Secretory vesicle formation and dynamics

The intracellular vesicle transport. The structural and metabolic stability of a eukaryotic cell depends on the trafficking of material between sub-cellular compartments. Intracellular trafficking requires the formation and correct targeting of small, membrane-bound vesicles. Vesicles are first created from a so-called donor compartment.  Once fully formed, the vesicles are then actively transported to, and subsequently fuse with, an acceptor compartment where they deliver their soluble cargo into the lumen of the acceptor compartment and release their membrane proteins into the acceptor compartment membrane.   

Membrane vesicle formation requires membrane fission. While much is known about the protein coats that sculpt membranes into vesicles, the mechanism by which vesicles are released, the so-called membrane fission reaction, remains poorly understood. We are using the release of transport carriers from the recycling endosome of C. elegans and from the plasma membrane of yeast as model systems for examining the mechanisms of membrane fission

Real-time observation of membrane fission with BAS. We have developed cell-free, single particle fission assays based on BAS and are using them to understand how nucleotide hydrolysis is coupled to membrane binding, rearrangement and fission. 

As an example of how membrane fission can be studied using BAS, we have examined the impact of the ENTH domain of the protein epsin on liposomes of defined composition. Liposomes are prepared by extrusion with a small mol fraction of fluorescent lipid. Upon addition of a fission (scission) system, fragmentation of a large liposome into many smaller fission products can be observed, and quantified, with BAS. The ENTH domain is a potent fission agent, driving product vesicle formation even at sub-micromolar protein concentrations.  See PloS One, 10: e0119563 (2015).

Department of Biochemistry and Biophysics                           Texas A&M University              2021