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First published online June 25, 2007
doi: 10.1242/10.1242/jcs.03464

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Microtubules offset growth site from the cell centre in fission yeast

¹ Cancer Research UK, Cell Cycle Lab, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK
² Oxford Centre for Integrative Systems Biology, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK
³ Rockefeller University, 1230 York Avenue, New York, NY 10021, USA

View larger version (34K): [in this window] [in a new window]   Fig. 1. Activation of new growth zones in bipolar G2 cells. (A) Branching cells during cdc25-22 block. Cells were blocked at 36.5°C for 1, 2 and 3 hours, prior to MBC/DMSO addition (length of block prior to MBC/DMSO addition shown in parentheses in the graph). Black arrows indicate time of drug treatment. The percentage of branched cells is an average of three experiments (unless otherwise stated) with s.e.m. values smaller than 5%. 100-200 cells were counted at each time point. (B) Brightfield images of blocked (3 hours) cells treated for 3 hours with DMSO (top left) or with 50 µg/ml MBC (top right and lower panels). Staining with TAT-1 antibody of MBC-treated cells, treated as above (bottom). (C) Cdc25-22 blocked (3 hours) cells treated with DMSO (left), MBC for 1 hour (middle) and for 3 hours (right), stained for actin (upper panel), with DAPI (middle panel) and with calcofluor (lower panel). (D) Frames from movie of cdc25-22 cdc13-YFP strains blocked at 36.5°C for 3 hours and then treated with MBC at t=0, at start of time-lapse. Frames were taken every 10 minutes, using an Axioplan Zeiss microscope. Bars, 5 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Activation of new growth zones in G1- and S phase-arrested cells. (A) Images of cdc10-129 cdc11-119 cells at 36.5°C, after treatment with 50 µg/ml MBC for 3 hours: Nomarski (top) and DAPI stained (bottom). (B) Scoring of branching cdc10-129 and cdc10-129 cdc11-119 cells. Both cdc10-129 and cdc10-129 cdc11-119 cells were blocked for 3 hours before MBC treatment. Black arrow indicates drug addition. (C) Scoring of branching cdc25-22 cells. Cells were blocked for 3 hours at the restrictive temperature to block cells in G2 prior to mitosis. After 3 hours cells were released to the permissive temperature (25°C) to allow mitosis in the presence of 12 mM HU to prevent S phase to proceed. After 1.5 hours DMSO/MBC was added to the media. The effect of temperature and HU treatment was controlled using a wild-type strain. Both treatments had no effect on wild-type cells (data not shown). (D) Scoring of monopolar and bipolar cells in cdc25-22 cells blocked at 36.5°C and then released to 25°C in 12 mM HU. Bars, 5 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 3. Nuclei determine position of branches. (A) Quantification of distance between nucleus and branch in individual cdc25-22-blocked cells 4 hours after centrifugation in the presence of MBC (black) or without centrifugation (grey). 82 cells were measured. DAPI staining of branching cdc25-22-blocked cells in MBC after centrifugation (lower panel). (B) Scoring of branching in cdc25-22- and cdc25-22 mto1-blocked cells in the presence of either DMSO or MBC. Prior to treatment cells were blocked for 3 hours at 36.5°C. (C) Branching cdc11-119 cells treated with DMSO or MBC over a 5-hour time course. Cells were blocked at the restrictive temperature for 4 hours prior to drug treatment. (D) Images of DAPI-(top) and actin (bottom)-stained cdc11-119 cells blocked at 36.5°C for 4 hours and then treated with MBC for 6 hours. Bar, 5 µm.

View larger version (112K): [in this window] [in a new window]   Fig. 4. Tea1 accumulates in the middle of the cell in the absence of microtubules. (A) Anti-Tea1 antibody staining of cdc25-22 cells blocked for 3 hours at 36.5°C and treated with DMSO (top) or MBC for 30, 45 and 60 minutes (below). Cells were fixed in TCA to allow visualization of Tea1 in the cell middle. Tea1 staining in the cell centre is lost with methanol fixation. (B) Brightfield images of cdc25-22 mid1 cells blocked for 3 hours at 36.5°C and then for 1 hour in 50 µg/ml MBC. Bar, 5 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Microtubules determine growth site position, but are not required for establishment of polarized growth. (A) Brightfield images of cdc25-22-blocked (3 hours prior to treatment) cells treated with MBC or DMSO. (B) Branch position at different MBC concentrations, measured as relative distance from the middle of the cell. Between 50 and 100 cells were scored for each MBC concentration. (C) Average microtubule length (bars) and branching efficiency (percentages) at different MBC concentrations. 25-35 cells were scored for each MBC concentration. Bar, 5 µm.

View larger version (11K): [in this window] [in a new window]   Fig. 6. Model for a morphogenetic mechanism in fission yeast. Microtubules transport Tea1 to cell ends where it recruits a spontaneous cell-polarization module. This module comprises a short-range activator, promoting growth zone formation, and a long-range lateral inhibitory component that prevents growth zone formation.