Figure 2.
Figure 2. IFs control the organization and dynamics of the actin network. (A) Immunofluorescence images of actin fibers in migrating astrocytes stained for nuclei (cyan) and actin (phalloidin, gray). (B) Graph showing the percentage of leader cells that present ITAs in si ctl and si triple IF cells. (C) Rose plot showing the frequency of angle distribution of actin fibers analyzed in follower cells (Fig. S3 D). Fibers with a 0° angle are perpendicular to the wound, and fibers with an angle close to 90° are parallel to the wound. (D) Schematics showing the position of the kymographs acquired in E–G. (E) Frames of migrating si ctl and si triple IF astrocytes transfected with LifeAct-mCherry. The white dotted line represents the wound, and the pink dotted lines represent the positions in which the kymographs were calculated. Kymographs (fire LUT) show the retrograde flow of actin on cell–cell junctions over a time period of 4 h. The graph shows the mean retrograde flow speed of actin cables measured at the level of the cell–cell junctions. (F) Frames from Video 4 of migrating astrocytes transfected with GFP-MRLC. The thick white dotted line represents the outline of nearby cells. The kymographs were obtained along the pink dotted lines. f and b on the side of the kymograph indicate the front and the rear of the line. Kymographs (fire LUT) show the retrograde flow of myosin at the front and at the rear of myosin longitudinal fibers over a time period of 15 min. The graph shows the mean speed of the myosin retrograde flow at the cell front and at the rear of the longitudinal fiber calculated from the kymographs. The white dotted lines indicate the position of the wound. (G) Immunofluorescence images from Video 5 showing GFP-N-cadherin–expressing astrocytes. Insets show the corresponding phase-contrast image. N, nucleus. The kymographs were obtained along the pink dotted lines. f and b on the side of the kymographs indicate the front and the rear of the line. Kymographs (fire LUT) show the retrograde flow of N-cadherin over a time period of 2 h. The graph shows the mean retrograde flow speed of the N-cadherin flow in si ctl and si triple IF migrating astrocytes. (H) Staining for N-cadherin (gray) and nuclei (cyan) in migrating astrocytes nucleofected with the indicated siRNAs. Histogram shows the mean percentage ± SEM of continuous junctions between adjacent leader cells. White dotted line indicates the position of the wound. Data are from n = 3 independent experiments. The sample size for each repeat: si ctl 64, 67, 92 and si triple IF 44, 96, 122 for B; si ctl 11, 10, eight stacks and si triple IF 12, 10, eight stacks for C; si ctl 30, 40, 32 and si triple IF 18, 33, 30 for E; si triple IF f 22, 27, 18 and si triple IF b 22, 24, 20 for F; si ctl 14, 38, 28 and si triple IF 38, 49, 14 for G; si ctl 104, 120, 178, si GFAP 134, 137, 125, si vimentin 113, 180, 133, si nestin 132, 190, 123, and si triple IF 122, 225, 112 for H. ***, P < 0.00. Bars: (main images) 10 µm; (kymographs and insets of H) 5 µm.

IFs control the organization and dynamics of the actin network. (A) Immunofluorescence images of actin fibers in migrating astrocytes stained for nuclei (cyan) and actin (phalloidin, gray). (B) Graph showing the percentage of leader cells that present ITAs in si ctl and si triple IF cells. (C) Rose plot showing the frequency of angle distribution of actin fibers analyzed in follower cells (Fig. S3 D). Fibers with a 0° angle are perpendicular to the wound, and fibers with an angle close to 90° are parallel to the wound. (D) Schematics showing the position of the kymographs acquired in E–G. (E) Frames of migrating si ctl and si triple IF astrocytes transfected with LifeAct-mCherry. The white dotted line represents the wound, and the pink dotted lines represent the positions in which the kymographs were calculated. Kymographs (fire LUT) show the retrograde flow of actin on cell–cell junctions over a time period of 4 h. The graph shows the mean retrograde flow speed of actin cables measured at the level of the cell–cell junctions. (F) Frames from Video 4 of migrating astrocytes transfected with GFP-MRLC. The thick white dotted line represents the outline of nearby cells. The kymographs were obtained along the pink dotted lines. f and b on the side of the kymograph indicate the front and the rear of the line. Kymographs (fire LUT) show the retrograde flow of myosin at the front and at the rear of myosin longitudinal fibers over a time period of 15 min. The graph shows the mean speed of the myosin retrograde flow at the cell front and at the rear of the longitudinal fiber calculated from the kymographs. The white dotted lines indicate the position of the wound. (G) Immunofluorescence images from Video 5 showing GFP-N-cadherin–expressing astrocytes. Insets show the corresponding phase-contrast image. N, nucleus. The kymographs were obtained along the pink dotted lines. f and b on the side of the kymographs indicate the front and the rear of the line. Kymographs (fire LUT) show the retrograde flow of N-cadherin over a time period of 2 h. The graph shows the mean retrograde flow speed of the N-cadherin flow in si ctl and si triple IF migrating astrocytes. (H) Staining for N-cadherin (gray) and nuclei (cyan) in migrating astrocytes nucleofected with the indicated siRNAs. Histogram shows the mean percentage ± SEM of continuous junctions between adjacent leader cells. White dotted line indicates the position of the wound. Data are from n = 3 independent experiments. The sample size for each repeat: si ctl 64, 67, 92 and si triple IF 44, 96, 122 for B; si ctl 11, 10, eight stacks and si triple IF 12, 10, eight stacks for C; si ctl 30, 40, 32 and si triple IF 18, 33, 30 for E; si triple IF f 22, 27, 18 and si triple IF b 22, 24, 20 for F; si ctl 14, 38, 28 and si triple IF 38, 49, 14 for G; si ctl 104, 120, 178, si GFAP 134, 137, 125, si vimentin 113, 180, 133, si nestin 132, 190, 123, and si triple IF 122, 225, 112 for H. ***, P < 0.00. Bars: (main images) 10 µm; (kymographs and insets of H) 5 µm.

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