RESUMO
Vertebrates usually have three class V myosin paralogues (MyoV) to control membrane trafficking in the actin-rich cell cortex, but their functional overlapping or differentiation through cargoes selectivity is yet only partially understood. In this work, we reveal that the globular tail domain of MyoVc binds to the active form of small GTPase Rab3A with nanomolar affinity, a feature shared with MyoVa but not with MyoVb. Using molecular docking analyses guided by chemical cross-linking restraints, we propose a model to explain how Rab3A selectively recognizes MyoVa and MyoVc via a distinct binding site from that used by Rab11A. The MyoVa/c binding interface involves multiple residues from both lobules (I and II) and the short helix at the α2-α3 link region, which is conserved between MyoVa and MyoVc, but not in MyoVb. This motif is also responsible for the selective binding of RILPL2 by MyoVa and potentially MyoVc. Together, these findings support the selective recruitment of MyoVa and MyoVc to exocytic pathways via Rab3A and expand our knowledge about the functional evolution of class V myosins. SIGNIFICANCE: Hormone secretion, neurotransmitter release, and cytoplasm membrane recycling are examples of processes that rely on the interaction of molecular motors and Rab GTPases to regulate the intracellular trafficking and tethering of vesicles. Defects in these proteins may cause neurological impairment, immunodeficiency, and other severe disorders, being fatal in some cases. Despite their crucial roles, little is known about how these molecular motors are selectively recruited by specific members of the large family of Rab GTPases. In this study, we unveil the interaction between the actin-based molecular motor Myosin Vc and the small GTPase Rab3A, a key coordinator of vesicle trafficking and exocytosis in mammalian cells. Moreover, we propose a model for their recognition and demonstrate that Rab3A specifically binds to the globular tail of Myosins Va and Vc, but not of Myosin Vb, advancing our knowledge about the molecular basis for the selective recruitment of class V myosins by Rab GTPases.
Assuntos
Exocitose , Miosina Tipo V/química , Proteína rab3A de Ligação ao GTP/química , Actinas/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Haplorrinos , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular/métodos , Miosina Tipo V/isolamento & purificação , Miosina Tipo V/metabolismo , Ligação Proteica , Homologia de Sequência de Aminoácidos , Proteína rab3A de Ligação ao GTP/isolamento & purificação , Proteína rab3A de Ligação ao GTP/metabolismoRESUMO
Sperm must undergo the regulated exocytosis of its dense core granule (the acrosome reaction, AR) to fertilize the egg. We have previously described that Rabs3 and 27 are organized in a RabGEF cascade within the signaling pathway elicited by exocytosis stimuli in human sperm. Here, we report the identity and the role of two molecules that link these secretory Rabs in the RabGEF cascade: Rabphilin3a and GRAB. Like Rab3 and Rab27, GRAB and Rabphilin3a are present, localize to the acrosomal region and are required for calcium-triggered exocytosis in human sperm. Sequestration of either protein with specific antibodies introduced into streptolysin O-permeabilized sperm impairs the activation of Rab3 in the acrosomal region elicited by calcium, but not that of Rab27. Biochemical and functional assays indicate that Rabphilin3a behaves as a Rab27 effector during the AR and that GRAB exhibits GEF activity toward Rab3A. Recombinant, active Rab27A pulls down Rabphilin3a and GRAB from human sperm extracts. Conversely, immobilized Rabphilin3a recruits Rab27 and GRAB; the latter promotes Rab3A activation. The enzymatic activity of GRAB toward Rab3A was also suggested by in silico and in vitro assays with purified proteins. In summary, we describe here a signaling module where Rab27A-GTP interacts with Rabphilin3a, which in turn recruits a guanine nucleotide-exchange activity toward Rab3A. This is the first description of the interaction of Rabphilin3a with a GEF. Because the machinery that drives exocytosis is highly conserved, it is tempting to hypothesize that the RabGEF cascade unveiled here might be part of the molecular mechanisms that drive exocytosis in other secretory systems.
Assuntos
Reação Acrossômica , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Espermatozoides/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Proteína rab3A de Ligação ao GTP/metabolismo , Acrossomo/metabolismo , Exocitose , Humanos , Masculino , Proteína rab3A de Ligação ao GTP/química , Rabfilina-3ARESUMO
At the final stage of exocytotis, a fusion pore opens between the plasma and a secretory vesicle membranes; typically, when the pore dilates the vesicle releases its cargo. Sperm contain a large dense-core secretory granule (the acrosome) whose contents are secreted by regulated exocytosis at fertilization. Minutes after the arrival of the triggering signal, the acrosomal and plasma membranes dock at multiple sites and fusion pores open at the contact points. It is believed that immediately afterward, fusion pores dilate spontaneously. Rab3A is an essential component of human sperm exocytotic machinery. Yet, recombinant, persistently active Rab3A halts calcium-triggered secretion when introduced after docking into streptolysin O-permeabilized cells; so does a Rab3A-22A chimera. Here, we applied functional assays, electron and confocal microscopy to show that the secretion blockage is due to the stabilization of open fusion pores. Other novel findings are that sperm SNAREs engage in α-SNAP/NSF-sensitive complexes at a post-fusion stage. Complexes are disentangled by these chaperons to achieve vesiculation and acrosomal contents release. Thus, post-fusion regulation of the pores determines their expansion and the success of the acrosome reaction.
