Differential requirements for clathrin-dependent endocytosis at sites of cell-substrate adhesion.

Molecular Biology of the Cell
Erika M Batchelder, Defne Yarar

Abstract

Clathrin-dependent endocytosis is a major route for the cellular import of macromolecules and occurs at the interface between the cell and its surroundings. However, little is known about the influences of cell-substrate attachment in clathrin-coated vesicle formation. Using biochemical and imaging-based methods, we find that cell-substrate adhesion reduces the rate of endocytosis. Clathrin-coated pits (CCPs) in proximity to substrate contacts exhibit slower dynamics in comparison to CCPs found more distant from adhesions. Direct manipulation of the extracellular matrix (ECM) to modulate adhesion demonstrates that tight adhesion dramatically reduces clathrin-dependent endocytosis and extends the lifetimes of clathrin structures. This reduction is in part mediated by integrin-matrix engagement. In addition, we demonstrate that actin cytoskeletal dynamics are differentially required for efficient endocytosis, with a stronger requirement for actin polymerization in areas of adhesion. Together, these results reveal that cell-substrate adhesion regulates clathrin-dependent endocytosis and suggests that actin assembly facilitates vesicle formation at sites of adhesion.

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Methods Mentioned

BETA
transfection
fluorescence microscopy
GTPase
confocal microscopy

Software Mentioned

MetaMorph
Minitab

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