RESUMO
The acrosome reaction is the regulated exocytosis of mammalian sperm's single secretory granule, essential for fertilization. It relies on small GTPases, the cAMP binding protein Epac, and the SNARE complex, among other components. Here, we describe a novel tool to investigate Rab27-related signaling pathways: a hybrid recombinant protein consisting of human Rab27A fused to TAT, a cell penetrating peptide. With this tool, we aimed to unravel the connection between Rab3, Rab27 and Rap1 in sperm exocytosis and to deepen our understanding about how isoprenylation and guanine nucleotides influence the behaviour of Rab27 in exocytosis. Our results show that TAT-Rab27A-GTP-γ-S permeated into live sperm and triggered acrosomal exocytosis per se when geraylgeranylated but inhibited it when not lipid-modified. Likewise, an impermeant version of Rab27A elicited exocytosis in streptolysin O-permeabilized - but not in non-permeabilized - cells when geranylgeranylated and active. When GDP-ß-S substituted for GTP-γ-S, isoprenylated TAT-Rab27A inhibited the acrosome reaction triggered by progesterone and an Epac-selective cAMP analogue, whereas the non-isoprenylated protein did not. Geranylgeranylated TAT-Rab27A-GTP-γ-S promoted the exchange of GDP for GTP on Rab3 and Rap1 detected by far-immunofluorescence with Rab3-GTP and Rap1-GTP binding cassettes. In contrast, TAT-Rab27A lacking isoprenylation or loaded with GDP-ß-S prevented the activation of Rab3 and Rap1 elicited by progesterone. Challenging streptolysin O-permeabilized human sperm with calcium increased the population of sperm with Rap1-GTP, Rab3-GTP and Rab27-GTP in the acrosomal region; pretreatment with anti-Rab27 antibodies prevented the activation of all three. The novel findings reported here include: the description of membrane permeant TAT-Rab27A as a trustworthy tool to unveil the regulation of the human sperm acrosome reaction by Rab27 under physiological conditions; that the activation of endogenous Rab27 is required for that of Rab3 and Rap1; and the connection between Epac and Rab27 and between Rab27 and the configuration of the SNARE complex. Moreover, we present direct evidence that Rab27A's lipid modification, and activation/inactivation status correlate with its stimulatory or inhibitory roles in exocytosis.
Assuntos
Reação Acrossômica , Exocitose , Nucleotídeos de Guanina/metabolismo , Prenilação , Proteínas de Ligação a Telômeros/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Humanos , Masculino , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Prenilação de Proteína , Proteínas Recombinantes/genética , Proteínas SNARE/metabolismo , Complexo Shelterina , Transdução de Sinais , Proteínas rab27 de Ligação ao GTP/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Exocytosis is a fundamental process used by eukaryotic cells to release biological compounds and to insert lipids and proteins in the plasma membrane. Specialized secretory cells undergo regulated exocytosis in response to physiological signals. Sperm exocytosis or acrosome reaction (AR) is essentially a regulated secretion with special characteristics. We will focus here on some of these unique features, covering the topology, kinetics, and molecular mechanisms that prepare, drive, and regulate membrane fusion during the AR. Last, we will compare acrosomal release with exocytosis in other model systems.
Assuntos
Reação Acrossômica/fisiologia , Acrossomo/metabolismo , Membrana Celular/metabolismo , Exocitose/fisiologia , Acrossomo/química , Animais , Cálcio/metabolismo , Membrana Celular/química , Regulação da Expressão Gênica , Cinética , Masculino , Fusão de Membrana/fisiologia , Camundongos , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transdução de Sinais , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTP , Proteínas rab3 de Ligação ao GTP/genética , Proteínas rab3 de Ligação ao GTP/metabolismoRESUMO
Two so-called "secretory Rabs," Rab3 and Rab27, regulate late steps during dense-core vesicle exocytosis in neuroendocrine cells. Sperm contain a single large dense-core granule that is released by regulated exocytosis (termed the acrosome reaction) during fertilization or on exposure to inducers in vitro. Sperm exocytosis uses the same fusion machinery as neurons and neuroendocrine cells, with an additional requirement for active Rab3. Here we show that Rab27 is also required for the acrosome reaction, as demonstrated by the inability of inducers to elicit exocytosis when streptolysin O-permeabilized human sperm were loaded with inhibitory anti-Rab27 antibodies or the Rab27-GTP binding domain of the effector Slac2-b. The levels of GTP-bound Rab27 increased on initiation of exocytosis, as did the proportion of GTP-bound Rab3A. We have developed a fluorescence microscopy-based method for detecting endogenous Rab3A-GTP and Rab27-GTP in the acrosomal region of human sperm. Challenge with an inducer increased the population of cells exhibiting GTP-bound Rabs in this subcellular domain. Interestingly, introducing recombinant Rab27A loaded with GTP-γ-S into sperm elicited a remarkable increase in the number of cells evincing GTP-bound Rab3A. In the converse condition, recombinant Rab3A did not modify the percentage of Rab27-GTP-containing cells. Furthermore, Rab27A-GTP recruited a Rab3 GDP/GTP exchange factor (GEF) activity. Our findings suggest that Rab27/Rab3A constitutes a Rab-GEF cascade in dense-core vesicle exocytosis.
