Contact

  • Academic
    jleitz@stanford.edu
    University - Academic staff Department: Department of Molecular and Cellular Physiology Position: Basic Life Research Scientist

Additional Info

  • Mail Code: 5345

All Publications

  • Structure and topography of the synaptic V-ATPase-synaptophysin complex. Nature Wang, C., Jiang, W., Leitz, J., Yang, K., Esquivies, L., Wang, X., Shen, X., Held, R., Adams, D. J., Basta, T., Hampton, L., Jian, R., Jiang, L., Stowell, M. H., Baumeister, W., Guo, Q., Brunger, A. T. 2024

    Abstract

    Synaptic vesicles are organelles with a precisely defined protein and lipid composition1,2, yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here, we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains3. The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the protein gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters4,5. Synaptophysin6 and its paralogs synaptoporin7 and synaptogyrin8 belong to a family of abundant synaptic vesicle proteins whose function is still unclear. We performed structural and functional studies of synaptophysin knockout mice, confirming the identity of synaptophysin as an interaction partner with the V-ATPase. Although there is little change in the conformation of the V-ATPase upon interaction with synaptophysin, the presence of synaptophysin in synaptic vesicles profoundly affects the copy number of V-ATPases. This effect on the topography of synaptic vesicles suggests that synaptophysin assists in their biogenesis. In support of this model, we observed that synaptophysin knockout mice exhibit severe seizure susceptibility, suggesting an imbalance of neurotransmitter release as a physiological consequence of the absence of synaptophysin.

    View details for DOI 10.1038/s41586-024-07610-x

    View details for PubMedID 38838737

  • Beyond the MUN domain, Munc13 controls priming and depriming of synaptic vesicles. Cell reports Leitz, J., Wang, C., Esquivies, L., Pfuetzner, R. A., Peters, J. J., Couoh-Cardel, S., Wang, A. L., Brunger, A. T. 2024; 43 (5): 114026

    Abstract

    Synaptic vesicle docking and priming are dynamic processes. At the molecular level, SNAREs (soluble NSF attachment protein receptors), synaptotagmins, and other factors are critical for Ca2+-triggered vesicle exocytosis, while disassembly factors, including NSF (N-ethylmaleimide-sensitive factor) and α-SNAP (soluble NSF attachment protein), disassemble and recycle SNAREs and antagonize fusion under some conditions. Here, we introduce a hybrid fusion assay that uses synaptic vesicles isolated from mouse brains and synthetic plasma membrane mimics. We included Munc18, Munc13, complexin, NSF, α-SNAP, and an ATP-regeneration system and maintained them continuously-as in the neuron-to investigate how these opposing processes yield fusogenic synaptic vesicles. In this setting, synaptic vesicle association is reversible, and the ATP-regeneration system produces the most synchronous Ca2+-triggered fusion, suggesting that disassembly factors perform quality control at the early stages of synaptic vesicle association to establish a highly fusogenic state. We uncovered a functional role for Munc13 ancillary to the MUN domain that alleviates an α-SNAP-dependent inhibition of Ca2+-triggered fusion.

    View details for DOI 10.1016/j.celrep.2024.114026

    View details for PubMedID 38809756

  • A new method for isolation and purification of fusion-competent inhibitory synaptic vesicles. Current research in physiology Gopal, N., Leitz, J., Wang, C., Esquivies, L., Pfuetzner, R. A., Brunger, A. T. 2024; 7: 100121

    Abstract

    Synaptic vesicles specific to inhibitory GABA-releasing neurons are critical for regulating neuronal excitability. To study the specific molecular composition, architecture, and function of inhibitory synaptic vesicles, we have developed a new method to isolate and purify GABA synaptic vesicles from mouse brains. GABA synaptic vesicles were immunoisolated from mouse brain tissue using an engineered fragment antigen-binding region (Fab) against the vesicular GABA transporter (vGAT) and purified. Western blot analysis confirmed that the GABA synaptic vesicles were specifically enriched for vGAT and largely depleted of contaminants from other synaptic vesicle types, such as vesicular glutamate transporter (vGLUT1), and other cellular organelles. This degree of purity was achieved despite the relatively low abundance of vGAT vesicles compared to the total synaptic vesicle pool in mammalian brains. Cryo-electron microscopy images of these isolated GABA synaptic vesicles revealed intact morphology with circular shape and protruding proteinaceous densities. The GABA synaptic vesicles are functional, as assessed by a hybrid (ex vivo/in vitro) vesicle fusion assay, and they undergo synchronized fusion with synthetic plasma membrane mimic vesicles in response to Ca2+-triggering, but, as a negative control, not to Mg2+-triggering. Our immunoisolation method could also be applied to other types of vesicles.

