2021 Fiscal Year Research-status Report
Synaptic vesicle transport revealed by electrophysiological and imaging studies
| Project/Area Number |
21K06445
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
MAHAPATRA Satyajit 沖縄科学技術大学院大学, 細胞分子シナプス機能ユニット, スタッフサイエンティスト (40832861)
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| Co-Investigator(Kenkyū-buntansha) |
DUTTA Soumyajit 沖縄科学技術大学院大学, 細胞分子シナプス機能ユニット, ポストドクトラルスカラー (20899372) [Withdrawn]
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| Project Period (FY) |
2021-04-01 – 2024-03-31
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| Keywords | SV transport / The calyx of Held / Hippocampal CA1 synapse / Endocytosis / Release-site clearance / STD / Recovery from STD / Physiological condition |
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| Outline of Annual Research Achievements |
To understand the synaptic vesicles (SVs) transport mechanism, I first tested how endocytic proteins (dynamin, clathrin) regulate short-term depression (STD) and recovery from STD through their release-site clearance effect using electrophysiology. Since the SV supply time includes SV transportation to nearby the release sites plus their docking/priming/fusion, blocking the SV docking would reveal the SV transport time and mechanism.
1) Initial findings show endocytic blockers (dynasore and pitstop-2) inhibit slow endocytosis and augment the STD at mice calyx synapse (p13-15, 37 C, 2.0 mM calcium). However, no delay in recovery from STD was observed, in contrast to the previous report.
2) I obtained similar results when testing its physiological relevancy (37 C, 1.3 mM calcium). Suggesting dynamin, clathrin in physiology regulates STD through their release-site clearance effect. However, some endocytic forms likely work independently, allowing synapses to recover without being delayed.
3) When tested in hippocampal CA1 Schaffer collateral-pyramidal cell synapse, endocytic block reduced synaptic facilitation. Thus, confirming endocytosis-mediated release-site clearance has broader implications.
4) Next, we probed the role of dynamin in regulating the fast form of endocytosis at calyx, using dynamin 1 PRD domain peptide. We found the application of peptide blocks partially and not wholly the rapid endocytosis, supporting our previous observation. To understand if dynamin 2, -3 and clathrin play any role, I'm currently testing the effect of dynasore/pitstop-2 on fast-endocytosis.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The electrophysiological assessment of the SV transport mechanism is going smoothly. Out of three functionally target groups, the first one (role of endocytic proteins) is nearing completion. For the first time, our data show that in physiological conditions how endocytic proteins (dynamin and clathrin) affect the SV release pattern on tens of milliseconds to seconds scale in two functionally distinct synapses owing to their release-site clearance property. The manuscript of this finding is getting ready to submit
These findings will next be compared in imaging studies using primary calyx-like and hippocampal cultures for live tracking of single SVs. However, as reported before, the Co-PI, who was supposed to carry out the imaging studies, left Japan to pursue his future career in the US. Therefore, no considerable progress in the imaging assessment of the SV transport mechanism was possible. Findings from electrophysiological studies on target identification and validation would set the platform for imaging studies to carry out next.
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| Strategy for Future Research Activity |
The physiological role of endocytic proteins in promoting SV docking through their release-site clearance property across synapses is now nearing completion. Clarifying the effect of dynamin and clathrin on modulating the fast-form of endocytic membrane recovery would complete the study.
Next, I would test the role cytoskeleton forming molecules (f-actin and microtubule), presynaptic G-proteins, and GTPases (small and large) play in regulating the SV transportation.
F-actin is thought to promote recovery of synaptic responses from activity-dependent STD through fast SV replenishment and assist in SV docking by clearing SV components from release sites. The microtubule, in comparison, is thought to support intra-terminal SV movements. Therefore, the role of cytoskeleton forming elements in SV-transport under physiological conditions requires thorough scrutiny.
GTP and GDP analogs play a role in G-protein activation, and a non-hydrolysable GDP analog is shown to delay recovery from STD, without affecting the endocytic membrane recovery. Suggesting a completely different role than endocytic proteins. Therefore, if GDP analogues, Rho- and Rab-family of GTPases play any role in SV-transport would be tested next.
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| Causes of Carryover |
Since Co-PI, who suppose to carry out the imaging part of the project, left Japan to pursue his future career in the US, the budgeted amount for the imaging expenses left unused.
Availability of this unused amount, allows for a better flexibility in terms of buying small equipments, reagents, and to pay off for the publication and travel costs.
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