scholarly journals Trans-synaptic assemblies link synaptic vesicles and neuroreceptors

2021 ◽  
Vol 7 (10) ◽  
pp. eabe6204
Author(s):  
Antonio Martinez-Sanchez ◽  
Ulrike Laugks ◽  
Zdravko Kochovski ◽  
Christos Papantoniou ◽  
Luca Zinzula ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
De Novo ◽  
Electron Tomography ◽  
Synaptic Function ◽  
Coupled Processes ◽  
Cryo Electron Tomography ◽  
Tightly Coupled ◽  
Synaptic Complexes ◽  
Transient Interactions ◽  
Complex Signaling

Synaptic transmission is characterized by fast, tightly coupled processes and complex signaling pathways that require a precise protein organization, such as the previously reported nanodomain colocalization of pre- and postsynaptic proteins. Here, we used cryo–electron tomography to visualize synaptic complexes together with their native environment comprising interacting proteins and lipids on a 2- to 4-nm scale. Using template-free detection and classification, we showed that tripartite trans-synaptic assemblies (subcolumns) link synaptic vesicles to postsynaptic receptors and established that a particular displacement between directly interacting complexes characterizes subcolumns. Furthermore, we obtained de novo average structures of ionotropic glutamate receptors in their physiological composition, embedded in plasma membrane. These data support the hypothesis that synaptic function is carried by precisely organized trans-synaptic units. It provides a framework for further exploration of synaptic and other large molecular assemblies that link different cells or cellular regions and may require weak or transient interactions to exert their function.

scholarly journals Trans-synaptic assemblies link synaptic vesicles and neuroreceptors

2020 ◽  
Author(s):  
Antonio Martinez-Sanchez ◽  
Ulrike Laugks ◽  
Zdravko Kochovski ◽  
Christos Papantoniou ◽  
Wolfgang Baumeister ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Synaptic Vesicles ◽  
Plasma Membranes ◽  
Spatial Organization ◽  
De Novo ◽  
Electron Tomography ◽  
Synaptic Function ◽  
Synaptic Proteins ◽  
Coupled Processes ◽  
Tightly Coupled

AbstractSynaptic transmission is characterized by fast, tightly coupled processes and complex signaling path-ways that require a distinctly non-random spatial organization of their components. Nanoscale organization of synaptic proteins at glutamatergic synapses was suggested to regulate synaptic plasticity, the process underlying learning and memory. Specifically, direct colocalization of pre- and postsynaptic proteins implicated that the alignment of neurotransmitter release sites with neurotransmitter receptors enables maximal synaptic response. However, direct visualization and the mechanistic understanding of this alignment is lacking. Here we used cryo-electron tomography to visualize synaptic complexes in their native environment with the full complement of their interacting partners, synaptic vesicles and plasma membranes on 2-4 nanometer scale. The application of our recent template-free detection and classification procedure showed that tripartite trans-synaptic assemblies (subcolumns) link synaptic vesicles to postsynaptic receptors, and established that a particular displacement between directly interacting complexes characterizes subcolumns. Furthermore, we obtained de novo average structures of ionotropic glutamate receptors in their physiological composition, embedded in lipid membranes. The data presented support the hypothesis that synaptic function is carried by precisely organized trans-synaptic units. It complements superresolution findings and provides a framework for further exploration of synaptic and other large molecular assemblies that link different cells or cellular regions and may require weak or transient interactions to exert their function.

scholarly journals Distinct nanoscale calcium channel and synaptic vesicle topographies contribute to the diversity of synaptic function

2018 ◽  
Author(s):  
Nelson Rebola ◽  
Maria Reva ◽  
Tekla Kirizs ◽  
Miklos Szoboszlay ◽  
Gael Moneron ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
Reaction Diffusion ◽  
Structural Data ◽  
Synaptic Function ◽  
Differential Sensitivity ◽  
Functional Heterogeneity ◽  
Electron Microscopic ◽  
Voltage Gated Calcium Channels ◽  
Tightly Coupled ◽  
Strength And Plasticity

