Actin dynamics coupled to clathrin-coated vesicle formation at the trans-Golgi network

Abstract

In diverse species, actin assembly facilitates clathrin-coated vesicle (CCV) formation during endocytosis. This role might be an adaptation specific to the unique environment at the cell cortex, or it might be fundamental, facilitating CCV formation on different membranes. Proteins of the Sla2p/Hip1R family bind to actin and clathrin at endocytic sites in yeast and mammals. We hypothesized that Hip1R might also coordinate actin assembly with clathrin budding at the trans-Golgi network (TGN). Using deconvolution and time-lapse microscopy, we showed that Hip1R is present on CCVs emerging from the TGN. These vesicles contain the mannose 6-phosphate receptor involved in targeting proteins to the lysosome, and the actin nucleating Arp2/3 complex. Silencing of Hip1R expression by RNAi resulted in disruption of Golgi organization and accumulation of F-actin structures associated with CCVs on the TGN. Hip1R silencing and actin poisons slowed cathepsin D exit from the TGN. These studies establish roles for Hip1R and actin in CCV budding from the TGN for lysosome biogenesis.

Figure 1.

Fluorescence microscopy analysis of Hip1R intracellular localization. (A–D) HeLa cells expressing Hip1R-GFP (green) (Engqvist-Goldstein et al., 1999) were fixed and immunostained for clathrin heavy chain (red) and TGN46 (blue). (A and B) Fluorescence data were collected using an API Deltavision DV4 Restoration microscope. Deconvolution was performed using the API SoftWoRx software. (B) Digital magnification of the TGN area boxed in A. (C and D) Three-dimensional volume (Top, top view; Bottom, side view) rendering of the area shown in B, made using 15 successive deconvolved planes using Imaris4.0 (Bitplane). (C) TGN46 (blue) and Hip1R (green). (D) TGN46 (blue) and clathrin (red). (E–H) Time-lapse microscopy imaging of live HeLa cells expressing (E) Hip1R-GFP (Engqvist-Goldstein et al., 1999), (F) dsRed-clathrin LC isoform a (LCa) (Gaidarov et al., 1999), (G) GalT-CFP (CLONTECH Laboratories, Inc.) as a TGN marker. (H) Merged image. (I) Magnification of the region boxed in H. Images were acquired using an API Deltavision DV4 microscope at the intervals indicated. The white dashed line in the merged image represents the edge of the GalT-CFP signal at the beginning of the time-lapse experiment. Arrows track individual Hip1R/clathrin vesicle arising from the TGN. The shift between Hip1R-GFP and dsRed-clathrin in I is due to the time delay for image acquisition between the two fluorescence channels (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200403120/DC1). (J–M) Time-lapse microscopy imaging of HeLa cells expressing (J) Hip1R-YFP or (K) CD-MPR-CFP (Barbero et al., 2002). (L) Merged image. (M) Magnification of the region boxed in L. Images were acquired using an API Deltavision DV4 microscope at the indicated intervals. Arrows track individual Hip1R vesicles aligned along tubulo-vesicular structures emerging from the TGN (see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200403120/DC1).

Figure 2.

Effect of Hip1R knock down on TGN morphology of HeLa cells. (A–C) HeLa cells treated with the indicated siRNA duplexes for 3 d were fixed, and then TGN46 (green) and nuclei (DAPI, blue) were labeled with antibodies and dye, respectively. Cells were imaged using an inverted Nikon Eclipse TE300 fluorescence microscope (Nikon) with a Nikon Plan Apo (1.4NA) 100× objective and a Hamamatsu ORCA cooled CCD camera (Hamamatsu). (D–F) Electron micrographs of HeLa cells treated with the InvA2 siRNA (D) and A2 siRNA (E). Cells depleted for Hip1R show an accumulation of lysosome (Ly) and of clathrin-coated buds or vesicles. (F) Magnification of the region boxed in E. Arrows, clathrin-coated buds or vesicles.

Figure 3.

Interaction between actin filaments and clathrin structures at the TGN of control and Hip1R knock down cells. (A–C) Association of F-actin structures with TGN membranes. HeLa cells treated for 3 d with the indicated siRNA duplexes were fixed and labeled for filamentous actin (red) and for TGN46 (green). Fluorescence data were collected and deconvolved as described in Fig. 1. Pictures were generated using 30 deconvolved planes with the blend section mode of Imaris3.2 (Bitplane) and represent a 3D projection of a 3-μm optical section. Inset, magnifications of deconvolved regions (white arrow on A–C) showing typical F-actin/TGN structures. (D and E) F-actin is associated with clathrin at the TGN. HeLa cells treated for 3 d with the indicated siRNA duplexes were fixed and stained for clathrin heavy chain (green), TGN46 (blue), and F-actin (red). Fluorescence data were collected and deconvolved as described in Fig. 1. Bottom panels, magnifications of boxed region. (F–H) Association of the Arp2/3 complex with clathrin-coated buds/vesicles at the TGN in untreated HeLa cells. HeLa cells were fixed and stained for clathrin heavy chain (red), TGN46 (blue), and the Arp2/3 complex (green) (detected using a p21-Arc:FITC antibody). (H) Digital magnification of the boxed region.

Figure 4.

Effect of Hip1R depletion or Jasplakinolide treatment on cathepsin D maturation. (A and B) Wild-type HeLa cells or (B) HeLa cells stably expressing a Hip1R “rescue” cDNA were treated with A2 or InvA2 siRNA duplexes for 3 d before the pulse-chase experiment. At the indicated chase times, the cells were lysed and intracellular cathepsin D was immunoprecipitated from the lysate. (C) The secreted cathepsin D from cells in A was immunoprecipitated from the medium. (D) Wild-type HeLa cells were treated with DMSO or 10 μg/ml Jasplakinolide during the chase. At the indicated chase times, the cells were lysed, and intracellular cathepsin D was immunoprecipitated from the lysate. White lines indicate that intervening lanes have been spliced out.