Molecular Interactions Decipher Cellular Identities During Neuronal Development

Abstract

Growing neurons extending their dendrites and axons in the developing brain encounter their neighbors as well as other parts of the same cell. At these contacts, interactions between cell surface proteins called clustered protocadherins allow cells to distinguish self from non-self. I used structural biology, sequence analyses, and cell-based assays to investigate the molecular details of clustered protocadherin interactions. In chapter 2, I describe how I designed, optimized, and built a reconstituted system to identify conditions that enable clustered protocadherin polymerization, which signals self-contacts. I found that His-tagged clustered protocadherins are laterally mobile when tethered to Ni-containing artificial membranes, forming a surface that is poised to interact with a transfected cell. I also found that an extracellular SNAP tag works well to enable specific labeling of surface delivered clustered protocadherins in transfected cells. In chapter 3, I describe the gain-of-function approach I took to investigate the role of the cis interaction interface in clustered protocadherin surface delivery. I found that there are isoform-specific differences for the role of the cis interaction in surface trafficking. I also developed a novel quantification strategy that revealed heterogeneity in surface delivery phenotypes between variants. My work highlights key questions for our understanding of self-avoidance, a crucial neurodevelopmental process, and cell-cell recognition more generally: (1) Which candidate interaction partners downstream of clustered protocadherins ultimately signal self-avoidance, and which correspond to other clustered protocadherin functions? (2) What supramolecular organization patterns or rearrangements can lead to cell signaling? (3) How does clustered protocadherin expression and surface trafficking vary between different cell types, contexts, and developmental stages, and how does that variability relate to different types of cell-cell interactions? The tools I developed for clustered protocadherins enable the field to begin to address these questions in vitro and in cell culture systems. In the future they could be extended to native neuronal contexts and applied to address similar questions for other families of cell recognition proteins.