SUMMARYThe nanoscale topographical arrangement of voltage-gated calcium channels (VGCC) and synaptic vesicles (SVs) determines synaptic strength and plasticity, but whether distinct spatial distributions underpin diversity of synaptic function is unknown. We performed single bouton Ca2+ imaging, Ca2+ chelator competition, immunogold electron microscopic (EM) localization of VGCCs and the active zone (AZ) protein Munc13-1, at two cerebellar synapses. Unexpectedly, we found that weak synapses exhibited 3-fold more VGCCs than strong synapses, while the coupling distance was 5-fold longer. Reaction-diffusion modelling could explain both functional and structural data with two strikingly different nanotopographical motifs: strong synapses are composed of SVs that are tightly coupled (∼10 nm) to VGCC clusters, whereas at weak synapses VGCCs were excluded from the vicinity (∼50 nm) of docked vesicles. The distinct VGCC-SV topographical motifs also confer differential sensitivity to neuromodulation. Thus VGCC-SV arrangements are not canonical across CNS synapses and their diversity could underlie functional heterogeneity.

scholarly journals Routine Single Particle CryoEM Sample and Grid Characterization by Tomography

2017 ◽  
Author(s):  
Alex J. Noble ◽  
Venkata P. Dandey ◽  
Hui Wei ◽  
Julia Brasch ◽  
Jillian Chase ◽  
...  
Keyword(s):  
Single Particle ◽  
Particle Distribution ◽  
De Novo ◽  
Protein Denaturation ◽  
Electron Tomography ◽  
Particle Collection ◽  
Cryo Electron Microscopy ◽  
Cryo Electron Tomography ◽  
Air Water Interface ◽  
Particle Alignment

AbstractSingle particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are freely floating away from the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1,000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

scholarly journals The roles of a flagellar HSP40 ensuring rhythmic beating

2019 ◽  
Vol 30 (2) ◽  
pp. 228-241 ◽  
Author(s):  
Xiaoyan Zhu ◽  
Emiliya Poghosyan ◽  
Lenka Rezabkova ◽  
Bridget Mehall ◽  
Hitoshi Sakakibara ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Biochemical Analysis ◽  
De Novo ◽  
Electron Tomography ◽  
Donor Cell ◽  
Central Pair ◽  
Radial Spoke ◽  
Cryo Electron Tomography ◽  
Rigid Conformation ◽  
Mechanical Feedback

HSP40s are regarded as cochaperones, perpetually shuttling client polypeptides to HSP70s for refolding. However, many HSP40s that are central for disparate processes diverge from this paradigm. To elucidate the noncanonical mechanisms, we investigated HSP40 in the radial spoke (RS) complex in flagella. Disruption of the gene by the MRC1 transposon in Chlamydomonas resulted in jerky flagella. Traditional electron microscopy, cryo-electron tomography, and sub-tomogram analysis revealed RSs of various altered morphologies that, unexpectedly, differed between the two RS species. This indicates that HSP40 locks the RS into a functionally rigid conformation, facilitating its interactions with the adjacent central pair apparatus for transducing locally varied mechanical feedback, which permits rhythmic beating. Missing HSP40, like missing RSs, could be restored in a tip-to-base direction when HSP40 mutants fused with a HSP40 donor cell. However, without concomitant de novo RS assembly, the repair was exceedingly slow, suggesting HSP40/RS-coupled intraflagellar trafficking and assembly. Biochemical analysis and modeling uncovered spoke HSP40’s cochaperone traits. On the basis of our data, we propose that HSP40 accompanies its client RS precursor when traveling to the flagellar tip. Upon arrival, both refold in concert to assemble into the mature configuration. HSP40’s roles in chaperoning and structural maintenance shed new light on its versatility and flagellar biology.

scholarly journals Routine single particle CryoEM sample and grid characterization by tomography

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Alex J Noble ◽  
Venkata P Dandey ◽  
Hui Wei ◽  
Julia Brasch ◽  
Jillian Chase ◽  
...  
Keyword(s):  
Single Particle ◽  
Particle Distribution ◽  
De Novo ◽  
Protein Denaturation ◽  
Electron Tomography ◽  
Particle Collection ◽  
Cryo Electron Microscopy ◽  
Cryo Electron Tomography ◽  
Air Water Interface ◽  
Particle Alignment

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

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