Figure 8
PAM-deficient Chlamydomonas have altered actin organization and upregulate levels of an actin paralogue. (A) Differential interference contrast (DIC) and fluorescence micrographs of empty vector (EV) control and PAM-deficient (PAM KD8) Chlamydomonas cells stained with Bodipy-phalloidin. The ciliated control cells exhibited diffuse cytoplasmic filamentous actin staining (red); no signal was present in the cilia. The PAM amiRNA cells, which only formed small ciliary stubs (arrows), show several bright foci or patches of filamentous actin staining in the cell body. (B) Integrated fluorescence intensities of Bodipy-phalloidin stained EV control and PAM KD8 cells were not significantly different in a two-way ANOVA (n = 19; P = 0.178). (C) Maximum fluorescence intensity of PAM KD8 cells was significantly different from EV controls in a two-way ANOVA (n = 19; P = 0.009). For the plots in both (B) and (C), the horizontal bar is the mean, boxes represent 95% confidence intervals, and whiskers show minimum to maximum range. (D) Immunoblots of cell lysates from two EV and two PAM-KD strains; equal protein loading was shown by Coomassie blue stain. The PAM-KD strains had reduced levels of PAM, whereas the canonical actin content was not altered. In contrast, levels of the actin paralogue, NAP, were significantly increased in the PAM-KD strains.
