Biology subjects

Krishnakumar, S. S.

Publications and source records attributed to Krishnakumar, S. S..

7 recordsLinked to original sources

bioRxiv2026

Evolutionary tuning of complexin-synaptotagmin fusion clamp governs regulated neurotransmitter release

Synaptic vesicles must be held in a fusion-ready but restrained state until calcium influx triggers release. Synaptotagmin-1 (Syt1) and Complexin (CPX) both contribute to this fusion clamp, yet removing either protein produces opposite effects in different species. Whether this reflects genuine divergence in their molecular roles, or species-specific tuning of a conserved mechanism, has remained unresolved. Here we address this by comparing mammalian and C. elegans orthologs in a reconstituted single-vesicle assay that isolates clamping from upstream docking and priming. We find that clamping in both species requires the combined action of CPX and Syt1, with neither sufficient on its own. CPX clamps only with its cognate SNARE complex, a specificity conferred entirely by its accessory helix. The Syt1 orthologs differ markedly in intrinsic clamping strength, which maps to C2B mediated higher-order oligomerization and sets the functional dependence on CPX in each system. These results support a dual-regulator model of exocytosis in which Syt1 and CPX act sequentially and cooperatively through discrete molecular states to control vesicle clamping and calcium-triggered release. This framework reconciles long-standing functional discrepancies across species and reveals how evolutionary divergence modulates conserved mechanisms governing synaptic fidelity.

neuroscience↗

bioRxiv2025

Synaptotagmin-7 is required for synchronous but not asynchronous facilitation of glutamate release at cortical boutons

Short-term synaptic plasticity and the kinetics of neurotransmitter release vary widely across synapses, but population measurements obscure the mechanisms that generate this diversity. While the Ca2+ sensor Synaptotagmin-7 (Syt7) has been implicated in facilitation, vesicle replenishment and asynchronous vesicle exocytosis, its precise contributions to these processes remain debated. We used quantal-resolution imaging to measure synchronous and asynchronous glutamate release at individual cortical boutons in wild type and Syt7-/- neurons. Stratifying boutons by release efficacy and applying failure-based analysis to isolate trials where the first action potential evoked no release allowed us to separate facilitation from vesicle depletion. Syt7 deletion selectively eliminated activity-dependent facilitation of synchronous release but left facilitation of asynchronous release intact, although its overall magnitude was reduced. We further show that synchronous and asynchronous events arise from functionally distinct vesicle populations. These findings demonstrate that activity-dependent facilitation of synchronous and asynchronous exocytosis are mechanistically separable, enabling synapses to independently tune distinct temporal components of neurotransmission.

neuroscience↗

bioRxiv2023

Novel Roles for Diacylglycerol in Synaptic Vesicle Priming and Release Revealed by Complete Reconstitution of Core Protein Machinery

Here we introduce the full functional reconstitution of genetically-validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca2+. Using this novel setup, we discover new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca2+-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca2+-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of ready-release vesicles. Dynamic single-molecule imaging of Complexin binding to ready-release vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by Munc13 and Munc18 chaperones. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 template complex is a functional intermediate in the production of primed, ready-release vesicles, which requires the coordinated action of Munc13 and Munc18. SIGNIFICANCE STATEMENTMunc13 and Munc18 are SNARE-associated chaperones that act as "priming" factors, facilitating the formation of a pool of docked, release-ready vesicles and regulating Ca2+-evoked neurotransmitter release. Although important insights into Munc18/Munc13 function have been gained, how they assemble and operate together remains enigmatic. To address this, we developed a novel biochemically-defined fusion assay which enabled us to investigate the cooperative action of Munc13 and Munc18 in molecular terms. We find that Munc18 nucleates the SNARE complex, while Munc13 promotes and accelerates the SNARE assembly in a DAG-dependent manner. The concerted action of Munc13 and Munc18 stages the SNARE assembly process to ensure efficient clamping and formation of stably docked vesicles, which can be triggered to fuse rapidly ([~]10 msec) upon Ca2+ influx.

