The CR landmark function is responsible for the Bgs1 ring persistence in the phase 2 septa and depends on the CR components Myo2 and Cdc12. (A) The onset of thick septa in cells with LatA carrying RFP-Bgs1 and Myo2-GFP was analyzed by time-lapse video microscopy as in Fig. 3 B (bottom panel). In control cells without LatA treatment, Myo2 remains in the CR during the entire septum ingression (top panel). In cells with LatA, Myo2 is lost coincident with the loss of the Bgs1 ring (arrow) and the onset of thick septa (bottom panel). Dashed rectangles, septum area shown in the time-lapse sequences of Myo2-GFP and CW series; red arrowhead, thick septum onset; green arrowheads, Myo2 ring loss; blue arrowhead, septum completion. Scale bars = 5 μm. (B) The persistence of CR proteins and glucan synthase rings in cells treated with LatA was analyzed. Persistence was quantified as percentage of open septa with the corresponding protein localized as a ring at the septum edge at the indicated times of LatA treatment. The localization analysis enabled the classification of the studied proteins into three persistence categories: low (including F-actin; lost after 5 min), medium (including all glucan synthases; highly reduced after 15 min), and high persistence (reduced only after 30 min) in the presence of LatA. Differences in the persistence of each group are marked (arrows). All proteins were analyzed simultaneously using Bgs1 as a control. Cells were grown in the presence of LatA as in Fig. 2 C (n = 837 septa). (C) Bgs1 depletion generates thick septa (arrows). 81X-bgs1⁺ cells carrying plasma membrane syntaxin GFP-Psy1 to visualize the septum thickness were grown in MM+S at 28°C and analyzed using GFP fluorescence microscopy at the indicated times of bgs1⁺ repression with thiamine (+T). Scale bars = 5 μm. (D) Bgs1 depletion promotes thick septation with normal Myo2 and Cdc12 localization as a ring (arrows). Cells were grown and analyzed by fluorescence microscopy as in C. Scale bar = 2 μm. (E) Loss of Myo2 function causes the loss of Cdc12 ring and vice versa, and both equally generate the simultaneous Bgs1 ring loss and thick septation (arrows; left panels of septum details). The mutant cells myo2-E1 or cdc12-112 were grown in YES+S at 25°C, the temperature was shifted as specified, and the cells were analyzed by fluorescence microscopy. There is a gradual decrease in fluorescence intensity of the Bgs1 and Cdc12 rings in myo2-E1 thermosensitive mutant cells (n = 140 septa) and of the Bgs1 and Myo2 rings in cdc12-112 thermosensitive mutant cells (n = 149 septa; right panels). The Bgs1 ring vanishes and Bgs1 spreads all over the septum membrane after the Cdc12 or Myo2 ring disappears (dotted red line). Scale bars = 5 μm. (F) Bgs1 depletion promotes the loss of Bgs4 and Ags1 rings and the spread of these proteins all over the membrane of the thick septa (arrows, septum details). Cells were grown and analyzed as in C. Scale bars = 2 μm. (G) Scheme of the protein pathway involved in the contractile and landmark functions of the CR. Error bars show SD. Data distribution was assumed to be normal, but this was not formally tested.