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 language | English |
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Pages (from-to) | 643-656 |
Number of pages | 14 |
Journal | Biophysical Journal |
Volume | 118 |
Issue number | 3 |
Early online date | 25 Dec 2019 |
DOIs | |
Publication status | Published - 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 ).
Funders | Funder number |
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Israeli National | |
National Institutes of Health | |
National Institute of Neurological Disorders and Stroke | R35NS097333 |
Welch Foundation | I-1304 |
Human Frontier Science Program | LT000419/2015 |
United Nations Educational, Scientific and Cultural Organization | |
Human Frontier Science Program | |
Nederlandse Organisatie voor Wetenschappelijk Onderzoek |