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. 2011 Nov 1;22(21):3986–3994. doi: 10.1091/mbc.E11-04-0379

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© 2011 Peng 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 4: — JLY arrests actin dynamics in other mammalian cells. We used FRAP to analyze the ability of JLY to arrest actin depolymerization, polymerization, and rearrangements in YFP-actin expressing (A–C) HT1080 and (D–F) NIH 3T3 cells. Cells imaged in oblique fluorescence illumination were first pretreated with JLY, then photobleached in a rectangular region. (B and E) FRAP is a measure of actin assembly, and the fluorescence intensity of the nonbleached region estimates depolymerization. (C and F) The persistence of the bleached area can assay large-scale actin rearrangements. (B) In HT1080 cells, 11% new actin polymerization (blue box) and 5% depolymerization (red box) occurred in the 10 min following JLY treatment (n = 20). (C) The line profile (green line) between unbleached and bleached regions indicates no detectable actin rearrangements in the 10 min after photobleaching for JLY-treated HT1080 cells. (E) NIH 3T3 cells display 8% recovery (blue box) in the 10 min after photobleaching (n = 14). NIH 3T3 cells show 5% loss of filamentous actin signal over 10 min (n = 14), as evaluated by the loss of fluorescence intensity of the unbleached region of filamentous actin (red box). (F) A line profile (green line) of the borders between unbleached and bleached regions indicates no detectable actin rearrangements in the 10 min after photobleaching for JLY-treated NIH 3T3 cells. Error bars show SEM. Scale bar: 10 μm.