Involvement of the Rho-mDia1 pathway in the regulation of Golgi complex architecture and dynamics

Abstract

In mammalian cells, the Golgi apparatus is a ribbon-like, compact structure composed of multiple membrane stacks connected by tubular bridges. Microtubules are known to be important to Golgi integrity, but the role of the actin cytoskeleton in the maintenance of Golgi architecture remains unclear. Here we show that an increase in Rho activity, either by treatment of cells with lysophosphatidic acid or by expression of constitutively active mutants, resulted in pronounced fragmentation of the Golgi complex into ministacks. Golgi dispersion required the involvement of mDia1 formin, a downstream target of Rho and a potent activator of actin polymerization; moreover, constitutively active mDia1, in and of itself, was sufficient for Golgi dispersion. The dispersion process was accompanied by formation of dynamic F-actin patches in the Golgi area. Experiments with cytoskeletal inhibitors (e.g., latrunculin B, blebbistatin, and Taxol) revealed that actin polymerization, myosin-II-driven contractility, and microtubule-based intracellular movement were all involved in the process of Golgi dispersion induced by Rho-mDia1 activation. Live imaging of Golgi recovery revealed that fusion of the small Golgi stacks into larger compartments was repressed in cells with active mDia1. Furthermore, the formation of Rab6-positive transport vesicles derived from the Golgi complex was enhanced upon activation of the Rho-mDia1 pathway. Transient localization of mDia1 to Rab6-positive vesicles was detected in cells expressing active RhoA. Thus, the Rho-mDia1 pathway is involved in regulation of the Golgi structure, affecting remodeling of Golgi membranes.

Figure 1:

Golgi dispersion induced by active RhoA is an mDia1-dependent process. (A, B) Control HeLa JW cells (con) and mDia1 knockdown cells (shDia1) were either (A) transfected with constitutively active Rho (RhoA-V14) and fixed 24 h later or (B) treated with RhoA activator LPA (12 μM) in serum-free medium and filmed for 2 h (see Supplemental Movie S2). (A) Golgi was visualized by transfection with trans-Golgi marker GalT-YFP (green; see also the enlarged insets) and actin by staining with phalloidin (red). RhoA-V14–transfected cells in A are marked by white asterisks. In B Golgi was visualized by ManII-GFP. Scale bars, (A) 10 μm, (B) 10 μm. (C, D) Degree of Golgi dispersion was quantified using a compactness (circularity) index calculated on the basis of morphometric measurements (Bard et al., 2003; see also Materials and Methods). Bars represent compactness values ± SEM. The p values were calculated according to Student's t test. (C) Compactness values for control (con) and mDia1-knockdown (shDia1) cells treated, or not treated, with LPA were measured on fixed specimens stained with p115 cis-Golgi marker. (D) Effect of RhoA-V14 on Golgi compactness in control (con) and Dia1-knockdown (shDia1) cells. (E) Western blot illustrating the depletion of mDia1 in HeLa JW cells expressing mDia1-targeted shRNA.

Figure 2:

An active form of mDia1 (mDia1ΔN3) induces Golgi dispersion in an actin polymerization–dependent manner. (A) Cells were transfected with Golgi marker ManII-GFP, shown in red (con), or cotransfected with ManII-GFP and constitutively active mDia1 (mDia1ΔN3). Latrunculin B at 2 μM concentration was added 24 h after transfection; cells were fixed 2 h later. F-Actin was visualized by phalloidin staining (green). Scale bar, 10 μm. (B) Dispersion of Golgi induced by mDia1ΔN3 is gradually reduced in cells treated with ascending concentrations of latrunculin B. Complete return of the compactness value to control levels was observed in 10 μM latrunculin B–treated, mDia1ΔN3-transfected cells. Error bars in B represent SEM values.

Figure 3:

Active Rho prevents fusion of Golgi elements into ribbon structures. (A) Cells expressing the Golgi marker ManII-GFP, alone or in combination with plasmids encoding shRNA against mDia1 or constitutively active Rho, were treated with nocodazole (2.5 μM) for 3 h until the Golgi elements were completely dispersed. Nocodazole was then washed out, and the process of Golgi recovery was filmed in a time-lapse manner. Frames from the three time-lapse movies taken for cells of each type at indicated time points are shown in montage A (see Supplemental Movies S5 and S6). Scale bar, 10 μm; time, minutes. Note that even though Golgi elements move centripetally in all cases, efficient fusion of dispersed elements into ribbon structures occurs in control and mDia1 knockdown cells but not in cells expressing active RhoA. (B) Quantification of average size and number of Golgi elements at different time points after nocodazole removal. (C) Quantification of the effect of latrunculin and constitutively active mDia1 (mDia1ΔN3) on the dynamics of Golgi recovery after nocodazole treatment. The corresponding movie frames are shown in Supplemental Figure S3. The data are normalized to their initial value at the zero time point. Particle size (in μm²) at the zero time point (average ± SD): control (1.3 ± 0.32), RhoV14 (1.3 ± 0.4), siDia1 (1.7 ± 0.4), mDia1ΔN3 (0.54 ± 0.14), and LatB (0.54 ± 0.15). Particle number at the zero time point (average ± SD): control (86 ± 15.8), RhoV14 (66 ± 12.27), siDia1 (64.4 ± 35.9), mDia1ΔN3 (106 ± 61.5), and LatB (57.6 ± 25). For each cell type, 7–10 time-lapse movies were taken for this analysis. Error bars show SD.

