Dynamin at actin tails - PubMed (original) (raw)

Dynamin at actin tails

Eunkyung Lee et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Dynamin, the product of the shibire gene of Drosophila, is a GTPase critically required for endocytosis. Some studies have suggested a functional link between dynamin and the actin cytoskeleton. This link is of special interest, because there is evidence implicating actin dynamics in endocytosis. Here we show that endogenous dynamin 2, as well as green fluorescence protein fusion proteins of both dynamin 1 and 2, is present in actin comets generated by Listeria or by type I PIP kinase (PIPK) overexpression. In PIPK-induced tails, dynamin is further enriched at the interface between the tails and the moving organelles. Dynamin mutants harboring mutations in the GTPase domain inhibited nucleation of actin tails induced by PIPK and moderately reduced their speed. Although dynamin localization to the tails required its proline-rich domain, expression of a dynamin mutant lacking this domain also diminished tail formation. In addition, this mutant disrupted a membrane-associated actin scaffold (podosome rosette) previously shown to include dynamin. These findings suggest that dynamin is part of a protein network that controls nucleation of actin from membranes. At endocytic sites, dynamin may couple the fission reaction to the polymerization of an actin pool that functions in the separation of the endocytic vesicles from the plasma membrane.

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Figures

Figure 1

Figure 1

Localization of full length dynamin and dynamin-PRD at actin tails of_Listeria_. (A) HeLa cells expressing dynamin 2-GFP were infected with Listeria and then observed by confocal microscopy at 20-sec time intervals. Arrows mark the same position in each frame to demonstrate the movement of a bacterium. (B) HeLa cells processed as in_A_ were fixed and counterstained for F-actin with phalloidin. Note the lack of dynamin staining on nonmoving_Listeria_, which are surrounded only by an actin shell (arrowheads). (C) Both dynamin 1-GFP and dynamin 2-GFP (green) colocalize with phalloidin staining (red) on_Listeria_ tails in HeLa cells. (D) PtK2 cells were infected with Listeria and then processed by dual labeling for endogenous dynamin 2 immunoreactivity (green) and for actin with phalloidin staining (red). (E) HeLa cells transfected with PRD-GFP were observed by confocal microscopy at 20-sec time intervals. (Bars = 5 μm for all fields.)

Figure 2

Figure 2

Presence of dynamin in actin tails of BHK/RSV cells.A–E are from cells cotransfected with dynamin constructs and with PIPK; F is from a cell not cotransfected with the kinase. (A) A series of images from a time-lapse movie of a cell cotransfected with dynamin-GFP and HA-PIPK (8-sec time intervals). Arrows point to the same position in each frame to demonstrate the movement of a vesicle. (B and C) Dynamin 2-GFP (B) and endogenous dynamin 2 immunoreactivity (C) (green) are compared with phalloidin stain (red) in tails induced by PIPK. Note the highest concentration of dynamin relative to actin at the heads of the comets. (D) Cells cotransfected with dynamin 2-GFP (green) and PIPK were imaged by confocal microscopy after a pulse–chase labeling with Bodipy Texas red-ceramide (red) or an incubation with TRITC-dextran (red) to label Golgi-derived vesicles and endocytic vesicles, respectively. (E) A series of images from a time-lapse movie of a cell cotransfected with PRD-GFP and HA-PIPK (8-sec time intervals). The PRD of dynamin 2 is present at low concentration throughout actin tails. The arrow indicates the same position in all frames. (F) Images from a time-lapse movie of a cell from a separate culture transfected with dynamin-GFP but not with PIPK (8-sec time intervals). A small tail-like structure moves across the field. The arrow indicates the same position in all frames. (Bars = 5 μm in A and D; 1 μm in B, C, E, and_F_.)

Figure 3

Figure 3

Effect of GTPase domain dynamin mutants on endocytosis and actin comets. (A) K44A dynamin 2-GFP inhibits transferrin uptake. K44Adynamin 2-GFP transfected cells were incubated with Cy3-transferrin for 10 min before fixation. Note the strong inhibition of transferrin uptake (red) in cells that expressK44Adynamin (green). (B)K44Adynamin 2-GFP is present at actin tails of cells cotransfected with PIPK and does not block tail motility. A series of images from a time-lapse movie (8-sec time intervals). Arrows mark the same position in each frame to demonstrate vesicle movement. (Bars = 20 μm in A and 5 μm in B.) (C) K44Adynamin 2-GFP andD208Ndynamin 2-GFP inhibit actin tail formation in PIPK-overexpressing cells. The data represent the number of tails per transfected (or GFP-fluorescent) cell expressed as percentage of the value observed with wild-type dynamin. The average and standard deviation of at least three separate experiments (total cells counted for each condition >180) are shown. Actin tails were counted in fixed cells after phalloidin stain. Cotransfection efficiency, as accessed by anti-HA staining for PIPK expression, was more than 90%. (D) K44Adynamin 2-GFP andD208Ndynamin 2-GFP slightly reduce motility of tails induced by PIPK cotransfection. The average tail speed in these cells was 4.9 μm/min for wild-type dynamin 2 (n = 120), 4.2 μm/min for K44Adynamin 2 (n = 124), and 3.5 μm/min (n = 49) for D208Ndynamin 2. Standard errors are indicated. The difference is statistically significant:P < 0.001 for K44Adynamin 2 and_P_ < 0.000002 for D208Ndynamin 2.

Figure 4

Figure 4

ΔPRD dynamin 2-GFP disrupts podosome rosettes of BHK/RSV cells. (A) Expression of ΔPRD dynamin 2-GFP (ΔPRD) in cells cotransfected with PIPK inhibits actin tail formation. Actin tails were counted in phalloidin-stained fixed cells and their number was calculated as in Fig. 3_C_. Values represent average and standard deviations derived from three separate experiments in which more than 250 cells were counted. (B) The PRD of dynamin is targeted to the podosome rosettes (arrowheads). BHK/RSV cells were transfected with a GFP fusion protein of the PRD of dynamin 2 (green) and stained with phalloidin (red). (C) Expression of ΔPRD dynamin 2-GFP (green) in BHK/RSV cells (arrows) results in loss of the podosome rosette previously shown to be positive for endogenous dynamin (35). (Bar = 20 μm.)

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