Cortactin is a component of clathrin-coated pits and participates in receptor-mediated endocytosis - PubMed (original) (raw)

Cortactin is a component of clathrin-coated pits and participates in receptor-mediated endocytosis

Hong Cao et al. Mol Cell Biol. 2003 Mar.

Abstract

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.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

Polyclonal antibodies raised against distinct cortactin peptides appear specific for the cortactin protein. (a) The diagram depicts different domains of the cortactin protein including the F-actin-binding sites, C-terminal tyrosines that are phosphorylated by Src kinases, and the SH3 domain that binds Dyn2. Areas that peptide antibodies were directed against are indicated (asterisks). To test the specificity of the antibodies, peptide affinity-purified serum was used for immunoblotting and immunoprecipitation. (b) An equal mass of total lysate from rat liver homogenate, cultured hepatocyte (clone 9 [C9]) lysate, or purified His6-cortactin (Cort) were immunoblotted with either the AB3, C-Tyr (C-Y), or a commercially available cortactin monoclonal antibody (4F11 [F11]; Upstate Biotechnology). The polyclonal anti-cortactin antibodies and the 4F11 antibody specifically detected cortactin in all conditions. (c) Cortactin was immunoprecipitated (IP) from clone 9 lysates with each antibody, and the precipitated protein was immunoblotted with the 4F11 antibody. All cortactin antibodies tested precipitated cortactin that was easily detected by immunoblotting. (d) Coomassie staining of the immunoprecipitation reactions demonstrated that a substantial amount of cortactin was obtained and that it was the major protein species present (compare the bands from the immunoprecipitations against 0.2 μg of purified His6-cortactin [Cort]). IgG, immunoglobulin G.

FIG. 2.

FIG. 2.

Cortactin antibodies stain numerous plasma membrane foci that colocalize with CCPs. (a to 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 to 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 to f) Gold particles can also be resolved on actin filaments and actin filament branches (arrows). Bars = 10 μm (a to c"). Magnification, ×72,750 (d to f).

FIG. 3.

FIG. 3.

Inhibition of clathrin-mediated endocytosis in cells expressing GTPase-deficient Dyn2 results in an abnormal distribution of both cortactin and clathrin. Clone 9 cells transiently expressing GTPase-deficient Dyn2K44A-GFP were fixed and labeled to visualize the clathrin (X-22 monoclonal antibody) and cortactin (C-Tyr). (a to a") Cells expressing the Dyn2 mutant (asterisks) have numerous clustered and clumpy-looking clathrin pits (a′ and b′, arrows) in comparison to neighboring nontransfected cells. The defective clathrin staining dramatically colocalized with cortactin (a" and b", arrows). Bar = 10 μm.

FIG. 4.

FIG. 4.

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 cortactin's SH3 domain for RME. Bar = 10 μm. (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.

FIG. 5.

FIG. 5.

Cortactin function is also required for the RME of LDL. (a and a′) A cortactin AB3 antibody-injected cell (asterisk) demonstrating a significant block in the level of internalized DiI-LDL compared to neighboring noninjected cells. (b and b′) Cells expressing the cortactin SH3 domain (asterisk) also demonstrated substantially reduced internalization of LDL compared to neighboring, nonexpressing cells. (c and d) Quantitation of the effect of cortactin antibody injection on LDL internalization. Nearly 80% of the cells injected with cortactin antibodies appeared blocked. Fluorescence intensity (FI) quantitation of internalized LDL in these antibody-injected cells showed only 40 to 50% uptake compared to buffer-injected, kinesin antibody-injected, or noninjected neighbors. (e and f) Quantitation of the effect of cortactin SH3 domain expression on LDL internalization. Over 80% of the cells expressing the SH3 domain appeared blocked (e). Fluorescence intensity quantitation of internalized LDL in these SH3 domain-expressing cells showed only 40 to 50% uptake compared to neighboring nontransfected cells (f). Bar = 10 μm. Cort wt, full-length cortactin; SH3, cortactin SH3 domain.

FIG. 6.

FIG. 6.

Inhibition of cortactin does not significantly affect fluid-phase endocytosis. (a and a′) Cortactin antibody (AB3)-injected cells (FITC-dextran) (asterisks) showed no obvious defect in the internalization of rhodamine-labeled dextran compared to their noninjected neighbors. (b) The number of cells with normal uptake was unaffected in cortactin antibody-injected cells, as reflected by the same number of dextran-positive cells compared to buffer- and kinesin antibody-injected cells. (c) In agreement with the immunocytochemistry experiments, quantitation of the mean dextran fluorescence intensity (FI) per unit area in cortactin antibody-injected cells revealed no significant difference in dextran uptake compared to control conditions. The results in this figure suggest that the reagents used in this study specifically affect cortactin function during RME and not during fluid-phase endocytosis. Bar = 10 μm.

FIG. 7.

FIG. 7.

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).

Similar articles

Cited by

References

    1. Ahn, S., S. Maudsley, L. M. Luttrell, R. J. Lefkowitz, and Y. Daaka. 1999. Src-mediated tyrosine phosphorylation of dynamin is required for beta2-adrenergic receptor internalization and mitogen-activated protein kinase signaling. J. Biol. Chem. 274:1185-1188. - PubMed
    1. Cao, H., F. Garcia, and M. McNiven. 1998. Differential distribution of dynamin isoforms in mammalian cells. Mol. Biol. Cell 9:2595-2609. - PMC - PubMed
    1. Cao, H., H. M. Thompson, E. W. Krueger, and M. A. McNiven. 2000. Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin. J. Cell Sci. 113:1993-2002. - PubMed
    1. Damke, H., D. D. Binns, H. Ueda, S. L. Schmid, and T. Baba. 2001. Dynamin GTPase domain mutants block endocytic vesicle formation at morphologically distinct stages. Mol. Biol. Cell 12:2578-2589. - PMC - PubMed
    1. Foster-Barber, A., and J. M. Bishop. 1998. Src interacts with dynamin and synapsin in neuronal cells. Proc. Natl. Acad. Sci. USA 95:4673-4677. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources