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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Budget End
Support Year
6
Fiscal Year
2016
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Indirect Cost
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U.S. National Heart Lung and Blood Inst
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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
Maldonado-Baez, Lymarie; Williamson, Chad; Donaldson, Julie G (2013) Clathrin-independent endocytosis: a cargo-centric view. Exp Cell Res 319:2759-69
Cole, Nelson B; Donaldson, Julie G (2012) Releasable SNAP-tag probes for studying endocytosis and recycling. ACS Chem Biol 7:464-9
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

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