Investigating the Influence of the Rho‐family GTPase TCL/RhoJ on Vesicular Trafficking

Abstract

TCL/RhoJ is a Rho family GTPase that was recently shown to have a novel N‐terminal sequence capable of regulating its localization to the cellular plasma membrane and vesicular compartments, as well as its ability to load with nucleotide. Additionally the protein has been shown to have a role in endosomal recycling pathways. To better understand how the N‐terminus of TCL may contribute to vesicular trafficking, WM‐115 cells will be used in transferrin uptake and recycling assays. WM‐115 cells are a continuous cell‐line derived from a human melanoma tumor and are believed to overexpress TCL. In order to manipulate TCL levels, the WM‐115 cells were initially tested for their transfection efficiency using DNA containing Venus fluorescent protein and were found to have a 60% transfection efficiency. To study vesicular trafficking, WM‐115 cells will be transfected with YFP‐TCL constructs corresponding to the wild‐type protein, an N‐terminal deletion mutant, as well as constitutively activating and dominant negative mutants of TCL. Transfected cells will be tested for transferrin uptake and recycling using Alexa‐647 transferrin and flow cytometry with gating on YFP‐positive cells. These experiments will help determine how transferrin recycling rates are affected by TCL and better illuminate its role in vesicular trafficking.