    View details for DOI 10.1016/j.crphys.2024.100121

    View details for PubMedID 38572021

    View details for PubMedCentralID PMC10990708

  • Neutral lysophosphatidylcholine mediates alpha-synuclein-induced synaptic vesicle clustering. Proceedings of the National Academy of Sciences of the United States of America Lai, Y., Zhao, C., Tian, Z., Wang, C., Fan, J., Hu, X., Tu, J., Li, T., Leitz, J., Pfuetzner, R. A., Liu, Z., Zhang, S., Su, Z., Burre, J., Li, D., Sudhof, T. C., Zhu, Z., Liu, C., Brunger, A. T., Diao, J. 2023; 120 (44): e2310174120

    Abstract

    alpha-synuclein (alpha-Syn) is a presynaptic protein that is involved in Parkinson's and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that alpha-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of alpha-Syn with anionic phospholipids. Here, we report that alpha-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates alpha-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of alpha-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the alpha-Syn-lysoPC interaction may play a role in alpha-Syn function.

    View details for DOI 10.1073/pnas.2310174120

    View details for PubMedID 37883437

  • The core complex of the Ca2+-triggered presynaptic fusion machinery. Journal of molecular biology Brunger, A. T., Leitz, J. 2022: 167853

    Abstract

    Synaptic neurotransmitter release is mediated by an orchestra of presynaptic proteins that precisely control and trigger fusion between synaptic vesicles and the neuron terminal at the active zone upon an action potential. Critical to this process are the neuronal SNAREs (Soluble N-ethylmaleimide sensitive factor Attachment protein REceptor), the Ca2+-sensor synaptotagmin, the activator/regulator complexin, and other factors. Here, we review the interactions between the SNARE complex and synaptotagmin, with focus on the so-called primary interface between synaptotagmin and the SNARE complex that has been validated in terms of its physiological relevance. We discuss several other but less validated interfaces as well, including the so-called tripartite interface, and we discuss the pros and cons for these possible alternative interfaces. We also present new molecular dynamics simulations of the tripartite interface and new data of an inhibitor of the primary interface in a reconstituted system of synaptic vesicle fusion.

    View details for DOI 10.1016/j.jmb.2022.167853

    View details for PubMedID 36243149

  • A feature-guided, focused 3D signal permutation method for subtomogram averaging. Journal of structural biology Jacob Peters, J., Leitz, J., Guo, Q., Beck, F., Baumeister, W., Brunger, A. T. 2022: 107851

    Abstract

    Advances in electron microscope instrumentation, cryo-electron tomography data collection, and subtomogram averaging have allowed for the in-situ visualization of molecules and their complexes in their native environment. Current data processing pipelines commonly extract subtomograms as a cubic subvolume with the key assumption that the selected object of interest is discrete from its surroundings. However, in instances when the object is in its native environment, surrounding densities may negatively affect the subsequent alignment and refinement processes, leading to loss of information due to misalignment. For example, the strong densities from surrounding membranes may dominate the alignment process for membrane proteins. Here, we developed methods for feature-guided subtomogram alignment and 3D signal permutation for subtomogram averaging. Our 3D signal permutation method randomizes and filters voxels outside a mask of any shape and blurs the boundary of the mask that encapsulates the object of interest. The randomization preserves global statistical properties such as mean density and standard deviation of voxel density values, effectively producing a featureless background surrounding the object of interest. This signal permutation process can be repeatedly applied with intervening alignments of the 3D signal-permuted subvolumes, recentering of the mask, and optional adjustments of the shape of the mask. We have implemented these methods in a new processing pipeline which starts from tomograms, contains feature-guided subtomogram extraction and alignment, 3D signal-permutation, and subtomogram visualization tools. As an example, feature-guided alignment and 3D signal permutation leads to improved subtomogram average maps for a dataset of synaptic protein complexes in their native environment.

