We have been studying clathrin-independent forms of endocytosis (CIE) and have identified a number of endogenous PM proteins that enter cells through this mechanism. We have begun to study these proteins in detail in an attempt to understand how they travel in cells and whether they specifically interact with cellular machinery. Last year, we described elaborate coordinate regulation of clathrin-mediated and clathrin-independent endocytosis (CME and CIE, respectively). We found that when CME is blocked, CIE continues but the itinerary of CIE cargo proteins is altered. Cargo proteins that typically do not go on to degradation (CD98 and CD147) now traffic to late endosomes and are degraded (Dutta and Donaldson, 2015). Rab35 signals that CME is functioning by recruiting an Arf6 GAP (ACAP) to inactivate Arf6. To further understand the mechanism or mechanisms of CIE, we have embarked on an siRNA screen using the Dharmacon Trafficking Library that targets 140 human genes. We are looking for hits that affect endocytosis of two CIE cargo proteins: MHCI (major histocompatibility complex Class I) and CD59 (a GPI-lipid anchored protein). We are at present validating the hits and hope to follow up on promising ones. In another study just begun, we are examining the role of glycosylation on cargo proteins and whether over or under glycosylation will affect rates of endocytosis and the trafficking of those proteins. Finally we looking at CIE in different cell types and also in cells that undergo a stimulated form of CIE, macropinocytosis. We previously described alterations in phosphoinositol lipids in HeLa cells expressing active forms of Ras and new work has shown that some cancer cells use macropinocytosis for nutrient uptake. We are now examining the trafficking and sorting of cargo in fibrosarcoma cells undergoing constitutive macropinocytosis.
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