Fig. 4. Actin-filament disassembly is required for formation of oriented actin-filament bundles and cell polarization. Cells infected and expressing GFP-actin (A-C) were imaged live by spinning-disc confocal time-lapse microscopy during normal cell polarization (after Lat A washout; A); or Lat A was washed off, cells left in culture medium for 15 minutes, then 0.5 µm jasplakinolide added and cells left in the continued presence of jasplakinolide (B,C); or Lat A was washed off uninfected cells, then 0.5 µm jasplakinolide added 15 minutes later and cells left in the continued presence of jasplakinolide then fixed in a time-course assay and stained for actin (D,E). (A-C) Time is hours:minutes:seconds and indicates total time after washout of Lat A. (A) As expected, in the control, actin initially forms a meshwork (by 0:12:00) then forms oriented actin-filament bundles (by 0:17:00). Stable retraction of the cell edge occurs (by 0:27:00) and the cell is fully polarized and migrating by 0:58:40. (B) In the jasplakinolide-treated cell, at 0:19:00 (19 minutes after Lat A washout), jasplakinolide has been present for the last 4 (0:15:00-0:19:00) minutes and, at 0:55:30, for the last 40 minutes, 30 seconds (0:15:00-0:40:30). Both formation of oriented actin-filament bundles and cell polarization fails. Note that delocalized protrusions still occur in the presence of jasplakinolide, as expected and similar to non-polarized cells (Cramer, 1999b) (see text for further details). (C, 1:07:30, last 0:52:30 in jasplakinolide) Prolonged treatment with 0.5 µm jasplakinolide causes formation of actin aggregates and disrupted bundles, as documented previously (see text). Note that this occurs well after the specific block in formation of oriented actin bundles, shown in B and D. (D, jasplakinolide added at 15 minutes after Lat A washout) Quantification of formation of oriented actin-filament bundles in cells fixed and stained for actin. (E, jasplakinolide added at 15 minutes after Lat A washout) Quantification of polarized cells in cells fixed at 30 and 60 minutes after washout of Lat A. Note the failure of jasplakinolide-treated cells to form oriented actin-filament bundles (D) and to polarize (E) when compared to the control cells. Graphs are expressed as mean ± s.e.m. for three independent experiments (n=600 cells counted for each condition). Bar, 10 µm.