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Morphologies of Synaptic Protein Membrane Fusion Interfaces

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Morphologies of Synaptic Protein Membrane Fusion Interfaces Morphologies of synaptic protein membrane fusion interfaces Preeti Gipsona,b,c,d,e, Yoshiyuki Fukudaf, Radostin Danevf, Ying Laia,b,c,d,e, Dong-Hua Chenc, Wolfgang Baumeisterf, and Axel T. Brungera,b,c,d,e,1 aDepartment of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305; bDepartment of Neurology and Neurological Sciences, Stanford University, Stanford, CA 94305; cDepartment of Structural Biology, Stanford University, Stanford, CA 94305; dDepartment of Photon Science, Stanford University, Stanford, CA 94305; eHoward Hughes Medical Institute, Stanford University, Stanford, CA 94305; and fDepartment of Molecular Structural Biology, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany Contributed by Axel T. Brunger, June 22, 2017 (sent for review May 23, 2017; reviewed by Abraham J. Koster and William T. Wickner) Neurotransmitter release is orchestrated by synaptic proteins, such Although high-resolution structures are available for some of the as SNAREs, synaptotagmin, and complexin, but the molecular key complexes of soluble fragments of synaptic proteins (6, 10, 13, mechanisms remain unclear. We visualized functionally active synap- 14), and the process has been reconstituted (11, 15, 16), the associ- tic proteins reconstituted into proteoliposomes and their interactions ationofthesecomplexeswiththesynapticandplasmamembranes in a native membrane environment by electron cryotomography remains unclear. Previous cryo-EM projection studies revealed only with a Volta phase plate for improved resolvability. The images membrane shapes but no protein densities (16–18). All synaptic revealed individual synaptic proteins and synaptic protein complex proteins are relatively small (ranging from 45 to 200 kDa) and form densities at prefusion contact sites between membranes. We ob- heterogeneous and dynamic complexes, posing challenges for imag- served distinct morphologies of individual synaptic proteins and ing by conventional electron tomography. Moreover, the complex their complexes. The minimal system, consisting of neuronal SNAREs nature of cellular synapses consisting of dynamic and dense networks and synaptotagmin-1, produced point and long-contact prefusion of protein–protein and protein–membrane interactions makes it dif- states. Morphologies and populations of these states changed as ficult to study the synaptic fusion protein machinery in vivo. There- the regulatory factors complexin and Munc13 were added. Com- fore, we used a bottom-up approach where we reconstituted synaptic plexin increased the membrane separation, along with a higher pro- proteins into liposomes in a stepwise fashion: Synaptobrevin-2 and pensity of point contacts. Further inclusion of the priming factor Syt1 were reconstituted into liposomes with a defined synthetic lipid Munc13 exclusively restricted prefusion states to point contacts, composition that mimics synaptic vesicles (referred to as “SV vesi- + all of which efficiently fused upon Ca2 triggering. We conclude cles”), and syntaxin-1A and SNAP-25A were reconstituted into li- that synaptic proteins have evolved to limit possible contact site posomes with a lipid composition that mimics the plasma membrane + assemblies and morphologies to those that promote fast Ca2 - obtained from lipid brain extracts (referred to as “PM vesicles”)(19). triggered release. To visualize the morphology of membrane-associated synaptic proteins and contact sites between membranes of these function- synaptic vesicle fusion | neurotransmitter release | Volta phase plate | ally active proteoliposomes, we used electron cryotomography electron cryotomography | Munc13 (cryo-ET), which allows the study of membranes (20) and he process of vesicle trafficking is central for transporting Significance Tmaterials both inside and outside of cells and is essential for maintaining cellular homeostasis (1, 2). Synaptic vesicle fusion Neurotransmitter release occurs upon fusion of synaptic vesicles releases neurotransmitter molecules into the synaptic cleft upon an with the plasma membrane, and it is orchestrated by synaptic action potential—afast(<5 ms) and highly regulated process. The proteins, including SNAREs, synaptotagmin, complexin, and other neuronal SNARE proteins synaptobrevin-2/VAMP2 and syntaxin- factors. The system is exquisitely fine-tuned to increase the 1A are anchored in the synaptic vesicle and plasma membranes, probability of membrane fusion by orders of magnitude upon respectively, whereas SNAP-25 is peripherally associated with Ca2+ bindingtoaCa2+ sensor, such as synaptotagmin. Although the plasma membrane. The N- to C-terminal zippering of the crystal structures are available for