30th Annual Student Research Conference
Optimizing Excitation Polarization to Probe Fusion Pore Dynamics Using TIRF Microscopy
Kasey Hancock
Dr. Vayujeet Gokhale, Faculty Mentor
During hormone or neurotransmitter release via exocytosis, fusion pores may flicker repeatedly before resealing or dilating irreversibly. Pore dynamics regulate kinetics of release and the mode of recycling. Due to lack of sensitive reconstituted assays, mechanisms governing pore dynamics are not understood. In this talk, we describe a polarized total internal reflection (pTIRF) microscopy assay to monitor fusion of proteoliposomes to planar lipid bilayers supported on soft polymer cushions with single molecule sensitivity and ~15 ms temporal resolution. Fluorescently labeled small unilamellar vesicles, reconstituted with exocytotic/neuronal v-SNAREs (v-SUVs), fuse with a supported bilayer containing cognate t-SNAREs (t-SBL). Fusion events are accompanied by changes in total fluorescence intensity surrounding the fusion site. Analysis of intensity changes, combined with a mathematical model, provides information on pore dynamics (Stratton et al. Biophys. J. 2016). We varied the polarization of the excitation field and quantified its contribution to intensity changes for different lipid-linked fluorophores.
Keywords: exocytosis, fusion pores, polarized total internal reflection microscopy, SNARE proteins, fluorescence intensity, lipid-linked fluorophores, membrane fusion
Topic(s):Physics
Biology
Mathematical Biology
Presentation Type: Oral Paper
Session: 206-3
Location: MG 2050
Time: 10:00