPxY motifs of N4WBP5A mediate interaction with WW domains of Nedd4 and that N4WBP5A can physically interact with the WW domains of several Nedd4-family proteins. N4WBP5A is ubiquitinated and ubiquitination does not significantly affect the turnover of N4WBP5A protein. Ubiquitination of N4WBP5A is enhanced by Nedd4 and Nedd4-2 expression. N4WBP5A localizes to the Golgi, vesicles associated with the Golgi complex and to multivesicular bodies. We show that the ectopic expression of N4WBP5A inhibits receptor-mediated endocytosis of labelled epidermal growth factor. N4WBP5A overexpression inhibits accumulation of EGF in large endocytic/lysosomal vesicles suggestive of a role for N4WBP5A in protein trafficking. We propose that N4WBP5A acts as an adaptor to recruit Nedd4 family ubiquitin-protein ligases to the protein trafficking machinery.