Munc13 supports fusogenicity of non-docked vesicles at synapses with disrupted active zones

Tan, Chung-I; Nola, Giovanni de; Qiao, Claire; Imig, Cordelia; Born, Richard T.; Kaeser, Pascal S.

doi:10.7554/elife.79077

Cited by 10 publications

(12 citation statements)

References 65 publications

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“…2c-e) at levels similar to (PDZ-C2A-P) or above (Zn-C2B) those for RIM1α, indicating targeting to the active zone. As described before 26,34,63 , the active zone was disrupted in cQKO R+E neurons with very low levels of Munc13-1, and robust reductions in Ca V 2.1 and RIM-BP2. The postsynaptic marker PSD-95 was unaltered, and Liprin-α3 was increased by ∼30%, in agreement with previous work 34 and with an upstream function 22,23,25 .…”

Section: Resultssupporting

confidence: 60%

“…Removing both proteins at the same time disrupts both complexes and induces strong synapse assembly defects 69 . We propose that the sub-structuring of the active zone that we describe here accounts for the resilience of synapse and active zone assembly that is often observed in loss-of-function studies 12,26,34,63,69,82,86 . (c-q) Example STED images, average line profiles and quantification of the peak intensities of HA and PSD-95 (c-e), Munc13-1 (f-h), CaV2.1 (i-k), Liprin-α3 (l-n) and RIM-BP2 (o-q) at excitatory side-view synapses identified by Synaptophysin (Syp) and PSD-95.…”

Section: Discussionmentioning

confidence: 80%

“…Three 5-minute washes with PBS were performed between steps. Primary antibodies used were: rabbit anti-Liprin-α3 (A232, 1:500; homemade, knockout-verified for STED 20 ), rabbit anti-RIM (A58, 1:500, RRID: AB_887774, knockout-verified for STED 34,61 ), rabbit anti-CaV2.1 (A46, 1:500; RRID: AB_2619841, knockout-verified for STED 50 ), rabbit anti-Munc13-1 (A72, 1:500; RRID: AB_887733, knockout-verified for STED 63 ), rabbit anti-RIM-BP2 (A126, 1:500; RRID: AB_2619739), mouse anti-HA (A12, 1:500; RRID: AB_2565006), mouse anti-Synaptophysin (A100, 1:500; RRID: AB_887824), mouse anti-PSD-95 (A152, 1:500; RRID: AB_10698024), and mouse anti-Gephyrin (A8, 1:500; RRID: AB_2232546). Secondary antibodies used were: goat anti-rabbit Alexa Fluor 488 (S5; 1:500, RRID: AB_2576217), goat anti-mouse IgG1 Alexa Fluor 488 (S7; 1:500, RRID: AB_2535764), goat anti-mouse IgG1 Alexa Fluor 555 (S19, 1:500, RRID: AB_2535769), goat anti-mouse IgG2a Alexa Fluor 633 (S30, 1:500, RRID: AB_1500826), goat anti-guinea pig Alexa Fluor 405 (S51, 1:500, RRID: AB_2827755).…”

Section: Immunofluorescence Staining For Sted and Confocal Microscopymentioning

confidence: 99%

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Molecular definition of distinct active zone protein machineries for Ca²⁺channel clustering and synaptic vesicle priming

Emperador-Melero,

Andersen,

Metzbower

et al. 2023

Preprint

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SummaryAction potentials trigger neurotransmitter release with minimal delay. Active zones mediate this temporal precision by co-organizing primed vesicles with CaV2 Ca2+channels. The presumed model is that scaffolding proteins directly tether primed vesicles to CaV2s. We find that CaV2 clustering and vesicle priming are executed by separate machineries. At hippocampal synapses, CaV2 nanoclusters are positioned at variable distances from those of the priming protein Munc13. The active zone organizer RIM anchors both proteins, but distinct interaction motifs independently execute these functions. In heterologous cells, Liprin-α and RIM from co- assemblies that are separate from CaV2-organizing complexes upon co-transfection. At synapses, Liprin-α1-4 knockout impairs vesicle priming, but not CaV2 clustering. The cell adhesion protein PTPσ recruits Liprin-α, RIM and Munc13 into priming complexes without co- clustering of CaV2s. We conclude that active zones consist of distinct complexes to organize CaV2s and vesicle priming, and Liprin-α and PTPσ specifically support priming site assembly.

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Section: Resultssupporting

confidence: 60%

Section: Discussionmentioning

confidence: 80%

Section: Immunofluorescence Staining For Sted and Confocal Microscopymentioning

confidence: 99%

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Molecular definition of distinct active zone protein machineries for Ca²⁺channel clustering and synaptic vesicle priming

Emperador-Melero,

Andersen,

Metzbower

et al. 2023

Preprint

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“…However, whether changing properties within single Munc13- 1 NCs affects functions in docking and priming remains an open question of intense interest. Though reducing Munc13 content by knockout alters both processes (47, 50), Karlocai et al (2021) argue that the high docked vesicle occupancy at release sites (5, 33) suggests that varying Munc13-1 content per release site may principally control release probability at these sites, consistent with a potential effect on vesicle priming (33). Our findings are also consistent with previous work that suggests Schaffer collateral synapses onto CA1 interneurons have a high probability of release due, in part, to a larger readily releasable pool (35).…”

Section: Discussionmentioning

confidence: 99%

Differential nanoscale organization of excitatory synapses onto excitatory vs inhibitory neurons