neuroscience↗

bioRxiv2021

Molecular Determinants of Complexin Clamping in Reconstituted Single-Vesicle Fusion

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al. 2020). Here using the same in vitro single-vesicle fusion assay, we establish the molecular details of the Complexin clamp and its physiological relevance. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins, analogous to the trans clamping model, enhances this functionality. The c-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Further, we find that Complexin likely contributes to rapid Ca2+-synchronized vesicular release by preventing un-initiated fusion rather than by directly facilitating vesicle fusion.

biophysics↗

bioRxiv2020

Munc13 binds and recruits SNAP25 to chaperone SNARE complex assembly

Synaptic vesicle fusion is mediated by membrane-bridging complexes formed by SNARE proteins - VAMP2 on the vesicle and Syntaxin-1/SNAP25 on the pre-synaptic membrane. Accumulating evidence suggest that chaperones Munc18-1 and Munc13-1 co-operatively catalyze SNARE assembly via an intermediate template complex containing Syntaxin-1 and VAMP2. How SNAP25 is chaperoned into this nascent complex remains a mystery. Here we report that Munc13-1 recruits SNAP25 to initiate the ternary SNARE complex assembly by direct binding, as judged by bulk FRET spectroscopy and single-molecule optical tweezer studies. Detailed structure-function analyses show that the binding is mediated by the Munc13-1 MUN domain and is specific for the SNAP25 linker region that connects the two SNARE motifs. Consequently, freely diffusing SNAP25 molecules on phospholipid bilayers are concentrated and presumably bound in ~1:1 stoichiometry by the self-assembled Munc13-1 nanoclusters. Our data suggests that Munc13-1s capacity to bind all three synaptic SNARE proteins likely underlie its chaperone function.

neuroscience↗

bioRxiv2019

Independent Yet Synergistic Roles of Synaptotagmin-1 and Complexin in Calcium Regulated Neuronal Exocytosis

Calcium (Ca2+)-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion of vesicles (clamping) and to trigger fusion following Ca2+-influx. The principal components involved, namely the vesicular fusion machinery (SNARE proteins) and the regulatory proteins (Synaptotagmin-1 and Complexin) are well-known. Here, we use a reconstituted single-vesicle fusion assay to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act independently but synergistically to establish Ca2+-regulated fusion. Under physiologically-relevant conditions, we find that Synaptotagmin-1 oligomers bind and clamp a limited number of central SNARE complexes via the primary binding interface, to introduce a kinetic delay in vesicle fusion mediated by the excess of free SNAREpins. This in turn enables Complexin to independently arrest the remaining free peripheral SNAREpins to produce stably clamped vesicles. Activation of the central SNAREpins associated with Synaptotagmin-1 by Ca2+ is sufficient to trigger rapid (<100 msec) and synchronous fusion of the docked vesicles.

neuroscience↗

bioRxiv2019

Structural Basis for the Clamping and Ca2+ Activation of SNARE-mediated Fusion by Synaptotagmin

Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to Ca2+-influx. To understand the underlying molecular mechanism, we determined the structure of the Syt1-SNARE complex on lipid membranes using cryo-electron microscopy. Under resting conditions, the Syt1 C2 domains adopt a novel membrane orientation with a Mg2+-mediated partial insertion of the aliphatic loops, alongside weak interactions with the anionic lipid headgroups. The C2B domain concurrently binds the SNARE bundle via the primary interface and is positioned between the SNAREpins and the membrane. In this configuration, Syt1 is projected to sterically delay the complete assembly of the associated SNAREpins and thus, contribute to clamping fusion. This Syt1-SNARE organization is disrupted upon Ca2+-influx as Syt1 reorients into the membrane, allowing the attached SNAREpins to complete zippering and drive fusion. Overall, we find cation (Mg2+/Ca2+) dependent membrane interaction is a key determinant of the dual clamp/activator function of Syt1.

neuroscience↗