Exploring the Potential of APEX2 tag in Electron Microscopy Studies of the PCP Protein, Celsr1

Abstract

Planar Cell Polarity (PCP) directs the local and global distribution and behavior of cellular structures. The PCP pathway is largely regulated by a set of asymmetrically distributed PCP proteins with Vang/Vangl2 and Pk on one side and Fz/Fzd6, Dsh, and Dgo on the opposite side while Fmi/Celsr1 is found on both sides of the cell. The intracellular and intercellular interactions among these PCP proteins are not only important to maintain the local PCP asymmetry but also critical to the propagation of PCP asymmetry across thousands of cells via intercellular communication. However, in highly proliferative tissues, such as the mammalian skin, rapidly dividing cells pose a significant risk to the maintenance and restoration of proper PCP during and post mitosis. It has been shown that the PCP signal remains unperturbed by internalizing the dividing cell’s transmembrane PCP proteins, a process in which Celsr1 plays a significant role. However, given the homophilic interaction between Celsr1-Celsr1 across cell junctions, the question remains as to how the dividing cells internalize the transmembrane Celsr1 protein during mitosis. Previous findings suggest that Celsr1 at the cell junctions are internalized via “trans-endocytosis” whereby the Celsr1 of the dividing cell and of the adjacent interphase cell are co-internalized into the same endosomes during mitosis. In this study, we aimed to elucidate the ultra-structure of the Celsr1-containing vesicle during mitotic internalization to shed more light on how the Celsr1 protein, located at cell junctions, behaves during mitotic internalization. Additionally, this study also explored the effectiveness of the recently engineered APEX2 peroxidase tag as an alternative method to probe for Celsr1 in fluorescence and electron microscopy studies.