Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Thursday Speaker Abstracts

MEMBRANE BINDING, BENDING AND REMODELING BY SYNAPTOTAGMIN-1 AND DOC2B Raya Sorkin 1,5 ; Margherita Marchetti 1 ; Emma Logtenberg 2 ; Melissa Piontek 3 ; Emma Kerklingh 1 ; Guy Brand 1 ; Rashmi Voleti 4 ; Josep Rizo 4 ; Wouter H Roos 3 ; Alexander J Groffen 2 ; Gijs J Wuite 1 ; 1 Vrije Universiteit , Department of Physics and Astronomy and LaserLab, Amsterdam, Zuid- Holland, The Netherlands 2 Vrije Universiteit and VU Medical Center, Department of Functional Genomics and Clinical Genetics , Amsterdam, Zuid-Holland, The Netherlands 3 Zernike Instituut, Rijksuniversiteit Groningen, Departement of Molecular Biophysics, Groningen, The Netherlands 4 UT Southwestern Medical Center, Departments of Biophysics, Biochemistry and Pharmacology, Dallas, Texas, USA 5 Tel Aviv University, Department of Physical Chemistry, Tel Aviv, Israel Ca 2+ triggered neurotransmitter release by synaptic exocytosis is a highly regulated process that enables neuronal communication. Synaptotagmin-1 (Syt1) is a calcium sensor protein that regulates synchronous neurotransmitter release. Despite extensive research, it remains a challenge to dissect the contribution of membrane remodeling induced by this protein to membrane fusion. Here, we use optical tweezers in combination with confocal fluorescence microscopy to quantify the protein-induced interactions between pairs of optically-trapped beads coated with synthetic membranes. We explore membrane remodeling in the presence of two proteins: Syt1 and Doc2b, a Ca 2+ sensor protein that triggers spontaneous neurotransmitter release. We find that Syt1 and Doc2b strongly affect the probability and strength of membrane- membrane interactions in a strictly Ca 2+ and protein-dependent manner. When comparing symmetrical (both sides) and asymmetrical (one side) presence of protein on the membranes, Syt1 favors an asymmetrical and Doc2b a symmetrical configuration, as inferred from higher tether probabilities and break forces. Further, we quantify the probability of hemifusion in the presence of Syt1 vs Doc2b, and find that Doc2b allows both hemifusion and membrane bridging, while Syt1 only bridges membranes. We also reveal lipid composition dependence of membrane remodeling by the calcium sensor proteins. To better understand how these proteins affect membranes, we probe membrane mechanical properties using a novel AFM-based method and reveal a decrease in membrane bending moduli following protein binding. Our results suggest an active role of C 2 AB domains in membrane remodeling during the fusion process: in addition to bringing the membranes into close proximity, Syt1-C 2 AB and Doc2b may also contribute to fusion by directly lowering the energy barrier. Overall, our approach provides new insights into the action mechanisms of calcium sensor proteins during neurotransmitter release, and can be readily extended to explore other membrane fusion events.

32