Role of Cytoskeleton in Cholera Toxin Diffusion and Endocytosis
Day, Charles Asher
The plasma membrane is highly organized as a means to regulate various processes including sorting of cargo for endocytosis. However, the mechanisms responsible for this organization are poorly understood. The B subunit of cholera toxin (CTxB) has become a popular tool to study membrane structure and function. CTxB binds 5 glycolipids on the plasma membrane to facilitate targeting of the holotoxin to the host cells for endocytosis via primarily clathrin-independent mechanisms. Previous studies indicate that CTxB has the ability to associate with and reorganize lipid rafts, regions of membrane having unique composition due to lipid-lipid interactions. Using quantitative microscopy approaches, I examined two unique characteristics of CTxB often attributed to its association with lipid rafts. Compared to other putative raft markers or lipid-anchored proteins, CTxB exhibits relatively slow diffusion across the plasma membrane. I evaluated previously proposed models of membrane organization that could impact the lateral diffusion of fluorescently labeled CTxB using fluorescence recovery after photobleaching (FRAP). These studies revealed no direct evidence that CTxB cross-linking of rafts was responsible for its slow diffusion. However, CTxB diffusion was most sensitive to actin disruption and ATP depletion (possibly due to ATP depletion remodeling cortical actin). This was unexpected as CTxB is bound to lipids on the outer leaflet and cannot make contact with actin. CTxB was the first cargo shown utilizing clathrin-independent endocytic pathways, many of which have not been well characterized. One prominent model for CTxB endocytosis states that the toxin crosslinks lipids to mechanically induce negative membrane curvature leading to the de novo formation of endocytic vesicles and that the only requirement from the cell in forming these vesicles is in scission. However, I discovered that this is an endogenous process which occurs in the absence of toxin. While it is an important regulator of vesicle formation, actin is not directly involved in generating the nascent vesicles. Unexpectedly, the driving force for tubule extension is supplied by the microtubule-based motor cytoplasmic dynein implicating a novel mechanism for generation of membrane curvature at the plasma membrane in clathrin-independent endocytosis.