Every living cell ingests and extrudes material by recycling part of its membrane to form vesicles that are internalized (endocytosis) or externalized (exocytosis). These processes are broadly important and carefully regulated in all cell types, but in electrically active cells like neurons, they form the basis for rapid intercellular communication (synaptic transmission) and are therefore under precise temporal control. Dysfunctions in synaptic transmission contribute to several neurologic disorders. Dr. Taraska studies how vesicles fuse with and are recaptured from the cell surface in excitable cells; he seeks to identify the proteins that control these processes and determine their impact on human health and disease.
Researchers have studied the proteins that underlie these fundamental processes primarily at two levels. On the one hand, beautiful static crystal structures reveal atomic scale information about individual proteins and their interactions; on the other, genetic manipulations combined with cell physiologic assays reveal the importance of particular proteins to exo- and endocytic subprocesses. Dr. Taraska aims to bridge these two levels of analysis to visualize how protein machines dynamically rearrange and move within a living cell. Through a multi-disciplinary approach, he hopes to reveal how individual molecular machines work in a complex cellular environment.
Focusing on techniques that utilize fluorescence as a reporter for the structure and activity of proteins, Dr. Taraska images the molecular behavior of proteins. These techniques include spectral microscopy, single molecule analysis, fluorescence resonance energy transfer, fluorometry, total internal reflection fluorescence microscopy, and electrophysiology. In parallel, using evanescent field, spectral, and confocal microscopy, he images the behavior of individual vesicles in real time.
His recent work has focused on the moments following vesicle fusion to the membrane to identify how proteins on the cell surface are gathering up the fused material and bringing it back in to the cell. During synaptic transmission, this rapid recycling process is critical to continued proper functioning of the cell. Dr. Taraska uses fluorescence imaging of individual candidate proteins in living cells to discern their kinetics and dynamics in relation to the recycling process.
Fluorescence tagging typically only represents a single pair of points on a highly complex protein. Dr. Taraska has been decorating proteins at multiple points with pairs of fluorescent tags to map the entire surface of the protein. The resulting matrix of distances can be used to model changes in protein conformation. Dr. Taraska hopes approaches such as these will one day reveal the entire molecular topology of the plasma membrane at an atomic level, so that regulation of cellular process like synaptic transmission—and ultimately, physiology processes like learning and memory—can be understood and manipulated at the atomic level.
Source: http://www.nhlbi.nih.gov/research/intramural/researchers/pi/taraska-justin
