Distinct impact of N- and C-terminal GFP-tagging of Bni5 on its recruitment timing and turnover kinetics at the division site. (A) Diagram of GFP-tagged Bni5 constructs. Bni5-C-GFP and Bni5-N-GFP indicate GFP inserted at their C and N terminus, respectively. Numbers indicate aa residues. Gray boxes represent predicted α-helical regions. (B) Time-lapse analysis of Bni5-C-GFP and Bni5-N-GFP accumulation at the budding site. Montages of GFP-tagged proteins with respect to Cdc3-mCherry were created from selected frames of time-lapse series taken with 1.5-min intervals. Arrowhead indicates the onset of Bni5-C-GFP accumulation. Strains used: YEF9336 (BNI5-C-GFP CDC3-mCherry), YEF10293 (BNI5-N-GFP CDC3-mCherry), YEF11750 (CDC10-GFP CDC3-mCherry), and YEF6108 (MYO1-GFP CDC3-mCherry). (C) Recruitment kinetics of GFP-tagged proteins during bud formation, quantified from cells shown in B. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. (D) Localization dynamics of Bni5-C-GFP and Bni5-N-GFP during cytokinesis. Montages were created as in B. The blue bar indicates the period of the septin HDR transition. Strains used: YEF9336 (BNI5-C-GFP CDC3-mCherry) and YEF10293 (BNI5-N-GFP CDC3-mCherry). (E) Localization kinetics Bni5-C-GFP and Bni5-N-GFP during cytokinesis, quantified from cells shown in D. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. The blue box indicates the period of the septin HDR transition. (F) FRAP analysis of Bni5-C-GFP and Bni5-N-GFP to assess turnover kinetics at the bud neck. Cells with a medium-sized bud and a short spindle were chosen for FRAP analysis. Top: Lines, symbols, and error bars represent recovery curves for indicated proteins, showing mean values and SD, respectively. n denotes the number of cells analyzed per strain. Bottom: Montages show selected frames from the time-lapse series acquired with 10-s intervals. Max and T1/2 indicate the estimated maximum amount of recovery and half-time of recovery. Strains used: YEF11624 (BNI5-C-GFP mScarlet-TUB1), YEF11546 (BNI5-N-GFP mScarlet-TUB1), YEF11753 (CDC10-GFP mScarlet-TUB1), and YEF11385 (MYO1-GFP mScarlet-TUB1).
Figure 1.

Distinct impact of N- and C-terminal GFP-tagging of Bni5 on its recruitment timing and turnover kinetics at the division site. (A) Diagram of GFP-tagged Bni5 constructs. Bni5-C-GFP and Bni5-N-GFP indicate GFP inserted at their C and N terminus, respectively. Numbers indicate aa residues. Gray boxes represent predicted α-helical regions. (B) Time-lapse analysis of Bni5-C-GFP and Bni5-N-GFP accumulation at the budding site. Montages of GFP-tagged proteins with respect to Cdc3-mCherry were created from selected frames of time-lapse series taken with 1.5-min intervals. Arrowhead indicates the onset of Bni5-C-GFP accumulation. Strains used: YEF9336 (BNI5-C-GFP CDC3-mCherry), YEF10293 (BNI5-N-GFP CDC3-mCherry), YEF11750 (CDC10-GFP CDC3-mCherry), and YEF6108 (MYO1-GFP CDC3-mCherry). (C) Recruitment kinetics of GFP-tagged proteins during bud formation, quantified from cells shown in B. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. (D) Localization dynamics of Bni5-C-GFP and Bni5-N-GFP during cytokinesis. Montages were created as in B. The blue bar indicates the period of the septin HDR transition. Strains used: YEF9336 (BNI5-C-GFP CDC3-mCherry) and YEF10293 (BNI5-N-GFP CDC3-mCherry). (E) Localization kinetics Bni5-C-GFP and Bni5-N-GFP during cytokinesis, quantified from cells shown in D. n denotes the number of cells analyzed per strain. Bold lines and shaded bands represent the mean and SD, respectively. The blue box indicates the period of the septin HDR transition. (F) FRAP analysis of Bni5-C-GFP and Bni5-N-GFP to assess turnover kinetics at the bud neck. Cells with a medium-sized bud and a short spindle were chosen for FRAP analysis. Top: Lines, symbols, and error bars represent recovery curves for indicated proteins, showing mean values and SD, respectively. n denotes the number of cells analyzed per strain. Bottom: Montages show selected frames from the time-lapse series acquired with 10-s intervals. Max and T1/2 indicate the estimated maximum amount of recovery and half-time of recovery. Strains used: YEF11624 (BNI5-C-GFP mScarlet-TUB1), YEF11546 (BNI5-N-GFP mScarlet-TUB1), YEF11753 (CDC10-GFP mScarlet-TUB1), and YEF11385 (MYO1-GFP mScarlet-TUB1).

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