Dharmasri,

Levy,

Blanpied

2023

Preprint

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A key feature of excitatory synapses is the existence of subsynaptic protein nanoclusters whose precise alignment across the cleft in a trans-synaptic nanocolumn influences the strength of synaptic transmission. However, whether nanocolumn properties vary between excitatory synapses functioning in different cellular contexts is unknown. We used a combination of confocal and DNA-PAINT super-resolution microscopy to directly compare the organization of shared scaffold proteins at two important excitatory synapses - those forming onto excitatory principal neurons (Ex→Ex synapses) and those forming onto parvalbumin-expressing interneurons (Ex→PV synapses). As in Ex→Ex synapses, we find that in Ex→PV synapses presynaptic Munc13-1 and postsynaptic PSD-95 both form nanoclusters that demonstrate alignment, underscoring synaptic nanostructure and the trans-synaptic nanocolumn as conserved organizational principles of excitatory synapses. Despite the general conservation of these features, we observed specific differences in the characteristics of pre- and postsynaptic Ex→PV nanostructure. Ex→PV synapses contained larger PSDs with fewer PSD-95 NCs when accounting for size than Ex→Ex synapses. Furthermore, the PSD-95 NCs were larger and denser. The identity of the postsynaptic cell also had a retrograde impact on Munc13-1 organization, as Ex→PV synapses hosted larger Munc13-1 puncta that contained less dense but larger and more numerous Munc13-1 NCs. Moreover, we measured the spatial variability of trans- synaptic alignment in these synapse types, revealing protein alignment in Ex→PV synapses over a distinct range of distances compared to Ex→Ex synapses. We conclude that while general principles of nanostructure and alignment are shared, cell-specific elements of nanodomain organization likely contribute to functional diversity of excitatory synapses. Understanding the rules of synapse nanodomain assembly, which themselves are cell-type specific, will be essential for illuminating brain network dynamics.

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“…For example, the readily releasable pool (RRP) often tightly correlates with the pool of docked vesicles (i.e. vesicles in the direct contact with AZ plasma membrane; Imig et al, 2014), but exceptions to this correlation have been also observed (Man et al, 2015; Wang et al, 2016; Tan et al, 2022). Further correlated biophysical-structural approaches are needed to address these questions.…”

Section: Introductionmentioning

confidence: 99%

Presynaptic cAMP-PKA-mediated potentiation induces reconfiguration of synaptic vesicle pools and channel-vesicle coupling at hippocampal mossy fiber boutons

Kim,

Okamoto,

Brose

et al. 2023

Preprint

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It is widely believed that information storage in neuronal circuits involves nanoscopic structural changes at synapses, resulting in the formation of synaptic engrams. However, direct evidence for this hypothesis is lacking. To test this conjecture, we combined chemical potentiation, functional analysis by paired pre-postsynaptic recordings, and structural analysis by electron microscopy (EM) and freeze-fracture replica labeling (FRL) at the hippocampal mossy fiber synapse, a key synapse in the trisynaptic circuit of the hippocampus. Biophysical analysis of synaptic transmission revealed that forskolin-induced chemical potentiation increased the readily releasable vesicle pool size (RRP) and vesicular release probability (Pr) by 146% and 49%, respectively. Structural analysis of mossy fiber synapses by EM and FRL demonstrated an increase in the number of vesicles close to the plasma membrane and the number of clusters of the priming protein Munc13-1, indicating an increase in the number of both docked and primed vesicles. Furthermore, FRL analysis revealed a significant reduction of the nearest neighbor distance (NND) between Munc13-1 and CaV2.1 Ca2+channels, suggesting reconfiguration of the channel-vesicle coupling nanotopography. Our results indicate that presynaptic plasticity is associated with structural reorganization of active zones (AZs). We propose that changes in potential nanoscopic organization at synaptic vesicle release sites may be correlates of learning and memory at a plastic central synapse.

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Munc13 supports fusogenicity of non-docked vesicles at synapses with disrupted active zones

Cited by 10 publications

References 65 publications

Molecular definition of distinct active zone protein machineries for Ca²⁺channel clustering and synaptic vesicle priming

Molecular definition of distinct active zone protein machineries for Ca²⁺channel clustering and synaptic vesicle priming

Differential nanoscale organization of excitatory synapses onto excitatory vs inhibitory neurons

Presynaptic cAMP-PKA-mediated potentiation induces reconfiguration of synaptic vesicle pools and channel-vesicle coupling at hippocampal mossy fiber boutons

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Munc13 supports fusogenicity of non-docked vesicles at synapses with disrupted active zones

Cited by 10 publications

References 65 publications

Molecular definition of distinct active zone protein machineries for Ca2+channel clustering and synaptic vesicle priming

Molecular definition of distinct active zone protein machineries for Ca2+channel clustering and synaptic vesicle priming

Differential nanoscale organization of excitatory synapses onto excitatory vs inhibitory neurons

Presynaptic cAMP-PKA-mediated potentiation induces reconfiguration of synaptic vesicle pools and channel-vesicle coupling at hippocampal mossy fiber boutons

Contact Info

Product

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Molecular definition of distinct active zone protein machineries for Ca²⁺channel clustering and synaptic vesicle priming

Molecular definition of distinct active zone protein machineries for Ca²⁺channel clustering and synaptic vesicle priming