Figure 3.
Figure 3. Kinetics of polarization to the DS and the initiation of tracking. (A) The time to tracking is the sum of the time it takes to detect a reporter at the DS following cytokinesis (PE) and the duration of its pause. (B) Use of Spa2-RFP as a marker for cytokinesis. MATa cells expressing in situ–tagged Spa2-RFP and GFP-Cdc3 were imaged in mating mixtures. Immediately after cytokinesis, marked by the splitting of the septin ring (filled cyan arrowhead), Spa2-RFP translocates from the bud neck to the DS (filled red arrowhead). (C–F) Representative time-lapse images. MATa cells expressing reporter genes tagged with GFP in situ were mixed with an equal number of MATα cells and imaged from cytokinesis to the initiation of tracking. The fluorescent images show the localization of each reporter as the MATa cells orient toward their mating partners. Unless indicated, the images were not deconvolved. The blue asterisk indicates reporter localization to the bud neck; dashed arrowheads indicate PE at the DS and mark the signal peak; closed arrowheads indicate redistribution (tracking) and mark the leading peak. The plots show the distribution of each reporter on the PM at the indicated time points (10-point rolling average). The dashed blue and green lines mark the DS peak and the tracking peak, respectively. Localization of Spa2-RFP and Ste2-GFP (C), GFP-Gβ (D), Far1-GFP (E), and Sst2-GFP (F; deconvolved). (G–I) Box scatterplots showing the distribution of the indicated measurements. n ≥ 50 for all strains and measurements; **, P < 0.0001; *, P < 0.002. (G) Distribution of PE values for the indicated reporters. Mean PE ± SEM in minutes: Far1-GFP = −1.9 ± 0.6; GFP-Gβ = 2.4 ± 0.4; Ste2-GFP = 6.1 ± 0.6; Sst2-GFP = 10.0 ± 0.5. (H) Distribution of Pause values for the indicated reporters. Mean Pause ± SEM in minutes: Far1-GFP = 13.5 ± 0.7; GFP-Gβ = 13.3 ± 0.8; Ste2-GFP = 9.2 ± 0.6; Sst2-GFP = 2.6 ± 0.4. (I) Distribution of times to tracking for the indicated reporters. Mean Times to tracking ± SEM in minutes: Far1-GFP = 11.6 ± 0.9; GFP-Gβ = 15.6 ± 0.8; Ste2-GFP = 15.3 ± 0.7; Sst2-GFP = 12.5 ± 0.5. (J) Signal intensity at the DS during pause. Mean intensity ± SEM, n = 25 for both reporters. F.I., fluorescence intensity.

Kinetics of polarization to the DS and the initiation of tracking. (A) The time to tracking is the sum of the time it takes to detect a reporter at the DS following cytokinesis (PE) and the duration of its pause. (B) Use of Spa2-RFP as a marker for cytokinesis. MATa cells expressing in situ–tagged Spa2-RFP and GFP-Cdc3 were imaged in mating mixtures. Immediately after cytokinesis, marked by the splitting of the septin ring (filled cyan arrowhead), Spa2-RFP translocates from the bud neck to the DS (filled red arrowhead). (C–F) Representative time-lapse images. MATa cells expressing reporter genes tagged with GFP in situ were mixed with an equal number of MATα cells and imaged from cytokinesis to the initiation of tracking. The fluorescent images show the localization of each reporter as the MATa cells orient toward their mating partners. Unless indicated, the images were not deconvolved. The blue asterisk indicates reporter localization to the bud neck; dashed arrowheads indicate PE at the DS and mark the signal peak; closed arrowheads indicate redistribution (tracking) and mark the leading peak. The plots show the distribution of each reporter on the PM at the indicated time points (10-point rolling average). The dashed blue and green lines mark the DS peak and the tracking peak, respectively. Localization of Spa2-RFP and Ste2-GFP (C), GFP-Gβ (D), Far1-GFP (E), and Sst2-GFP (F; deconvolved). (G–I) Box scatterplots showing the distribution of the indicated measurements. n ≥ 50 for all strains and measurements; **, P < 0.0001; *, P < 0.002. (G) Distribution of PE values for the indicated reporters. Mean PE ± SEM in minutes: Far1-GFP = −1.9 ± 0.6; GFP-Gβ = 2.4 ± 0.4; Ste2-GFP = 6.1 ± 0.6; Sst2-GFP = 10.0 ± 0.5. (H) Distribution of Pause values for the indicated reporters. Mean Pause ± SEM in minutes: Far1-GFP = 13.5 ± 0.7; GFP-Gβ = 13.3 ± 0.8; Ste2-GFP = 9.2 ± 0.6; Sst2-GFP = 2.6 ± 0.4. (I) Distribution of times to tracking for the indicated reporters. Mean Times to tracking ± SEM in minutes: Far1-GFP = 11.6 ± 0.9; GFP-Gβ = 15.6 ± 0.8; Ste2-GFP = 15.3 ± 0.7; Sst2-GFP = 12.5 ± 0.5. (J) Signal intensity at the DS during pause. Mean intensity ± SEM, n = 25 for both reporters. F.I., fluorescence intensity.

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