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First published online 8 January 2003
doi: 10.1242/jcs.00288

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Microtubule involvement in NIH 3T3 Golgi and MTOC polarity establishment

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

View larger version (59K): [in a new window]   Fig. 4. ß-COP labeling of wound-edge NIH 3T3 cells microinjected with different pEGFP-mDia1 expression constructs. (A) The main domains contained in the four mDia1 constructs used in this study are shown. Full-length mDia1 contains the following domains from N- to C-terminus: N3, FH1 and FH2. All constructs are tagged with GFP as indicated. (B) The microinjected cells expressing the GFP-fusion protein are shown as indicated (top panels): mDia-deltaN3 (left), mDia F2 (middle) and mDia-deltaN3KA3 (right). Cells were fixed and stained for the Golgi apparatus with the anti-ß-COP antibody (bottom panels). Bar, 40 µm.

View larger version (88K): [in a new window]   Fig. 1. MTOC polarization over the time course of wound closure using NIH 3T3 fibroblasts. (A) Co-labeling of wounded NIH 3T3 monolayers for tubulin and pericentrin. NIH 3T3 fibroblasts were fixed and stained for MTOC concomitantly with the anti-tubulin antibody (top panels) and with the anti-pericentrin antibody (bottom panels), 3 hours post wounding. Enlarged pictures of positively scored MTOC polarization are shown on the left panels and correspond to a region from the middle panel as indicated. Using white dotted lines, we indicate the 120° sector facing the wound. (B) The percentage of wound-edge cells with a MTOC orientated towards the wound was determined at 3, 5 and 7 hours post wounding. (C) Quantification data. The first number corresponds to the number of MTOC observed; the second, in parentheses, is the number of separate experiments. Bar, 40 µm.

View larger version (68K): [in a new window]   Fig. 2. Nocodazole, cytochalasin D and taxol effect on Golgi and MTOC polarization in wound-edge NIH 3T3 cells. Percentage of Golgi (A) or MTOC (B) orientated towards the wound was determined at 3 hours post wounding in cell monolayers treated with nocodazole (blue bars), cytochalasin D (pink bars) or taxol (green bars). Cell polarity of wound-edge cells of non-drug treated monolayers was included as a control of Golgi polarization (grey bar) (A) or MTOC polarization (white bar) (B) (see also legend at the bottom of the graphs). (C) Quantification data. The first number corresponds to the number of Golgi (A) or MTOC (B) observed and the second, in parentheses, is the number of separate experiments. (D) Golgi polarization of wound-edge cells treated with nocodazole at 300 nM and 16 µM. Cells were fixed and stained for tubulin (top panels) and for the Golgi apparatus with anti-ß-COP antibody (bottom panels).

View larger version (66K): [in a new window]   Fig. 3. Arp2/3 and WASP-family proteins are not involved in MTOC polarization of wound-edge NIH 3T3 cells. MTOC polarity, at the edge of the wound, of cells expressing Scar1-WCA, Scar1-2-3-deltaA and N-WASP-deltaA: MTOC reorientation to face the wound was evaluated when wound-edge NIH 3T3 cells were microinjected with the five expression vectors encoding for the WASP-family deletion constructs described earlier (Magdalena et al., 2003). (A) The microinjected cells expressing the GFP-fusion protein are shown (top panels, from left to right): Scar1-deltaA, Scar2-deltaA and Scar3-deltaA. Cells were fixed and stained for the MTOC with the anti-pericentrin antibody (bottom panels). (B) The percentage of MTOC orientated towards the wound was determined at 3 hours post wounding for cells microinjected with the expression vectors encoding for the WASP-family deletion constructs. MTOC polarization of non-microinjected wound-edge cells was included as a control (white bar) (see legend on the right side of the graph; n is the number of MTOC observed and the second number, after the slash, indicates the number of separate experiments). Bar, 40 µm.

View larger version (85K): [in a new window]   Fig. 6. Overexpression of the mDia1 domains does not interfere with MTOC polarization in wound-edge NIH 3T3 cells. MTOC polarity, at the edge of the wound, of mDia-deltaN3-, mDia F2- and mDia-deltaN3KA3-expressing cells: MTOC reorientation to face the wound was evaluated when wound-edge NIH 3T3 cells were microinjected with the three corresponding pEGFP-mDia1 expression constructs (see Fig. 4). (A) The microinjected cells expressing the GFP-fusion protein are shown (upper panels): mDia-deltaN3 (left), mDia F2 (middle) and mDia-deltaN3KA3 (right). Cells were fixed and stained for the MTOC with the anti-pericentrin antibody (lower panels). (B) The percentage of MTOC orientated towards the wound was determined at 3 hours post wounding for cells microinjected with the three mDia1 expression vectors. MTOC polarization of non-microinjected wound-edge cells was included as a control (see legend on the right side of the graph; n is the number of MTOC observed and the second number, after the slash, indicates the number of separate experiments). Bar, 40 µm.