Assuntos
Reação Acrossômica/fisiologia , Acrossomo/fisiologia , Exocitose/fisiologia , Vesículas Secretórias/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Bactérias , Western Blotting , Eletroforese em Gel de Poliacrilamida , Técnica Indireta de Fluorescência para Anticorpo , Glutationa Transferase , Guanosina Trifosfato/metabolismo , Humanos , Masculino , Microscopia de Fluorescência , Prenilação , Proteínas Recombinantes/metabolismo , Vesículas Secretórias/metabolismo , Sefarose , Estreptolisinas , Proteínas rab27 de Ligação ao GTPRESUMO
PURPOSE: Anti-oxidation and exocytosis are important for maintaining exocrine tissue homeostasis. During aging, functional and structural alterations occur in the lacrimal gland (LG), including oxidative damage to proteins, lipids, and DNA. The aims of the present study were to determine in the aging LG: a) the effects of aging on LG structure and secretory activity and b) changes in the expression of oxidative stress markers. METHODS: To address these goals, tear secretion composition and corneal impression cytology were compared between male Wistar rats of 2 (control) and 24 (aged) months. LG morphology and the expression levels of vitamin E and malonaldehyde (MDA) were evaluated to determine the anti-oxidant activity and lipid peroxidation, respectively. RT-PCR and western blot analysis were used for the analysis of Ras related in brain GTPase protein (Rab) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the secretory machinery (i.e.; Rab 3d, Rab 27, vesicle-associated membrane protein-2 (Vamp-2), and syntaxin). RESULTS: Histological analysis of aged rats revealed a higher frequency of corneal epithelia metaplasia. In the acinar cells, organelles underwent degeneration, and lipofucsin-like material accumulated in the cytoplasm along with declines in the anti-oxidant marker vitamin E. Rab3d and Rab27b mRNA levels fell along with Rab3d protein expression, whereas syntaxin levels increased. CONCLUSIONS: These findings indicate that exocytotic and anti-oxidant mechanisms become impaired with age in the rat LG. In parallel with these structural alterations, functional declines may contribute to the pathophysiology caused by tear film modification in dry eye disease.
Assuntos
Envelhecimento/metabolismo , Expressão Gênica , Aparelho Lacrimal/metabolismo , Envelhecimento/genética , Animais , Biomarcadores/metabolismo , Western Blotting , Córnea/citologia , Córnea/metabolismo , Epitélio Corneano/citologia , Epitélio Corneano/metabolismo , Aparelho Lacrimal/citologia , Peroxidação de Lipídeos , Masculino , Malondialdeído/metabolismo , Estresse Oxidativo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Lágrimas/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Vitamina E/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/genética , Proteínas rab3 de Ligação ao GTP/metabolismoRESUMO
Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggested as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have central roles in membrane docking.
