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. 2017 Aug 15;28(17):2251–2259. doi: 10.1091/mbc.E17-04-0238

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© 2017 Lacy et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Single-molecule recovery after photobleaching of SNAP-tag labeled Pil1p. Cells expressing Pil1p-SNAP were labeled with SNAP-SiR647 at 0.5 µM for 15 h, washed, and imaged in TIRF. (A) Average intensity projection of the first 5 frames (0.5 s) of a movie reveals linear eisosomes. (B) Maximum intensity projection of frames 50–200 (5–20 s) of the same movie shows single-molecule recovery events. Orange lines i–iv are the line traces used for the kymographs in E. (C) Example intensity traces of SRAP spots show stepwise photobleaching and single-molecule recovery of Pil1p-SiR. (D) The positions of single-molecule recovery events (SRAP spots, magenta) are mapped on the visible eisosomes. (E) Kymographs of line traces along eisosomes (i–iv as labeled in A and B), with bars indicating the time spans for the projection images. (F) Cells expressing Pil1p-mEGFP imaged in TIRF are similar in appearance to SNAP-tag–labeled cells in A. (G) Comparison of eisosome lengths measured in cells with Pil1p-mEGFP (black, 1250 ± 650 nm for 304 eisosomes) or labeled Pil1p-SiR (gray, 1240 ± 580 nm for 275 eisosomes) show no significant difference by two-sample Kolmogorov–Smirnov test (p = 0.33). (H) Comparison of number of eisosome objects visible in cells expressing Pil1p-mEGFP (black, 15.7 ± 2.2) or labeled Pil1p-SiR (gray, 14.8 ± 2.7) shows no significant difference by two-sample Kolmogorov–Smirnov test (p = 0.89). Scale bar, 5 µm (A, B, D, F). Cell outlines are drawn in orange dashed lines. Mean ± SD reported for at least 16 cells in each measurement.