Figure 1.
Figure 1. The dynein–dynactin complex controls microtubule dynamics and the polarized approach of microtubules at the leading edge plasma membrane of migrating astrocytes. (A) Superimposition of microtubule staining visualized by TIRF (green) and wide-field epifluorescence (red, left panels), and TIRF images of microtubules (red) in cytoplasmic GFP-expressing cells (right panels). Microtubules specifically approach the basal plasma membrane at the front of migrating cells independently of plasma membrane adhesion to the substrate. (B) Average (red) and individual microtubule z-profiles (black) in a migrating astrocyte (top graph). The inset shows the cell from which 16 individual microtubule z-profiles were calculated and analyzed. Cell-averaged microtubule morphology in migrating astrocytes (bottom graph). 6 cells were analyzed (see Materials and methods). Note the difference in scale between the horizontal and vertical axes. (C) Combined dual-color TIRF microscopy and IRM (interference reflection microscopy) of a migrating astrocyte. Superimposition of IRM image (blue), microtubule (green), and actin (red) stainings and individual channels (gray) are shown. In IRM, darker signals reflect strong cell adhesion to the substrate, whereas in TIRF the fluorescence increases close to the substrate. Note that the contrast was inverted in microtubule and actin TIRF images. The dashed region shows the region of microtubule close approach toward the plasma membrane. The graph plots the intensity profiles of the IRM image, the microtubule, and the actin stainings (inverted contrast) along a leading edge microtubule (dotted line) from the leading edge toward the cell body. (D) Drawing showing the relative positions of the lamellipodia, the actin-rich strong adhesion region, and the zone where microtubules approach in close proximity with the plasma membrane at the leading edge of a migrating astrocyte. (E) EB1 dynamics in control (Control and si GFP) and DHC-depleted (si DHC) cells. Two frames were superimposed: the first frame is color coded in green and the second frame (separated from the first one by a time interval, Δt = 1.6, 1.7, and 1.3 s for control, si GFP, and si DHC, respectively) is color coded in red. In control cells, red EB1 clusters position in front of the corresponding green EB1 cluster toward the cell leading edge. Abnormal EB1 dynamics in the DHC-depleted cell is shown in ellipses. See Videos 6–8. (F) Superimposition of TIRF (green) and wide-field epifluorescence (red) images of microtubules in control (si GFP, left), DHC-depleted (si DHC, top right), and p150Glued-depleted (si p150, bottom right) cells. (G) Quantification of polarized microtubule z-profile. 54–329 cells from three independent experiments were scored. Error bars represent SEM. Dashed lines indicate the orientation of the wound. Bars, 5 µm.

The dynein–dynactin complex controls microtubule dynamics and the polarized approach of microtubules at the leading edge plasma membrane of migrating astrocytes. (A) Superimposition of microtubule staining visualized by TIRF (green) and wide-field epifluorescence (red, left panels), and TIRF images of microtubules (red) in cytoplasmic GFP-expressing cells (right panels). Microtubules specifically approach the basal plasma membrane at the front of migrating cells independently of plasma membrane adhesion to the substrate. (B) Average (red) and individual microtubule z-profiles (black) in a migrating astrocyte (top graph). The inset shows the cell from which 16 individual microtubule z-profiles were calculated and analyzed. Cell-averaged microtubule morphology in migrating astrocytes (bottom graph). 6 cells were analyzed (see Materials and methods). Note the difference in scale between the horizontal and vertical axes. (C) Combined dual-color TIRF microscopy and IRM (interference reflection microscopy) of a migrating astrocyte. Superimposition of IRM image (blue), microtubule (green), and actin (red) stainings and individual channels (gray) are shown. In IRM, darker signals reflect strong cell adhesion to the substrate, whereas in TIRF the fluorescence increases close to the substrate. Note that the contrast was inverted in microtubule and actin TIRF images. The dashed region shows the region of microtubule close approach toward the plasma membrane. The graph plots the intensity profiles of the IRM image, the microtubule, and the actin stainings (inverted contrast) along a leading edge microtubule (dotted line) from the leading edge toward the cell body. (D) Drawing showing the relative positions of the lamellipodia, the actin-rich strong adhesion region, and the zone where microtubules approach in close proximity with the plasma membrane at the leading edge of a migrating astrocyte. (E) EB1 dynamics in control (Control and si GFP) and DHC-depleted (si DHC) cells. Two frames were superimposed: the first frame is color coded in green and the second frame (separated from the first one by a time interval, Δt = 1.6, 1.7, and 1.3 s for control, si GFP, and si DHC, respectively) is color coded in red. In control cells, red EB1 clusters position in front of the corresponding green EB1 cluster toward the cell leading edge. Abnormal EB1 dynamics in the DHC-depleted cell is shown in ellipses. See Videos 6–8. (F) Superimposition of TIRF (green) and wide-field epifluorescence (red) images of microtubules in control (si GFP, left), DHC-depleted (si DHC, top right), and p150Glued-depleted (si p150, bottom right) cells. (G) Quantification of polarized microtubule z-profile. 54–329 cells from three independent experiments were scored. Error bars represent SEM. Dashed lines indicate the orientation of the wound. Bars, 5 µm.

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