Figure 8.
Figure 8. Septum ingression in the absence of F-actin depends on Cdc42 GTPase and the exocyst complex. (A) Kymographs of time-lapse videos to visualize the onset of stable localization of different septum proteins (green arrowheads) with respect to Bgs1 ring localization (red arrowheads) and the onset of septum synthesis (T = 0). Time = 0 was the time immediately before the first CW-stained septum detection (blue arrowheads). Cells were visualized by simultaneous GFP (septum protein), RFP (Bgs1), and CW (PS structure) fluorescence time-lapse video microscopy in each case (n = 95 cells). Kymographs simultaneously detecting chitin synthase homologue Chs2, Bgs1, and CW are shown as an example. For simplicity, only the GFP kymograph of the other analyzed proteins indicating the start of stable GFP localization (green arrowheads) is shown, and the corresponding RFP-Bgs1 and CW detections are shown by the red and blue arrowheads, respectively (RFP and CW kymographs not depicted). The kymographs were aligned with respect to the start of CW detection (blue arrowheads). Scale bar = 2 µm. (B) Scheme of the timing (minute ± SD) of stable localization of the analyzed septum proteins in the time-lapse video series from A with respect to the simultaneous localizations of the Bgs1 ring (red), onset of septation (T = 0), and detection of the CW-stained septum (T = +0.5, blue; n = 95 cells). The timing of anaphase B, spindle disassembly, and telophase was selected from data previously reported in similar time-lapse video analyses (Cortés et al., 2018). (C) Cdc42 GTPase, Gef1 exchange factor, Hob3 BAR protein, Shk1 effector kinase, and the exocyst complex are essential for the ingression of phase 2 septa (>0.30 µm) in cells deprived of F-actin (arrows). The number of phase 2 open septa was quantified at the indicated times after treatment with LatA (n = 4,554 open septa). Values are the relative percentage of phase 2 open septa with respect to the total number of cells and were normalized, giving a value of 100% to the initial percentage of phase 2 open septa without LatA treatment. Cells were grown in YES at 28°C in the case of constitutive mutants, or at 25°C and shifted to the indicated temperatures in the case of thermosensitive mutants, transferred to YES containing LatA and analyzed by RFP-Bgs1 and CW fluorescence microscopy. The temperatures used correspond to the highest permissive temperature at which the septum ingression of thermosensitive mutant cells and control WT cells without LatA treatment proceeded as normal (not depicted). A scheme of the protein pathway involved in the control of septum ingression is shown. CRIB, Cdc42/Rac-interacting binding (probe for active Cdc42-GTP). Error bars show SD.

Septum ingression in the absence of F-actin depends on Cdc42 GTPase and the exocyst complex. (A) Kymographs of time-lapse videos to visualize the onset of stable localization of different septum proteins (green arrowheads) with respect to Bgs1 ring localization (red arrowheads) and the onset of septum synthesis (T = 0). Time = 0 was the time immediately before the first CW-stained septum detection (blue arrowheads). Cells were visualized by simultaneous GFP (septum protein), RFP (Bgs1), and CW (PS structure) fluorescence time-lapse video microscopy in each case (n = 95 cells). Kymographs simultaneously detecting chitin synthase homologue Chs2, Bgs1, and CW are shown as an example. For simplicity, only the GFP kymograph of the other analyzed proteins indicating the start of stable GFP localization (green arrowheads) is shown, and the corresponding RFP-Bgs1 and CW detections are shown by the red and blue arrowheads, respectively (RFP and CW kymographs not depicted). The kymographs were aligned with respect to the start of CW detection (blue arrowheads). Scale bar = 2 µm. (B) Scheme of the timing (minute ± SD) of stable localization of the analyzed septum proteins in the time-lapse video series from A with respect to the simultaneous localizations of the Bgs1 ring (red), onset of septation (T = 0), and detection of the CW-stained septum (T = +0.5, blue; n = 95 cells). The timing of anaphase B, spindle disassembly, and telophase was selected from data previously reported in similar time-lapse video analyses (Cortés et al., 2018). (C) Cdc42 GTPase, Gef1 exchange factor, Hob3 BAR protein, Shk1 effector kinase, and the exocyst complex are essential for the ingression of phase 2 septa (>0.30 µm) in cells deprived of F-actin (arrows). The number of phase 2 open septa was quantified at the indicated times after treatment with LatA (n = 4,554 open septa). Values are the relative percentage of phase 2 open septa with respect to the total number of cells and were normalized, giving a value of 100% to the initial percentage of phase 2 open septa without LatA treatment. Cells were grown in YES at 28°C in the case of constitutive mutants, or at 25°C and shifted to the indicated temperatures in the case of thermosensitive mutants, transferred to YES containing LatA and analyzed by RFP-Bgs1 and CW fluorescence microscopy. The temperatures used correspond to the highest permissive temperature at which the septum ingression of thermosensitive mutant cells and control WT cells without LatA treatment proceeded as normal (not depicted). A scheme of the protein pathway involved in the control of septum ingression is shown. CRIB, Cdc42/Rac-interacting binding (probe for active Cdc42-GTP). Error bars show SD.

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