The project is a study of the assembly and organization of the eyespot of the unicellular alga, Chlamydomonas. The eyespot is a light-sensing organelle which gives rise to characteristic phototactic behavior through control of the flagella which this organism has. Several features of the eyespot make it an extremely interesting system. It includes components of both the plasma membrane and a portion of the underlying, closely apposed membranes of the chloroplast. Associated with this region of the chloroplast membranes are layers of carotenoid pigment granules which reflect light to the apposed rhodopsin light-sensing region of the plasma membrane. Light activation of the rhodopsin leads to gated entry into the cell of calcium which initiates a signal transduction pathway that culminates in patterned flagellar movements to move the organism toward or away from the light source. The calcium channel has not been characterized and may in fact be the Chlamydomonas rhodopsin. Also fascinating is the fact that the eyespot is precisely positioned on the surface of the cell with respect to the complex rootlet structures of the flagella. During cell division, the eyespot first disappears. Then two new eyespots form, each 90 degrees from the division plane and 180 degrees from each other. The system poses numerous interesting questions of how expression of individual components is regulated and how these components are assembled into the highly ordered, functional structures. In this sabbatical study, three specific aims will be pursued. The aims are (1) to use biophysical techniques to analyze the physiology and behavior of eyespot-assembly mutants, (2) to analyze multi-eye mutants which presumably have defects in the positioning system for eyespot assembly during cell division, and (3) to use immunofluorescence light microscopy, GFP-protein techniques and immunoelectron microscopy to determine the organization of some of the major components in the eyespot structure of both normal and mutant organisms. This is a POWRE project which will significantly enhance the scientific training and expertise of the investigator.
