Not Your Average Cadherin: Optimization of Expression, Purification, and Initial Structural Analysis of the Atypical Celsr1

Abstract

Cadherin EGF LAG seven-pass G-type receptor 1 (Celsr1) is an atypical cadherin that is central to the planar cell polarity (PCP) pathway. The PCP pathway organizes polarization across the tissue plane, and is involved in a myriad of biological functions, from patterning stereocilia in the inner ear to organizing neural tube closure during fetal development. When this pathway malfunctions, various health conditions can arise, such as congenital heart disease, neural tube defects, and cancer. Celsr1 binds to itself in trans to form bridges between adjacent cells at cell-cell junctions, and asymmetrically recruits fellow membrane proteins Frizzled6 and Vangl2 to propagate polarization. Although Celsr1 is a critical part of an important biological pathway, very little is known about its structure, binding mechanism, or even the ratios in which it binds itself, Frizzled6, and Vangl2. Determining a high-resolution structure of Celsr1 should elucidate the residues involved in its homophilic binding activity, providing insight into its mechanism of binding and potentially mechanisms of inhibition, which would further understanding of Celsr1 and the PCP pathway as a whole. This thesis focuses on optimizing expression and purification of recombinant Celsr1 for sample preparation for cryogenic electron microscopy (cryo-EM), and analysis of initial structural data obtained from imaging prepared samples. Determining the optimal construct, expression system, and purification protocol resulted in enough highly concentrated pure protein to create a low-resolution map of Celsr1, which with further data collection should yield a high resolution solved structure. This thesis represents exciting steps on the path to solving the structure of Celsr1.