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Fig. 2. Two main types of actin organization are detectable in cells during cell polarization. Cells were treated with 1 µm Lat A for 1 hour (A), then washed in medium (B-E) (see Materials and Methods) to initiate cell polarization, then fixed at various time points and stained with Alexa-Fluor-594-conjugated phalloidin to visualize actin organization. (A) Rounded-up cell, after 1 hour of Lat A treatment, prior to initiation of cell polarization with actin aggregates (note that no visible actin-filament bundles are present). (B,B') Re-spread cells early in cell polarization: actin is organized as a non-oriented actin-filament meshwork (B) or non-oriented actin-filament bundles (B'). Red arrows (B') indicate different directions of unaligned filament bundles. (C,C') Re-spread cells later in cell polarization: actin is organized as oriented actin-filament bundles. Bundles are more than 70% (C) to 100% (C') aligned with each other (red arrow, C') ±0-30°, as in fully polarized and moving cells (Cramer et al., 1997) (and see E). (D) The cell edge is retracted (arrow) and actin remains organized as oriented actin-filament bundles. (E, fully polarized cell) Actin-filament bundles within the cell body are oriented in the direction of migration. (F) Quantitative analysis of actin organization in fixed and stained polarizing cells at fixed time points during the cell-polarization assay (0-60 minutes after Lat A washout). (G) Stacked histogram illustrating actin organization in fixed cells as a function of cell shape during cell polarization. Graphs (F,G) are expressed as mean ± s.e.m. (n=600 cells per time point, triplicate experiments). Bar, 10 µm.
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