Figure 4.
Figure 4. The CR landmark function is essential for the maintenance of the glucan synthase rings, the shape, and the ingression rate of the phase 2 long septa. (A) After long treatments with LatA (15–30 min), the phase 2 ingressing septa became thick and aberrant, with Bgs1, Bgs4, and Ags1 spread all over the septum membrane. Cells were grown in the presence of LatA and analyzed as in Fig. 2 C. Scale bar = 2 μm. (B) The thick septa formed in cells treated with LatA can slowly ingress to completion. The emergence of thick septum morphology coincides with the loss of Bgs1 ring and the spreading of Bgs1 all over the septum membrane (arrows), but not with the loss of the Rlc1 ring, which remains for a longer period. Cells were grown and imaged using time-lapse video microscopy as in Fig. 3 B. Upper panel, control nontreated cells (CR diameter at time = 0 of Rlc1 = 0.81 µm); middle and lower panels, LatA-treated cells (middle, CR diameter at time = 0 of Rlc1 = 0.90 µm; bottom, Bgs1 ring diameter at time = 0 of 0.74 µm). Dashed rectangles, septum area shown in the time-lapse sequences of Rlc1, Bgs1 and Rlc1 merge, and CW series. Red arrowheads, thick septum onset; green arrowheads, Rlc1 ring loss; blue arrowheads, septum completion detected by RFP-Bgs1 septum membrane closure. Scale bars = 5 μm. (C) The length and timing of thin and thick septa were analyzed from time-lapse videos as in Fig. 3 B and Fig. 4 B. Images were captured at 2-min intervals. Left: septa longer than 60% of the total length at the time of LatA addition are completed as thin septa, whereas septa shorter than 60% of the length change morphology and are completed as thick septa (n = 38 cells). Middle: the Bgs1 ring loss and septum change to become thick occur ∼15 min after LatA addition (n = 19 cells). Right: the septum progression from the start of LatA addition to the Bgs1 ring loss and emergence of thick morphology is ~24% of the total septum length (n = 29 septa). (D and E) Ultrastructure of thick septa formed after 15–30 min (D) or 2 h (E) of LatA treatment. The septa are thick, blunt structures (arrows) with twisted PSs, likely due to the absence of the Bgs1 ring (landmark defect) and to a contractile defect. The complete long-term thick septa (E) present an additional ICW probably made by Bgs1, Bgs4, and Ags1 spread all over the septum membrane, generating abnormal five-layered septa. Cells were grown in the presence of LatA as in Fig. 2 A and examined by TEM. The control septa formed without LatA are in Fig. 2 E. Arrows, whole thick septum structure; line points inside the PS, SS, and ICW. Scale bars = 2 µm. GS, glucan synthase. Bars in graphs show the average, and error bars indicate SD. Data distribution was assumed to be normal, but this was not formally tested.

The CR landmark function is essential for the maintenance of the glucan synthase rings, the shape, and the ingression rate of the phase 2 long septa. (A) After long treatments with LatA (15–30 min), the phase 2 ingressing septa became thick and aberrant, with Bgs1, Bgs4, and Ags1 spread all over the septum membrane. Cells were grown in the presence of LatA and analyzed as in Fig. 2 C. Scale bar = 2 μm. (B) The thick septa formed in cells treated with LatA can slowly ingress to completion. The emergence of thick septum morphology coincides with the loss of Bgs1 ring and the spreading of Bgs1 all over the septum membrane (arrows), but not with the loss of the Rlc1 ring, which remains for a longer period. Cells were grown and imaged using time-lapse video microscopy as in Fig. 3 B. Upper panel, control nontreated cells (CR diameter at time = 0 of Rlc1 = 0.81 µm); middle and lower panels, LatA-treated cells (middle, CR diameter at time = 0 of Rlc1 = 0.90 µm; bottom, Bgs1 ring diameter at time = 0 of 0.74 µm). Dashed rectangles, septum area shown in the time-lapse sequences of Rlc1, Bgs1 and Rlc1 merge, and CW series. Red arrowheads, thick septum onset; green arrowheads, Rlc1 ring loss; blue arrowheads, septum completion detected by RFP-Bgs1 septum membrane closure. Scale bars = 5 μm. (C) The length and timing of thin and thick septa were analyzed from time-lapse videos as in Fig. 3 B and Fig. 4 B. Images were captured at 2-min intervals. Left: septa longer than 60% of the total length at the time of LatA addition are completed as thin septa, whereas septa shorter than 60% of the length change morphology and are completed as thick septa (n = 38 cells). Middle: the Bgs1 ring loss and septum change to become thick occur ∼15 min after LatA addition (n = 19 cells). Right: the septum progression from the start of LatA addition to the Bgs1 ring loss and emergence of thick morphology is ~24% of the total septum length (n = 29 septa). (D and E) Ultrastructure of thick septa formed after 15–30 min (D) or 2 h (E) of LatA treatment. The septa are thick, blunt structures (arrows) with twisted PSs, likely due to the absence of the Bgs1 ring (landmark defect) and to a contractile defect. The complete long-term thick septa (E) present an additional ICW probably made by Bgs1, Bgs4, and Ags1 spread all over the septum membrane, generating abnormal five-layered septa. Cells were grown in the presence of LatA as in Fig. 2 A and examined by TEM. The control septa formed without LatA are in Fig. 2 E. Arrows, whole thick septum structure; line points inside the PS, SS, and ICW. Scale bars = 2 µm. GS, glucan synthase. Bars in graphs show the average, and error bars indicate SD. Data distribution was assumed to be normal, but this was not formally tested.

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