Biology subjects

Rothman, J. E.

Publications and source records attributed to Rothman, J. E..

17 recordsLinked to original sources

bioRxiv2025

The Golgi Rim is a Precise Tetraplex of Golgin Proteins that Can Self-Assemble into Filamentous Bands

Golgin proteins have long been suspected to be organizers of the Golgi stack. Using three-dimensional super-resolution microscopy, we comprehensively localize the human golgin family at the rim of the Golgi apparatus at 10-20 nm resolution in situ. Unexpectedly, we find that the golgins are precisely organized into a tetraplex with four discrete layers, each containing a specific set of rim golgins. We observe no golgins inside the stack between its membrane-bound cisternae. Biochemically characterizing most of the golgins as isolated proteins, we find that they form anti-parallel dimers and further self-assemble into bands of multi-micron-long filaments. Based on our findings, we propose an "outside-in" physical model, the Golgin Organizer Hypothesis, in which the Golgi stack of cisternae and its overall ribbon morphology directly result from bending circumferential bands of rim golgin filaments onto a membrane surface, explaining stack formation without the need for special "stacking proteins."

cell biology↗

bioRxiv2024

A proteome-wide quantitative guide for nanoscale spatially resolved extraction of membrane proteins into native nanodiscs

The intricate molecular environment of the native membrane profoundly influences every aspect of membrane protein (MP) biology. Despite this, the most prevalent method of studying MPs uses detergent-like molecules that disrupt and remove this vital local membrane context. This severely impedes our ability to quantitatively decipher the local molecular context and comprehend its regulatory role in the structure, function, and biogenesis of MPs. Using a library of membrane-active polymers we have developed a platform for the high-throughput analysis of the membrane proteome. The platform enables near-complete spatially resolved extraction of target MPs directly from their endogenous membranes into native nanodiscs that maintain the local membrane context. We accompany this advancement with an open-access database that quantifies the polymer-specific extraction variability for 2065 unique mammalian MPs and provides the most optimized condition for each of them. Our method enables rapid and near-complete extraction and purification of target MPs directly from their endogenous organellar membranes at physiological expression levels while maintaining the nanoscale local membrane environment. Going beyond the plasma membrane proteome, our platform enables extraction from any target organellar membrane including the endoplasmic reticulum, mitochondria, lysosome, Golgi, and even transient organelles such as the autophagosome. To further validate this platform, we took several independent MPs and demonstrated how our resource can enable rapid extraction and purification of target MPs from different organellar membranes with high efficiency and purity. Further, taking two synaptic vesicle MPs, we show how the database can be extended to capture multiprotein complexes between overexpressed MPs. We expect these publicly available resources to empower researchers across disciplines to efficiently capture membrane nano-scoops containing a target MP and interface with structural, functional, and other bioanalytical approaches. We demonstrate an example of this by combining our extraction platform with single-molecule TIRF imaging to demonstrate how it can enable rapid determination of homo-oligomeric states of target MPs in native cell membranes.

biophysics↗

bioRxiv2023

Two successive oligomeric Munc13 assemblies scaffold vesicle docking and SNARE assembly to support neurotransmitter release

The critical presynaptic protein Munc13 serves numerous roles in the process of docking and priming synaptic vesicles. Here we investigate the functional significance of two distinct oligomers of the Munc13 core domain (Munc13C) comprising C1-C2B-MUN-C2C. Oligomer interface point mutations that specifically destabilized either the trimer or lateral hexamer assemblies of Munc13C disrupted vesicle docking, trans-SNARE formation, and Ca2+-triggered vesicle fusion in vitro and impaired neurotransmitter secretion and motor nervous system function in vivo. We suggest that a progression of oligomeric Munc13 complexes couples vesicle docking and assembly of a precise number of SNARE molecules to support rapid and high-fidelity vesicle priming.

neuroscience↗

bioRxiv2023

Synaptophysin Chaperones the Assembly of 12 SNAREpins under each Ready-Release Vesicle

