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. 2019 Feb 5;26(6):1654–1667.e7. doi: 10.1016/j.celrep.2019.01.027

Figure 1.

Figure 1

Orb6 Inhibition Leads to Increased Cell Width, Cell-Separation Defects, and Cessation of Polarized Elongation in Both Wild-Type and gef1Δ Cells

(A) Video time points of Cdc42-GTP reporter CRIB-3mCitrine after Orb6 inhibition (3-BrB-PP1 treatment of orb6-as2) in indicated strains. 3-BrB-PP1 was added after the 0 time point. Both strains show cell-width increase and cell-separation defects. Video S1 shows more cells, with Lifeact-3mCherry. Green arrowheads indicate ectopic CRIB-3mCitrine on cell sides in orb6-as2 cells, but not orb6-as2 gef1Δ cells. Red arrowheads indicate CRIB-3mCitrine at the midzone during early stages of septation in orb6-as2 cells. Blue arrowheads indicate the absence of CRIB-3mCitrine at comparable stages in orb6-as2 gef1Δ cells (Video S1).

(B) Morphology of indicated strains at 25°C and after 5 h at 37°C, shown by fluorescein-dextran exclusion. Images of this type were used for cell-width measurements in (C).

(C) Cell width in indicated strains after 5 h of the indicated treatment and/or condition. Diagrams illustrate how width was measured. Red lines and error bars indicate median and interquartile range.

(D) Kymographs of CRIB-3mCitrine in indicated strains, starting 1 h after 3-BrB-PP1 addition. Video S4 shows the same cells. Orb6-inhibited cells do not elongate, despite Cdc42-GTP enrichment at cell tips.

(E–G) Rates of cell elongation (E), cell widening (F), and cell-volume expansion (G) from experiments as in (D). Red lines and error bars indicate mean and SD. Cell-volume expansion is derived from the other two parameters (see STAR Methods). Videos show that slightly positive values for cell elongation rates in orb6-as2 and orb6-as2 gef1Δ cells are due to cell swelling in all directions and not to polarized elongation (Video S4).

n shows the number of cells scored. Scale bars, 5 μm. p values were determined by two-tailed unpaired Mann-Whitney test. See also Figure S1 and Videos S2 and S3. Three biological replicates were performed for (A) and (D). Measurements for (E)–(G) were made from one of the replicates. Imaging experiments for (B) and (C) were performed once.