Dissertations, Theses, and Capstone Projects

Date of Degree

9-2022

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Greg Phillips

Committee Members

ChangHui Shen

Jimmie Fata

Anthony Esposito

Raffaella Diotti

Subject Categories

Biology | Cognitive Neuroscience | Developmental Neuroscience | Molecular and Cellular Neuroscience | Neuroscience and Neurobiology | Other Cell and Developmental Biology

Keywords

Clustered protocadherins ubiquitination, Clustered protocadherins surface expression, ubiquitination and phosphorylation, cytoplasmic domain (VCD), Pcdh self-avoidance

Abstract

Clustered protocadherins (Pcdhs) are a family of 60 adhesion-like molecules forming a neural barcode. In vertebrate neurons, 60 Pcdhs are coded by a large gene cluster. Numerous axons in the cluster are coding for the different extracellular, transmembrane, variable portion of the cytoplasmic and constant cytoplasmic domains where their expression is controlled epigenetically. These proteins mediate interactions between axons, dendrites, and glial cells during neural development. Yet, Pcdhs are not strictly adhesion molecules. In the amacrine cells of the retina, Pcdhs promote avoidance of the same cell dendrites, where in the cortex Pcdhs promote interactions between dendrites and astrocytes. In the current study, we propose that part of the mechanism of self-avoidance may involve endocytosis and removal of the adhesive complex from the surface. Ubiquitination is a known factor in endocytosis. A covalent transfer of ubiquitin protein to lysine residues of the target proteins have been implicated in the mechanism of endocytosis and it has been observed as a sorting signal of proteins at endosomes, targeting proteins to multiple vesicular bodies (MVB). In previous studies it was shown that Pcdh intracellular trafficking depends on the variable portion of the cytoplasmic domain (VCD). In this study it was found that Pcdh utilizes endosomal trafficking via the VCD which is ubiquitinated and affected by a potential phosphorylation site to control surface expression in HEK cells. This ubiquitination was not lysine dependent but rather serine and threonine dependent. Further mapping indicated that ubiquitination of a serine at position 754 and threonine at position 756 in Pcdh-gamma-A3 may be controlled by phosphorylation of serines (Ser) at positions 759 and 762. Both sites are highly conserved throughout the Pcdh-gamma-A subfamily. Endogenously expressed Pcdh-gamma-A3 in a stable HEK 293 cell line revealed that phosphorylation is a time dependent process. In Neuro-2A cells, ubiquitination deficient Pcdh-gamma-A3 causes extensive surface expression. In cultured neurons, Pcdh-gamma-A3 accumulates on the hair like structures or bridges among and between dendrites connection. These results suggest that ubiquitination and endocytosis of Pcdh-gamma-As may be a factor in the mechanism of self-avoidance in dendrites. Determining the molecular mechanism that controls Pcdh trafficking will help us understand how neurons interact with each other in development and may shed light on the nature of neuropsychiatric diseases, such as autism spectrum disorders.

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