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. 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 in the process of validating the hits and will follow up on promising ones. Interestingly, we find that the hits that affect MHCI vs. CD59 endocytosis are different, suggesting that the requirements for internalization of these 2 CIE cargo proteins are somewhat distinct. In another study, 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. Here again, the effect of glycosylation of cargo and presence of extracellular lectins can alter the landscape to be permissive or not for certain cargo. Finally we are 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 and the role that the microtubule cytoskeleton and motors play in this process. In collaborations with Ed Korn's group in NHLBI, we provide evidence that myosin II associates with anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate. Additionally in a collaborative study with Dr. Jia Song at the University of Delaware, we helped establish a role for Arf6 in sea urchin development.

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Support Year
7
Fiscal Year
2017
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U.S. National Heart Lung and Blood Inst
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Mathew, Mohit P; Donaldson, Julie G (2018) Distinct cargo-specific response landscapes underpin the complex and nuanced role of galectin-glycan interactions in clathrin-independent endocytosis. J Biol Chem 293:7222-7237
Stepicheva, Nadezda A; Dumas, Megan; Kobi, Priscilla et al. (2017) The small GTPase Arf6 regulates sea urchin morphogenesis. Differentiation 95:31-43
Donaldson, Julie G; Johnson, Debra L; Dutta, Dipannita (2016) Rab and Arf G Proteins in Endosomal Trafficking & Cell Surface Homeostasis. Small GTPases :0
Liu, Xiong; Shu, Shi; Billington, Neil et al. (2016) Mammalian Nonmuscle Myosin II Binds to Anionic Phospholipids with Concomitant Dissociation of the Regulatory Light Chain. J Biol Chem 291:24828-24837
Dutta, Dipannita; Donaldson, Julie G (2015) Rab and Arf G proteins in endosomal trafficking. Methods Cell Biol 130:127-38
Dutta, Dipannita; Donaldson, Julie G (2015) Sorting of Clathrin-Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin-Mediated Endocytosis. Traffic 16:994-1009
Mayor, Satyajit; Parton, Robert G; Donaldson, Julie G (2014) Clathrin-independent pathways of endocytosis. Cold Spring Harb Perspect Biol 6:
Maldonado-Báez, Lymarie; Donaldson, Julie G (2013) Hook1, microtubules, and Rab22: mediators of selective sorting of clathrin-independent endocytic cargo proteins on endosomes. Bioarchitecture 3:141-6
Dutta, Dipannita; Williamson, Chad D; Cole, Nelson B et al. (2012) Pitstop 2 is a potent inhibitor of clathrin-independent endocytosis. PLoS One 7:e45799
Dutta, Dipannita; Donaldson, Julie G (2012) Search for inhibitors of endocytosis: Intended specificity and unintended consequences. Cell Logist 2:203-208

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