Par6 alpha interacts with the dynactin subunit p150 Glued and is a critical regulator of centrosomal protein recruitment

Abstract

The centrosome contains proteins that control the organization of the microtubule cytoskeleton in interphase and mitosis. Its protein composition is tightly regulated through selective and cell cycle-dependent recruitment, retention, and removal of components. However, the mechanisms underlying protein delivery to the centrosome are not completely understood. We describe a novel function for the polarity protein Par6α in protein transport to the centrosome. We detected Par6α at the centrosome and centriolar satellites where it interacted with the centriolar satellite protein PCM-1 and the dynactin subunit p150(Glued). Depletion of Par6α caused the mislocalization of p150(Glued) and centrosomal components that are critical for microtubule anchoring at the centrosome. As a consequence, there were severe alterations in the organization of the microtubule cytoskeleton in the absence of Par6α and cell division was blocked. We propose a model in which Par6α controls centrosome organization through its association with the dynactin subunit p150(Glued).

Figure 1.

Par6α is a component of the centrosome and of centriolar satellites. (A) Immunofluorescence microscopy analysis of HeLa cells costained with antibodies to Par6α (red) and centrin, γ-tubulin or PCM-1 (all in green). The insets show magnified images of the boxed area. (B) Total lysates of control or Par6α-depleted HeLa cells were analyzed by Western blotting with antibodies that recognize only Par6α (left) or that detects both isoforms, Par6α and Par6β (right). α-tubulin served as a loading control. (C) Analysis of control and Par6α-depleted cells with antibodies to Par6α and γ-tubulin. (D) HeLa cell lysates were fractionated on sucrose gradients to enrich for centrosomes. Fractions were analyzed by Western blotting with antibodies to Par6α, centrin, γ-tubulin, and PCM-1. (E) Cell cycle–dependent localization of Par6α was determined by staining a nonsynchronous population of HeLa cells with antibodies to Par6α and PCM-1. The organization of γ-tubulin and DNA was used to mark the centrosome and to determine the specific cell cycle stage, respectively. Scale bars, (A, C, and E), 5 μm.

Figure 2.

Par6α-depleted cells contain disorganized, nonfunctional centrosomes. (A) Control and Par6α-depleted cells were stained with antibodies to Par6α (red, 6) to confirm protein depletion, to γ-tubulin (γ, cyan) and to PCM-1 (P), centrin (C), CPAP (C), ninein (N), and Cep170 (7; all in green) to monitor centrosomal protein organization. Scale bar, 5 μm. (B) Quantifications of the fluorescence intensity at the centrosome are shown. The control was defined as 100% and represents the fluorescent signal detected for each marker protein at the centrosome of control siRNA-transfected cells. Fifteen cells were analyzed per experiment, and three independent experiments were performed. The p values for each experiment were <0.005 (paired t test). (C) Microtubule organization was examined by staining control and Par6α-depleted cells with antibodies to Par6α and α-tubulin. Scale bar, 10 μm. (D) Staining with a specific antibody to phospho-Histone H3 was used to determine the percentage of cells in mitosis (n = 3), *p < 0.001, paired t test. (E) Mitotic control and Par6α-depleted cells were stained with antibodies to phospho-Histone H3, and to α-tubulin and the DNA dye, Hoechst, to visualize spindle organization or DNA alignment, respectively. The percentage of mitotic cells with aberrant spindles is shown (n = 3), *p < 0.001, paired t test. Scale bar, 5 μm.

Figure 3.

Par6α interacts with PCM-1 and directs its localization. (A) Total HeLa cell lysates were subjected to immunoprecipitations with polyclonal antibodies to Par6α (Par6α IgG), PCM-1 (PCM-1 IgG), or to β-PIX or the FLAG-epitope (control IgG) as negative controls. Immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with antibodies to Par6α and PCM-1. The input corresponds to 50 μg of total lysates, whereas each immunoprecipitation was performed from 2 mg of total lysate. (B) Schematic representation of the mitochondria-targeted constructs. (C) HeLa cells expressing mitochondrially-targeted GFP (Mito-GFP) or mitochondrially-targeted GFP-tagged full-length Par6α (Mito-GFP-Par6α) were incubated with MitoTracker or fixed and stained with antibodies to PCM-1. The localization of GFP (G), Par6α (P) and mitochondria (M) are shown at larger magnifications. Scale bar, 5 μm. (D) HeLa cells expressing Mito-GFP or Mito-GFP-Par6α were subjected to immunoprecipitations with a GFP-specific antibody, followed by Western blot analysis with antibodies to GFP and PCM-1.