The synaptic vesicle protein Synaptophysin has long been known to form a complex with the v-SNARE VAMP, but a more specific molecular function or mechanism of action in exocytosis has been lacking because gene knockouts have minimal effects. Utilizing fully-defined reconstitution and single-molecule measurements, we now report that Synaptophysin functions as a chaperone that determines the number of SNAREpins assembling between a ready-release vesicle and its target membrane bilayer. Specifically, Synaptophysin directs the assembly of 12 {+/-} 1 SNAREpins under each docked vesicle, even in the face of an excess of SNARE proteins. The SNAREpins assemble in successive waves of 6 {+/-} 1 and 5 {+/-} 2 SNAREpins, respectively, tightly linked to oligomerization of and binding to the vesicle Ca++ sensor Synaptotagmin. Templating of 12 SNAREpins by Synaptophysin is likely the direct result of its hexamer structure and its binding of VAMP2 dimers, both of which we demonstrate in detergent extracts and lipid bilayers. Significance StatementSynaptophysin is the most abundant protein and a unique constituent of synaptic vesicles, yet it has no known function, due to minimal genetic phenotypes and the lack of biochemical assays. Here, we directly establish using two independent methods that the synaptic vesicle protein Synaptophysin forms a hexameric complex containing 12 copies of the v-SNARE VAMP2. These v-SNAREs assemble into SNAREpins as ready-release vesicles are formed in a fully-defined cell-free system, and do so in two equal waves organized by oligomerization of the Ca++ sensor Synaptotagmin. In the absence of Synaptophysin, two waves are also observed, but the number of SNAREpins in each varies widely. We suggest that a single Synaptophysin hexamer in each vesicle symmetrically organizes 6 pairs of peripheral and central SNAREpins, the latter being directly bound to the Synaptotagmin ring. This gives rise to the symmetrical ring-like arrangement of densities observed by cryo-EM tomography under each synaptic vesicle (1, 2).

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↗

bioRxiv2023

Unraveling cellular complexity with unlimited multiplexed super-resolution imaging

Mapping the intricate spatial relationships between the many different molecules inside a cell is essential to understanding cellular functions in all their complexity. Super-resolution fluorescence microscopy offers the required spatial resolution but struggles to reveal more than four different targets simultaneously. Exchanging labels in subsequent imaging rounds for multiplexed imaging extends this number but is limited by its low throughput. Here we present a novel imaging method for rapid multiplexed super-resolution microscopy of a nearly unlimited number of molecular targets by leveraging fluorogenic labeling in conjunction with Transient Adapter-mediated switching for high-throughput DNA-PAINT (FLASH-PAINT). We demonstrate the cell biological versatility of FLASH-PAINT in mammalian cells in four applications: i) mapping nine proteins in a single mammalian cell, ii) elucidating the functional organization of primary cilia by nine-target imaging, iii) revealing the changes in proximity of twelve different targets in unperturbed and dissociated Golgi stacks and iv) investigating inter-organelle contacts at 3D super-resolution.

cell biology↗

bioRxiv2023

Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer

Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components/properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP-lipid complexes directly from intact and customizable lipid membranes. Both the lipid composition and membrane properties (such as curvature, tension, fluidity) of these bilayers can be precisely customized to a target membrane. Subsequent direct nMS analysis of these intact proteo-lipid vesicles can yield the oligomeric states of the embedded IMPs, identify bound lipids, and determine the membrane properties that can regulate the observed IMP-lipid organization. Applying this, we show how lipid binding regulates neurotransmitter release and how membrane composition regulates the functional oligomeric state of a transporter.

biophysics↗

bioRxiv2022

All-optical visualization of specific molecules in the ultrastructural context of brain tissue

Understanding the molecular anatomy and neural connectivity of the brain requires imaging technologies that can map the 3D nanoscale distribution of specific proteins in the context of brain ultrastructure. Light and electron microscopy (EM) enable visualization of either specific labels or anatomical ultrastructure, but combining molecular specificity with anatomical context is challenging. Here, we present pan-Expansion Microscopy of tissue (pan-ExM-t), an all-optical mouse brain imaging method that combines [~]24-fold linear expansion of biological samples with fluorescent pan-staining of protein densities (providing EM-like ultrastructural context), and immunolabeling of protein targets (for molecular imaging). We demonstrate the versatility of this approach by imaging the established synaptic markers Homer1, Bassoon, PSD-95, Synaptophysin, the astrocytic protein GFAP, myelin basic protein (MBP), and anti-GFP antibodies in dissociated neuron cultures and mouse brain tissue sections. pan-ExM-t reveals these markers in the context of ultrastructural features such as pre and postsynaptic densities, 3D nanoarchitecture of neuropil, and the fine structures of cellular organelles. pan-ExM-t is adoptable in any neurobiological laboratory with access to a confocal microscope and has therefore broad applicability in the research community. HighlightsO_LIpan-ExM-t visualizes proteins in the context of synaptic ultrastructure C_LIO_LILipid labeling in pan-ExM-t reveals organellar and cellular membranes C_LIO_LIAll-optical, easily accessible alternative to correlative light/electron microscopy C_LIO_LIHigh potential for high throughput connectomics studies C_LI

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↗

bioRxiv2021

Synaptotagmin rings as high sensitivity regulators of synaptic vesicle docking and fusion

