Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Wednesday Speaker Abstracts

AN UNBIASED CLASSIFICATION APPROACH REVEALS MULTIPLE FUSION CATEGORIES OF VAMP2-MEDIATED EXOCYTOSIS Fabio Urbina 1 ; Stephanie L Gupton 1 ; 1 University of North Carolina , Cell Biology and Physiology, Chapel Hill, North Carolina, USA Exocytosis is a fundamental process that secretes cargo into the extracellular space and potentially inserts lipids and proteins into the plasma membrane. A pH-sensitive variant of GFP (pHluorin) fused to the luminal end of a vesicle-SNARE protein, such as VAMP2, provides a fluorescent intensity readout of the fusion pore opening during exocytosis and subsequent fate of the v-SNARE. We previously reported an automated analysis platform that identifies such exocytic events, and records several parameters of their fusion, such as the frequency, half-life fluorescence decay, and spatio-temporal distribution. This led us to hypothesize there were discrete types of exocytic events based on the behavior of the v-SNARE after fusion pore opening. Here, we introduce a novel machine-learning method to automatically classify TIRF images of VAMP2-pHluorin exocytic events in developing murine cortical neurons. We used multiple classifiers to categorize exocytic events in an unsupervised way. Using a majority-rule committee of 28 indices, run with each of the classifiers individually, we determined the most likely number of classes. The committee always selected four discrete classes of fusion, with each classifier similarly grouping exocytic events. This was surprising, as classically, two modes of fusion are recognized. During full-vesicle-fusion (FVF), the fusion pore dilates after opening and the vesicle collapses into the membrane. During kiss-and-run (KNR) exocytosis a transient fusion pore closes after cargo secretion. We discuss the validation of these four distinct modes of fusion and provide examples of biological manipulations that alter the proportion of events and their distribution in neurons, and how this affects neuronal morphogenesis.

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