Abstract
For a long time, it remained an unresolved question how the fusion pore is regulated to close after fusion occurs during kiss-and-run. Our study focuses on the physiological function of NSF in modulating single vesicle fusion events in live cells, using whole-cell capacitance recording coupled with live-cell confocal imaging. Our results demonstrates that in both male Sprague-Dawley rat adrenal chromaffin endocrine cells and dorsal root ganglion neurons, inhibiting or diminishing the ATPase activity of NSF significantly impedes fusion pore closure, inhibits various forms of endocytosis, and disrupts the replenishment of readily releasable pool. Interestingly, NSF is exclusively required for calcium-dependent exo-endocytosis but not for the calcium-independent process. In response to calcium influx, NSF disassembles the trans-SNARE complex intermediate via its ATPase activity, which regulates fusion pore closure and subsequently mediates slow, fast, and overshoot endocytosis. Our findings revealed a key mechanism for fusion pore regulation in calcium-evoked exo-endocytosis coupling.
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Data availability
The datasets generated and analyzed during the current study are included in the supplementary materials of this article. Original, uncropped, and unedited Western blot images are presented in Supplementary Figs. 7 and 8 within the Supplementary Information. Supplementary Data 1 provides a comprehensive list of nucleotide and peptide sequences, including all sgRNA, siRNAs, primers, and inhibitory peptide amino acid sequences. Supplementary Data 2 includes the numerical source data underlying the main and Supplementary Figs. Supplementary Data 3 contains the original Sanger sequencing trace files validating the genome editing results. Plasmids generated in this study have been deposited in Addgene with the following Addgene IDs: 252600, 252601, 252602, 252603, and 252604. Additional data supporting the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
This work was supported by the Key Project of Gansu Province Science and Technology, China [Grant No. 22ZD6FA053]; National Natural Science Foundation of China [Grant No. 82073825]; Natural Science Foundation of Gansu Province, China [20JR10RA619] and Medical Innovation and Development Project of Lanzhou University [Grant No. lzuyxcx-2022-197].
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Y.W.: Writing - original draft, Formal analysis, Methodology. S.M.: Formal analysis, Methodology, Validation. S.S.: Writing - original draft, Formal analysis, Visualization. C.Z.: Investigation. Yu Wang: Methodology, Validation. B.L.: Resources. W.H.: Validation. H.L.: Writing - review & editing, Supervision. X.W.: Writing - review & editing, Methodology, Supervision. D.Z.: Writing - original draft, Writing - review & editing, Supervision, Project administration. D.W.: Writing - review & editing, Project administration, Funding acquisition, Conceptualization.
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Wang, Y., Ma, S., Song, S. et al. ATPase N-ethylmaleimide-sensitive factor mediated calcium dependent fusion pore closure and endocytosis. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09743-3
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DOI: https://doi.org/10.1038/s42003-026-09743-3
