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First published online 20 January 2004
doi: 10.1242/jcs.00925

Journal of Cell Science 117, 689-700 (2004)
Published by The Company of Biologists 2004
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Role of microtubules and tea1p in establishment and maintenance of fission yeast cell polarity

Kenneth E. Sawin* and Hilary A. Snaith

Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, Michael Swann Building, University of Edinburgh, Edinburgh, EH9 3JR, UK



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  		Fig. 1. Assay for cell branching in fission yeast. cdc10-129 mutant cells are arrested in G1 by a temperature shift, drugs are added, and the temperature is subsequently shifted down to allow cells to reenter the cell cycle. Cells are shown 2 hours after release from cell-cycle arrest, in the presence of (A) control DMSO or (B) 100 µg/ml TBZ.

 


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  		Fig. 4. TBZ but not MBC causes transient depolarization of the cortical actin cytoskeleton and arrest of cell elongation. (A) Percentage of cdc10-129 arrested cells with a polarized actin cytoskeleton 45 minutes after addition of DMSO, TBZ, MBC or TBZ plus MBC. (B) Time-course showing the percentage of cdc10-129 arrested cells with polarized ral3-GFP, after addition of TBZ, MBC or TBZ plus MBC. (C) Mean cell length versus time in a cdc10-129 block-and-release experiment, using cells treated either with DMSO, TBZ or MBC. (D) Mean cell length versus time in wild-type elutriated cells treated either with DMSO, TBZ or MBC. (E-H) Images of cdc10-129 cells showing Rhodamine-Phalloidin staining of (E) polarized actin and (F) depolarized actin, and live-cell fluorescence of (G) polarized ral3-GFP and (H) depolarized ral3-GFP.

 


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  		Fig. 2. MBC is a more potent microtubule inhibitor than TBZ. Anti-tubulin immunofluorescence images of wild-type cells (A-G) treated with (A) DMSO, (B) 5 µg/ml MBC, (C) 10 µg/ml MBC, (D) 25 µg/ml MBC, (E) 50 µg/ml MBC, (F) 50 µg/ml TBZ or (G) 100 µg/ml TBZ; and benzimidazole-resistant nda3-TB101 beta-tubulin mutant cells (H-J) treated with (H) DMSO, (I) 50 µg/ml MBC or (J) 100 µg/ml TBZ. Similar microtubule distributions were seen at both 10 and 90 minutes of drug treatment (not shown).

 


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  		Fig. 9. Tea1p shows a non-uniform association with microtubules in mod5{Delta} tea2-1 mutants before cell branching. Anti-tubulin (A,D) and anti-tea1p (B,E) immunofluorescence, and merged images (C,F) in tea2-1 (A-C) and mod5{Delta} tea2-1 (D-F) mutants 60 minutes after temperature shift to 36°C. Note that, in mod5{Delta} tea2-1 cells, tea1p often appears more concentrated on microtubules towards the cell middle (yellow in merged panel F), relative to the total microtubule signal (arrows in D-F). Also shown are merged images of branching mod5{Delta} tea2-1 mutants 90 minutes after temperature shift (H), as well as wild-type cells without a temperature shift (G), to show the relatively low tea1p signal in these mutants as compared with wild-type cells.

 


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  		Fig. 3. TBZ but not MBC causes high levels of cell branching. The frequency of cell branching was measured in temperature-shift block-and-release experiments, using cdc10-129 mutants (see Fig. 1) and different concentrations of (A) TBZ or (B) MBC.

 


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  		Fig. 5. In tea1+ cells, the formation of branches after TBZ-induced cell depolarization requires the presence of short microtubules but, in tea1{Delta} cells, formation of branches after TBZ treatment is independent of microtubule distribution. The frequency of cell branching was measured in G1 block-and-release experiments using cdc10-129 mutants that were either tea1+ (A,C,E) or tea1{Delta} (B,D,F). The genotype of each strain is indicated in each panel (A-F). In one set of experiments (A,B), cells were treated with either TBZ or MBC, or with TBZ plus MBC. In a second set (C,D), cells were treated with DMSO or TBZ, or with TBZ followed by wash-out of the drug after release from the G1 arrest. In a third set, a benzimidazole-resistant beta-tubulin mutant background (nda3-TB101) was used, and cells were treated with either DMSO or TBZ. The baseline frequency of branched cells is higher in tea1{Delta} mutants because they branch in response to the temperature shift required for the cdc10-129 arrest.

