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1.  Dynamic Control of Excitatory Synapse Development by a Rac1 GEF/GAP Regulatory Complex 
Developmental cell  2014;29(6):701-715.
The small GTPase Rac1 orchestrates actin-dependent remodeling essential for numerous cellular processes including synapse development. While precise spatiotemporal regulation of Rac1 is necessary for its function, little is known about the mechanisms that enable Rac1 activators (GEFs) and inhibitors (GAPs) to act in concert to regulate Rac1 signaling. Here we identify a regulatory complex composed of a Rac-GEF (Tiam1) and a Rac-GAP (Bcr) that cooperate to control excitatory synapse development. Disruption of Bcr function within this complex increases Rac1 activity and dendritic spine remodeling, resulting in excessive synaptic growth that is rescued by Tiam1 inhibition. Notably, EphB receptors utilize the Tiam1-Bcr complex to control synaptogenesis. Following EphB activation, Tiam1 induces Rac1-dependent spine formation, whereas Bcr prevents Rac1-mediated receptor internalization, promoting spine growth over retraction. The finding that a Rac-specific GEF/GAP complex is required to maintain optimal levels of Rac1 signaling provides an important insight into the regulation of small GTPases.
PMCID: PMC4111230  PMID: 24960694
2.  A dynamin homolog promotes the transition from hemifusion to content mixing in intracellular membrane fusion 
Traffic (Copenhagen, Denmark)  2014;15(5):558-571.
The convergence of the antagonistic reactions of membrane fusion and fission at the hemifusion/hemifission intermediate has generated a captivating enigma of whether SNAREs and dynamin have unusual counter-functions in fission and fusion respectively. SNARE-mediated fusion and dynamin-driven fission are fundamental membrane flux reactions known to occur during ubiquitous cellular communication events such as exocytosis, endocytosis and vesicle transport. Here we demonstrate the influence of the dynamin homolog Vps1 on lipid mixing and content mixing properties of yeast vacuoles, and on the incorporation of SNAREs into fusogenic complexes. We propose a novel concept that Vps1, through its oligomerization and SNARE domain binding, promotes the hemifusion-content mixing transition in yeast vacuole fusion by increasing the number of trans-SNAREs.
PMCID: PMC3976715  PMID: 24471450
Vps1; Dynamin superfamily; SNAREs; Vam3; Syntaxin; membrane fusion; hemifusion; neurotransmitter release; exocytosis; endocytosis
3.  Nicotinamide Adenine Dinucleotide-Dependent Binding of the Neuronal Ca2+ Sensor Protein GCAP2 to Photoreceptor Synaptic Ribbons 
Guanylate cyclase activating protein 2 (GCAP2) is a recoverin-like Ca2+-sensor protein known to modulate guanylate cyclase activity in photoreceptor outer segments. GCAP2 is also present in photoreceptor ribbon synapses where its function is unknown. Synaptic ribbons are active zone-associated presynaptic structures in the tonically active photoreceptor ribbon synapses and contain RIBEYE as a unique and major protein component. In the present study, we demonstrate by various independent approaches that GCAP2 specifically interacts with RIBEYE in photoreceptor synapses. We show that the flexible hinge 2 linker region of RIBEYE(B) domain that connects the nicotinamide adenine dinucleotide (NADH)-binding subdomain with the substrate-binding subdomain (SBD) binds to the C terminus of GCAP2. We demonstrate that the RIBEYE–GCAP2 interaction is induced by the binding of NADH to RIBEYE. RIBEYE–GCAP2 interaction is modulated by the SBD. GCAP2 is strongly expressed in synaptic terminals of light-adapted photoreceptors where GCAP2 is found close to synaptic ribbons as judged by confocal microscopy and proximity ligation assays. Virus-mediated overexpression of GCAP2 in photoreceptor synaptic terminals leads to a reduction in the number of synaptic ribbons. Therefore, GCAP2 is a prime candidate for mediating Ca2+-dependent dynamic changes of synaptic ribbons in photoreceptor synapses.