    View details for DOI 10.1016/j.jsb.2022.107851

    View details for PubMedID 35346811

  • Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides. Nature Lai, Y., Fois, G., Flores, J. R., Tuvim, M. J., Zhou, Q., Yang, K., Leitz, J., Peters, J., Zhang, Y., Pfuetzner, R. A., Esquivies, L., Jones, P., Frick, M., Dickey, B. F., Brunger, A. T. 2022

    Abstract

    Membrane fusion triggered by Ca2+ is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes1-4. For neurotransmitter release, the Ca2+ sensitivity is introduced by interactions between the Ca2+ sensor synaptotagmin and the SNARE complex5, and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion6. Disruption of Ca2+-triggered membrane fusion by a pharmacological agent would have therapeutic value for mucus hypersecretion as it is the major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, chronic obstructive pulmonary disease and cystic fibrosis7-11. Here we designed a hydrocarbon-stapled peptide that specifically disrupts Ca2+-triggered membrane fusion by interfering with the so-called primary interface between the neuronal SNARE complex and the Ca2+-binding C2B domain of synaptotagmin-1. In reconstituted systems with these neuronal synaptic proteins or with their airway homologues syntaxin-3, SNAP-23, VAMP8, synaptotagmin-2, along with Munc13-2 and Munc18-2, the stapled peptide strongly suppressed Ca2+-triggered fusion at physiological Ca2+ concentrations. Conjugation of cell-penetrating peptides to the stapled peptide resulted in efficient delivery into cultured human airway epithelial cells and mouse airway epithelium, where it markedly and specifically reduced stimulated mucin secretion in both systems, and substantially attenuated mucus occlusion of mouse airways. Taken together, peptides that disrupt Ca2+-triggered membrane fusion may enable the therapeutic modulation of mucin secretory pathways.

    View details for DOI 10.1038/s41586-022-04543-1

    View details for PubMedID 35322233

  • Screening of Hydrocarbon-Stapled Peptides for Inhibition of Calcium-Triggered Exocytosis. Frontiers in pharmacology Lai, Y., Tuvim, M. J., Leitz, J., Peters, J., Pfuetzner, R. A., Esquivies, L., Zhou, Q., Czako, B., Cross, J. B., Jones, P., Dickey, B. F., Brunger, A. T. 2022; 13: 891041

    Abstract

    The so-called primary interface between the SNARE complex and synaptotagmin-1 (Syt1) is essential for Ca2+-triggered neurotransmitter release in neuronal synapses. The interacting residues of the primary interface are conserved across different species for synaptotagmins (Syt1, Syt2, Syt9), SNAP-25, and syntaxin-1A homologs involved in fast synchronous release. This Ca2+-independent interface forms prior to Ca2+-triggering and plays a role in synaptic vesicle priming. This primary interface is also conserved in the fusion machinery that is responsible for mucin granule membrane fusion. Ca2+-stimulated mucin secretion is mediated by the SNAREs syntaxin-3, SNAP-23, VAMP8, Syt2, and other proteins. Here, we designed and screened a series of hydrocarbon-stapled peptides consisting of SNAP-25 fragments that included some of the key residues involved in the primary interface as observed in high-resolution crystal structures. We selected a subset of four stapled peptides that were highly alpha-helical as assessed by circular dichroism and that inhibited both Ca2+-independent and Ca2+-triggered ensemble lipid-mixing with neuronal SNAREs and Syt1. In a single-vesicle content-mixing assay with reconstituted neuronal SNAREs and Syt1 or with reconstituted airway SNAREs and Syt2, the selected peptides also suppressed Ca2+-triggered fusion. Taken together, hydrocarbon-stapled peptides that interfere with the primary interface consequently inhibit Ca2+-triggered exocytosis. Our inhibitor screen suggests that these compounds may be useful to combat mucus hypersecretion, which is a major cause of airway obstruction in the pathophysiology of COPD, asthma, and cystic fibrosis.

