Synaptotagmin-1 and Doc2b Exhibit Distinct Membrane-Remodeling Mechanisms

Raya Sorkin, Margherita Marchetti, Emma Logtenberg, Melissa C. Piontek, Emma Kerklingh, Guy Brand, Rashmi Voleti, Josep Rizo, Wouter H. Roos, Alexander J. Groffen, Gijs J.L. Wuite*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

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Abstract

Synaptotagmin-1 (Syt1) is a calcium sensor protein that is critical for neurotransmission and is therefore extensively studied. Here, we use pairs of optically trapped beads coated with SNARE-free synthetic membranes to investigate Syt1-induced membrane remodeling. This activity is compared with that of Doc2b, which contains a conserved C2AB domain and induces membrane tethering and hemifusion in this cell-free model. We find that the soluble C2AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca2+- and protein-dependent manner. Single-membrane loading of Syt1 yielded the highest probability and force of membrane interactions, whereas in contrast, Doc2b was more effective after loading both membranes. A lipid-mixing assay with confocal imaging reveals that both Syt1 and Doc2b are able to induce hemifusion; however, significantly higher Syt1 concentrations are required. Consistently, both C2AB fragments cause a reduction in the membrane-bending modulus, as measured by a method based on atomic force microscopy. This lowering of the energy required for membrane deformation may contribute to Ca2+-induced fusion.

Original languageEnglish
Pages (from-to)643-656
Number of pages14
JournalBiophysical Journal
Volume118
Issue number3
Early online date25 Dec 2019
DOIs
Publication statusPublished - 4 Feb 2020

Funding

The authors thank Michael Kozlov for insightful discussions and Ineke Brouwer for useful discussions and technical advice. R.S. acknowledges support through the Human Frontier Science Program (HFSP) postdoctoral fellowship LT000419/2015 , as well as support through the Israeli National Postdoctoral Award for Advancing Women in Science and the L`Oreal UNESCO award for advancing women in science. W.H.R. acknowledges the support of the Nederlandse Organisatie voor Wetenschappelijk Onderzoek Vidi grant. J.R. acknowledges the support of the Welch Foundation (grant I-1304 ) and the National Institutes of Health (Research Project Award R35 NS097333 ).

FundersFunder number
Israeli National
National Institutes of Health
National Institute of Neurological Disorders and StrokeR35NS097333
Welch FoundationI-1304
Human Frontier Science ProgramLT000419/2015
United Nations Educational, Scientific and Cultural Organization
Human Frontier Science Program
Nederlandse Organisatie voor Wetenschappelijk Onderzoek

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