Figure 7.
Figure 7. Prolonged Swe1 overexpression causes elongated buds, multiple buds, spindle mispositioning, and septin defects during mitosis. (A and B) Image showing an elongated bud, aberrant spindle position, and septin localized in the bud after Swe1 overexpression for 360 min. (C) Time-lapse images of a cell without Swe1 overexpression with time in minutes after initial recruitment of septins. (D) Mean bud length after 360 min of Swe1 overexpression or without Swe1 overexpression at the time of anaphase onset (error bars indicate SD; n = 100 each). (E) Septin localization in the bud, spindle migration to the bud, second bud formation, and nuclear division in WT cells overexpressing Swe1 (n = 100). (F) Superresolution images of bud-neck septin structures with Swe1 overexpression for 360 min or without Swe1 overexpression at anaphase onset. (G) Percentage of cells with defective versus normal bud-neck septin structures analyzed by superresolution imaging at individual time points. Septins were imaged at early bud formation and at metaphase delay (400 min after nutrient addition for hsl1Δ and nap1Δ; 360 min after SWE1 induction; n = 50 each). Septins in a culture without Swe1 overexpression were imaged at log phase (n = 50 each). Differences are statistically significant (Two-tailed Fisher’s exact test; P < 0.0001). (H) Mean fluorescence intensity of Cdc3-GFP at the bud neck measured at bud formation and at a metaphase delay (210 min later) for hsl1Δ, nap1Δ, and Swe1 overexpression (error bars indicate SEM). WT control cells were imaged in early log phase and 210 min later (n = 30 each). (D and H) Differences were statistically significant (Mann-Whitney test with computation of two-tailed exact p-value; ****, P < 0.0001) unless marked ns. (I) Time-lapse images of persistent GTP-Cdc42 localization at the bud tip and subsequent recruitment of septins in a cell overexpressing Swe1 during mitosis with time in minutes after galactose induction. White arrows indicate Gic2-PDB-RFP localization; blue arrows indicate mCherry-Tub1. Bars: (A–C and I) 3 µm; (F) 1 µm.

Prolonged Swe1 overexpression causes elongated buds, multiple buds, spindle mispositioning, and septin defects during mitosis. (A and B) Image showing an elongated bud, aberrant spindle position, and septin localized in the bud after Swe1 overexpression for 360 min. (C) Time-lapse images of a cell without Swe1 overexpression with time in minutes after initial recruitment of septins. (D) Mean bud length after 360 min of Swe1 overexpression or without Swe1 overexpression at the time of anaphase onset (error bars indicate SD; n = 100 each). (E) Septin localization in the bud, spindle migration to the bud, second bud formation, and nuclear division in WT cells overexpressing Swe1 (n = 100). (F) Superresolution images of bud-neck septin structures with Swe1 overexpression for 360 min or without Swe1 overexpression at anaphase onset. (G) Percentage of cells with defective versus normal bud-neck septin structures analyzed by superresolution imaging at individual time points. Septins were imaged at early bud formation and at metaphase delay (400 min after nutrient addition for hsl1Δ and nap1Δ; 360 min after SWE1 induction; n = 50 each). Septins in a culture without Swe1 overexpression were imaged at log phase (n = 50 each). Differences are statistically significant (Two-tailed Fisher’s exact test; P < 0.0001). (H) Mean fluorescence intensity of Cdc3-GFP at the bud neck measured at bud formation and at a metaphase delay (210 min later) for hsl1Δ, nap1Δ, and Swe1 overexpression (error bars indicate SEM). WT control cells were imaged in early log phase and 210 min later (n = 30 each). (D and H) Differences were statistically significant (Mann-Whitney test with computation of two-tailed exact p-value; ****, P < 0.0001) unless marked ns. (I) Time-lapse images of persistent GTP-Cdc42 localization at the bud tip and subsequent recruitment of septins in a cell overexpressing Swe1 during mitosis with time in minutes after galactose induction. White arrows indicate Gic2-PDB-RFP localization; blue arrows indicate mCherry-Tub1. Bars: (A–C and I) 3 µm; (F) 1 µm.

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