Extended Data Fig. 2: Use of the kinetochore protein Fta3 as a standard for ratiometric determination of ESCRT protein copy number at the NE.

a, Fluorescence micrographs of representative mixture of a GFP–ESCRT strain (Ist1–GFP; MKSP3365) and one expressing Fta3–GFP (MKSP3186). Overview micrograph: top left, strain expressing Ist1–GFP (green), mCherry–Atb2 (magenta), and Sad1–mCherry (magenta); bottom right, Fta3–GFP strain (green). Dotted white lines indicate the cell outline. The blue and orange boxes in the overview micrograph outline the ROIs magnified to the right of image as single channel images for Ist1–GFP and Fta3–GFP, as indicated. b, Fluorescence micrographs of representative Fta3–GFP expressing cells (MKSP3186) at indicated cell-cycle stage (top). The black boxes in overview micrographs outline the ROI magnified below. Schematic representation of Fta3–GFP appearance in the different cell-cycle stages (bottom). Number of molecules based on Lawrimore and colleagues²⁷. c, x–y plot of Fta3–GFP focus fluorescence intensity against the expected number of molecules per focus in MKSP3186 based on Lawrimore and colleagues²⁷. Points represent median of all values collected for that stage of the cell cycle (see b); error bars represent the 95% confidence interval. The red line represents a simple linear regression with the R² shown; n = 366 foci across three biological replicates with at least 50 foci per replicate. Scale bars, 1 µm (overview) and 250 nm (ROI). Source numerical data are provided.

Source data