Figure 4.

PCM-1 is required for the centrosomal localization of Par6α. (A) HeLa cells expressing GFP-tagged full-length PCM-1 (PCM-1-GFP) or an N-terminal portion of PCM-1 corresponding to amino acids 1-1468 (GFP-PCM-1ΔC) were fixed and stained with antibodies to Par6α. (B) Total lysates of control and PCM-1—depleted HeLa cells were analyzed by Western blotting with antibodies to PCM-1 and Par6α. α-Tubulin served as a loading control. (C) Staining of control and PCM-1—depleted HeLa cells with antibodies to PCM-1 (P, green) and Par6α (6, red). γ-tubulin (γ, cyan) served to visualize the centrosome in the same cell. Scale bar, 5 μm.

Figure 5.

The centrosomal localization of Par6α depends on microtubules and the dynein–dynactin complex. (A) HeLa cells treated for 4 h with the microtubule-depolymerizing agent, nocodazole, or DMSO as a negative control were fixed and stained with antibodies to α-tubulin to verify microtubule depolymerization and to Par6α (6, red), PCM-1 (P, green) and γ-tubulin (γ, green) to monitor centrosomal protein organization. Insets show a magnification of the boxed area in the large image. (B) HeLa cells were transfected with HA-tagged p150^Glued (left) or with myc-tagged p50-dynamitin (right) to inhibit dynein or dynactin function, respectively. Empty vectors served as negative controls. Cells were fixed and stained with antibodies to HA or myc (green) and Par6α (6, red). We also stained with antibodies to γ-tubulin (γ, cyan) to mark the position of the centrosome. The two highlighted areas in the main images show the two disconnected γ-tubulin foci which are magnified in the insets. Insets that are marked by an asterisk are magnified images of the boxed area that is labeled with the asterisk. Scale bar, 5 μm. (C) Transfected cell lysates were analyzed by Western blotting with antibodies to HA, myc, and Par6α. α-Tubulin served as a loading control.

Figure 6.

Par6α interacts with p150^Glued and is required for its centrosomal localization. (A) Total HeLa cell lysates were subjected to immunoprecipitations with antibodies to Par6α (Par6α IgG), PCM-1(PCM-1 IgG) p150^Glued (p150^Glued IgG). Antibodies to β-PIX or the FLAG-epitope (control IgG) were used as negative controls. Par6α, PCM-1, and p150^Glued in these samples were detected with specific antibodies. (B) Par6α, p150^Glued, or β-PIX as a negative control, were immunoprecipitated from control or PCM-1—depleted HeLa cells. Immunoprecipitates were analyzed by Western blotting with antibodies to Par6α, p150^Glued, and PCM-1. (C) Control or Par6α-depleted HeLa cells were stained with antibodies to Par6α (red) and to p150^Glued (green). γ-Tubulin staining (cyan) revealed the position of the centrosome in the same cell (left panel). In a parallel experiment, control or PCM-1—depleted cells were stained with antibodies to PCM-1 (red), p150^Glued (green), and γ-tubulin (cyan). Scale bar, 5 μm. (D) Quantifications of the fluorescent intensity are shown as percentage of the fluorescent signal at the centrosome of control siRNA-transfected cells. Fifteen cells were analyzed per experiment, and three independent experiments were performed (*p < 0.005).

Figure 7.

Model of Par6α function at the centrosome. (A) Par6α associates with PCM-1, the dynactin subunit, p150^Glued, and cargo proteins at centriolar satellites. This complex is loaded onto the dynein motor complex for transport to the centrosome. (B) In Par6α-depleted cells, PCM-1 and p150^Glued are unable to associate with the dynein motor complex resulting in a bloc in cargo delivery to the centrosome. (C) In PCM-1—depleted cells, Par6α associates with p150^Glued and cargo proteins, which can associate with dynein for transport to the centrosome. However, in the absence of PCM-1, Par6α is not retained at the centrosome.