RNAi-mediated Hip1R silencing results in stable association between the endocytic machinery and the actin assembly machinery

Abstract

Actin filaments transiently associate with the endocytic machinery during clathrin-coated vesicle formation. Although several proteins that might mediate or regulate this association have been identified, in vivo demonstration of such an activity has not been achieved. Huntingtin interacting protein 1R (Hip1R) is a candidate cytoskeletal-endocytic linker or regulator because it binds to clathrin and actin. Here, Hip1R levels were lowered by RNA interference (RNAi). Surprisingly, rather than disrupting the transient association between endocytic and cytoskeletal proteins, clathrin-coated structures (CCSs) and their endocytic cargo became stably associated with dynamin, actin, the Arp2/3 complex, and its activator, cortactin. RNAi double-depletion experiments demonstrated that accumulation of the cortical actin-endocytic complexes depended on cortactin. Fluorescence recovery after photobleaching showed that dynamic actin filament assembly can occur at CCSs. Our results provide evidence that Hip1R helps to make the interaction between actin and the endocytic machinery functional and transient.

Figure 1.

Hip1R siRNA duplexes reduce Hip1R expression in HeLa cells. (A) Western blots of siRNA-transfected cells using antibodies against Hip1R, Hip1, clathrin heavy chain (CL HC). (B) Western blot to quantify the reduction in Hip1R expression in A1 and A2 cells. In lanes 4–7 different amounts of extracts were loaded (10–100%) as indicated in the figure. (C) Hip1R immunofluoresence of cells treated with invA2, A1, or A2 (top panels). Bottom: DAPI staining of nuclei. Bars, 10 μm.

Figure 2.

Hip1R “knock-down” cells have endocytic and actin defects. (A) Visual endocytosis assay. HeLa cells treated with siRNA (A1, A2, or invA2 as indicated) were labeled with Texas Red tranferrin for 30 min to visualize endocytic compartments. Top: DAPI staining of nuclei; bottom: transferrin labeling. (B) Biochemical endocytosis assay. The graph shows percent transferrin uptake as a function of total surface bound transferrin over time in A2-treated and mock-treated cells (control). Three independent experiments were performed for each data point. (C) The bar graph shows percent inhibition in biochemical transferrin uptake assay in A2 cells compared with control cells at different time points as indicated. (D) HeLa cells treated with siRNA (A1, A2, A3, or invA2 as indicated) were stained with DAPI (top) and Texas Red-X phalloidin (middle and bottom) to visualize F-actin. Bottom panels are enlargements of boxes in the middle panels. Bars, 10 μm.

Figure 3.

Cortactin and the Arp2/3 complex are specifically recruited to the cortical F-actin structures. HeLa cells treated with siRNA (A1 or invA2) for 3 days were stained with fluorescent phalloidin or different antibodies as indicated. (A) Texas Red-X phalloidin (left panels), cortactin (middle panels), merge (right panels). Boxes in the bottom right corners are enlarged regions of the micrographs. Bars, 10 μm. (B) Cortactin (left), Arp3 (middle), merge (right). Bars, 10 μm. (C) This figure shows the different types of F-actin/cortactin structures observed in A1-treated cells. Left panels show low magnification views of cells wherein the periphery of each cell is marked. Red arrows indicate individual tail-like F-actin/cortactin structures (top). Blue arrows indicate ring-like F-actin/cortactin structures (bottom). Yellow arrows indicate more complex F-actin/cortactin structures (bottom). Bars, 2.5 μm.

Figure 4

The F-actin structures that accumulate in the absence of Hip1R are associated with endocytic components. HeLa cells treated with siRNA (A1 or invA2) for 3 days were stained with different antibodies as indicated. (A) Clathrin (green) colocalizes with the F-actin (red) structures in A1 cells. The middle panel shows A1 cells containing several F-actin spots. Arrows indicate colocalization between clathrin and F-actin. Arrowheads indicate spots that only stain with clathrin. Bars, 10 μm. (B) Coincidence of clathrin and actin in A1 cells containing complex F-actin structures (see arrows). Top two panels show Texas Red-X phalloidin staining (red) and clathrin staining (green). Bars, 10 μm. Bottom panel shows Arp3 staining (green) and clathrin staining (red). Asterisk indicates a stress fiber. Bar, 10 μm. (C) Clathrin, AP-2, and dynamin (green) localize to one end of the tail-like F-actin structures (red) in A1 cells (see arrows). Bars, 2 μm.

