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. 2019 Apr 3;8:e45284. doi: 10.7554/eLife.45284

Figure 4. Heh1 is required to induce the focal accumulation of Chm7.

(A) Schematic of ‘Fluorescence ESCRT Targeting and Activation Assay’ where Heh2 is expressed as a fusion to GFP-binding protein (GBP; red). Chm7-GFP is expressed by the addition of galactose to the growth medium. The focal accumulation of Heh2-GBP-mCherry is interpreted as Chm7 activation. (B) Deconvolved fluorescence micrographs of Chm7-GFP and Heh2-GBP-mCherry at the indicated timepoints after addition of galactose to the growth medium. Scale bar is 5 μm. (C) As a metric for the clustering of Heh2-GBP-mCherry, a coefficient of variation (CV) of the Heh2-GBP-mCherry fluorescence along the nuclear envelope in a mid-section was calculated over time. Mean and SD normalized to 0 timepoint are shown. n = 25.

Figure 4.

Figure 4—figure supplement 1. Heh1 is required to induce the focal accumulation of Chm7.

Figure 4—figure supplement 1.

(A) Overexpression of Chm7-GFP does not impact the distribution of Heh2-mCherry. Deconvolved fluorescence micrographs of Heh2-mCherry and Chm7-GFP under conditions where Chm7-GFP was produced (galactose) or repressed (glucose). Merge of green and red fluorescence also shown. Asterisks denote vacuolar autofluorescence. Scale bar 5 μm. (B) Plot of the CV of Heh2-mCherry fluorescence along the nuclear envelope from images and conditions in (A). Error bars represent SD of the mean from three independent replicates of >50 cells. P-values from Student’s t-test where ns is p≥0.05. (C) Overexpression of Chm7-GFP in the context of Heh2-GBP leads to the accumulation of Heh1-mCherry in Chm7-GFP foci at the nuclear envelope. Deconvolved fluorescence micrographs of Heh1-mCherry and Chm7-GFP (in a strain also expressing Heh2-GBP) under conditions where Chm7-GFP is produced (galactose) or repressed (glucose). Merge of green and red fluorescence also shown. Asterisks denote vacuolar autofluorescence. Scale bar 5 μm.