Assuntos
Exocitose , Espermatozoides/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteína rab3A de Ligação ao GTP/metabolismo , Acrossomo/metabolismo , Cálcio/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Masculino , Proteínas rab de Ligação ao GTP/genética , Proteína rab3A de Ligação ao GTP/genéticaRESUMO
Fusion of cortical granules with the oocyte plasma membrane is the most significant event to prevent polyspermy. This particular exocytosis, also known as cortical reaction, is regulated by calcium and its molecular mechanism is still not known. Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in calcium-dependent exocytosis and is not yet clear whether Rab3A participates in cortical granules exocytosis. Here, we examine the involvement of Rab3A in the physiology of cortical granules, particularly, in their distribution during oocyte maturation and activation, and their participation in membrane fusion during cortical granule exocytosis. Immunofluorescence and Western blot analysis showed that Rab3A and cortical granules have a similar migration pattern during oocyte maturation, and that Rab3A is no longer detected after cortical granule exocytosis. These results suggested that Rab3A might be a marker of cortical granules. Overexpression of EGFP-Rab3A colocalized with cortical granules with a Pearson correlation coefficient of +0.967, indicating that Rab3A and cortical granules have almost a perfect colocalization in the egg cortical region. Using a functional assay, we demonstrated that microinjection of recombinant, prenylated and active GST-Rab3A triggered cortical granule exocytosis, indicating that Rab3A has an active role in this secretory pathway. To confirm this active role, we inhibited the function of endogenous Rab3A by microinjecting a polyclonal antibody raised against Rab3A prior to parthenogenetic activation. Our results showed that Rab3A antibody microinjection abolished cortical granule exocytosis in parthenogenetically activated oocytes. Altogether, our findings confirm that Rab3A might function as a marker of cortical granules and participates in cortical granule exocytosis in mouse eggs.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Exocitose , Oócitos/citologia , Oócitos/metabolismo , Proteína rab3A de Ligação ao GTP/metabolismo , Animais , Feminino , Proteínas de Fluorescência Verde/metabolismo , Cavalos , Humanos , Metáfase , Camundongos , Microinjeções , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Exocytosis of mammalian sperm dense-core secretory granule relies on the same fusion molecules as all other secretory cells; one such molecule is the small GTPase Rab3A. Here, we report an in-depth biochemical characterization of the role of Rab3A in secretion by scrutinizing the exocytotic response of streptolysin O-permeabilized human sperm to the acute application of a number of Rab3A-containing constructs and correlating the findings with those gathered with the endogenous protein. Full length, geranylgeranylated, and active Rab3A elicited human sperm exocytosis per se. With Rab3A/Rab22A chimeric proteins, we demonstrated that the carboxy-terminal domain of the Rab3A molecule was necessary and sufficient to promote exocytosis, whereas its amino-terminus prevented calcium-triggered secretion. Interestingly, full length Rab3A halted secretion when added after the docking of the acrosome to the plasma membrane. This effect depended on the inability of Rab3A to hydrolyze GTP. We combined modified immunofluorescence and acrosomal staining protocols to detect membrane fusion and the activation status of endogenous Rab3 simultaneously in individual cells, and found that GTP hydrolysis on endogenous Rab3 was mandatory for fusion pores to open. Our findings contribute to establishing that Rab3 modulates regulated exocytosis differently depending on the nucleotide bound and the exocytosis stage under study.
Assuntos
Membrana Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Exocitose/fisiologia , Guanosina Trifosfato/metabolismo , Espermatozoides/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteína rab3A de Ligação ao GTP/metabolismo , Acrossomo/metabolismo , Adulto , Sequência de Aminoácidos , Cálcio/metabolismo , Imunofluorescência , Humanos , Masculino , Fusão de Membrana/fisiologia , Dados de Sequência Molecular , Receptores Androgênicos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Adulto Jovem , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Acrosomal exocytosis involves a massive fusion between the outer acrosomal and the plasma membranes of the spermatozoon triggered by stimuli that open calcium channels at the plasma membrane. Diacylglycerol has been implicated in the activation of these calcium channels. Here we report that this lipid promotes the efflux of intraacrosomal calcium and triggers exocytosis in permeabilized human sperm, implying that diacylglycerol activates events downstream of the opening of plasma membrane channels. Furthermore, we show that calcium and diacylglycerol converge in a signaling pathway leading to the production of phosphatidylinositol 4,5-bisphosphate (PIP(2)). Addition of diacylglycerol promotes the PKC-dependent activation of PLD1. Rescue experiments adding phosphatidic acid or PIP(2) and direct measurement of lipid production suggest that both PKC and PLD1 promote PIP(2) synthesis. Inhibition of different steps of the pathway was reverted by adenophostin, an agonist of IP(3)-sensitive calcium channels, indicating that PIP(2) is necessary to keep these channels opened. However, phosphatidic acid, PIP(2), or adenophostin could not trigger exocytosis by themselves, indicating that diacylglycerol must also activate another factor. We found that diacylglycerol and phorbol ester stimulate the accumulation of the GTP-bound form of Rab3A. Together our results indicate that diacylglycerol promotes acrosomal exocytosis by i) maintaining high levels of IP(3) - an effect that depends on a positive feedback loop leading to the production of PIP(2) - and ii) stimulating the activation of Rab3A, which in turn initiates a cascade of protein interactions leading to the assembly of SNARE complexes and membrane fusion.
