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First published online 25 January 2005
doi: 10.1242/jcs.01668

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Regulation of cortactin/dynamin interaction by actin polymerization during the fission of clathrin-coated pits

Department of Pathology, Greenebaum Cancer Center, University of Maryland School of Medicine, 15601 Crabbs Branch Way, Rockville, MD 20855, USA

View larger version (42K): [in a new window]   Fig. 1. Knockdown of cortactin expression attenuates transferrin uptake. (A) Cells grown in a six-well plate were transfected with cortactin siRNAs H1, H2, H3 and GFP siRNA as described in Materials and Methods. After 24 hours of transfection, cortactin expression in the treated cells was examined by immunoblot analysis. The same samples were also analyzed for actin and dynamin expression as negative controls. (B) Cells transfected with cortactin siRNA H1 (a and b), H2 (c and d), H3 (e and f) and GFP siRNA (g and h) were incubated with Oregon Green-labeled transferrin at 37°C for 30 minutes, fixed and stained with cortactin antibody (a,c,e and g) and DAPI (b,d,f and h). Internalized transferrin was seen as green dots in the cytoplasm. Magnification x600. (C) Percentage uptake of transferrin was quantified based on the fluorescence intensity of internalized transferrin in treated cells. The data shown are mean ± s.e.m. (n>400).

View larger version (27K): [in a new window]   Fig. 2. Cortactin is required for the formation of clathrin-coated vesicles. (A) Rat brain extracts were depleted with beads conjugated with cortactin antibody as described in Materials and Methods. Depletion of cortactin was verified by cortactin immunoblot analysis. Non-treated brain extracts at different amounts were also analyzed (lane 1, 12 µl; lane 2, 6 µl; lane 3, 3 µl). Lane 4, 12 µl of cortactin depleted extracts; lane 5, cortactin antibody conjugated beads that had been used to absorb brain extracts; lane 6, unused cortactin antibody beads. (B) 3T3-L1 cells were permeabilized by freezing and thawing. The permeabilized cells were incubated with B-SS-Tfn at 4°C for 20 minutes and then mixed with mock-depleted brain extracts (Mock), recombinant cortactin protein in the same buffer as extracts plus 1% BSA (Cort only), cortactin-depleted extracts (Cort D), or cortactin-depleted extracts supplemented with recombinant wild-type cortactin protein (Cort D + Cort), respectively. The mixtures were incubated at 37°C for 20 minutes. The cell pellets were treated with MesNa followed by quenching with iodoacetic acid and further analyzed for the presence of the remaining Biotin-SS-Tfn in the lysates, which represents MesNa resistance and transferrin internalized into CCVs. Data shown are the mean±s.d. of three experiments.

View larger version (36K): [in a new window]   Fig. 3. Cytochalasin D disrupts the association of cortactin with dynamin 2 at endocytic sites. (A) NIH3T3 cells were treated with DMSO or 10 µM cytochalasin D in serum-free medium. After 1 hour of treatment, cells were treated with 20 µg/ml biotin transferrin and incubated for 10 minutes. In a parallel experiment, cytochalasin D was removed washed once with serum-free medium and the cells were incubated for additional 1 hour and followed by adding biotin-labeled transferrin. The treated cells were fixed, permeabilized and stained for dynamin 2 (green) and cortactin (red). The boxed regions shown in panels a, b and c were magnified and are presented in panels a', b' and c', respectively. N, nucleus. Arrows in a' indicate typical colocalization of dynamin and cortactin. Magnification x1000. (B) Colocalization of endogenous cortactin and dynamin 2 in NIH3T3 cells was quantified using Optimas 5.2 image analysis software. The data shown are the mean±s.e.m. (n=20).

View larger version (33K): [in a new window]   Fig. 4. Quantitative analysis of the effect of cytochalasin D on the interaction between cortactin and dynamin 2. (A) NIH3T3 cells were treated with 10 µM cytochalasin D or DMSO for 0, 30, 60 or 90 minutes, followed by transferrin treatment for addition 10 minutes. The treated cells were lysed and immunoprecipitated with dynamin antibody. The immune complex was subjected to SDS-PAGE and immunoblot assay using cortactin antibody. To measure dynamin, filters were stripped and reblotted with dynamin 2 antibody. In a parallel experiment, internalization of biotin-labeled transferrin was performed in cytochalasin D-treated cells. The internalized transferrin was detected by immunoblot analysis with biotin antibody. (B) Quantification of the interaction between cortactin and dynamin was performed based on two independent experiments and the mean±s.d. is shown.

