Fig. 3.

Caldesmon overexpression and CALI of MLC reveals requirement of NMII in cytoplasm coherence. (A) Left panels: time-lapse sequential DIC images (within 20 minutes) of spreading cells with a high level of GFP-caldesmon overexpression, showing the dynamic formation of ‘C’ and dendritic shapes. Right panels: DIC and GFP epifluorescence images of the cells on the left panels at 40 minutes after spreading. Scale bars: 20 μm. (B-F) TIRF and (G-K) DIC images of the same cell expressing MLC-eDHFR labeled with fluorescein-conjugated TMP before (B,G) and after (C-F, H-K) laser irradiation. Negative times signify time before irradiation. White circles denote the irradiated region. A region devoid of fluorescence forms (arrow in C) after laser irradiation, which is enlarged with time. Meanwhile, fluorescence recovers (arrows in D-F). The decrease of cytoplasmic coherence is clearly displayed by the changes of shapes and positions of actin cables (used as markers) within or close to the irradiated region (G-K) in DIC images. For instance, the upper portion of the actin cable denoted by a green line is curved and clearly moves left, whereas the upper portion of the actin cable denoted by a red line is curved but moves right (H-K). The space between them becomes larger. (L-P) TIRF images of a cell. (Q-U) DIC images of another cell. Both cells expressed eDHFR that was labeled with fluorescein-conjugated TMP. Images L and Q are pre-irradiation images. Images M-P and R-U are post-irradiation images. eDHFR diffuses in the cell. Following laser irradiation (white circle), the eDHFR fluorescence recovers completely within 5 seconds (M-O). There is no indication of decrease of cytoplasmic coherence because the shapes and positions of marker actin cables (R-U) appear not to change.