Clathrin-mediated endocytosis is a critical process through which a wide variety of extracellular material is internalized. The primary component, clathrin, forms a cargo-selective lattice at the plasma membrane, as well as on endosomes and the TGN, though the cargo-selective components are incompletely defined. An ideal tool for understanding the spatio-temporal dynamics of both the clathrin coat and the cargo selected is total internal reflection fluorescence microscopy (TIR-FM), which permits selective imaging of events closely apposed to the ventral plasma membrane. Previously, observation of the clathrin coat has shown both static and dynamic populations, with some dynamic structures undergoing microtubule-dependent motion; the 70-110 nm decay constant of the TIR-FM field has led to the assumption that these are all representative of coated pits. Here, I demonstrate that the dynamic population of clathrin is primarily endosomal, as it lacks colocalization with the plasma membrane-specific endocytic adaptor AP-2, but colocalizes with large, internalized low density lipoprotein (LDL) and transferrin positive structures. Other clathrin-associated sorting proteins (CLASPs) remain in relatively static structures as well. One such CLASP, autosomal recessive hypercholesterolemia (ARH) protein, is the defective protein in ARH, which is typified by the failure of hepatic LDL receptor internalization, despite no LDL receptor mutations. ARH interacts with AP-2 via the novel, helical FXX[FL]XXXR motif present in its C-terminus. Here, I demonstrate the importance of this motif for targeting ARH to coated pits in cells and LDL uptake. As knockdown of ARH is insufficient to block LDL receptor endocytosis in fibroblasts, I show that the CLASP Disabled-2 (Dab2) works with ARH to sort the LDL receptor. Ablation of these two components using RNAi halts LDL receptor endocytosis, and either exogenous ARH or Dab2 rescue this phenotype. The endocytic defect in the liver of ARH patients is due to the lack of Dab2 expression in hepatocytes, making this cell type sensitive to ARH levels for LDL uptake. This work formally validates the CLASP hypothesis, and demonstrates that these CLASPs are general components of the clathrin-coated pit that are regulated in a tissue-specific fashion.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-08232006-130824 |
| Date | 13 September 2006 |
| Creators | Keyel, Peter Andrew |
| Contributors | Adam Linstedt, Peter Drain, Simon Watkins, Meir Aridor, Linton Traub |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf, video/x-msvideo, video/quicktime |
| Source | http://etd.library.pitt.edu/ETD/available/etd-08232006-130824/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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