Abstract
BackgroundThe notion that AP-2 clathrin adaptor is an essential component of an endocytic clathrin coat appears to conflict with recent observations that substantial AP-2 depletion, using RNA interference with synthesis of AP-2 subunits, fails to block uptake of certain ligands known to internalize through a clathrin-based pathway.Methodology/Principal FindingsWe report here the use of in vivo imaging data obtained by spinning-disk confocal microscopy to study the formation of clathrin-coated structures at the plasma membranes of BSC1 and HeLa cells depleted by RNAi of the clathrin adaptor, AP-2. Very few clathrin coats continue to assemble after AP-2 knockdown. Moreover, there is a total absence of clathrin-containing structures completely lacking AP-2 while all the remaining coats still contain a small amount of AP-2. These observations suggest that AP-2 is essential for endocytic coated-pit and coated-vesicle formation. We also find that AP-2 knockdown strongly inhibits light-density lipoprotein (LDL) receptor-mediated endocytosis, as long as cells are maintained in complete serum and at 37°C. If cells are first incubated with LDL at 4°C, followed by warming, there is little or no decrease in LDL uptake with respect to control cells. LDL uptake at 37°C is also not affected in AP-2 depleted cells first deprived of LDL by incubation with either serum-starved or LDL-starved cells for 24 hr. The LDL-deprived cells display a significant increase in endocytic structures enriched on deeply invaginated tubes that contain LDL and we suggest that under this condition of stress, LDL might enter through this alternative pathway.Conclusions/SignificanceThese results suggest that AP-2 is essential for endocytic clathrin coated-pit and coated-vesicle formation. They also indicate that under normal conditions, functional endocytic clathrin coated pits are required for LDL internalization. We also show that under certain conditions of stress, cells can upregulate alternative endocytic structures with the potential to provide compensatory trafficking pathways.
Highlights
Heterotetrameric clathrin adaptor complexes (APs) are major components of clathrin-coated vesicles, second in abundance only to clathrin itself [1]
The notion that AP-2 is an essential component of an endocytic clathrin coat appears to conflict with the recent observation that substantial AP-2 depletion, using RNA interference with synthesis of m2, b2- or a-adaptin, fails to block uptake of certain ligands known to internalize through a clathrin-based pathway: lightdensity lipoprotein (LDL) [5,6,7,8], epidermal growth factor (EGF)
After AP-2 depletion, can we find clathrin coats devoid of AP-2, or does the reduced number of coats that form always contain a detectable amount of the residual AP-2 not eliminated by the RNAi treatment? Second, what happens to receptor-mediated endocytosis of LDL in AP-2 depleted cells, if the cells remain in physiological conditions throughout the experiment? Using livecell fluorescence microscopy imaging, we confirm the severe reduction in the number of clathrin coats forming in cells severely depleted of AP-2
Summary
Heterotetrameric clathrin adaptor complexes (APs) are major components of clathrin-coated vesicles, second in abundance only to clathrin itself [1]. The notion that AP-2 is an essential component of an endocytic clathrin coat appears to conflict with the recent observation that substantial AP-2 depletion, using RNA interference with synthesis of m2-, b2- or a-adaptin, fails to block uptake of certain ligands known to internalize through a clathrin-based pathway: lightdensity lipoprotein (LDL) [5,6,7,8], epidermal growth factor (EGF). The notion that AP-2 clathrin adaptor is an essential component of an endocytic clathrin coat appears to conflict with recent observations that substantial AP-2 depletion, using RNA interference with synthesis of AP-2 subunits, fails to block uptake of certain ligands known to internalize through a clathrin-based pathway
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