Research SummaryUnderstanding the dynamics of the components of the GEEC endocytic pathway using single molecule visualization techniques
Receptor-mediated endocytosis is triggered by active events that occur at the cell membrane causing it to invaginate and eventually form vesicles. Unlike the well characterized clathrin and dynamin-dependent pathways, in this project I will be studying a clathrin and dynamin independent pathway. This pathway is an ARF1 dependent-CDC42-regulated pathway where the endosomes are enriched with glycosyl-phosphatidylinositol-anchored proteins (GPI-AP Early Endosomal Compartment (GEEC)). This is the main route for fluid and folic acid uptake as well as for toxins like Cholera toxin B and plant protein ricin to enter cells.
While molecular players involved in GEEC endocytosis are being identified, the mechanics and dynamics of formation of the initial clathrin independent compartment (CLIC) are yet to be established. I will be developing assays to perform live imaging of formation of CLICs using a pH sensitive GFP-GPI. The spatial and temporal observations obtained of the distribution of endosomes will give us preliminary characteristics of this pathway in a whole cell context. Using endocytic assays we will identify molecular players involved in the initial formation of these endosomes. Subsequently, dual color live imaging we can correlate these molecular players to the formation of the CLICs. Using these assays we will also investigate effect of mechanical stresses like fluid shear and surface tension in the endocytic frequency and spatial distribution.