Rab3-GEF controls active zone development at the Drosophila neuromuscular junction

Synaptic signaling involves the release of neurotransmitter from presynaptic active zones (AZs). Proteins that regulate vesicle exocytosis cluster at AZs, composing the cytomatrix at the active zone (CAZ). At the Drosophila neuromuscular junction (NMJ), the small GTPase Rab3 controls the distribution of CAZ proteins across release sites, thereby regulating the efficacy of individual AZs. Here we identify Rab3-GEF as a second protein that acts in conjunction with Rab3 to control AZ protein composition. At rab3-GEF mutant NMJs, Bruchpilot (Brp) and Ca(2+) channels are enriched at a subset of AZs, leaving the remaining sites devoid of key CAZ components in a manner that is indistinguishable from rab3 mutant NMJs. As the Drosophila homologue of mammalian DENN/MADD and Caenorhabditis elegans AEX-3, Rab3-GEF is a guanine nucleotide exchange factor (GEF) for Rab3 that stimulates GDP to GTP exchange. Mechanistic studies reveal that although Rab3 and Rab3-GEF act within the same mechanism to control AZ development, Rab3-GEF is involved in multiple roles. We show that Rab3-GEF is required for transport of Rab3. However, the synaptic phenotype in the rab3-GEF mutant cannot be fully explained by defective transport and loss of GEF activity. A transgenically expressed GTP-locked variant of Rab3 accumulates at the NMJ at wild-type levels and fully rescues the rab3 mutant but is unable to rescue the rab3-GEF mutant. Our results suggest that although Rab3-GEF acts upstream of Rab3 to control Rab3 localization and likely GTP-binding, it also acts downstream to regulate CAZ development, potentially as a Rab3 effector at the synapse

Epac, Rap and Rab3 act in concert to mobilize calcium from sperm's acrosome during exocytosis

BACKGROUND: Exocytosis of sperm's single secretory granule or acrosome (acrosome reaction, AR) is a highly regulated event essential for fertilization. The AR begins with an influx of calcium from the extracellular milieu and continues with the synthesis of cAMP and the activation of its target Epac. The cascade bifurcates into a Rab3-GTP-driven limb that assembles the fusion machinery and a Rap-GTP-driven limb that mobilizes internal calcium. RESULTS: To understand the crosstalk between the two signaling cascades, we applied known AR inhibitors in three experimental approaches: reversible, stage-specific blockers in a functional assay, a far-immunofluorescence protocol to detect active Rab3 and Rap, and single cell-confocal microscopy to visualize fluctuations in internal calcium stores. Our model system was human sperm with their plasma membrane permeabilized with streptolysin O and stimulated with external calcium. The inhibition caused by reagents that prevented the activation of Rap was reversed by mobilizing intracellular calcium pharmacologically, whereas that caused by AR inhibitors that impeded Rab3's binding to GTP was not. Both limbs of the exocytotic cascade joined at or near the stage catalyzed by Rab3 in a unidirectional, hierarchical connection in which the intra-acrosomal calcium mobilization arm was subordinated to the fusion protein arm; somewhere after Rab3, the pathways became independent. CONCLUSIONS: We delineated the sequence of events that connect an external calcium signal to internal calcium mobilization during exocytosis. We have taken advantage of the versatility of the sperm model to investigate how cAMP, calcium, and the proteinaceous fusion machinery coordinate to accomplish secretion. Because the requirement of calcium from two different sources is not unique to sperm and fusion proteins are highly conserved, our findings might contribute to elucidate mechanisms that operate in regulated exocytosis in other secretory cell types.Fil: Ruete, María Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Lucchesi, Ornella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Bustos, Matias Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Tomes, Claudia Nora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

The rab3A-22A chimera prevents sperm exocytosis by stabilizing open fusion pores

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.Fil: Quevedo, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Lucchesi, Ornella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Bustos, Matias Alberto. John Wayne Cancer Institute; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Pocognoni, Cristián Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: De la Iglesia, Paola X.. Hospital Italiano; ArgentinaFil: Tomes, Claudia Nora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentin

Rab3D is critical for secretory granule maturation in PC12 cells.

