The objectives of this proposal are to determine the mechanisms of photoreceptor protein compartmentalization. Retinal photoreceptors are polarized neurons whose major functions, include receiving and transmitting signals, are compartmentalized into discrete subcellular domains. Compartmentalization is critical for normal photoreceptor activity and reduced vision or blindness result from improper segregation of proteins. Despite their importance, the mechanisms underlying protein compartmentalization in photoreceptors, or any other neuron, remain poorly understood. Essential for understanding compartmentalization are the biophysical properties of the photoreceptor cytoplasm, the biophysical properties of the proteins that are destined to be compartmentalized and the forces that drive accumulation of proteins, against significant concentration gradients, into the specific compartments. We have uncovered a fundamental biophysical mechanism that may be central to protein transport and segregation in all electrically active cells: transport of charged proteins within the electrical field generated by the photoreceptor neuronal activity. We call this novel mechanism axial dynamic electrophoretic protein transport (ADEPT). We will use state of the art live cell fluorescence imaging tools developed in our lab, powerful transgenic and gene editing techniques in Xenopus, and sophisticated biochemical and cell biological approaches to address the following aims:
Aim 1 : Map the axial cytoplasmic electric field, Eax, in rod photoreceptors.
Aim 2 : Determine the impact of ADEPT on photoreceptor protein transport and compartmentalization in living photoreceptors.
Aim 3 : Determine the locations and influence of Arrestin interactions on their distributions and dynamics in living rods and cones.
This work seeks to understand the molecular mechanisms of retinal photoreceptor protein compartmentalization. Retinal degeneration and blindness may be caused by improper delivery of proteins to specialized regions within photoreceptors, the light detecting cells in our eyes. Understanding the mechanisms that control photoreceptor construction, and what goes wrong with this process in blinding diseases, will help find new therapies to extend or restore vision.
