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. 2020 Nov 12;9:e60419. doi: 10.7554/eLife.60419

Figure 1. Wsp1 plays a role in the initiation of endocytic actin patches.

(A) Equatorial plane images of Fim1-GFP in Schizosaccharomyces pombe cells taken using spinning disk confocal microscopy. Scale Bar: 5 µm. (B) Plot showing the relative Fim1-GFP intensity in S. pombe mutant endocytic patches over their lifetimes. Traces represent the average of 16–18 endocytic patches. Intensity plots were aligned based on their peak values and normalized based on the peak value of the Fim1-GFP signal in the wild-type strain. Standard deviation is shown as shaded region around each trace. (C) Plot comparing the assembly time of Fim1-GFP in endocytic patches in wild-type cells to wsp1∆CA, dip1∆, and wsp1∆CA dip1∆ mutants. Error bars: standard error from 16 to 18 patches. (D) Plot comparing the endocytic patch initiation rate in wild-type cells to wsp1∆CA, dip1∆, and wsp1∆CA dip1∆ mutants. Error bars: standard error from 10 cells. (E) Plot comparing the endocytic actin patch density in wild-type cells to wsp1∆CA, dip1∆, and wsp1∆CA dip1∆ mutants as determined based on the number of Fim1-GFP-marked cortical puncta. Error bars: standard error from 10 cells. (F) Plot showing the percentage of endocytic patches internalized in wild-type and mutant S. pombe cells. Error bars: standard error from 10 to 22 cells. p-values: *<0.05, **<0.01, ***<0.001.

Figure 1—source data 1. Analysis of spinning disk confocal videos of S. pombe source data.

Figure 1.

Figure 1—figure supplement 1. Nucleation promoting factor (NPF) mutations do not deplete the pool of soluble actin monomers in the cytoplasm.

Figure 1—figure supplement 1.

(A) Central confocal slices of wild-type and mutant strains expressing GFP-actin via the arp3 promoter at the leu1 locus. Scale bar: 2 µm. (B) Quantification of the mean cytoplasmic intensity of GFP-actin in wild-type and mutant strains in a central confocal slice. The contribution of autofluorescence was eliminated by subtracting the cytoplasmic signal from an image of an untagged wild-type strain collected with the same parameters. (C) Quantification of the mean total cell intensity of GFP-actin for wild-type and mutant strains in a central confocal slice, corrected as described in B. (D) Quantification of the ratio of total fluorescence intensity of GFP-actin in patches versus the total GFP-actin intensity in the cell. In all panels, error bars show the standard deviation from the mean. p-values: **<0.01, ***<0.001.
Figure 1—video 1. Spinning disk confocal microscopy video of Fim1-GFP in wild-type Schizosaccharomyces pombe cells.
Download video file (959.9KB, mp4)
Images of the cell medial plane were taken once every second and are displayed here at 10× collection speed (related to Figure 1). Scale bar: 5 µM. 10 fps.
Figure 1—video 2. Spinning disk confocal microscopy video of Fim1-GFP in dip1∆ Schizosaccharomyces pombe cells.
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Images of the cell medial plane were taken once every second and are displayed here at 10× collection speed (related to Figure 1). Scale bar: 5 µM. 10 fps.
Figure 1—video 3. Spinning disk confocal microscopy video of Fim1-GFP in wsp1∆CA Schizosaccharomyces pombe cells.
Download video file (989.8KB, mp4)
Images of the cell medial plane were taken once every second and are displayed here at 10× collection speed (related to Figure 1). Scale bar: 5 µM. 10 fps.
Figure 1—video 4. Spinning disk confocal microscopy video of Fim1-GFP in dip1∆ wsp1∆CA Schizosaccharomyces pombe cells.
Download video file (1,006.5KB, mp4)
Images of the cell medial plane were taken once every second and are displayed here at 10× collection speed (related to Figure 1). Scale bar: 5 µM. 10 fps.