Figure 2. Determining the maximum number of fluorescently-tagged endocytic proteins at endocytic sties by ratiometric comparison of fluorescence intensities to the intensity of 120-sfGFP-tagged nanocages.

(A) Comparison of ratios of maximum protein levels at endocytic sites for the two yeasts in this study (green) vs in previous studies (gray). (B) The mean fluorescence intensity of 120-sfGFP- tagged nanocages (n = 367) is linearly proportional to exposure time. Images obtained for indicated exposure times are presented at the top of the graph. (C) Ratiometric comparison between the fluorescence intensity of 120-sfGFP-tagged nanocages (n = 305) and maximum intensity of scSla1-sfGFP at endocytic sites (n = 58). Left panel, image of 120-sfGFP-tagged nanocages. Right panel, single frame from a processed movie of scSLA1-sfGFP cells. The nanocage image and the scSLA1-sfGFP movie has been imaged using the same imaging system and the images were processed and analyzed using the same particle tracking parameters. Red circles indicate the sites automatically detected by the tracking program. The intensity (middle) and molecular numbers (bottom) were determined and plotted. (D) Maximum molecular number of scMyo5 (n = 210), scSla1 (n = 152) and scLas17 (n = 112) at endocytic sites as well as the molecular number of scCse4 (n = 17) on the kinetochore clusters. The molecular numbers were calculated by the ratiometric fluorescence intensity comparison of the indicated sfGFP-tagged proteins and 120-sfGFP-tagged nanocages. (E-G) Maximum molecular numbers for indicated proteins at endocytic sites in budding (blue) and fission (brown) yeast. The molecular numbers were calculated by the ratiometric fluorescence intensity comparisons using scSla1, scLas17, or scMyo5 as standards (Figure 2—figure supplement 2, Table 2). For each indicated protein, at least 50 endocytic sites were examined. The scale bars on the images are 2 µm.

Figure 2—figure supplement 1. Quantitative comparison of 120-sfGFP-tagged nanocages prepared on different days.

120-sfGFP-tagged nanocages were prepared and imaged three different days using the same method and conditions. The fluorescence intensities of the 120-sfGFP-tagged nanocages were measured and compared. Scale bars are 2 µm.

Figure 2—figure supplement 2. Quantitative comparison of the maximum number of homologous proteins at endocytic sites in budding and fission yeast.

(A-I) Cells expressing indicated proteins were mixed and then imaged over time. The resulting movies were analyzed using a particle tracking program. The maximum fluorescence intensities were measured and compared for the indicated protein pairs.

Figure 2—figure supplement 3. Determining the maximum number of Myo3-GFP molecules using Sla1-GFP and Las17-GFP as standards.

(A) Determining the maximum number of Myo3-GFP molecules using Las17-GFP as a standard. Two-color image of MYO3-GFP ABP1-RFP cells and LAS17-GFP cells (left panel). Single frame of a movie of MYO3-GFP ABP1-RFP cells and LAS17-GFP cells simultaneously imaged in the GFP channel (right panel). The dynamics of GFP-labeled protein patches were analyzed using the Particle Tracker plugin in ImageJ software and the maximum fluorescence intensity was measured and compared between the indicated proteins (C). (B) Determining the maximum protein numbers of scMyo3-GFP using scMyo5-GFP molecules as a standard. Two-color image of MYO5-GFP ABP1-RFP cells and MYO3-GFP cells (left panel). Single frame from a movie of MYO5-GFP ABP1-RFP and MYO3-GFP cells imaged simultaneously in the GFP channel (right panel). The dynamics of GFP-labeled protein patches were analyzed using the Particle Tracker plugin in ImageJ software and the maximum fluorescence intensity was measured and compared between the indicated proteins (C). (C) The molecular numbers were calculated by ratiometric fluorescence intensity comparison using data acquired in A and B. The asterisks represent LAS17-GFP cells in (A) and represent MYO3-GFP cells in (B). Scale bars are 2 µm.