In the brains of organisms as diverse as fruit flies to humans, developing neurons generally recognize one another through specific types of receptors. It is well-known that The surfaces of growing neurons can adhere to other neurons and glial cells through receptors called cell adhesion molecules. However, axons and dendrites from the same neuron must avoid each other so that they do not cross. The protocadherins (Pcdhs) are proteins that are related to conventional cell adhesion molecules, but appear to mediate the self-avoidance process in the vertebrate nervous system. This project aims to study the mechanism of the anti-adhesive process by which neurons achieve self-avoidance. This project will recruit undergraduate researchers, under the guidance of the principal investigator and experienced graduate students and/or research associates, to study Pcdh mediated anti-adhesion in cells using cutting-edge molecular biological and cellular imaging techniques. The project will contribute to the understanding of neural development. The experience that these undergraduates receive from this project will be an essential part of their training for careers in education, biology research, as well as preparation for medical and dental schools and other postgraduate programs in health care-related fields.
This project will study how an interesting class of neural cell receptors, the clustered protocadherins (Pcdhs), hallmarks of complex vertebrate brains, function in wiring neurons during development. The Pcdhs resemble cell surface adhesion molecules but are prominently trafficked in internal compartments including the endosome/lysosome system. It is hypothesized that this intracellular trafficking might be related to the anti-adhesive properties of Pcdhs. An amino acid segment of the cytoplasmic domain from one Pcdh family member that controls intracellular trafficking (termed the VCD motif) has been mapped. The function of this segment and similar segments in other Pcdh family members needs to be elaborated and how Pcdhs cause cell membranes to detach needs to be studied. In this project, VCD motifs from representatives of Pcdh subfamilies will be studied by site-directed mutagenesis and trafficking assays. The ability of these motifs to physically interact will also be characterized. Critical residues for VCD motif function will be identified and used to create trafficking defective Pcdh mutants that will be used to study the requirements for correct intracellular trafficking in the anti-adhesive process. The ability of wild-type and mutant Pcdhs to undergo trans-endocytosis from one interacting cell to the other, a proposed mechanism for anti-adhesion, will be characterized by correlative light and electron microscopy. Finally, how Pcdh mediated cell-cell interaction affects the levels of other cell-surface adhesion molecules by altering the rate of lysosomal degradation will be characterized and proteins up- or down-regulated by Pcdh interaction identified by quantitative proteomics. Completion of these experiments will lead to a better understanding of how Pcdhs affect interacting cells in neural development.
