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Dynamics and Cargo Selectivity of Endocytic Adaptor Proteins

Keyel, Peter Andrew (2006) Dynamics and Cargo Selectivity of Endocytic Adaptor Proteins. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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[img] Video (QuickTime) (Movie 4-1: live cell confocal following DiI-LDL and Tfn488 internalization in control and Dab2+ARH siRNA treated HeLa cells)
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[img] Video (AVI) (Movie 2-1: live cell TIR-FM)
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[img] Video (QuickTime) (Movie 4-2: DiI-LDL internalization in YFP-Dab2 transfected HeLa cells)
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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.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Keyel, Peter
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairTraub, Lintontraub@pitt.eduTRAUB
Committee ChairAridor, Meiraridor@pitt.eduARIDOR
Committee MemberLinstedt,
Committee MemberDrain, Peterdrain@pitt.eduDRAIN
Committee MemberWatkins, Simonswatkins@pitt.eduSWATKINS
Date: 13 September 2006
Date Type: Completion
Defense Date: 21 August 2006
Approval Date: 13 September 2006
Submission Date: 23 August 2006
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Cell Biology and Molecular Physiology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: AP-2; CLASP; transferrin
Other ID:, etd-08232006-130824
Date Deposited: 10 Nov 2011 20:01
Last Modified: 15 Nov 2016 13:49


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