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Research in my laboratory is aimed at understanding the mechanisms of signal dependent protein localization and transport in neurons. Dynamic protein localization within cells is often determined by environmental stimuli. In rod photoreceptors of the retina transitions between darkness and light result in the massive translocation of several key signal transduction proteins between two major compartments. The second and third most abundant proteins in rods, arrestin and transducin, respectively, move in opposite directions. Arrestin, a protein that has been identified to be important for regulation of heterotrimeric G protein coupled receptors, is found primarily in the inner segment compartment of dark adapted rods, but essentially its entire compliment is transported to the outer segment compartment upon exposure to light. Transducin, the G protein in rod visual transduction, is found in the rod outer segment in dark-adapted rods and moves to the inner segment with the onset of light. A third phototransduction protein - the calcium binding protein, recoverin - also shifts its position toward the synaptic end of rods when they are exposed to steady light. To further complicate the story, all of these proteins are transported through a thin connecting cilium, within tens of minutes of light onset.
The functions of, and mechanisms underlying these massive protein redistributions that occur day in and day out are not known despite decades of study. A major handicap to investigation has been the use of static, histological methods to examine dynamic processes. In living cells proteins are ever moving, faster or slower, in one direction or randomly. It is the net movement of populations of individual molecules that determines their average distributions within cells. Thus, it stands to reason that the only way to determine the mechanism of cellular localization and transport of proteins is to observe their dynamic behavior in the living, functioning cells they normally inhabit.
We employ multiphoton & confocal microscopy and the expression of fluorescently labeled proteins in transgenic animals to monitor protein dynamics in living cells in real time, particularly after changes in the cell's signaling state. Furthermore, we monitor intracellular signaling using fluorescent indicators such that the changes in protein dynamics may be correlated with the signaling state of the cell. Many disease causing genetic mutations result in the improper localization of proteins in cells. Studying the mechanisms by which proteins are localized and transported in living cells may lead to strategies for therapeutic interventions that will correct or alleviate disorders caused by such mutations.