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- Farsi, Zohreh, et al.
(författare)
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Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity
- 2018
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Ingår i: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling.
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| 2. |
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| 3. |
- Juranek, Judyta Karolina, et al.
(författare)
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Active zone protein expression changes at the key stages of cerebellar cortex neurogenesis in the rat
- 2013
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Ingår i: Acta Histochemica. - : Elsevier BV. - 0065-1281. ; 115:6, s. 616-625
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Tidskriftsartikel (refereegranskat)abstract
- Signal transduction and neurotransmitter release in the vertebrate central nervous system are confined to the structurally complex presynaptic electron dense projections called "active zones." Although the nature of these projections remains a mystery, genetic and biochemical work has provided evidence for the active zone (AZ) associated proteins i.e. Piccolo/Aczonin, Bassoon, RIM1/Unc10, Munc13/Unc13, Liprin-alpha/SYD2/Dliprin and ELKS/CAST/BRP and their specific molecular functions. It still remains unclear, however, what their precise contribution is to the AZ assembly. In our project, we studied in Wistar rats the temporal and spatial distribution of AZ proteins and their colocalization with Synaptophysin in the developing cerebellar cortex at key stages of cerebellum neurogenesis. Our study demonstrated that AZ proteins were already present at the very early stages of cerebellar neurogenesis and exhibited distinct spatial and temporal variations in immunoexpression throughout the course of the study. Colocalization analysis revealed that the colocalization pattern was time-dependent and different for each studied protein. The highest collective mean percentage of colocalization (>85%) was observed at postnatal day (PD) 5, followed by PD10 (>83%) and PD15 (>80%). The findings of our study shed light on AZ protein immunoexpression changes during cerebellar cortex neurogenesis and help frame a hypothetical model of AZ assembly. (C) 2013 Elsevier GmbH. All rights reserved.
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| 4. |
- Zhao, Ying, et al.
(författare)
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All SNAP25 molecules in the vesicle–plasma membrane contact zone change conformation during vesicle priming
- 2024
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences (PNAS). - 0027-8424 .- 1091-6490. ; 121:2
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Tidskriftsartikel (refereegranskat)abstract
- In neuronal cell types, vesicular exocytosis is governed by the SNARE (soluble NSF attachment receptor) complex consisting of synaptobrevin2, SNAP25, and syntaxin1. These proteins are required for vesicle priming and fusion. We generated an improved SNAP25- based SNARE COmplex Reporter (SCORE2) incorporating mCeruelan3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. This construct rescues vesicle fusion with properties indistinguishable from fusion in wild- type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR- FRET) imaging reveals a rapid FRET increase preceding indi-vidual fusion events by 65 ms. The experiments are performed under conditions of a steady- state cycle of docking, priming, and fusion, and the delay suggests that the FRET change reflects tight docking and priming of the vesicle, followed by fusion after ~65 ms. Given the absence of wt SNAP25, SCORE2 allows determination of the number of molecules at fusion sites and the number that changes conformation. The number of SNAP25 molecules changing conformation in the priming step increases with vesicle size and SNAP25 density in the plasma membrane and equals the number of copies present in the vesicle–plasma membrane contact zone. We estimate that in wt cells, 6 to 7 copies of SNAP25 change conformation during the priming step.
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