Dataset Information

Endocytic trafficking of activated EGFR is AP-2 dependent and occurs through preformed clathrin spots.

ABSTRACT: The removal of the epidermal growth factor receptor (EGFR) from the cell surface by endocytosis is triggered by receptor activation, but many facets of EGFR trafficking remain unresolved. We employed total internal fluorescence microscopy to elucidate the dynamics of activated EGFR at the cell surface through live-cell imaging. The results of these studies demonstrate that: (1) EGFR does not localize to caveolae in live cells either before or after activation; (2) EGFR does localize to clathrin-coated pits, but only after activation; (3) activation does not result in the formation of new clathrin-coated pits; (4) activated EGFR clusters at sites of preformed clathrin lattices; (5) The AP-2 complex is involved in the internalization of activated EGFR. Using imaging techniques to show the endocytic sorting of activated EGFR for the first time in live cells, these studies suggest a refinement of the model for EGFR entry.

SUBMITTER: Rappoport JZ

PROVIDER: S-EPMC2721002 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Publications

Endocytic trafficking of activated EGFR is AP-2 dependent and occurs through preformed clathrin spots.

Rappoport Joshua Z JZ Simon Sanford M SM

Journal of cell science 20090407 Pt 9

The removal of the epidermal growth factor receptor (EGFR) from the cell surface by endocytosis is triggered by receptor activation, but many facets of EGFR trafficking remain unresolved. We employed total internal fluorescence microscopy to elucidate the dynamics of activated EGFR at the cell surface through live-cell imaging. The results of these studies demonstrate that: (1) EGFR does not localize to caveolae in live cells either before or after activation; (2) EGFR does localize to clathrin- ...[more]

PMID: 19351721

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Project description:UnlabelledBacteria shed a diverse set of outer membrane vesicles that function as transport vehicles to deliver effector molecules and virulence factors to host cells. Helicobacter pylori is a gastric pathogen that infects half of the world's population, and in some individuals the infection progresses into peptic ulcer disease or gastric cancer. Here we report that intact vesicles from H. pylori are internalized by clathrin-dependent endocytosis and further dynamin-dependent processes, as well as in a cholesterol-sensitive manner. We analyzed the uptake of H. pylori vesicles by gastric epithelial cells using a method that we refer to as quantification of internalized substances (qIS). The qIS assay is based on a near-infrared dye with a cleavable linker that enables the specific quantification of internalized substances after exposure to reducing conditions. Both chemical inhibition and RNA interference in combination with the qIS assay showed that H. pylori vesicles enter gastric epithelial cells via both clathrin-mediated endocytosis and additional endocytic processes that are dependent on dynamin. Confocal microscopy revealed that H. pylori vesicles colocalized with clathrin and dynamin II and with markers of subsequent endosomal and lysosomal trafficking. Interestingly, however, knockdown of components required for caveolae had no significant effect on internalization and knockdown of components required for clathrin-independent carrier (CLIC) endocytosis increased internalization of H. pylori vesicles. Furthermore, uptake of vesicles by both clathrin-dependent and -independent pathways was sensitive to depletion, but not sequestering, of cholesterol in the host cell membrane suggesting that membrane fluidity influences the efficiency of H. pylori vesicle uptake.ImportanceBacterial vesicles act as long-distance tools to deliver toxins and effector molecules to host cells. Vesicles can cause a variety of host cell responses via cell surface-induced cell signaling or internalization. Vesicles of diverse bacterial species enter host cells via different endocytic pathways or via membrane fusion. With the combination of a fluorescence-based quantification assay that quantifies internalized vesicles in a large number of cells and either chemical inhibition or RNA interference, we show that clathrin-mediated endocytosis is the major pathway for uptake of Helicobacter pylori vesicles and that lipid microdomains of the host cell membrane affect uptake of vesicles via clathrin-independent pathways. Our results provide important insights about membrane fluidity and its important role in the complex process that directs the H. pylori vesicle to a specific endocytic pathway. Understanding the mechanisms that operate in vesicle-host interactions is important to fully recognize the impact of vesicles in pathogenesis.

Dataset Information

Endocytic trafficking of activated EGFR is AP-2 dependent and occurs through preformed clathrin spots.

Publications

Endocytic trafficking of activated EGFR is AP-2 dependent and occurs through preformed clathrin spots.

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