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Fig. 5. Modulation of the actin cytoskeleton in response to osmotic stress. (A-J) Wild-type cells were grown as described in Fig. 1A, fixed and stained with rhodamine-phalloidin to localize F-actin (B,C,E,F,H,I) or DAPI and Calcofluor to localize chromatin and septa/SICS, respectively (A,D,G), at the indicated time points after the addition of sorbitol to a final concentration of 1.2 M. Captured images are presented either unprocessed (B,E,H) or with identical degrees of image processing applied (C,F,I) such that the signal in the processed images of stressed cells (F,I) was similar to that seen in the unstressed, unprocessed image (B). Specifically, using the levels function in Adobe Photoshop CS2, the brightest pixel in the unprocessed image shown in H was mapped to the pure-white level. This adjustment was saved and then applied to the unprocessed images shown in B and E. A contrast adjustment of +20 was then applied to each image. This two-step processing resulted in the images shown in C, F and I. (I) Asterisks highlight cells with actin patches that were relocalized to cell tips. (J) Quantification of the appearance of cells with SICS at cell tips and the appearance of cells with actin delocalized from tips. Scale bar: 5 µm.
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