Lipids and proteins form a variety of complexes and domains in cellular membranes. The underlying principles that govern the assembly and function of these structures remain enigmatic. To address this gap in knowledge, we have focused on critically testing key predictions of a prevalent model of membrane domain organization: the lipid raft hypothesis. Current models propose that raft domains normally exist as nanoscale compositional fluctuations at steady state in cells, but can be stabilized by proteins to form functional rafts. The non-toxic membrane binding subunit of cholera toxin (CTxB) is an example of a protein that can cluster raft-associated glycolipids to assembled stabilized raft domains. In the previous funding period, we examined the mechanisms by which toxin-stabilized domains form and asked whether they function to mechanically deform cell membranes to facilitate CTxB uptake by clathrin independent endocytosis. We tested this using a novel variant of CTxB that is unable to cluster glycolipids. Our results led to the unexpected discover that microtubules, dynein, and dynactin generate pulling forces at sites of clathrin independent uptake of CTxB. Our goal for the upcoming funding period is to better understand how microtubules and dynein/dynactin participate in clathrin- independent endocytosis and how CTxB is selectively sorted into these specialized clathrin- independent pathways. Using a combination of cell biological and live cell imaging approaches, we will tackle these questions through three specific aims. First, we will determine if stabilized rafts sort CTxB into clathrin independent endocytic pathways. Second, we will test the hypothesis that multiple clathrin- independent pathways exploit microtubules and motors to tubulate and scission the plasma membrane. Finally, we will identify cellular machinery responsible for linking microtubules and dynein/dynactin to nascent clathrin-independent carriers. Successful completion of this work will provide fundamental insights into the functions of stabilized rafts and uncover new mechanism by which toxins manipulate and hijack cellular machinery for their own purposes. It will also advance our understanding of how microtubules and microtubule-associated motors function in membrane trafficking events and reveal novel role(s) for microtubules, dynein, and dynactin at the plasma membrane. Finally, it will define new mechanisms and machinery that contribute to the assembly of clathrin independent endocytic carriers.