Browsing by Subject "Clathrin"
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Item PI(4)P-Dependent Recruitment of Clathrin Adaptors to the Trans-Golgi Network(2005-04-29) Wang, Jing; Yin, Helen L.The Trans Golgi Network (TGN) is the cell's central sorting station, and the complex trafficking patterns are organized by many types of trafficking adaptors. These include the heterotetrameric adaptor protein complexes (APs) and the monomeric Golgi-localized, gamma-ear containing, Arf-binding proteins (GGAs). The fundamental question of how these adaptors are recruited to TGN membrane remains unclear. Previous studies have shown that adaptor recruitment to the TGN is absolutely dependent on the small GTPase ADP ribosylation factor 1 (Arf1), but paradoxically, Arf1 has a broader intracellular distribution than these adaptors. We found that the Golgi is particularly enriched in phosphatidylinositol 4 phosphate [PI(4)P] and that the clathrin adaptor AP-1 binds PI(4)P directly, suggesting that PI(4)P binding may specify the TGN-specific recruitment in conjunction with Arf1. My studies showed that another monomeric clathrin adaptor GGA also binds PI(4)P and Arf1 independently. The C-terminal "triple helix bundle" of the GGA GAT domain is a polyfunctional module that interacts with multiple partners including PI(4)P and ubiquitin, and ubiquitin may provide a recognition signal for GGAs to control protein sorting. We found that PI(4)P increases wild type GAT binding to ubiquitin-conjugated agarose beads, but has no effect on a mutant GAT that does not bind PI(4)P. Therefore, PI(4)P may be an allosteric regulator of GGAs which enhances ubiquitin binding to GGAs. Based on these results, we conclude: (1) PI(4)P defines the TGN organelle identity by recruiting TGN-targeted adaptors; (2) TGN-enriched adaptors are recruited to the Golgi by binding to both PI(4)P and Arf1, and neither alone is sufficient; (3) PI(4)P acts as a scaffold, and may also be an allosteric regulator for GGAs that modulates GGA function with other ligands. We propose that the integration of combinatorial inputs from PI(4)P, Arf1 and ubiquitin may coordinately specify clathrin adaptor TGN recruitment through multiple low-affinity interactions.Item Regulation of Clathrin Mediated Endocytosis and Its Role in Cancer Progression(2017-05-15) Connelly, Sarah Elkin; Minna, John D.; Schmid, Sandra; Shay, Jerry W.; Danuser, GaudenzMetastasis is a multistep process requiring cancer cell signaling, invasion, migration, survival, and proliferation. These processes require dynamic modulation of cell surface proteins by endocytosis. Given this functional connection, it has been suggested that endocytosis is dysregulated in cancer. To test this, we developed In-Cell ELISA assays to measure three different endocytic pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin-independent endocytosis and compared these activities in 29 independently isolated non-small cell lung cancer (NSCLC) cell lines to determine whether there were systematic changes in the three different endocytic pathways. However we observed significant heterogeneity. Nonetheless, using hierarchical clustering based on their combined endocytic properties we identified two phenotypically distinct clusters of NSCLCs. One co-clustered with mutations in KRAS, a mesenchymal phenotype, increased invasion through collagen and decreased growth in soft agar, whereas the second was enriched in cells with an epithelial phenotype. We also used the In-Cell ELISA assay to characterize Ikarugamycin (IKA), a previously discovered antibiotic, which inhibits the uptake of oxidized low-density lipoproteins in macrophages, as well as clathrin-mediated endocytosis (CME) in plant cell lines. However, detailed characterization of IKA had yet been performed. Therefore, we performed biochemistry and microscopy experiments to further characterize the effects of IKA on CME. We showed that IKA acutely inhibits CME, but not other endocytic pathways with an IC50 of 2.7 μM. Although long-term incubation with IKA has cytotoxic effects, the short-term inhibitory effects on CME were reversible. Thus, IKA can be a useful tool for probing routes of endocytic trafficking. Finally, we investigated possible mechanisms that lead to altered endocytosis in cancer cells. We discovered that dynamin 1 (Dyn1), previously thought to be neuron specific is frequently upregulated and postranslationally regulated in cancer cells. Dyn1 expression alters the proliferation rates, growth in soft agar, and tumor growth of cancer cells. We hypothesize that these changes are due to alteration in cell surface protein expression and downstream signaling pathways and have developed protocols to test these hypothesizes. Taken together, our results suggest that endocytic alterations in cancer cells can significantly influence cancer-relevant phenotypes.