Figure 11.

DdLIS1-D327H cells, MBP-DdLIS1 overexpressors, and DICΔC overexpressors often show altered F-actin distribution and alterations in actin dynamics similar to control cells treated with low concentrations of latrunculin A. Immunofluorescence labeling of control cells (A), DdLIS1-D327H cells (B), and DICΔC overexpressors (C) with anti-α-tubulin (green) and phalloidin-AlexaFluor568 (Molecular Probes) as an F-actin labeling probe (red). Nuclei were stained with TOPRO3 (blue). Note the broad F-actin distribution at the bottom of the cells along with disrupted microtubule cytoskeletons in (B and C). Cells were fixed with glutaraldehyde. Bar, 5 μm. Note that phalloidin-labeling intensities in these images do not illustrate the F-actin content, because all images were acquired at microscope settings allowing coverage of the full dynamic range of the 8-bit scale. Live analysis of actin dynamics was performed with GFP-actin control cells (D, Supplementary Movie7), GFP-actin/MBP-DdLIS1 overexpressors (E, Supplementary Movie8), GFP-actin cells treated with 0.2 μM latrunculin A (F, Supplementary Movie9) and GFP-actin cells carrying DdLIS1-D327H mutation (G, Supplementary Movie10). Each image represents a brightest point z-projection of three confocal slices with a distance of 0.8 μm each. One a single, representative time frame of each movie is shown in D–F, whereas G represents a time sequence.