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. 2015 Jul 17;4(8):1040–1051. doi: 10.1242/bio.012609

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© 2015. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 2. — Morphology and cytoskeleton organization of cdc8^wt, cdc8^R121A, cdc8^D131A, cdc8^E138A. (A) Fixed wildtype (SH30) and mutant cells stained with Calcofluor to show septum and septal material and DAPI to show nuclei. The cdc8^R121A cells (SH39) had disorganized septa and abnormal cell shapes. Cells with up to four nuclei were observed. cdc8^D131A (SH43) and cdc8^E138A (SH34) appeared normal. (B) Staining of the actin cytoskeleton using Alexa-phalloidin (green), and nuclei with DAPI (blue) showed dispersed patches, the absence of actin cables and poorly condensed actin rings in cdc8^R121A. The other strains appeared normal. (C) cdc8^wt-rlc1-mCherry (SH49) and cdc8^R121A-rlc1-mCherry (SH52) cells fixed and stained with Alexa-phalloidin (green) to visualize the actin cytoskeleton. m-Cherry (red) shows localization of myosin II in the contractile ring in cdc8^wt. In cdc8^R121A-rlc1-mCherry myosin II assembles into contractile rings despite the absence of organized actin in contractile rings or cables. For comparison, in cdc8-27-rlc1-mCherry (SH81), a temperature-sensitive mutant, neither the actin (Alexa phalloidin) nor myosin II (rlc1-mCherry) assembled into a contractile ring at the restrictive temperature (35°C). Other cells were grown and fixed at 30°C. (D) Formin-GFP (Cdc12p-GFP) fluoresence. Formin localized in rings in wildtype (SH57) but, like Rlc1p-mCherry, formin (Cdc12p) assembled into abnormal rings in cdc8^R121A (SH60) (live cells). No midline localization was observed in a temperature sensitive strain (SH87) at the restrictive temperature (fixed cells at 35°C). (E) Fimbrin-GFP fluorescence (live cells confocal image). Fim1p-GFP localized in the midline region in cdc8^wt (SH77) and cdc8^R121A cells (SH75) (live cells), but not in the temperature-sensitive strain (SH83) at the restrictive temperature (cells grown and fixed at 35°C). The localization of fluorescent proteins was normal in cdc8^D131A and cdc8^E138A strains (supplementary material Fig. S3).