    View details for DOI 10.3389/fphar.2022.891041

    View details for PubMedID 35814209

  • Molecular Characterization of AMPA-Receptor-Containing Vesicles. Frontiers in molecular neuroscience Peters, J. J., Leitz, J., Oses-Prieto, J. A., Burlingame, A. L., Brunger, A. T. 2021; 14: 754631

    Abstract

    Regulated delivery of AMPA receptors (AMPARs) to the postsynaptic membrane is an essential step in synaptic strength modification, and in particular, long-term potentiation (LTP). While LTP has been extensively studied using electrophysiology and light microscopy, several questions regarding the molecular mechanisms of AMPAR delivery via trafficking vesicles remain outstanding, including the gross molecular make up of AMPAR trafficking organelles and identification and location of calcium sensors required for SNARE complex-dependent membrane fusion of such trafficking vesicles with the plasma membrane. Here, we isolated AMPA-containing vesicles (ACVs) from whole mouse brains via immunoisolation and characterized them using immunoelectron microscopy, immunoblotting, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified several proteins on ACVs that were previously found to play a role in AMPAR trafficking, including synaptobrevin-2, Rabs, the SM protein Munc18-1, the calcium-sensor synaptotagmin-1, as well as several new candidates, including synaptophysin and synaptogyrin on ACV membranes. Additionally, we identified two populations of ACVs based on size and molecular composition: small-diameter, synaptobrevin-2- and GluA1-containing ACVs, and larger transferrin- receptor-, GluA1-, GluA2-, and GluA3-containing ACVs. The small-diameter population of ACVs may represent a fusion-capable population of vesicles due to the presence of synaptobrevin-2. Because the fusion of ACVs may be a requisite of LTP, this population could represent trafficking vesicles related to LTP.

    View details for DOI 10.3389/fnmol.2021.754631

    View details for PubMedID 34720876

    View details for PubMedCentralID PMC8554035

  • The pre-synaptic fusion machinery. Current opinion in structural biology Brunger, A. T., Choi, U. B., Lai, Y., Leitz, J., White, K. I., Zhou, Q. 2019; 54: 179–88

    Abstract

    Here, we review recent insights into the neuronal presynaptic fusion machinery that releases neurotransmitter molecules into the synaptic cleft upon stimulation. The structure of the pre-fusion state of the SNARE/complexin-1/synaptotagmin-1 synaptic protein complex suggests a new model for the initiation of fast Ca2+-triggered membrane fusion. Functional studies have revealed roles of the essential factors Munc18 and Munc13, demonstrating that a part of their function involves the proper assembly of synaptic protein complexes. Near-atomic resolution structures of the NSF/alphaSNAP/SNARE complex provide first glimpses of the molecular machinery that disassembles the SNARE complex during the synaptic vesicle cycle. These structures show how this machinery captures the SNARE substrate and provide clues as to a possible processing mechanism.

    View details for PubMedID 30986753

  • The pre-synaptic fusion machinery CURRENT OPINION IN STRUCTURAL BIOLOGY Brunger, A. T., Choi, U. B., Lai, Y., Leitz, J., White, K., Zhou, Q. 2019; 54: 179–88

    View details for DOI 10.1016/j.sbi.2019.03.007

    View details for Web of Science ID 000473554100023

  • Ca2+-Triggered Synaptic Vesicle Fusion Initiated by Release of Inhibition. Trends in cell biology Brunger, A. T., Leitz, J. n., Zhou, Q. n., Choi, U. B., Lai, Y. n. 2018

    Abstract

    Recent structural and functional studies of the synaptic vesicle fusion machinery suggest an inhibited tripartite complex consisting of neuronal soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), synaptotagmin, and complexin prior to Ca2+-triggered synaptic vesicle fusion. We speculate that Ca2+-triggered fusion commences with the release of inhibition by Ca2+ binding to synaptotagmin C2 domains. Subsequently, fusion is assisted by SNARE complex zippering and by active membrane remodeling properties of synaptotagmin. This additional, inhibitory role of synaptotagmin may be a general principle since other recent studies suggest that Ca2+ binding to extended synaptotagmin C2 domains enables lipid transport by releasing an inhibited state of the system, and that Munc13 may nominally be in an inhibited state, which is released upon Ca2+ binding to one of its C2 domains.

