F-actin is dispensable for the ingression of the longer septa of phase 2. (A) After short LatA treatment (5–15 min), the longer septa of phase 2 remain thin and with Bgs1, Bgs4, and Ags1 correctly concentrated as a ring at the septum edge. Cells were grown in the presence of LatA and analyzed as in Fig. 2 C. Scale bar = 2 μm. (B) Time-lapse video microscopy of phase 2 septum ingression in cells grown either in the absence (control, top panel) or in the presence of LatA (middle and bottom panels). RFP-Bgs1, myosin-II regulatory light chain Rlc1-GFP, and CW fluorescence enabled the detection of the septum membrane, CR, and PS, respectively. Cells were grown in YES without or with LatA added at time = 0 and analyzed as in Fig. 2 D by RFP, GFP, and CW fluorescence microscopy. CR diameter at time = 0 measured as Rlc1 fluorescence was 0.61 µm (top panel), 0.64 µm (middle panel), and 0.81 µm (bottom panel). Dashed rectangles, septum area shown in the time-lapse sequences of Rlc1, Bgs1 and Rlc1 merge, and CW series; green arrowheads, Rlc1 ring loss; blue arrowhead, septum completion. Scale bars = 5 μm. (C) Ultrastructure of thin septa formed in cells after a short LatA treatment. The septa slightly increase in thickness and finish in a sharp edge (arrows). The PS is twisted, suggesting a misdirected ingression due to the absence of CR tension. Cells were grown as in Fig. 2 A in the presence of LatA and analyzed by TEM. The control septa formed without LatA are shown in Fig. 2 E. Line points inside the PS and SS. Scale bars = 2 μm. (D) Model of thin septum ingression in the absence of F-actin. Short LatA treatments in phase 2 ingressing septa (>0.30 µm in length) promote a slight reduction in the ingression rate, but the glucan synthase rings and CR Rlc1 remain until completion. GS, glucan synthase.