Assuntos
Acrossomo/fisiologia , Exocitose , Proteínas de Ligação ao GTP/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Proteína rab3A de Ligação ao GTP/metabolismo , Acrossomo/metabolismo , Acrossomo/ultraestrutura , Cálcio/farmacologia , Cálcio/fisiologia , Membrana Celular/metabolismo , Humanos , Masculino , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/genética , Permeabilidade , Ligação ProteicaRESUMO
Stimulus-secretion coupling is a complex set of intracellular reactions initiated by an external stimulus that result in the release of hormones and neurotransmitters. Under physiological conditions this signaling process takes a few milliseconds, and to minimize delays cells have developed a formidable integrated network, in which the relevant molecules are tightly packed on the nanometer scale. Active zones, the sites of release, are composed of several different proteins including voltage-gated Ca(2+) (Ca(V)) channels. It is well acknowledged that hormone and neurotransmitter release is initiated by the activation of these channels located close to docked vesicles, though the mechanisms that enrich channels at release sites are largely unknown. Interestingly, Rab3 binding proteins (RIMs), a diverse multidomain family of proteins that operate as effectors of the small G protein Rab3 involved in secretory vesicle trafficking, have recently identified as binding partners of Ca(V) channels, placing both proteins in the center of an interaction network in the molecular anatomy of the active zones that influence different aspects of secretion. Here, we review recent evidences providing support for the notion that RIMs directly bind to the pore-forming and auxiliary ß subunits of Ca(V) channels and with RIM-binding protein, another interactor of the channels. Through these interactions, RIMs regulate the biophysical properties of the channels and their anchoring relative to active zones, significantly influencing hormone and neurotransmitter release.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Neurotransmissores/metabolismo , Vesículas Secretórias/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , HumanosRESUMO
OBJECTIVE: Oral and ocular dryness are frequent and serious symptoms of Sjögren's syndrome (SS) that reflect problems in secretion due to glandular dysfunction. Exocytosis, an important process in the secretory pathway, requires the participation of Rab family GTPases. This study was undertaken to analyze the expression and localization of Rab3D and Rab8A and to examine their correlation with acinar cell polarity and glandular secretory function. METHODS: Nineteen patients with SS and 17 controls were evaluated. Levels of Rab3D and Rab8A messenger RNA (mRNA) and protein were determined by real-time polymerase chain reaction and Western blotting. Subcellular localization of proteins was determined by indirect immunofluorescence analysis. RESULTS: In patients with SS, total Rab3D protein levels decreased significantly, while mRNA levels remained unchanged. For Rab8A, no changes in either mRNA or protein levels were detected. In serous acini of labial salivary glands from patients with SS, the following 4 patterns of Rab3D staining were distinguishable: severely decreased, distribution throughout the cytoplasm, distribution throughout the cytoplasm combined with loss of nuclear polarity, and normal apical localization. Basal localization of Rab8A was not modified. Rab3D changes were accompanied by apicobasolateral redistribution of ezrin, loss of nuclear polarity, thicker Golgi stacks, and mucin 7 accumulation in the cytoplasm. Finally, low Rab3D protein levels correlated with alterations in scintigraphy measurements. CONCLUSION: Our findings indicate that Rab3D regulates the exocytosis of many components critical for the maintenance of oral physiology. Hence, the changes observed in Rab3D expression and distribution are likely to contribute to the decrease in or loss of saliva components (i.e., mucins), which may explain the variety of oral and ocular symptoms associated with SS.
Assuntos
Polaridade Celular/fisiologia , Glândulas Salivares/metabolismo , Síndrome de Sjogren/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome de Sjogren/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/genéticaRESUMO
The acrosome is an exocytic granule that overlies the spermatozoan nucleus. In response to different stimuli, it undergoes calcium-regulated exocytosis. Freshly ejaculated mammalian sperm are not immediately capable of undergoing acrosome reaction. The acquisition of this ability is called capacitation and involves a series of still not well-characterized changes in the sperm physiology. Plasma membrane cholesterol removal is one of the sperm modifications that are associated with capacitation. However, how sterols affect acrosomal exocytosis is unknown. Here, we show that short incubations with cyclodextrin, a cholesterol removal agent, just before stimulation promote acrosomal exocytosis. Moreover, the effect was also observed in permeabilized cells stimulated with calcium, indicating that cholesterol plays a direct role in the calcium-dependent exocytosis associated with acrosome reaction. Using a photo-inhibitable calcium chelator, we show that cholesterol affects an early event of the exocytic cascade rather than the lipid bilayers mixing. Functional data indicate that one target for the cholesterol effect is Rab3A. The sterol content does not affect the Rab3A activation-deactivation cycle but regulates its membrane anchoring. Western blot analysis and immunoelectron microscopy confirmed that cholesterol efflux facilitates Rab3A association to sperm plasma membrane. Our data indicate that the cholesterol efflux occurring during capacitation optimizes the conditions for the productive assembly of the fusion machinery required for acrosome reaction.