View larger version (34K): [in a new window]   Fig. 5. Dynamin proline-rich domain binds directly to cortactin. (A) Far-western blot analysis of interaction between cortactin and dynamin PRD. Recombinant His-cortactin, Arp2/3 complex and actin protein were subjected to SDS-PAGE followed by Coomassie Blue staining (upper panel) or transferred to a cellulose membrane and then incubated with a fusion protein containing dynamin PRD (GST-Dyn-PRD). Binding of GST-Dyn-PRD to cortactin was detected by anti-GST antibody (lower panel). (B) GST-Dyn-PRD or GST were immobilized on glutathione beads and incubated with cytosolic extracts of NIH3T3 cells at 4°C for 2 hours. Cortactin binding to beads was detected by immunoblot. The amino acid sequence of PRD in GST-Dyn-PRD, corresponding to rat dynamin 2 C-terminus, is presented in the lower panel.

View larger version (23K): [in a new window]   Fig. 6. Actin polymerization regulates the interaction between cortactin and dynamin. (A) Purified GST-Dyn-PRD (30 nM) was mixed with immobilized His-cortactin ranging from 0-200 nM in actin polymerization buffer plus G-actin (2 µM) and Arp2/3 complex (200 nM), and incubated at room temperature for 30 minutes. As a control, the reaction was also performed in the same buffer without G-actin and Arp2/3 complex. The samples were briefly centrifuged and the supernatants were analyzed for the presence of remaining GST-Dyn-PRD by immunoblotting with GST antibody. Amounts of GST-Dyn-PRD on the blot were quantified by digital scanning and normalized to the percentage of depletion. The resulting data were used to fit a rectangular hyperbola, yielding apparent Kd values as indicated. Inset, showing that GST has little binding activity to cortactin as compared to GST-Dyn-PRD. (B) The interaction of GST-Dyn-PRD and cortactin was carried out with G-actin plus Arp2/3 complex or G-actin without Arp2/3 complex. Cortactin was also preincubated with G-actin and Arp2/3 complex for 20 minutes prior to incubation with GST-Dyn-PRD for additional 20 minutes.

View larger version (63K): [in a new window]   Fig. 7. Binding to Arp2/3 complex is required for cortactin to recruit to dynamin 2. (A) MDA-MB-231 cells expressing cortactin-GFP or Cort(1-23)-GFP were grown in 10% serum medium and immunostained with GFP (a and d) or dynamin 2 antibody (b and e) as indicated. The stained cells were examined by confocal microscopy. Magnified images corresponding to the indicated boxes in merged pictures (c and f) are shown in c' and f'. Two dynamin structures that were closely associated with cortactin-GFP in panel c' are indicated by arrows. Magnification x1000. (B) Quantification of colocalization of cortactin-GFP and endogenous dynamin 2 using Optimas 5.2 image-analysis software. The data shown represent the mean±s.d. (n=20). (C) Cells expressing cortactin GFP variants were incubated with bio-transferrin for 10 minutes, lysed and subjected to immunoprecipitation with GFP antibody and further analyzed for the presence of cortactin and dynamin 2 by immunoblot assay. A representative blot is shown in the inset. The positions for dynamin 2 and cortactin GFP proteins are indicated. The density of the bands on the blot was digitalized and quantified. The data shown are the mean±s.d. of two independent experiments.

View larger version (14K): [in a new window]   Fig. 8. Arp2/3 complex binding is required for cortactin to interact with dynamin 2. (A) Expression of cortactin fusion proteins was analyzed by immunoblotting whole cell lysates with cortactin antibody. (B) 2x105 Cells expressing cortactin-GFP fusions were seeded on a 12-well plate, grown in a serum-free medium at 37°C for 30 minutes and incubated with biotin-transferrin for 40 minutes on ice to allow sufficient binding. Endocytosis was initiated by incubation at 37°C for the times as indicated and terminated by placing on ice. The treated cells were then subjected to ELISA-based biotin-transferrin uptake assay as described in Materials and Methods.

View larger version (17K): [in a new window]   Fig. 9. A model for cortactin- and dynamin-mediated fission of CCV. Step 1, binding of an extracellular ligand to its membrane receptor induces formation of a clathrin-coated pit and recruitment of dynamin to the neck of an invaginated vesicle where it is oligomerized to form a ring structure (for simplicity, only one dynamin ring is shown). The ligand/receptor interaction also triggers assembly of actin (orange line) by activation of WASP family proteins, the Arp2/3 complex and cortactin, resulting ultimately in a tight association of cortactin/Arp2/3 complex at a branched site (yellow circle). Step 2, while the actin polymerization is taking place, the SH3 domain of cortactin at a branching site becomes accessible for interaction with the PRD of dynamin at vesicles. Detail of the interactions between F-actin, cortactin, Arp2/3 complex and dynamin is presented. Step 3, elongation of actin filaments makes the complex of cortactin and dynamin away from the barbed end or its associated plasma membrane and eventually results in the separation of the vesicle from the plasma membrane (Step 4).

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