Figure 6.
Figure 6. Localization of Sec3, Sla1, and Sst2 relative to the receptor in mating cells. (A and B) Representative time-lapse images. MATa cells coexpressing in situ–tagged Ste2-GFP and Sec3-RFP or Sla1-RFP were mixed with an equal number of MATα cells and imaged from cytokinesis to fusion. The fluorescent images show the localization of Ste2-GFP and Sec3-RFP (A) or Sla1-RFP (B) as the MATa cell orients toward its mating partner. During tracking, closed arrowheads indicate the leading Ste2-GFP and Sec3-RFP peaks and the lagging Sla1-RFP peak. After stabilization, the filled arrowheads indicate the center Ste2-GFP and Sec3-RFP peaks and the flanking Sla1-RFP peaks. The plots show the distribution on the PM of Ste2-GFP (green) and Sec3-RFP (red) or Sla1-RFP (red), respectively, at the indicated time points. The dashed blue, green, and red lines mark the DS peak, leading peak, and CS peak, respectively. (C–F) Average PM distribution of Ste2-GFP and Sec3-RFP or Sla1-RFP in mating cells during tracking and at the prezygote stage. The PM signals of 30 cells two time points before shmoo emergence (tracking) and one time point before fusion (prezygote) were quantified with ImageJ, normalized for cell size, and averaged as described in Materials and methods. The plots show the mean signal distribution ± SEM (light shadow) of Ste2-GFP (green) and Sec3-RFP (red) or Sla1-RFP (red). The dashed green lines mark the leading receptor peak and the shmoo tip during tracking and in prezygote; the dashed red lines mark the peaks of Sla1-RFP. Average distributions of Ste2-GFP and Sec3-RFP during tracking (C), Ste2-GFP and Sla1-RFP during tracking (D), Ste2-GFP and Sec3-RFP in prezygotes (E), and Ste2-GFP and Sla1-RFP in prezygotes (F). (G and H) Sst2 surrounds the growth site after shmoo formation. Time-lapse images generated for the experiment represented in Fig. 2 were used to determine the distribution of Sst2-GFP in MATa cells at the prezygote stage. (G) Representative DIC and fluorescent images of a shmooing MATa Sst2-GFP cell and corresponding quantification of the PM signal. The filled yellow arrowheads and dashed red lines indicate the peaks of the Sst2-GFP PM signal; the closed green arrowhead and dashed green line indicate the shmoo tip. (H) Average distribution of Sst2-GFP during shmoo formation. The plot shows the average distribution of Sst2-GFP in 21 cells. The dashed green lines mark the shmoo tip; the dashed red lines mark the Sst2-GFP peaks. F.I., fluorescence intensity.

Localization of Sec3, Sla1, and Sst2 relative to the receptor in mating cells. (A and B) Representative time-lapse images. MATa cells coexpressing in situ–tagged Ste2-GFP and Sec3-RFP or Sla1-RFP were mixed with an equal number of MATα cells and imaged from cytokinesis to fusion. The fluorescent images show the localization of Ste2-GFP and Sec3-RFP (A) or Sla1-RFP (B) as the MATa cell orients toward its mating partner. During tracking, closed arrowheads indicate the leading Ste2-GFP and Sec3-RFP peaks and the lagging Sla1-RFP peak. After stabilization, the filled arrowheads indicate the center Ste2-GFP and Sec3-RFP peaks and the flanking Sla1-RFP peaks. The plots show the distribution on the PM of Ste2-GFP (green) and Sec3-RFP (red) or Sla1-RFP (red), respectively, at the indicated time points. The dashed blue, green, and red lines mark the DS peak, leading peak, and CS peak, respectively. (C–F) Average PM distribution of Ste2-GFP and Sec3-RFP or Sla1-RFP in mating cells during tracking and at the prezygote stage. The PM signals of 30 cells two time points before shmoo emergence (tracking) and one time point before fusion (prezygote) were quantified with ImageJ, normalized for cell size, and averaged as described in Materials and methods. The plots show the mean signal distribution ± SEM (light shadow) of Ste2-GFP (green) and Sec3-RFP (red) or Sla1-RFP (red). The dashed green lines mark the leading receptor peak and the shmoo tip during tracking and in prezygote; the dashed red lines mark the peaks of Sla1-RFP. Average distributions of Ste2-GFP and Sec3-RFP during tracking (C), Ste2-GFP and Sla1-RFP during tracking (D), Ste2-GFP and Sec3-RFP in prezygotes (E), and Ste2-GFP and Sla1-RFP in prezygotes (F). (G and H) Sst2 surrounds the growth site after shmoo formation. Time-lapse images generated for the experiment represented in Fig. 2 were used to determine the distribution of Sst2-GFP in MATa cells at the prezygote stage. (G) Representative DIC and fluorescent images of a shmooing MATa Sst2-GFP cell and corresponding quantification of the PM signal. The filled yellow arrowheads and dashed red lines indicate the peaks of the Sst2-GFP PM signal; the closed green arrowhead and dashed green line indicate the shmoo tip. (H) Average distribution of Sst2-GFP during shmoo formation. The plot shows the average distribution of Sst2-GFP in 21 cells. The dashed green lines mark the shmoo tip; the dashed red lines mark the Sst2-GFP peaks. F.I., fluorescence intensity.

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