View larger version (57K): [in a new window]   Fig. 8. ST638 inhibits wound closure as well as Golgi polarization but does not prevent the MTOC polarization of wound-edge NIH 3T3 cells. (A) Wounded NIH 3T3 monolayers not treated (left panel) or treated with ST638 (right panel) were fixed and stained for actin 9 hours post wounding. (B) Percentage of Golgi polarization and (C) percentage of MTOC polarization: Golgi and MTOC orientated towards the wound were determined at 3 hours following wounding for cells treated with ST638 (pink bars). Golgi polarization (B) and MTOC polarization (C) of non-drug treated wound-edge cells were included as a control: grey bars (B) and white bars (C), respectively (see also legend on the right side of the graph; n is the number of Golgi (B) or MTOC (C) observed and the second number, after the slash, indicates the number of separate experiments). Bar, 80 µm.

View larger version (61K): [in a new window]   Fig. 10. Y27632 accelerates wound closure and inhibits both Golgi polarization and MTOC polarization of wound-edge NIH 3T3 cells. (A) Wounded NIH 3T3 monolayers not treated (left panel) or treated with Y27632 (right panel) were fixed and stained for actin 15 minutes post wounding. (B) As described in (A) but fixation and labeling were performed 4 hours post wounding. (C) Percentage of Golgi polarization and (D) percentage of MTOC polarization: Golgi and MTOC orientated towards the wound were determined at 3 hours post wounding for cells treated with Y27632 (short hashed bars). Golgi polarization (C) and MTOC polarization (D) of non-drug treated woundedge cells were included as a control: blue-grey bars (C) and white bars (D), respectively (see also legend on the right side of the graph; n is the number of Golgi (C) or MTOC (D) observed and the second number, after the slash, indicates the number of separate experiments). Bar, 80 µm.

View larger version (57K): [in a new window]   Fig. 5. Overexpression of the mDia1 FH2 domain interferes with Golgi polarization in wound-edge NIH 3T3 cells. Golgi polarity, at the edge of the wound, of cells expressing full-length mDia1, mDia-deltaN3, mDia F2 and mDia-deltaN3KA3: Golgi reorientation to face the wound was evaluated when wound-edge NIH 3T3 cells were microinjected with the four different pEGFP-mDia1 expression constructs (see Fig. 4). (A) The microinjected cells expressing the GFP-fusion protein are shown (top panels): mDia-deltaN3 (left), mDia F2 (middle) and mDia-deltaN3KA3 (right). Cells were fixed and stained for the Golgi apparatus with the anti-ß-COP antibody (bottom panels). (B) The percentage of Golgi orientated towards the wound was determined at 3 hours post wounding for cells microinjected with the four mDia1 expression vectors. Golgi polarization, over the time course of wound closure, of non-microinjected wound-edge cells was included as a control (blue-grey bars) (see also legend on the right side of the graph). (C) Quantification data. The first number corresponds to the number of Golgi observed and the second, in parentheses, is the number of separate experiments. Bar, 40 µm.

View larger version (81K): [in a new window]   Fig. 7. Motility of the microinjected wound-edge NIH 3T3 cells within the cell sheet during wound closure. Cells were microinjected with pEGFP-expression vectors encoding mDia-deltaN3 (left panels), mDia F2 (middle panels) and mDia-deltaN3KA3 (right panels). Cells were fixed 4 hours after microinjection and labeled for actin. (A) GFP-mDia-deltaN3-, GFP-mDia F2- and GFP-mDia-deltaN3KA3-expressing cells are shown in the top panels (left to right panels, respectively, as indicated). Actin labeling is shown under the corresponding top panel and merges of the two top panels are shown at the bottom of the corresponding column. Cells expressing mDia-deltaN3 and mDia F2 were left behind the wound edge (left and middle columns). Cells expressing mDia-deltaN3KA3 were motile as they persisted at the margin of the closing wound (right column). (B) The table includes the percentage of migrating cells for each expression construct microinjected in wound-edge cells and the number (n) of microinjected cells observed. Quantification was performed from three separate experiments for each construct. Bar, 40 um.

View larger version (87K): [in a new window]   Fig. 9. Overexpression of IRSp53 does not prevent Golgi polarization, but inhibits lamellipodia formation and migration of the wound-edge NIH 3T3 cells. The microinjected NIH 3T3 cells expressing the myc-tagged IRSp53 are shown in the top panels of sections A, B and C. Cells were fixed at 2 hours post wounding and labeled for Golgi (lower panel) (A) or actin (lower panel) (B). (C) Cells were fixed at 4 hours post wounding and labeled for actin (middle panel), and the upper and middle panels were merged and shown at the bottom. Bar, 40 µm.

View larger version (25K): [in a new window]   Fig. 11. Involvement of microtubules in Golgi and MTOC polarity establishment in wound-edge NIH 3T3 cells. (A) Summary of results obtained in this study: the effect on Golgi polarization, MTOC polarization and cell migration of different drugs (nocodazole, taxol, cytochalasin D, Y27632, ST638) and/or different expression constructs (WASP family WCA, several deletion constructs of mDia1 and IRSp53). (B) Proteins involved in MTOC polarity, Golgi polarity and/or cell motility. (C) Rho, ROCK, mDia, IRSp53 and Src proteins are shown inside rectangles, and distributed as shown on the basis of the pathway proposed and described in the literature. It is shown at which level of the pathway and which proteins are important for MTOC polarity, for Golgi polarity and for cell motility.

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