Figure 3.
Figure 3. F-actin and microtubules control the enrichment of ACA at the back of cells. (A, left) Confocal fluorescent images of ACA-YFP/aca− cells treated with 5 μM LatA for the designated lengths of time. (inset) Confocal fluorescent image of an ACA-YFP/aca− cell treated with LatA for 45 min showing the bleached area (white) and the side (blue) and middle (red) boxes where the fluorescence recovery is monitored. The graphs depict the recovery of ACA-YFP. Data is presented as a mean of five cells ± SEM. See Fig. 2 B for details. (B, left) Fluorescent images of GFP–α-tubulin/WT cells with or without 60 μM Noco. (inset) Confocal fluorescent image of an ACA-YFP/aca− cell treated with Noco showing the bleached area. The graphs depict the recovery of ACA-YFP. Data is presented as a mean of five cells ± SEM. See Fig. 2 B for details. (C) Deconvoluted fluorescent image showing ACA-YFP (green) and α-tubulin (red) in fixed ACA-YFP/aca− cells. Also see Video 3. (D) Fluorescent image showing ACA-YFP (green), α-tubulin (red), and DAPI (blue) in fixed ACA-YFP/aca− cells. The position of the MTOC relative to the nucleus was quantified in 63 cells. We find that in 62% of migrating cells the MTOC is localized behind the nucleus and that 76% of cells have either none or one microtubule filament extending to the leading edge. The position of the MTOC was confirmed by labeling centrosomes with anti–γ-tubulin antibodies (not depicted). (E) Fluorescent maximum intensity projections of ACA-YFP/aca− cells (top) or GFP–α-tubulin/WT cells (bottom) chemotaxing to a micropipette filled with 1 μM cAMP in the presence or absence of Noco. The asterisk shows the position of the micropipette. (F) Montage of bright field images of ACA-YFP/aca− cells chemotaxing to a micropipette filled with 1 μM cAMP in the presence or absence of Noco. Also see Video 4. Identical results were observed with WT cells. Videos are available.

F-actin and microtubules control the enrichment of ACA at the back of cells. (A, left) Confocal fluorescent images of ACA-YFP/aca− cells treated with 5 μM LatA for the designated lengths of time. (inset) Confocal fluorescent image of an ACA-YFP/aca− cell treated with LatA for 45 min showing the bleached area (white) and the side (blue) and middle (red) boxes where the fluorescence recovery is monitored. The graphs depict the recovery of ACA-YFP. Data is presented as a mean of five cells ± SEM. See Fig. 2 B for details. (B, left) Fluorescent images of GFP–α-tubulin/WT cells with or without 60 μM Noco. (inset) Confocal fluorescent image of an ACA-YFP/aca− cell treated with Noco showing the bleached area. The graphs depict the recovery of ACA-YFP. Data is presented as a mean of five cells ± SEM. See Fig. 2 B for details. (C) Deconvoluted fluorescent image showing ACA-YFP (green) and α-tubulin (red) in fixed ACA-YFP/aca− cells. Also see Video 3. (D) Fluorescent image showing ACA-YFP (green), α-tubulin (red), and DAPI (blue) in fixed ACA-YFP/aca− cells. The position of the MTOC relative to the nucleus was quantified in 63 cells. We find that in 62% of migrating cells the MTOC is localized behind the nucleus and that 76% of cells have either none or one microtubule filament extending to the leading edge. The position of the MTOC was confirmed by labeling centrosomes with anti–γ-tubulin antibodies (not depicted). (E) Fluorescent maximum intensity projections of ACA-YFP/aca− cells (top) or GFP–α-tubulin/WT cells (bottom) chemotaxing to a micropipette filled with 1 μM cAMP in the presence or absence of Noco. The asterisk shows the position of the micropipette. (F) Montage of bright field images of ACA-YFP/aca− cells chemotaxing to a micropipette filled with 1 μM cAMP in the presence or absence of Noco. Also see Video 4. Identical results were observed with WT cells. Videos are available.

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