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First published online 18 December 2007
doi: 10.1242/jcs.014449

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Cdc42 and the Ste20-like kinase Don3 act independently in triggering cytokinesis in Ustilago maydis

Christian Böhmer, Maik Böhmer^*, Michael Bölker and Björn Sandrock

Department of Biology, Philipps-University Marburg, Karl-von-Frisch-Str. 8, 35032 Marburg, Germany

View larger version (31K): [in this window] [in a new window]   Fig. 1. The analogue-sensitive Don3M157A kinase is active in vivo. (A) Alignment of the ATP-binding pocket of the U. maydis (Um) Don3 kinase domain with corresponding sequences of other yeast (Sc) and human (Hs) kinases, which have been used to create analogue-sensitive kinases. The gatekeeper amino acids (positions 140 and 157) are bold and the position of the amino acid exchange is indicated. (B) Pcrg::don3 and Pcrg::don3M157G/A expression cassettes. The kinase domain (KD) is indicated, and the site of the amino acid exchange is marked by an asterisk. (C) don3 cells expressing Don3, Don3M157G and Don3M157A under control of the inducible Pcrg-promoter. DIC-images illustrate the repressed state (glucose) and the induced state (arabinose). Scale bars, 10 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Inhibition of don3-as cells with NA-PP1 results in a reversible cytokinesis arrest. (A) In the presence of 1 µM NA-PP1 don3-as cells form large cell clusters. Scale bar, 10 µm. (B) don3-as cells were labelled with NHC-LS-biotin and the activity of Don3-as was inhibited with 1 µM NA-PP1. After 5 hours cells were washed and further incubated without inhibitor. Fluorescence and DIC images were taken at the time-points indicated. Scale bars, 10 µm. Diagrams indicate the abundance of single budding cells (1-2 cells) and cell clusters consisting of three or more cells in percent (n=200) (>3 cells).

View larger version (32K): [in this window] [in a new window]   Fig. 3. Don3 and Cdc42 act independently to trigger secondary septum initiation. (A) Strain CB46 (don3-as; Pcrg::cdc42) was grown in glucose-containing medium, in which the Pcrg promoter is inactive (Cdc42, no activity). The cell clusters were washed and labelled with biotin. At the same time (0 h) Don3 activity was blocked by adding 1 µM NA-PP1 to the medium (Don3, no activity). Cdc42 expression was induced after 90 minutes by switching the carbon source of the NA-PP1-containing medium from glucose to arabinose (Cdc42, increasing activity). The fate of the cell clusters were monitored at the given time-points using fluorescence and DIC microscopy. Scale bars, 10 µm. (B) GFP-fusion of Cdc42 (upper panel) or Don3 (lower panel) expressed from the constitutive Etef-promotor in wild type cells (left panel), in the respective complementary deletion strain (central panel) and in don1 cells (right panel). Boxed areas are magnified, and arrowheads mark the localisation of GFP-Cdc42 at both septa in the wild-type or the primary septum in the mutant strains. Scale bars, 10 µm.

View larger version (57K): [in this window] [in a new window]   Fig. 4. Don3 and Cdc42 activities are required for assembly of the cytokinetic contractile actomyosin ring. (A-O) don3-as cells expressing Cdc15-GFP (A-E) were stained with 10 µg/ml Calcofluor White (F-J). Merged images of the septation zone are shown as magnifications (K-O), GFP (green) and Calcofluor White (red). Different stages of actomyosin ring assembly and septum formation are shown. (A,F,K) Cdc15-GFP assembles at the neck of the mother bud. (B,G,L) Cdc15-GFP accumulates in the actomyosin ring during primary septum formation. (C,H,M) After completion of the first septum Cdc15-GFP disappeared. (D,I,N) A second actomyosin ring is visible before secondary septum intitiation. (E,J,O) During secondary septum formation Cdc15-GFP colocalises with the inward growing septum. (P-T) don3-as cells expressing Cdc15-GFP were grown in the presence of 1 µM NA-PP1 and released from inhibition after 8 hours. (P,S) In the presence of the inhibitor, Cdc15-GFP was detectable only in the primary septa of the newly formed buds (arrows). (Q,T) Upon inhibitor release, Cdc15-GFP accumulation in all cells at the site where the secondary septum was formed (arrow heads in the magnification). (R,U) Cdc15-GFP expressed in Pcrg::cdc42 cells. Under repressing conditions Cdc15-GFP behaves similar as shown in panel M. After shifting the cells to arabinose-containing medium cells start to build the secondary septum. In cells whose the second septum is stained with Calcofluor-White (U), Cdc15-GFP can be observed there, too (R) (arrowheads in the magnification).