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| TR |
TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
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MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 608 |
Dynamics
and Cargo Selectivity of Endocytic Adaptor Proteins |
Peter
Andrew Keyel |
endocytosis,
clathrin, total internal reflection fluorescence microscopy (TIR-FM), clathrin-associated sorting
proteins (CLASPs), autosomal recessive hypercholesterolemia (ARH) protein |
For immunofluorescence, the cells
were plated onto 12 mm glass coverslips prior to transfection, while for TIR-FM experiments
cells were plated onto glass-bottom 35-mm tissue culture dishes (MatTek, Ashland, MA).
|
total
internal reflection fluorescence microscopy (TIR-FM) |
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|
HeLa |
| Abstract |
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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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