Bni5 regulates septin hourglass architecture and its timely remodeling into a double ring. (A) Representative fluorescent images of Cdc3-GFP in WT and bni5Δ cells, taken 1.5 h after release from a 2.5-h α-factor arrest. Arrowheads indicate discontinuities in the septin hourglass. Strains used: YEF9333 (bar1Δ CDC3-GFP) and YEF9767 (bni5Δ bar1Δ CDC3-GFP). (B) Quantification of septin hourglass morphology. Patterns of septin structures in the WT and bni5Δ cells were quantified from imaging data shown in A. n denotes the number of cells analyzed per strain. (C) Representative PREM images of Cdc3-labeled septin hourglass structures from WT and bni5Δ cells after α-factor arrest and release. Septin structures were immunogold-labeled using an 18-nm gold-conjugated secondary antibody and a primary anti-GFP antibody to detect Cdc3-GFP. The inset shows the magnified region of the white box. In the inset, septin filaments and gold particles are pseudo-colored green and pink, respectively. See also Fig. S3 A. (D) Quantification of septin structures in WT and bni5Δ cells from PREM imaging shown in C. n denotes the number of septin structures analyzed per strain. (E) Time-lapse analysis of the septin HDR transition in WT and bni5Δ. Top: Montages of Cdc3-GFP created from time-lapse series taken with 1.5-min intervals. Bottom: Cdc3-GFP localization at the bud neck during the HDR transition. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. Strains used: YEF9180 (CDC3-GFP mRuby2-TUB1) and YEF9618 (bni5Δ CDC3-GFP mRuby2-TUB1). (F) Time-lapse analysis of Bni5 lacking its IDR. Montages and localization plots were created as in E. Asterisk indicates Bni5 variants. Strains used: YEF11546 (mScarlet-TUB1 GFP-BNI5) and YEF11553 [bni5Δ mScarlet-TUB1 GFP-bni5(Δ41-305)]. (G) Time-lapse analysis of the septin HDR transition in WT and bni5(ΔIDR) cells. Montages of Cdc10-mScarlet and corresponding localization plots were created as in E. P values were determined using a two-sided Mann–Whitney U test. Arrowheads indicate the onset of HDR transition. Strains used: YEF11772 [CDC10-mScarlet Venus-TUB1 GFP-BNI5(FL)] and YEF11773 [CDC10-mScarlet Venus-TUB1 GFP-bni5(Δ41-305)].
Figure 4.

Bni5 regulates septin hourglass architecture and its timely remodeling into a double ring. (A) Representative fluorescent images of Cdc3-GFP in WT and bni5Δ cells, taken 1.5 h after release from a 2.5-h α-factor arrest. Arrowheads indicate discontinuities in the septin hourglass. Strains used: YEF9333 (bar1Δ CDC3-GFP) and YEF9767 (bni5Δ bar1Δ CDC3-GFP). (B) Quantification of septin hourglass morphology. Patterns of septin structures in the WT and bni5Δ cells were quantified from imaging data shown in A. n denotes the number of cells analyzed per strain. (C) Representative PREM images of Cdc3-labeled septin hourglass structures from WT and bni5Δ cells after α-factor arrest and release. Septin structures were immunogold-labeled using an 18-nm gold-conjugated secondary antibody and a primary anti-GFP antibody to detect Cdc3-GFP. The inset shows the magnified region of the white box. In the inset, septin filaments and gold particles are pseudo-colored green and pink, respectively. See also Fig. S3 A. (D) Quantification of septin structures in WT and bni5Δ cells from PREM imaging shown in C. n denotes the number of septin structures analyzed per strain. (E) Time-lapse analysis of the septin HDR transition in WT and bni5Δ. Top: Montages of Cdc3-GFP created from time-lapse series taken with 1.5-min intervals. Bottom: Cdc3-GFP localization at the bud neck during the HDR transition. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. Strains used: YEF9180 (CDC3-GFP mRuby2-TUB1) and YEF9618 (bni5Δ CDC3-GFP mRuby2-TUB1). (F) Time-lapse analysis of Bni5 lacking its IDR. Montages and localization plots were created as in E. Asterisk indicates Bni5 variants. Strains used: YEF11546 (mScarlet-TUB1 GFP-BNI5) and YEF11553 [bni5Δ mScarlet-TUB1 GFP-bni5(Δ41-305)]. (G) Time-lapse analysis of the septin HDR transition in WT and bni5(ΔIDR) cells. Montages of Cdc10-mScarlet and corresponding localization plots were created as in E. P values were determined using a two-sided Mann–Whitney U test. Arrowheads indicate the onset of HDR transition. Strains used: YEF11772 [CDC10-mScarlet Venus-TUB1 GFP-BNI5(FL)] and YEF11773 [CDC10-mScarlet Venus-TUB1 GFP-bni5(Δ41-305)].

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