Assuntos
Acrossomo/metabolismo , Diglicerídeos/farmacologia , Exocitose/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase D/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Exocitose/fisiologia , Humanos , Masculino , Fusão de Membrana/efeitos dos fármacos , Fusão de Membrana/fisiologia , Proteínas SNARE/metabolismo , Transdução de Sinais/fisiologia , Proteína rab3A 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
The reversible phosphorylation of tyrosyl residues in proteins is a cornerstone of the signaling pathways that regulate numerous cellular responses. Protein tyrosine phosphorylation is controlled through the concerted actions of protein-tyrosine kinases and phosphatases. The goal of the present study was to unveil the mechanisms by which protein tyrosine dephosphorylation modulates secretion. The acrosome reaction, a specialized type of regulated exocytosis undergone by sperm, is initiated by calcium and carried out by a number of players, including tyrosine kinases and phosphatases, and fusion-related proteins such as Rab3A, alpha-SNAP, N-ethylmaleimide-sensitive factor (NSF), SNAREs, complexin, and synaptotagmin VI. We report here that inducers were unable to elicit the acrosome reaction when permeabilized human sperm were loaded with anti-PTP1B antibodies or with the dominant-negative mutant PTP1B D181A; subsequent introduction of wild type PTP1B or NSF rescued exocytosis. Wild type PTP1B, but not PTP1B D181A, caused cis SNARE complex dissociation during the acrosome reaction through a mechanism involving NSF. Unlike its non-phosphorylated counterpart, recombinant phospho-NSF failed to dissociate SNARE complexes from rat brain membranes. These results strengthen our previous observation that NSF activity is regulated rather than constitutive during sperm exocytosis and indicate that NSF must be dephosphorylated by PTP1B to disassemble SNARE complexes. Interestingly, phospho-NSF served as a substrate for PTP1B in an in vitro assay. Our findings demonstrate that phosphorylation of NSF on tyrosine residues prevents its SNARE complex dissociation activity and establish for the first time a role for PTP1B in the modulation of the membrane fusion machinery.
Assuntos
Exocitose/fisiologia , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas SNARE/metabolismo , Espermatozoides/fisiologia , Reação Acrossômica/fisiologia , Animais , Cálcio/metabolismo , Humanos , Masculino , Proteínas Sensíveis a N-Etilmaleimida/genética , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas SNARE/genética , Espermatozoides/citologia , Tirosina/metabolismo , Proteína rab3A de Ligação ao GTP/genética , Proteína rab3A de Ligação ao GTP/metabolismoRESUMO
The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli it undergoes a special type of Ca2+-dependent exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that alpha-soluble NSF-attachment protein (alpha-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of alpha-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca2+ and Rab3A-triggered ARs are inhibited by anti-alpha-SNAP antibodies. Third, bacterially expressed alpha-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for alpha-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca2+. These results suggest a key role for alpha-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis.
Assuntos
Reação Acrossômica/fisiologia , Espermatozoides/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/fisiologia , Acrossomo/química , Acrossomo/metabolismo , Anticorpos/farmacologia , Cálcio/metabolismo , Cálcio/farmacologia , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Humanos , Masculino , Proteínas SNARE , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Espermatozoides/química , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteína rab3A de Ligação ao GTP/metabolismo , Proteína rab3A de Ligação ao GTP/farmacologiaRESUMO
The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli, sperm undergo calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Protein tyrosine phosphorylation and dephosphorylation are a mechanisms by which multiple cellular events are regulated. Here we report that calcium induces tyrosine phosphorylation in streptolysin O (SLO)-permeabilized human sperm. As expected, pretreatment with tyrphostin A47-a tyrosine kinase inhibitor-abolishes the calcium effect. Interestingly, the calcium-induced increase in tyrosine phosphorylation has a functional correlate in sperm exocytosis. Masking of phosphotyrosyl groups with a specific antibody or inhibition of tyrosine kinases with genistein, tyrphostin A47, and tyrphostin A51 prevent the acrosome reaction. By reversibly sequestering intra-acrosomal calcium with a photo-inhibitable chelator, we show a requirement for protein tyrosine phosphorylation late in the exocytotic pathway, after the efflux of intra-acrosomal calcium. Both mouse and human sperm contain highly active tyrosine phosphatases. Importantly, this activity declines when sperm are incubated under capacitating conditions. Inhibition of tyrosine phosphatases with pervanadate, bis(N,N-dimethylhydroxoamido)hydroxovanadate, ethyl-3,4-dephostatin, and phenylarsine oxide prevents the acrosome reaction. Our results show that both tyrosine kinases and phosphatases play a central role in sperm exocytosis.