Figure 4:

Dynamics of Golgi fragmentation and dispersion induced by Taxol in active mDia1-expressing cells. Sequences from time-lapse movies illustrating the effect of Taxol (24 μM) on microtubules (green) and Golgi (red) in a control cell (top) and an mDia1ΔN3-expressing cell (bottom). Microtubules and Golgi were visualized by transfection of cherry-α-tubulin and ManII-GFP, respectively. Time after addition of Taxol is shown in minutes. Note that Taxol treatment leads to the formation of prominent microtubule bundles in both control and mDia1-expressing cells. In control cells, Golgi membranes remain associated with the ends of these bundles and do not undergo fragmentation. In mDia1ΔN3-transfected cells, a large Golgi fragment can move either together with moving bundles (arrows) or along such bundles (arrowheads). The process of fragmentation continues during the course of such movement (see Supplemental Movie S7). Scale bar, 5 μm.

Figure 5:

Golgi dispersion induced by active mDia1 is enhanced by Taxol in an actin- and myosin II–dependent manner. HeLa JW cells transfected with GalT-YFP Golgi marker (green) alone (con) or together with active mDia1 (mDia1ΔN3) were either left untreated or incubated for 3 h with the microtubule-stabilizing drug Taxol (24 μM) alone (Taxol), with Taxol in combination with blebbistatin (50 μM) (Taxol + Bleb), or with Taxol and latrunculin B (2 μM) (Taxol + LatB). Microtubules (red) and F-actin (black and white photos) were visualized in the same cells by staining with antibody against tubulin and with phalloidin, respectively. Note that Taxol treatment does not induce significant Golgi dispersion in and of itself but strongly stimulates it in cells expressing active mDia1. Both inhibition of actin polymerization by latrunculin B and inhibition of myosin II activity by blebbistatin abolished this effect. Scale bar, 10 μm.

Figure 6:

Dispersed Golgi elements preserve a ministack structure. (A) The Golgi compartments were labeled by cell transfection with the trans-Golgi marker GalT-YFP (green) and immunofluorescence staining with antibody to the cis-Golgi marker p115 (red). Nonfragmented Golgi in control or Taxol-treated cells display typical cis and trans cisternae, forming the ribbon structure. Fragmented Golgi elements in RhoA-V14– or mDia1ΔN3-expressing cells, and even the smallest fragments in RhoA-V14–expressing cells treated with taxol, still preserve joint cis and trans markers. Scale bar, 10 μm. (B) Transmission electron microscopy of control cells and cells expressing mDia1ΔN3. Arrows indicate the Golgi stacks; n, nucleus. Scale bar, 500 nm. Golgi elements in mDia1ΔN3 cells preserved a stacked structure.

Figure 7:

Production of Rab6-positive transport carriers is enhanced by active RhoA and inhibited by mDia1 knockdown. (A) Rab6A-GFP was transiently expressed in control HeLa JW cells (con), in cells expressing active RhoA (RhoA-V14), in mDia1 knockdown cells (shDia1), and in mDia1 knockdown cells expressing active RhoA. Rab6A-GFP localizes to Golgi-derived vesicular and tubular carriers. Note the numerous vesicles in control cells expressing RhoA-V14 and the elongated (tubular) morphology of Rab6 membranes in mDia1 knockdown cells with or without active RhoA (insets). Scale bars, 10 and 4 μm (insets). (B) The density of Rab6-positive carriers increases in control but not in mDia1 knockdown cells expressing active RhoA. Error bars show SD.

Figure 8:

Colocalization of mDia1 and Rab6A′ in cells expressing constitutively active RhoA. Cells were triple transfected with GFP-mDia1 (green), Cherry-Rab6A′ (red), and RhoA-V14-VSV (not shown). The colocalization regions in the merged images are colored yellow. The images of the same cell at two different time points are shown in top and bottom rows, respectively. Some sites of colocalization are indicated by arrowheads and numbered. Insets on the right show magnified images corresponding to these colocalization events. Scale bars, 10 and 1.5 μm (insets).