The actin cytoskeleton is believed to contribute to the formation of clathrin-coated pits, although the specific components that connect actin filaments with the endocytic machinery are unclear. Cortactin is an F-actin-associated protein, localizes within membrane ruffles in cultured cells, and is a direct binding partner of the large GTPase dynamin. This direct interaction with a component of the endocytic machinery suggests that cortactin may participate in one or several endocytic processes. Therefore, the goal of this study was to test whether cortactin associates with clathrin-coated pits and participates in receptor-mediated endocytosis. Morphological experiments with either anti-cortactin antibodies or expressed red fluorescence protein-tagged cortactin revealed a striking colocalization of cortactin and clathrin puncta at the ventral plasma membrane. Consistent with these observations, cells microinjected with these antibodies exhibited a marked decrease in the uptake of labeled transferrin and low-density lipoprotein while internalization of the fluid marker dextran was unchanged. Cells expressing the cortactin Src homology three domain also exhibited markedly reduced endocytosis. These findings suggest that cortactin is an important component of the receptor-mediated endocytic machinery, where, together with actin and dynamin, it regulates the scission of clathrin pits from the plasma membrane. Thus, cortactin provides a direct link between the dynamic actin cytoskeleton and the membrane pinchase dynamin that supports vesicle formation during receptor-mediated endocytosis.

Molecular and Cellular Biology. 2003 March; 23 (6): 2162?2170.
Cortactin Is a Component of Clathrin-Coated Pits and Participates in Receptor-Mediated Endocytosis
Hong Cao, James D. Orth, Jing Chen, Shaun G. Weller, John E. Heuser, and Mark A. McNiven

Cortactin antibodies stain numerous plasma membrane foci that colocalize with CCPs. (a - c") Immunofluorescence microscopy of cultured clone 9 cells stained with cortactin antibodies directed to the C-Tyr (a) and the actin-binding (AB3) (b) domains. Both antibodies labeled small punctate foci on the basal plasma membrane that colocalize significantly (arrows) with clathrin (a" and b"). Fluorescence images of clone 9 cells expressing cortactin-RFP and costained for clathrin are shown (c - c"). As for the antibody staining, significant levels of the tagged cortactin protein associated with numerous punctate foci along the ventral membrane. (c") Higher-magnification images of the boxed regions show that a substantial number of the cortactin-RFP- and clathrin-positive puncta overlap or are in close association. (d to f) Immunoelectron microscopy of the inner plasma membrane of unroofed COS7 cells labeled with the C-Tyr (d and e) and AB3 (f) cortactin antibodies. Flat or curved clathrin lattices as well as actin filaments can be seen associated with the plasma membrane. Numerous immuno-gold particles (yellow) can be seen specifically labeling the clathrin cage and appear to cluster in a concentric ring along the pit base, as opposed to the bulb, as the membrane invaginates (g). (d - f) Gold particles can also be resolved on actin filaments and actin filament branches (arrows). Bars = 10 mm (a - c"). Magnification, X 72,750 (d - f).
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Cortactin antibody injection and expression of the SH3 domain significantly inhibit RME of transferrin. (a) Transferrin fluorescence images of cultured cells microinjected (asterisk) with purified antibodies or expressing the cortactin SH3 domain. All injectate solutions contained FITC-dextran to confirm successful injections. Injection of kinesin antibody had no effect on the internalization of Alexa 594-transferrin. (b and c) In contrast, cells microinjected with either the AB3 or C-Tyr purified cortactin antibodies showed significant inhibition of the RME of Alexa 594-transferrin. (d) Expression of the SH3 domain also potently blocked transferrin internalization, demonstrating the importance of cortactinís SH3 domain for RME. Bar = 10 mm. (e) The antibody-injected and SH3 domain-expressing cells showing a block in endocytosis were counted. Whereas the number of cells with normal uptake is not affected in buffer and kinesin antibody-injected cells, only 20% of cells injected with cortactin antibody or expressing the SH3 domain had normal uptake. (f and g) Fluorescence intensity (FI) quantitation of internalized transferrin confirmed that, when compared to controls, there is a significant 60% block in the antibody-injected cells (f) and a 70% block in the cells expressing the SH3 domain (g). Cort wt = full-length cortactin, SH3 = cortactin SH3 domain. Click here for a larger version.

Dynamin and cortactin as key components of the cytoskeletal-endocytic machinery. Dynamin interacts with numerous proteins that function to regulate clathrin-dependent endocytosis. Oligomers of dynamin function to restrict or pinch the neck of the invaginating membrane while multiple extended PRDs interact directly with a variety of other proteins, including cortactin, that serve to link Dyn2 to the actin cytoskeleton and the Arp2/3 complex. The complex cytoskeletal network also includes interactions with other actin-binding proteins such as syndapin, N-WASp, Abp1, and Hip1R (some not shown for simplification). Click here for a larger version.