A primary role for Golgi positioning in directed secretion, cell polarity, and wound healing

Abstract

Peri-centrosomal positioning of the mammalian Golgi apparatus is known to involve microtubule-based motility, but its importance for cellular physiology is a major unanswered question. Here, we identify golgin-160 and GMAP210 as proteins required for centripetal motility of Golgi membranes. In the absence of either golgin, peri-centrosomal positioning of the Golgi apparatus was disrupted while the cytoskeleton remained intact. Although secretion persisted with normal kinetics, it was evenly distributed in response to wounding rather than directed to the wound edge. Strikingly, these cells also completely failed to polarize. Further, directionally persistent cell migration was inhibited such that wound closure was impaired. These findings not only reveal novel roles for golgin-160 and GMAP210 in conferring membrane motility but also indicate that Golgi positioning has an active role in directed secretion, cell polarity, and wound healing.

Figure 1.

Depletion of golgin-160 and GMAP210 fragments the Golgi apparatus. (A–I) HeLa cells expressing GalNAc T2-GFP were treated with control siRNA (A–C), golgin-160 siRNA (D–F), or GMAP210 siRNA (G–I) and then costained with anti-golgin 160 and anti-GMAP210 antibodies. Bar, 10 μm. (J) Cell lysates prepared from cells 0, 1, or 2 d after transfections with golgin-160 or GMAP210 siRNAs analyzed by immunoblotting to assay golgin-160, GMAP210, and tubulin levels. The inset shows representative immunoblots, and the average extent of knockdown is plotted for multiple experiments (±SEM, n = 3). (K) The number of Golgi objects determined by automated fluorescent object counting was plotted for control, golgin-160, or GMAP210 knockdown cells. Values are averages (±SEM, n = 15).

Figure 2.

Depletion of golgin-160 or GMAP210 yields dispersed Golgi ministacks. (A–D) Cells treated with control siRNA (A), 0.5 μg/ml nocodazole for 3 h (B), golgin-160 siRNA (C), or GMAP210 siRNA (D) were analyzed by transmission electron microscopy. Bars, (A–D) 0.5, 0.1, 0.1, and 0.1 μm, respectively. (E–P) Control siRNA-treated cells (E–G) or cells depleted of golgin-160 (H–J), GMAP210 (K–M), or p115 (N–P) were stained and imaged to detect GalNAcT2-GFP and giantin. Merged images are also shown. Bar, 10 μm. (Q) Marker segregation was determined using the colocalization plugin of ImageJ, and the percentage of coincident pixels was compared. P values are indicated for comparison to the siRNA control sample and support persistent segregation after golgin, but not p115, knockdown.

Figure 3.

Integrity of the microtubule and actin cytoskeleton. (A–I) The microtubule cytoskeleton was examined in cells 72 h after treatment with control siRNA (A–C), golgin-160 siRNA (D–F), or GMAP210 siRNA (G–I) and then imaged to reveal the Golgi (GalNAcT2-GFP) and tubulin staining. Merged images are also shown. Bar, 10 μm. (J–R) The actin cytoskeleton was examined in cells treated as above (A–I) and imaged to reveal the Golgi (GalNAcT2-GFP) and phalloidin staining. Bar, 10 μm.

Figure 4.

Golgin-160 and GMAP210 are required for Golgi membrane motility. (A–D) GalNAcT2-expressing cells were treated with control siRNA (A), golgin-160 siRNA (B), or GMAP210 siRNA (C), and then live imaging was carried after nocodazole washout. Nocodazole treatment was at 0.5 μg/ml for 3 h and washout was initiated by perfusion with fresh growth medium. Excerpted frames show final time points overlaid by tracks indicating frame-by-frame movement of Golgi objects during the entire imaging period. Bar, 10 μm. See Supplemental Material for the original movies. Golgi object movement was quantified for each condition by determining the net displacement of each object from the first to last frame (D). Average values are plotted (±SEM, n = 3).

Figure 5.