Figure 4

The F-actin structures that accumulate in the absence of Hip1R are associated with endocytic components. HeLa cells treated with siRNA (A1 or invA2) for 3 days were stained with different antibodies as indicated. (A) Clathrin (green) colocalizes with the F-actin (red) structures in A1 cells. The middle panel shows A1 cells containing several F-actin spots. Arrows indicate colocalization between clathrin and F-actin. Arrowheads indicate spots that only stain with clathrin. Bars, 10 μm. (B) Coincidence of clathrin and actin in A1 cells containing complex F-actin structures (see arrows). Top two panels show Texas Red-X phalloidin staining (red) and clathrin staining (green). Bars, 10 μm. Bottom panel shows Arp3 staining (green) and clathrin staining (red). Asterisk indicates a stress fiber. Bar, 10 μm. (C) Clathrin, AP-2, and dynamin (green) localize to one end of the tail-like F-actin structures (red) in A1 cells (see arrows). Bars, 2 μm.

Figure 5.

EGF colocalizes with the cortical actin structures. HeLa cells treated with invA2 or A1 were incubated with EGF-rhodamine at 4°C to label CCPs. Left: EGF staining (red); middle: cortactin staining (green); and right: merged images. Bar, 10 μm.

Figure 6.

The F-actin/cortactin structures are tethered to the cell cortex via association with the endocytic machinery. (A and B) HeLa cells treated with A3 were stained Texas Red-X phallodin (red) and anti–AP-2 antibodies (green). (A) A gallery of images showing the first 15 deconvolved focal planes (0.1 μm/section) of actin/AP-2 structures. The top left image shows the first plane from the ventral surface. Bar, 1 μm. (B) Two orientations of a volume rendering of the actin/AP2 structures shown in A. Renderings are oriented with plasma membrane at bottom (bottom image) or in the plane of the viewer (top image). Arrow shows a tail-like F-actin structure connected to one AP-2 spot. Arrowhead shows a tail-like actin structure, wherein several AP-2 spots are connected to F-actin. Small grid boxes, 200 × 200 nm. (C) A gallery of “deep etch” electron micrographs of A3-treated HeLa cells. These micrographs show anaglyph stereo views of the inner surface of “unroofed” cells. Bar, 0.5 μm.

Figure 7.

The F-actin structures that accumulate in the absence of Hip1R contain rapidly polymerizing actin. (A) Time lapse videomicroscopy of invA2- or A1-treated HeLa cells expressing GFP-actin. The F-actin structures in A1 cells were observed at 3-s intervals. Top panel shows low magnification views of the cells. Bars, 10 μm. Bottom panel is enlargements of boxes in the top panel showing the first frame of movie 1 and movie 2. Bars, 2.5 μm. (B) FRAP analysis of A1-treated HeLa cells expressing GFP-actin. Top (FRAP 1): analysis of small spots/tail-like F-actin structures; bottom (FRAP 2): analysis of a larger ring-like structures. We observed the F-actin structures in A1 cells at 3-s intervals >40 frames. The cells were photobleached in a defined area after two frames as indicated in the figure (see asterisk). The graph shows fluorescence intensity (arbitrary units) as a function of time (s), where the data have been normalized against a reference region that was not photobleached. Bars, 10 μm.

Figure 8.

Phenotypes associated with A1 and A3 are due to loss of Hip1R function. (A and B) Stable HeLa cell lines expressing mouse Hip1R-6myc or 6myc (control) were treated with A1 or invA2 for 3 days as indicated. (A) Cells were fixed and stained with DAPI (top) and anticortactin antibodies (middle and bottom). (B) Western blotting of cell extracts from these cells. Black arrow (top) indicates exogenous Hip1R-6myc, and gray arrow (bottom) indicates endogenous Hip1R.