some of the key complexes of trans SNARE complex between synaptic and plasma mem- soluble fragments of synaptic proteins, and the process has been branes provides the force for membrane juxtaposition and fusion reconstituted, the association of these complexes with the syn- + (3), but SNAREs alone are not Ca2 -sensitive. Synaptotagmins aptic and plasma membranes remains unclear. We visualized + are membrane-tethered proteins containing two Ca2 -binding functionally active synaptic proteins reconstituted into proteoli- C2 domains, and a subset of the 16 isoforms of mammalian syn- posomes and their interactions in a native membrane environ- + aptotagmins act as Ca2 sensors for neurotransmitter release: e.g., ment by electron cryotomography with a Volta phase plate for + synaptotagmin-1 (Syt1) is the Ca2 sensor for evoked synchronous improved resolvability. neurotransmitter release (4). The system is exquisitely fine-tuned to increase the probability of fusion between synaptic vesicles and Author contributions: P.G. and A.T.B. designed research; P.G., Y.F., R.D., Y.L., and D.-H.C. 2+ performed research; P.G., Y.F., R.D., Y.L., W.B., and A.T.B. analyzed data; and P.G. and the plasma membrane by orders of magnitude upon Ca binding A.T.B. wrote the paper with input from all authors. to Syt1. Synaptotagmins simultaneously interact with anionic Reviewers: A.J.K., Leiden University Medical Center; and W.T.W., Geisel School of Medi- phospholipid membranes (5) and the neuronal SNARE complex cine at Dartmouth College. (6, 7). The SNARE complex also interacts with complexin (Cpx), a The authors declare no conflict of interest. small soluble protein that both activates evoked release and reg- Freely available online through the PNAS open access option. ulates spontaneous release (8, 9). Moreover, Cpx forms a tripartite Data deposition: Representative tomograms have been deposited in the Electron Micros- complex with the SNARE complex and Syt1 (10). This tripartite copy Data Bank (accession nos. EMD-8807–EMD-8811). complex is part of the prefusion, “primed” state of the synaptic See Commentary on page 8920. vesicle fusion machinery. Both Munc13 and Munc18 are impor- 1To whom correspondence should be addressed. Email: [email protected]. tant for priming, and, at the molecular level, they facilitate proper This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. SNARE complex assembly (11, 12). 1073/pnas.1708492114/-/DCSupplemental. 9110–9115 | PNAS | August 22, 2017 | vol. 114 | no. 34 www.pnas.org/cgi/doi/10.1073/pnas.1708492114 Downloaded by guest on September 24, 2021 membrane proteins under close to native conditions (21). How- protruding densities made it possible to uniquely identify the par- ever, low-dose imaging of radiation-sensitive biological samples ticular type of vesicle in mixtures of PM and SV vesicles. SEE COMMENTARY leads to poor contrast (that is, poor signal-to-noise ratio) of the We trapped prefusion protein–membrane contact sites by in- + images. The conventional method of artificially defocusing im- cubating a mixture of PM and SV vesicles in the absence of Ca2 proves the phase contrast of the images, but at the expense of for 1 min before freezing. Cryo-ET of the mixture revealed con- inducing contrast transfer function-related artifacts that have to be nected densities between many pairs of docked vesicles. These dealt with during image processing. This limitation becomes even contact sites are strong densities as seen in 2D slice views (gray left more important for imaging of the relatively small synaptic pro- subpanels in Fig. 2 B–D, Fig. S4,andMovie S1) and in isosurface teins. Introduction of defocus fundamentally limits the achievable 3D maps (Fig. 2 B–D, green surface subpanels). The isosurface resolution of EM images unless accurately corrected, but, in to- contour level of the 3D maps was adjusted such that the membrane mography, the contrast transfer function (CTF) correction is or membrane-associated proteins, such as Syt1, were visible and technically challenging due to the tilt-induced complexity in the matched the expected size. sample (22). Consequently, alignments become very sensitive to An analysis of tomograms with several hundred contact sites of + defocus artifacts exaggerating resolution loss in the original image the SV PM vesicle mixtures revealed a limited number of distinct when reconstructed in 3D, more so in small particles due to poor contact site morphologies. We characterized them based on the contact site length (Methods). The majority of the observed contact alignment features. To overcome the limitations of conventional ∼ cryo-ET, we used phase contrast imaging with the Volta phase sites are tight point contacts (site length 115 to 160 Å, 48%), along with smaller populations of loose point contacts (site length plate (VPP),
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