Large amounts of protein are trafficked between the inner and outer segments of photoreceptor cells, along the axonemal linkage. Gene mutations that intefere with this trafficking typically cause retinal degenerations and often syndromic ciliopathies. The Williams laboratory is studying ciliary proteins that have transport or regulatory roles in delivering opsin and other outer segment proteins. Approaches include microscopy (such as live cell imaging and EM) and biochemistry, using cell culture and in vivo analysis.
Fig. 1. Schematic of a vertebrate photoreceptor cell, illustrating the pathway for the turnover of opsin (and other outer segment membrane proteins).
Roles of Motor Proteins in the Transport of Organelles in the RPE
Melanosomes, phagosomes, and lysosomes are transported to key locations in the RPE cells. Dr. Williams’ laboratory is investigating the roles of motor proteins in the transport of these organelles. Both actin and microtubule motor proteins are involved in the transport. A major focus is on how the action of the different motors is coordinated. Approaches include microscopy (such as live cell imaging and EM) and biochemistry, using primary cultures of RPE cells as well as in vivo analysis.
Gene Therapy for Retinal Degeneration in Usher Syndrome
Dr. Williams’ laboratory is investigating gene therapy for preventing the vision loss associated with different types of Usher Syndrome, which is an inherited recessive disorder caused by mutations in any one of eleven genes. Studies were initiated with Usher 1B, which is caused by mutations in the MYO7A gene. In the retina, this gene is expressed in both the photoreceptor cells and the retinal pigment epithelium (RPE). The approach involved the subretinal delivery of MYO7A cDNA in a viral vector to treat these two cell types. These studies have led to Phase I clinical trials for gene replacement therapy of Usher 1B from MYO7A mutations. More recent studies concern refining methods for the delivery of MYO7A, and exploring gene therapy strategies for some of the other types of Usher syndrome.
Fig. 2. Primary culture of RPE cells, showing the melanosome autofluorescence in red, due to excitation with 633-nm light, and phalloidin labeling of actin filaments in green. From Gibbs et al., 2009.
Fig. 3. Movies of melanosomes in WT and MYO7A-null RPE cells. From Gibbs et al., 2004.
Studies on RPE cells derived from stem cells
In collaboration with the Broad Stem Cell Research Center at UCLA and clinical ophthalmologists, the Williams laboratory is studying RPE cells that have been derived from stem cells, including induced pluripotent stem cells (iPS cells) from patients with retinal degenerations. The objectives of these studies are to increase our understanding of the disease pathogenesis, and to develop cell lines that can be used for the transplantation of RPE cells into diseased retinas.