Neuropeptide- and hormone-containing secretory granules (SGs) are synthesized at the trans-Golgi network (TGN) as immature secretory granules (ISGs) and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs). Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I) decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs

Localization versus function of Rab3 proteins - Evidence for a common regulatory role in controlling fusion

Rab3A, Rab3B, Rab3C, and Rab3D constitute a family of GTP- binding proteins that are implicated in regulated exocytosis. Various localizations and distinct functions have been proposed for different and occasionally even for the same Rab3 protein. This is exemplified by studies demonstrating that deletion of Rab3A in knock-out mice results in dysregulation of the final stages of exocytosis, whereas overexpression of Rab3A in neuroendocrine cells causes nearly complete inhibition of Ca2+- triggered exocytosis. We have now examined the properties of all Rab3 proteins in the same assays, with the long-term goal of identifying a common conceptual framework for their functions. Using quantitative immunoblotting, we found that all four Rab3 proteins were expressed in brain and endocrine tissues, although at widely different levels. Rab3A, Rab3B, and Rab3C co-localized to synaptic and secretory vesicles consistent with potential redundancy, whereas Rab3D was expressed at high levels only in the endocrine pituitary (where it was more abundant than Rab3A, Rab3B, and Rab3C combined), in exocrine glands, and in adipose tissue. In transfected PC12 cells, all four Rab3 proteins strongly inhibited Ca2+-triggered exocytosis. Except for a mutation that fixes Rab3 into a permanently GDP-bound state, all Rab3 mutations tested had no effect on this inhibition, including a mutation in the calmodulin-binding site that was described as inactivating (Coppola, T., Perret-Menoud, V., Luthi, S., Farnsworth, C. C., Glomset, J. A., and Regazzi, R. (1999) EMBO J. 18, 5885- 5891).:Unexpectedly, overexpression of wild type Rab3A and permanently GTP-bound mutant Rab3A in PC12 cells caused a loss of secretory vesicles and an increase in constitutive, Ca2+- independent exocytosis that correlated with the inhibition of regulated Ca2+-triggered exocytosis. Our data indicate that overexpression of Rab3 in PC12 cells impairs the normal control of the final step in exocytosis, thereby converting the regulated secretory pathway into a constitutive pathway. These results offer an hypothesis that reconciles Rab3 transfection and knockout studies by suggesting that Rab3 functions as a gatekeeper of a late stage in exocytosis

The RAB3-RIM Pathway Is Essential for the Release of Neuromodulators

Neurons secrete neuromodulators/neuropeptides from dense-core vesicles (DCVs) by a largely unknown mechanism. Persoon et al. identify RAB3 and RIM1/2 as essential factors. RAB3’s indispensable role is the first distinct feature of DCV secretion as compared to synaptic vesicle secretion

Insulin regulates Rab3-Noc2 complex dissociation to promote GLUT4 translocation in rat adipocytes.

AIMS/HYPOTHESIS: The glucose transporter GLUT4 is present mainly in insulin-responsive tissues of fat, heart and skeletal muscle and is translocated from intracellular membrane compartments to the plasma membrane (PM) upon insulin stimulation. The transit of GLUT4 to the PM is known to be dependent on a series of Rab proteins. However, the extent to which the activity of these Rabs is regulated by the action of insulin action is still unknown. We sought to identify insulin-activated Rab proteins and Rab effectors that facilitate GLUT4 translocation. METHODS: We developed a new photoaffinity reagent (Bio-ATB-GTP) that allows GTP-binding proteomes to be explored. Using this approach we screened for insulin-responsive GTP loading of Rabs in primary rat adipocytes. RESULTS: We identified Rab3B as a new candidate insulin-stimulated G-protein in adipocytes. Using constitutively active and dominant negative mutants and Rab3 knockdown we provide evidence that Rab3 isoforms are key regulators of GLUT4 translocation in adipocytes. Insulin-stimulated Rab3 GTP binding is associated with disruption of the interaction between Rab3 and its negative effector Noc2. Disruption of the Rab3-Noc2 complex leads to displacement of Noc2 from the PM. This relieves the inhibitory effect of Noc2, facilitating GLUT4 translocation. CONCLUSIONS/INTERPRETATION: The discovery of the involvement of Rab3 and Noc2 in an insulin-regulated step in GLUT4 translocation suggests that the control of this translocation process is unexpectedly similar to regulated secretion and particularly pancreatic insulin-vesicle release

Neuropeptide secretion principles:Vesicle populations, characteristics and fusion mechanisms

