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First published online 17 June 2008
doi: 10.1242/jcs.027052

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Dual role for microtubules in regulating cortical contractility during cytokinesis

Department of Biology and Program in Molecular and Cellular Biology, University of Massachusetts at Amherst, N. Pleasant Street, Amherst, MA 01003, USA

View larger version (82K): [in this window] [in a new window]   Fig. 1. Differential stability of microtubules following anaphase onset. LLC-Pk1 cells stably expressing GFP-tubulin are shown before and at various times after the addition of 33 µM nocodazole. (A) Addition of nocodazole within 2 minutes of anaphase onset induces rapid disassembly of microtubules and prevents cytokinesis. (B) Addition of nocodazole >2 minutes after anaphase onset results in disassembly of the majority of astral microtubules in the polar region and most astral microtubules in the equatorial region, but not of interzonal microtubules. Time is shown in minutes:seconds relative to the addition of nocodazole. Scale bars: 10 µm.

View larger version (133K): [in this window] [in a new window]   Fig. 2. Microtubule disassembly induces changes in the organization of cortical actin. LLC-Pk1 cells stably expressing GFP-actin are shown before and at various times after the addition of 33 µM nocodazole. (A-D) Contractile-ring formation and cytokinesis in a control cell (A) and in cells to which nocodazole was added either within 2 minutes of (B) or >2 minutes after (C,D) anaphase onset. In B, both contractile-ring assembly and cytokinesis fail, whereas in C and D, furrow formation and ingression proceed. Wave-like behavior of cortical actin is shown in the inset panels in B. Arrowheads mark wave-like behavior in B and C. In D, actin from distal regions of the cortex flows towards the equatorial region and contributes to the contractile ring; arrows mark the direction and region contributing to flow. Time is shown in minutes:seconds and is relative to the addition of nocodazole in B-C. Scale bars: 10 µm.

View larger version (125K): [in this window] [in a new window]   Fig. 3. Localization of GFP-RhoA in control and in nocodazole-treated cells. Fluorescence images of living LLC-Pk1 cells transfected with GFP-tagged C. elegans RhoA (GFP-CeRhoA). In control cells (A) and nocodazole-treated cells that assemble a contractile ring (C), GFP-CeRhoA localizes to the equatorial cortex during cytokinesis. In nocodazole-treated cells that fail to assemble a contractile ring (B), GFP-CeRhoA fails to accumulate at the equatorial region; asterisks mark the location of chromosomes. In both control and nocodazole-treated cells, GFP-CeRhoA also localizes to membranous folds between neighboring cells. Time is shown in minutes:seconds and is relative to the addition of nocodazole in B and C. Scale bars: 10 µm.

View larger version (89K): [in this window] [in a new window]   Fig. 4. Actin polymerization and cortical contractions contribute to wave-like behavior of cortical actin in nocodazole-treated anaphase cells. LLC-Pk1 cells expressing GFP-actin were followed until anaphase onset, nocodazole was added and photobleaching performed. Areas enclosed by a red broken line indicate the photobleached regions; the white box indicates the region that is shown in A'; asterisks mark the location of chromosomes; arrows mark the site of cortical ingression. Time is shown in minutes:seconds relative to photobleaching. Scale bars: 10 µm.

View larger version (178K): [in this window] [in a new window]   Fig. 5. Mislocalization of TDRFP-MRLC in nocodazole-treated anaphase cells. (A-C) LLC-Pk1 cells stably expressing TDRFP-MRLC. (A) In control cells, TDRFP-MRLC accumulates at the equatorial cortex. (B) In nocodazole-treated cells that fail to assemble a contractile ring, TDRFP-MRLC is diffusely distributed along the ventral cortex. (C) In nocodazole-treated cells that assemble a contractile ring, myosin accumulates at the equatorial cortex and shows transient accumulation in the polar cortex (arrows) (compare the 00:30 time point with 03:50). The first two frames are focused on the accumulation of MRLC at the equator and, as soon as contractile activity is observed away from the ring at 3:50, the focus shifts to the non-equatorial cortex. Time is shown in minutes:seconds after the addition of nocodazole. Asterisks mark the position of the chromosomes. Scale bars: 10 µm.

View larger version (133K): [in this window] [in a new window]   Fig. 6. Rho is mislocalized and its activity is required for wave-like behavior of cortical actin in nocodazole-treated cells. (A) GFP-CeRhoA is mislocalized to the polar region in nocodazole-treated cells; arrowheads mark an area of GFP-CeRhoA fluorescence in the polar cortex. White boxes indicate the region that is shown to the right. (B,C) LLC-Pk1 cells expressing GFP-actin were treated with nocodazole in anaphase and then injected with C3 transferase (B) or treated with Y-27632 (C). White boxes indicate the region that is shown in B',C'. Addition of either C3 transferase or Y-27632 suppresses wave-like behavior (B',C'); arrows show sites at which cortical actin accumulated during wave-like behavior following treatment with nocodazole. Time of addition of C3 transferase or Y27632 is indicated. Time is shown in minutes:seconds. Scale bars: 10 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Model of microtubule-dependent regulation of cortical actomyosin. Red lines, differentially stable microtubules; green lines, dynamic microtubules; blue lines, F-actin; yellow and red complexes, centralspindlin and RhoGEF. Dumbbells represent myosin II in the inactive (green) and active (pink) form. See text for details.

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