 


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  		Fig. 6. Microtubule disruption during return-to-growth of tea2-1 cells reduces the frequency of branching in tea1+ but not in tea1{Delta} genetic backgrounds. (A) The percentage of branched cells in the indicated wild-type and mutant strains is shown, 3 hours after dilution of stationary cultures into fresh medium containing either DMSO, 25 µg/ml MBC or 50 µg/ml MBC. (B,C) tea2-1 mutants 2 hours after dilution into either (B) DMSO or (C) 25 µg/ml MBC.

 


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  		Fig. 7. Transient depolymerization of the actin cytoskeleton by latrunculin B (LatB) triggers microtubule-dependent branching in cdc10-129 tea2-1 mutants in a tea1+ background but microtubule-independent branching in a tea1{Delta} background. Percent branched cells in cdc10-129 block-and-release experiments after either a LatB pulse followed by wash-out or a LatB plus MBC pulse followed by wash-out into MBC. (A) cdc10-129 tea2-1, (B) cdc10-129 nda3-TB101 tea2-1, (C) cdc10-129 tea1{Delta} tea2-1, (D) cdc10-129 tea1{Delta}.

 


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  		Fig. 8. Tea1p is required for a high frequency of branching after a mild depolarization of the cortical actin cytosksleton. (A) Temperature shift in mod5{Delta} tea2-1 double mutants produces a synergy in cell branching that depends on tea1p. The percentage of branched cells in the indicated strains is shown 2 hours after shift-up from 32°C to 36°C. (B) Percentage of branched cells in a cdc10-129 block-and-release experiment, using a TBZ wash-out (Fig. 5) and either cdc10-129 nda3-TB101 tea2-1 triple mutants or cdc10-129 nda3-TB101 tea1{Delta} tea2-1 quadruple mutants.

 


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  		Fig. 10. Model for regulation of cell polarity establishment by microtubules and tea1p (see text for additional details). Patterns of polarity establishment are shown for tea1+ cells (A-E) and tea1{Delta} mutants (F-J). During steady-state growth in tea1+ cells (A), a growth zone, including polarized cortical actin (red), is at cell tips. tea1p (blue) is targeted to cell tips by association with the plus ends of growing microtubules (green). (B) Representation of a generic state of microtubule depolymerization in conjunction with depolarization of the cortical actin cytoskeleton, which is achieved alternatively by TBZ treatment (which simultaneously affects microtubules), by growth to an extended stationary phase, or by depolymerization of actin with LatB. Under these conditions, some tea1p can remain at cell tips to provide residual cortical landmark cues for polarity re-establishment. During polarity re-establishment (C-E), cells with normal-length microtubules can re-target tea1p back to cell tips (C), whereas cells with short microtubules (e.g. after TBZ treatment, or tea2-1 mutants) target tea1p ectopically to the cell middle (E). This targeting of tea1p helps to set up the new polarity axis. If microtubules are strongly disrupted and thus unable to direct tea1p to the cortex, the residual tea1p-dependent landmarks direct polarity re-establishment back to cell tips (D). In tea1{Delta} mutants, microtubules are normal and actin is polarized at cell tips during steady-state growth (F), but the absence of tea1p means that, upon cell depolarization and microtubule depolymerization, cortical landmarks for polarity establishment are not available (G). In addition, because microtubule signalling to the cortex depends on tea1p, tea1{Delta} mutants squander the opportunity to retarget the polarity machinery back to cell tips through a microtubule-based mechanism. As a result, polarity is re-established either randomly or following additional unknown cues (H,I,J).

 

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© The Company of Biologists Ltd 2004