Our brain consists of a complex network of neurons which communicate by secreting signaling molecules as neurotransmitters and neuromodulators. For fast communication, neurotransmitters are released by synaptic vesicles (SVs) at the contact points between neurons, the synapses. Neuromodulators are secreted by dense-core vesicles (DCVs) to regulate processes such as development, circadian rhythm, metabolism, behavior and emotions. Defects in neuromodulator signaling result in brain disorders as depression, schizophrenia, autism or obesity, but how neuromodulators are released by neurons is largely unknown. The general aim of this thesis was to characterize the mechanism of neuromodulator secretion by DCVs. We have determined the number, location and release characteristics of DCVs in neurons. Furthermore, we show that two proteins, RAB3 and RIM proteins, are essential for DCV fusion. Finally, we identified two new proteins present on DCVs and developed new tools to study DCVs in the future. The fundamental knowledge and new methods provided in this thesis will contribute to new directions for treatments of disorders related to neuromodulator signaling. Mammalian neurons contain a large number of DCVs, ranging from 1,400-18,000 per neuron, which are distributed equally throughout the neuron. DCV fusion, like SV fusion, is triggered by calcium influx but requires more prolonged repetitive stimulation to release only a small fraction. While SV fusion mechanisms have been extensively studied, providing a molecular framework of proteins organizing the release of neurotransmitters, much less is known about proteins that regulate neuromodulator secretion. We now show that RIM proteins are indispensable organizers of DCV fusion by positioning the active zone protein MUNC13 and interacting with DCVs via RAB3. Together, these data identified an essential mechanism that brings DCVs to their release site for fusion. The identification of the essential function of RAB3 in DCV fusion resolved a longstanding question in the field. In 1980, a breakthrough study by Novick and Schekman identified SEC4, the yeast RAB3, as one of the essential proteins required for fusion. Many of the proteins they identified have been shown to be essential for SV fusion in the mammalian brain, but surprisingly, RAB3 proteins are not required for SV fusion. However, removal of all RAB3 proteins is lethal for mice, suggesting RAB3 proteins had an unidentified important function. Our data now shows that RAB3 is an indispensableregulator of DCV fusion. The crucial role of RAB3 in DCV fusion shows the first distinct feature between DCV and SV fusion, and provides new explanations for the multiple, severe problems in mice which lack RAB3. This thesis contributes with fundamental insights into different aspects of neuromodulator secretion, but we currently do not have a consistent overview of what is on and inside a DCV. Therefore, we developed a new approach to isolate intact DCVs from neurons. This approach could be used for future studies to determine the composition of DCVs, which will identify important molecules that function in the DCV pathway, providing new directions to study neuromodulator secretion and new leads for diagnostics and treatments of neuromodulator signaling related disorders

The Rab3-interacting molecule RIM is expressed in pancreatic β-cells and is implicated in insulin exocytosis

AbstractThe putative Rab3 effector RIM (Rab3-interacting molecule) was detected by Northern blotting, RT-PCR and Western blotting in native pancreatic β-cells as well as in the derived cell lines INS-1E and HIT-T15. RIM was localized on the plasma membrane of INS-1E cells and β-cells. An involvement of RIM in insulin exocytosis was indicated by transfection experiments of INS-1E cells with the Rab3 binding domain of RIM. This domain enhanced glucose-stimulated secretion in intact cells and Ca2+-stimulated exocytosis in permeabilized cells. Co-expression of Rab3A reversed the effect of RIM on exocytosis. These results suggest an implication of RIM in the control of insulin secretion

RIM promotes calcium channel accumulation at active zones of the Drosophila neuromuscular junction

Synaptic communication requires the controlled release of synaptic vesicles from presynaptic axon terminals. Release efficacy is regulated by the many proteins that comprise the presynaptic release apparatus, including Ca(2+) channels and proteins that influence Ca(2+) channel accumulation at release sites. Here we identify Drosophila RIM and demonstrate that it localizes to active zones at the larval neuromuscular junction. In Drosophila RIM mutants, there is a large decrease in evoked synaptic transmission, due to a significant reduction in both the clustering of Ca(2+) channels and the size of the readily releasable pool of synaptic vesicles at active zones. Hence, RIM plays an evolutionarily conserved role in regulating synaptic calcium channel localization and readily releasable pool size. Since RIM has traditionally been studied as an effector of Rab3 function, we investigate whether RIM is involved in the newly identified function of Rab3 in the distribution of presynaptic release machinery components across release sites. Bruchpilot (Brp), an essential component of the active zone cytomatrix T bar, is unaffected by RIM disruption, indicating that Brp localization and distribution across active zones does not require wild type RIM. In addition, larvae containing mutations in both RIM and rab3 have reduced Ca(2+) channel levels and a Brp distribution that is very similar to that of the rab3 single mutant, indicating that RIM functions to regulate Ca(2+) channel accumulation but is not a Rab3 effector for release machinery distribution across release sites