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. 2019 Oct 24;20(12):e47743. doi: 10.15252/embr.201947743

Figure 7. Somatic F‐actin puncta are Formin but not Arp2/3 dependent.

Figure 7

  1. DIV 1 neurons were incubated for two hours with DMSO, 50 μM CK666, 25 μM SMIFH2, or 50 μM CK666 + 25 μM SMIFH2. Cells were fixed and stained with anti‐pericentrin antibody and Phalloidin.
  2. Total number of cytosolic F‐actin puncta per μm3 from cells treated with DMSO = 1.000 ± 0.2559, CK666 = 0.7740 ± 0.0693, SMIFH2 = 0.3308 ± 0.0605, and CK666 + SMIFH2 = 0.4067 ± 0.0554. P = 0.0036 by one‐way ANOVA, post hoc Dunnett's test; *P < 0.05, **P < 0.01. Mean ± SEM; n = 11 for SMIFH2, n = 10 cells each for DMSO, CK666, and SMIFH2 + CK666 groups from at least two different cultures.
  3. 3D graphs show the distribution of cytosolic F‐actin puncta around the centrosome in DMSO, CK666, SMIFH2, and CK666 + SMIFH2‐treated cells. The coordinates of the centrosome and F‐actin puncta are obtained from the confocal images. The coordinates of centrosomes from all the cells, indicated as a black cube, are positioned at the center (x, y, z = 0) and the color‐coded F‐actin puncta are plotted with respect to the position of the centrosome from the respective cell.
  4. PaGFP‐UtrCH and mMaroon1 co‐transfected DIV1 rat hippocampal neuron treated with DMSO (upper panel) SMIFH2 (37.5 μM for 2 h; lower panel) photoactivated in the soma with 405 nm laser (red circle with a diameter of 5.146 μm).
  5. Neurites from cells in (D; insets 1, 2) show the reach of the photoactivated signal at the end of the time lapse (146 s).
  6. Left panel: normalized intensity values in the photoactivated area (soma) of DMSO‐ and SMIFH2‐treated PaGFP‐UtrCH expressing cells. Inset graph: half‐time (t½) values in seconds for DMSO‐treated cells = 52.00 ± 9.317 (n = 7), SMIFH2‐treated cells = 85.33 ± 15.17 (n = 8). P = 0.0942 by unpaired Student's t‐test. Middle panel: photoactivated signal in the neurite tip over time relative to the average initial signal from illuminated area for DMSO‐ and SMIFH2‐treated PaGFP‐UtrCH expressing cells. Right panel: neurite tip to soma intensity ratio of photoactivated for DMSO and SMIFH2‐treated PaGFP‐UtrCH expressing cells. All panels: mean ± SEM; n = 9 for DMSO and n = 13 cells for SMIFH2‐treated PaGFP‐UtrCH expressing cells, from at least two different cultures.
Data information: Scale bar: 5 μm (A), 10 μm (D).