ER to surface trafficking of VSVG-GFP. (A–I) Control siRNA- (A and B), golgin-160 siRNA- (C and D), GMAP210 siRNA- (E and F), and nocodazole- (G and H) treated HeLa cells stably expressing ts045 VSVG-GFP were shifted to the permissive temperature for VSVG trafficking for 60 min before staining surface-exposed VSVG. After fixation the cells were stained using anti-giantin antibodies to reveal the Golgi structure. The plot shows the ratio of surface to total VSVG-GFP fluorescence for the indicated time points (I).

Figure 6.

Golgi dispersal associates with impaired polarized secretion. (A–C) HeLa cells expressing GalNAcT2-GFP and transfected with control siRNA (A), golgin-160 siRNA (B), or GMAP210 siRNA (C) were wounded and, after 6 h the Golgi (GalNAcT2-GFP in red) and the nuclei (Hoechst staining in blue) were imaged. White line indicates wound edge. Examples of best-fit circles and quadrants with the 180° radian perpendicular to the wound edge are shown. Bar, 10 μm. (D) Golgi position polarity was quantified by plotting the normalized intensity of GalNAcT2-GFP fluorescence for each degree radian of the best-fitting circle. The hemicycle facing the leading edge is indicated. Values are averages (±SEM, n = 3) with control, golgin-160, and GMAP210 siRNA treatments plotted in red, black, and gray, respectively. (E–M) HeLa cells were transfected with control siRNA (E–G), golgin-160 siRNA (H–J), or GMAP210 siRNA (K–M), and then, after 52 h the cells were induced at 40°C with doxycycline to accumulate VSVG-GFP in the ER for 20 h. Then the cells were wounded (white line indicates wound edge), and after 6 h the cells were incubated at the permissive temperature for 30 min and processed to reveal surface VSVG staining and localization of the Golgi marker GRASP65. Bar, 10 μm. (N) Secretion polarity was quantified by plotting the normalized intensity of VSVG-GFP surface staining for each degree radian of the best-fitting circle encompassing the cell. Radian degrees corresponding to the leading edge are indicated. Values are averages (±SEM, n = 3) with control, golgin-160, and GMAP210 siRNA treatments plotted in red, black, and gray, respectively.

Figure 7.

Actin loses its asymmetric localization in the absence of Golgi pericentrosomal positioning. (A–I) HeLa cells expressing GalNAcT2-GFP and transfected with control siRNA (A–C), golgin-160 siRNA (D–F), or GMAP210 siRNA (G–I) were wounded and, after 6 h, were stained with phalloidin to visualize the polymerized actin network. White line indicates wound edge. Bar, 10 μm. (J) The percentage of cells at the wound edge with a polarized distribution of actin facing the leading edge is plotted. Values are averages (±SEM, n = 3).

Figure 8.

Randomized orientation of acetylated microtubules upon golgin knockdown. (A–I) HeLa cells expressing GalNAcT2-GFP were stained with an anti-acetylated tubulin antibody 72 h after transfection with control (A–C), golgin-160 (D–F), or GMAP210 (G–I) siRNA and 6 h after wounding. Bar, 10 μm. (J–L) Acetylated tubulin polarity was quantified by plotting the normalized intensity of staining for each degree radian of the best-fitting circle encompassing the cell (J). Radian degrees corresponding to the leading edge are indicated. Values are averages (±SEM, n = 3) with control, golgin-160, and GMAP210 siRNA treatments plotted in red, black, and gray, respectively. The percentage of cells with a wound edge oriented acetylated microtubule population is also plotted (K). Values are averages (±SEM, n = 3). The mean intensity of acetylated tubulin staining per cell in the absence of wounding and at the wound edge was also determined for each knockdown treatment (L). Golgin knockdown caused a randomization in orientation but not a loss of stable microtubules.

Figure 9.

Wound healing is defective in cells with dispersed Golgi membranes. (A–D) HeLa cells treated with control siRNA (A), golgin-160 siRNA (C), or GMAP210 siRNA (D) were grown in a monolayer and wounded and, after 17 h, were fixed and processed to visualize the Golgi (Giantin) and nuclei (Hoechst shown in blue). The same experiment was also performed on nontransfected cells treated with 0.5 μg/ml nocodazole 3 h before wounding and during the wound healing incubation (B). Bar, 200 μm. (E) An estimate of migration speed (μm/h) for each condition is plotted. Values are averages (±SEM, n = 3).