PMCID: PMC3900572  PMID: 20463219
4.  Dynamin-SNARE interactions control trans-SNARE formation in intracellular membrane fusion 
Nature communications  2013;4:1704.
The fundamental processes of membrane fission and fusion determine size and copy numbers of intracellular organelles. While SNARE proteins and tethering complexes mediate intracellular membrane fusion, fission requires the presence of dynamin or dynamin-related proteins. Here we study these reactions in native yeast vacuoles and find that the yeast dynamin homolog Vps1 is not only an essential part of the fission machinery, but also controls membrane fusion by generating an active Qa SNARE- tethering complex pool, which is essential for trans-SNARE formation. Our findings provide new insight into the role of dynamins in membrane fusion by directly acting on SNARE proteins.
PMCID: PMC3630463  PMID: 23591871
5.  A tethering complex dimer catalyzes trans-SNARE complex formation in intracellular membrane fusion 
Bioarchitecture  2012;2(2):59-69.
SNARE complexes mediate membrane fusion in the endomembrane system. They consist of coiled-coil bundles of four helices designated as Qa, Qb, Qc and R. A critical intermediate in the fusion pathway is the trans-SNARE complex generated by the assembly of SNAREs residing in opposing membranes. Mechanistic details of trans-SNARE complex formation and topology in a physiological system remain largely unresolved. Our studies on native yeast vacuoles revealed that SNAREs alone are insufficient to form trans-SNARE complexes and that additional factors, potentially tethering complexes and Rab GTPases, are required for the process. Here we report a novel finding that a HOPS tethering complex dimer catalyzes Rab GTPase-dependent formation of a topologically preferred QbQcR-Qa trans-SNARE complex.
PMCID: PMC3383723  PMID: 22754631
HOPS tethering complex dimer; QbQcR-Qa trans-SNARE complex; Rab GTPase
6.  Sequential Analysis of Trans-SNARE Formation in Intracellular Membrane Fusion 
PLoS Biology  2012;10(1):e1001243.
SM proteins stabilize cis-SNARE complexes leading to a specific preferred topology for trans-SNARE formation.
SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Qa, Qb, and Qc) from target (t)-SNAREs and one (R) from the vesicular (v)-SNARE. NSF/Sec18 disrupts these cis-SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Qa SNARE, leaving behind a QbcR subcomplex. This subcomplex serves as an acceptor for a Qa SNARE from the opposite membrane, leading to Qa-QbcR trans-complexes. Activity tests of vacuoles with diagnostic distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the QbcR cis-complex and the formation of the Qa-QbcR trans-complex are both sensitive to the Rab-GTPase inhibitor, GDI, and to mutations in the vacuolar tether complex, HOPS (HOmotypic fusion and vacuolar Protein Sorting complex). This suggests that the vacuolar Rab-GTPase, Ypt7, and HOPS restrict cis-SNARE disassembly and thereby bias trans-SNARE assembly into a preferred topology.
Author Summary
Cellular components often travel between organelles in vesicular entities. This intracellular traffic usually involves production of a vesicle containing cargo from one organelle membrane, movement of the vesicle to its destination, and then fusion of the vesicle with the target organelle. Thus, membrane fusion is a fundamental process required for these intracellular trafficking events. SNARE proteins and SM proteins mediate this fusion process. SNAREs form complexes that are either located on the same membrane or vesicle (called cis-SNARE complexes) or bridge two membrane compartments or vesicles (trans-SNARE complexes). The cis-SNARE complexes must be activated before trans-SNARE complexes can form and allow the membranes to fuse. We investigated the mechanism of cis-SNARE activation and trans-SNARE formation by studying the fusion of highly purified yeast vacuoles. We found that cis-SNARE activation involves the selective removal of a single SNARE protein from a pre-existing cis-SNARE complex, which is replaced by a similar SNARE originating from the other fusion partner. The activated cis-SNARE complexes depended on SM proteins for their stability. Thus, we have shown that the preferred topology of trans-SNARE formation is determined by cis-SNARE–SM protein interactions.
PMCID: PMC3260307  PMID: 22272185

Results 1-6 (6)