    View details for PubMedID 29706534

  • Molecular Mechanisms of Fast Neurotransmitter Release ANNUAL REVIEW OF BIOPHYSICS, VOL 47 Brunger, A. T., Choi, U. B., Lai, Y., Leitz, J., Zhou, Q., Dill, K. A. 2018; 47: 469–97

    Abstract

    This review summarizes current knowledge of synaptic proteins that are central to synaptic vesicle fusion in presynaptic active zones, including SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), synaptotagmin, complexin, Munc18 (mammalian uncoordinated-18), and Munc13 (mammalian uncoordinated-13), and highlights recent insights in the cooperation of these proteins for neurotransmitter release. Structural and functional studies of the synaptic fusion machinery suggest new molecular models of synaptic vesicle priming and Ca2+-triggered fusion. These studies will be a stepping-stone toward answering the question of how the synaptic vesicle fusion machinery achieves such high speed and sensitivity.

    View details for PubMedID 29792815

  • Molecular Mechanisms of Synaptic Vesicle Priming by Munc13 and Munc18 NEURON Lai, Y., Choi, U. B., Leitz, J., Rhee, H., Lee, C., Altas, B., Zhao, M., Pfuetzner, R. A., Wang, A. L., Brose, N., Rhee, J., Brunger, A. T. 2017; 95 (3): 591-+

    Abstract

    Munc13 catalyzes the transit of syntaxin from a closed complex with Munc18 into the ternary SNARE complex. Here we report a new function of Munc13, independent of Munc18: it promotes the proper syntaxin/synaptobrevin subconfiguration during assembly of the ternary SNARE complex. In cooperation with Munc18, Munc13 additionally ensures the proper syntaxin/SNAP-25 subconfiguration. In a reconstituted fusion assay with SNAREs, complexin, and synaptotagmin, inclusion of both Munc13 and Munc18 quadruples the Ca2+-triggered amplitude and achieves Ca2+ sensitivity at near-physiological concentrations. In Munc13-1/2 double-knockout neurons, expression of a constitutively open mutant of syntaxin could only minimally restore neurotransmitter release relative to Munc13-1 rescue. Together, the physiological functions of Munc13 may be related to regulation of proper SNARE complex assembly.

    View details for PubMedID 28772123

    View details for PubMedCentralID PMC5747255

  • C-terminal domain of mammalian complexin-1 localizes to highly curved membranes. Proceedings of the National Academy of Sciences of the United States of America Gong, J., Lai, Y., Li, X., Wang, M., Leitz, J., Hu, Y., Zhang, Y., Choi, U. B., Cipriano, D., Pfuetzner, R. A., Südhof, T. C., Yang, X., Brunger, A. T., Diao, J. 2016

    Abstract

    In presynaptic nerve terminals, complexin regulates spontaneous "mini" neurotransmitter release and activates Ca(2+)-triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using single-particle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca(2+)-independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents.

    View details for PubMedID 27821736

  • Intrinsic and membrane-facilitated alpha-synuclein oligomerization revealed by label-free detection through solid-state nanopores SCIENTIFIC REPORTS Hu, R., Diao, J., Li, J., Tang, Z., Li, X., Leitz, J., Long, J., Liu, J., Yu, D., Zhao, Q. 2016; 6

    Abstract

    α-Synuclein (α-Syn) is an abundant cytosolic protein involved in the release of neurotransmitters in presynaptic terminal and its aberrant aggregation is found to be associated with Parkinson's disease. Recent study suggests that the oligomers formed at the initial oligomerization stage may be the root cause of cytotoxicity. While characterizing this stage is challenging due to the inherent difficulties in studying heterogeneous and transient systems by conventional biochemical technology. Here we use solid-state nanopores to study the time-dependent kinetics of α-Syn oligomerization through a label-free and single molecule approach. A tween 20 coating method is developed to inhibit non-specific adsorption between α-Syn and nanopore surface to ensure successful and continuous detection of α-Syn translocation. We identify four types of oligomers formed in oligomerization stage and find an underlying consumption mechanism that the formation of large oligomers consumes small oligomers. Furthermore, the effect of lipid membrane on oligomerization of α-Syn is also investigated and the results show that 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS) small unilamellar vesicles (SUVs) dramatically enhances the aggregation rate of α-Syn while do not alter the aggregation pathway.

    View details for DOI 10.1038/srep20776

    View details for Web of Science ID 000369898800002

    View details for PubMedID 26865505