Synchronous release at neuronal synapses is accomplished by a machinery that senses calcium influx and fuses the synaptic vesicle and plasma membranes to release neurotransmitters. Previous studies suggested the calcium sensor Synaptotagmin (Syt) is a facilitator of vesicle docking and both a facilitator and inhibitor of fusion. On phospholipid monolayers, the Syt C2AB domain spontaneously oligomerized into rings that are disassembled by Ca2+, suggesting Syt rings may clamp fusion as membrane-separating "washers" until Ca2+-mediated disassembly triggers fusion and release (Wang et al., 2014). Here we combined mathematical modeling with experiment to measure mechanical properties of Syt rings and to test this mechanism. Consistent with experiment, the model quantitatively recapitulates observed Syt ring-induced dome and volcano shapes on phospholipid monolayers, and predicts rings are stabilized by anionic phospholipid bilayers or bulk solution with ATP. The selected ring conformation is highly sensitive to membrane composition and bulk ATP levels, a property that may regulate vesicle docking and fusion in ATP-rich synaptic terminals. We find the Syt molecules hosted by a synaptic vesicle oligomerize into a halo, unbound from the vesicle, but in proximity to sufficiently PIP2-rich plasma membrane (PM) domains the PM-bound trans Syt ring conformation is preferred. Thus, the Syt halo serves as landing gear for spatially directed docking at PIP2-rich sites that define the active zones of exocytotic release, positioning the Syt ring to clamp fusion and await calcium. Our results suggest the Syt ring is both a Ca2+-sensitive fusion clamp and a high-fidelity sensor for directed docking. SignificanceSynchronous neurotransmitter release relies on directed docking of synaptic vesicles at active zones in axon terminals, where calcium influx activates membrane fusion and release. In vitro, the calcium sensor Synaptotagmin oligomerizes into rings disassembled by calcium. Here, experiment and modeling suggest the Synaptotagmin molecules hosted by an undocked vesicle oligomerize into a tethered, unbound halo in ATP-rich synaptic terminals. The halo directs vesicle docking to PIP2-rich plasma membrane domains in active zones, where the trans-bound ring conformation is favored, interposed between the membranes to clamp fusion until calcium triggers ring disassembly and neurotransmitter release. The mechanism exploits the extreme sensitivity of Synaptotagmin ring binding preferences to solution and membrane composition, with ~15 -fold-enhanced sensitivity for rings of ~15 molecules.

neuroscience↗

bioRxiv2020

3D Adaptive Optical Nanoscopy for Thick Specimen Imaging at sub-50 nm Resolution

Understanding cellular organization demands the best possible spatial resolution in all three dimensions (3D). In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here, we have developed a nanoscope that, by utilizing an advanced adaptive optics strategy, achieves sub-50 nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue.

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↗

bioRxiv2020

Vesicle capture by discrete self-assembled clusters of membrane-bound Munc13

Munc13 is a large banana-shaped soluble protein that is involved in the regulation of synaptic vesicle docking and fusion. Recent studies suggested that multiple copies of Munc13 form nanoassemblies in active zones of neurons. However, it is not known if such clustering is an inherent self-assembly property of Munc13 or whether Munc13 clusters indirectly by multivalent binding to synaptic vesicles or specific plasma membrane domains at docking sites in the active zone. The functional significance of putative Munc13 clustering is also unknown. Here we report that nano-clustering is an inherent property of Munc13, and is indeed required for vesicle binding to bilayers containing Munc13. Pure Munc13 reconstituted onto supported lipid bilayers assembled into clusters containing from 2 to [~]20 copies as revealed by a combination of quantitative TIRF microscopy and step-wise photobleaching. Surprisingly, only clusters a minimum of 6 copies of Munc13 were capable of efficiently capturing and retaining small unilamellar vesicles. The C-terminal C2C domain of Munc13 is not required for Munc13 clustering, but is required for efficient vesicle capture.

biochemistry↗

bioRxiv2020

TANGO1 membrane helices create a lipid diffusion barrier at curved membranes

We have previously shown TANGO1 organises membranes at the interface of the endoplasmic reticulum (ER) and ERGIC/Golgi (Raote et al., 2018). TANGO1 corrals retrograde membranes at ER exit sites to create an export conduit. Here the retrograde membrane is, in itself, an anterograde carrier. This mode of forward transport necessitates a mechanism to prevent membrane mixing between ER and the retrograde membrane. TANGO1 has an unusual membrane helix organisation, composed of one membrane-spanning helix (TM) and another that penetrates the inner leaflet (IM). We have reconstituted these membrane helices in model membranes and shown that TM and IM together reduce the flow of lipids at a region of defined shape. We have also shown that the helices align TANGO1 around an ER exit site. We suggest this is a mechanism to prevent membrane mixing during TANGO1-mediated transfer of bulky secretory cargos from the ER to the ERGIC/Golgi via a tunnel.

cell biology↗

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

Nanoscale subcellular architecture revealed by multicolor 3D salvaged fluorescence imaging

Combining the molecular specificity of fluorescent probes with three-dimensional (3D) imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a super-resolution light microscope that enables simultaneous multicolor imaging of whole mammalian cells at ~20 nm 3D resolution. We show its power for cell biology research with fluorescence images that resolved the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy.\n\nOne Sentence SummaryComplex cellular structures previously only resolved by electron microscopy can now be imaged in multiple colors by 4Pi-SMS.

biophysics↗

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↗