FIGURE 4:

Tm1A colocalizes with myosin-II but only partially overlaps with Diaphanous in the cleavage furrow, where Diaphanous is required for retention, but not recruitment, of Tm1A. (A–C) During interphase, mCherry-Tm1A and myosin-GFP localize to retracting, but not expanding, blebs at the cell periphery. See also Supplemental Figure 4 Video 1. (B) Zoomed-in single-color frames from time-lapse imaging. (C) Graph of total fluorescence intensity in the example bleb over time during bleb expansion (light gray) and bleb retraction (darker gray). (D) During mitosis, Tm1A and myosin-II colocalize at the equatorial cortex (arrow) during early anaphase and to retracting blebs (arrowhead), but not expanding blebs (asterisks), at the cell poles during late anaphase and telophase. Bottom, cartoon schematic illustrating localization of Tm1A (purple) and myosin (blue) in corresponding upper images. See also Supplemental Figure 4 Video 2. (E) During metaphase, Tm1A localizes to the cortex before Dia. As the cell progresses through mitosis, Tm1A occupies a larger, expanding region of the cortex around the periphery of the cell, whereas in contrast, Dia occupies a smaller region in the middle of the cell that seems to concentrate at the site of cleavage furrow ingression. Bottom, schematic diagram illustrating differential localizations of Tm1A (purple) and Dia (orange). See also Supplemental Figure 4 Video 3. (F) Dia is required for retention, not recruitment, of Tm1A to the equatorial cortex during anaphase. mCherry-Tm1A in a dividing cell after depletion of Dia with RNAi. Tm1A still initially localizes to the equatorial cortex (arrowheads), but it does not remain there. As the cell proceeds through anaphase, Tm1A localizes to sites of contraction (arrows) around the periphery of the cell during shape instability. See also Supplemental Figure 4 Video 4.