The overall goal of this proposal is to define new molecular and cellular mechanisms that enable vertebrate photoreceptors to adapt to constantly changing conditions of ambient illumination. We will focus on a novel mechanism of photoreceptor light adaptation based on a massive light-dependent translocation of the photoreceptor-specific G protein transducin from the outer segments to other cellular compartments. Our recent work shows that this translocation reduces rod sensitivity under conditions of bright illumination and extends the operating range of rod vision. We plan to continue these studies in two major directions. First, we will determine whether transducin translocation is also present in cones. Cones might benefit from such a mechanism even more than rods because they are better suited to work at the high light intensities that cause transducin movement.
In Aim 1, we will test whether this phenomenon takes place in cones using two independent animal models, a nocturnal model (mouse) and a diurnal model (ground squirrel). If transducin translocation takes place in cones, we will quantify this process and assess its functional consequences for cone light adaptation. Our second direction is to address the molecular and cellular mechanisms that underlie transducin translocation.
In Aim 2 we will study the role of phosducin in this process. We will test the hypotheses that phosducin assists transducin betagamma subunits in their light-dependent translocation from rod outer segments to other parts of the cell and that phosducin phosphorylation in darkness may serve as the signal for transducin to return back to the outer segements. The experiments will utilize an array of biochemical approaches and will take advantage of a phosducin knockout mouse model recently developed in our laboratory.
In Aim 3 we will assess the role of cytoskeleton and molecular motors in light-dependent transducin movement. We will use a pharmacological approach where major cytoskeletal structures will be disrupted by specific drugs, a genetic approach using mice bearing mutations in molecular transport systems, and a microscopic approach determining whether transducin subunits co-localize with major cytoskeletal elements and molecular motors upon their movement. The proposed experiments are relevant to understanding the molecular and cellular mechanisms that regulate normal photoreceptor activity, mechanisms that may be perturbed in several degenerative retinal diseases.
Showing the most recent 10 out of 44 publications
