Single Particle Fluorescence Burst Analysis of Membrane Fission

Abstract

The ability to catalyze the fission step of endocytosis is critical to many biological processes including cell communication and synaptic transmission. This activity must be tightly regulated for cellular homeostasis. Limitations of current approaches used for the study of this process, in conjunction with a historical focus on neuronal systems that may not generalize to somatic cells, have left the field torn between several conflicting models. Functional redundancy of several key players complicate interpretation of in vivo studies, stressing the need for a sensitive in vitro approach capable of revealing attenuated activity of tightly regulated machinery. In this document, we introduce Burst Analysis Spectroscopy (BAS) as a simple reagent sparing approach for the investigation of vesicle membrane fission. Using BAS, we accurately map liposome distributions across several orders of magnitude and observe subtle shifts in liposome population as functions of time, temperature, and protein concentration using the fission potent ENTH domain of the protein epsin. We proceed to further uncover an unrecognized fission activity of full-length epsin, consistent with a mechanism in which membrane fission proceeds as a consequence of amphipathic helix insertion into the lipid bilayer.