Figure 5.
Figure 5. ACA trafficking requires de novo ACA synthesis. (A) Western analysis showing the expression of ACA-YFP from ACA-YFP/aca− cells or cAR1-YFP from cAR1-YFP/car1/3− cells in the presence or absence of 400 μM CHX. Similar findings were observed when we monitored endogenous levels of ACA in WT cells. See Materials and methods for details. Representative data of at least three independent experiments are shown. (B) Fluorescent maximum intensity projections of ACA-YFP/aca− cells chemotaxing to a micropipette filled with 1 μM cAMP in the presence or absence of CHX (1-h treatment). The asterisk represents the position of the micropipette. (C, inset) Confocal fluorescent images of ACA-YFP/aca− or cAR1-YFP/car1/3− cells treated with CHX (1 h) 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 and cAR1-YFP. See Fig. 2 B for details. (D) Bright field images of ACA-YFP/aca− cells chemotaxing to a micropipette containing 1 μM cAMP in the presence or absence of CHX (1-h treatment). Also see Video 5. Identical results were observed with WT cells. (E, top) Confocal fluorescent images of ACA-YFP/aca− or cAR1-YFP/car1/3− migrating cells before and after complete bleach. The numbers represent the time (in seconds) after the bleach. Also see Video 6. The graphs depict the total fluorescent recovery of ACA-YFP or cAR1-YFP from the entire cell under various conditions. Data are presented as a mean of five cells ± SEM. It is the short maturation time of YFP that has allowed us to visualize the quick recovery of ACA. Cells expressing ACA-GFP show no recovery after total bleaching (not depicted). (F, right) Radiographs depicting the incorporation of 35S-Translabel into ACA-YFP or cAR1-YFP at various time points after adding it to ACA-YFP/aca− and cAR1-YFP/car1/3− migrating cells (see Materials and Methods for details). The input lane shows a Western analysis (using an α-GFP antibody) of pulled down ACA-YFP and cAR1-YFP samples and depicts the starting amounts of protein for both ACA-YFP and cAR1-GFP. The graph shows the optical density (OD) values from the radiographs. Representative data of at least three independent experiments are shown. Videos are available.

ACA trafficking requires de novo ACA synthesis. (A) Western analysis showing the expression of ACA-YFP from ACA-YFP/aca− cells or cAR1-YFP from cAR1-YFP/car1/3− cells in the presence or absence of 400 μM CHX. Similar findings were observed when we monitored endogenous levels of ACA in WT cells. See Materials and methods for details. Representative data of at least three independent experiments are shown. (B) Fluorescent maximum intensity projections of ACA-YFP/aca− cells chemotaxing to a micropipette filled with 1 μM cAMP in the presence or absence of CHX (1-h treatment). The asterisk represents the position of the micropipette. (C, inset) Confocal fluorescent images of ACA-YFP/aca− or cAR1-YFP/car1/3− cells treated with CHX (1 h) 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 and cAR1-YFP. See Fig. 2 B for details. (D) Bright field images of ACA-YFP/aca− cells chemotaxing to a micropipette containing 1 μM cAMP in the presence or absence of CHX (1-h treatment). Also see Video 5. Identical results were observed with WT cells. (E, top) Confocal fluorescent images of ACA-YFP/aca− or cAR1-YFP/car1/3− migrating cells before and after complete bleach. The numbers represent the time (in seconds) after the bleach. Also see Video 6. The graphs depict the total fluorescent recovery of ACA-YFP or cAR1-YFP from the entire cell under various conditions. Data are presented as a mean of five cells ± SEM. It is the short maturation time of YFP that has allowed us to visualize the quick recovery of ACA. Cells expressing ACA-GFP show no recovery after total bleaching (not depicted). (F, right) Radiographs depicting the incorporation of 35S-Translabel into ACA-YFP or cAR1-YFP at various time points after adding it to ACA-YFP/aca− and cAR1-YFP/car1/3− migrating cells (see Materials and Methods for details). The input lane shows a Western analysis (using an α-GFP antibody) of pulled down ACA-YFP and cAR1-YFP samples and depicts the starting amounts of protein for both ACA-YFP and cAR1-GFP. The graph shows the optical density (OD) values from the radiographs. Representative data of at least three independent experiments are shown. Videos are available.

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