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. 2005 Oct;16(10):4485-94.
doi: 10.1091/mbc.e05-04-0305. Epub 2005 Jul 12.

Induction of cytokinesis is independent of precisely regulated microtubule dynamics

Laila I Strickland  1 , Erin J Donnelly, David R Burgess
Affiliations

Induction of cytokinesis is independent of precisely regulated microtubule dynamics

Laila I Strickland et al. Mol Biol Cell. 2005 Oct.

Abstract

Astral microtubules (MTs) emanating from the mitotic apparatus (MA) during anaphase are required for stimulation of cytokinesis in eggs. We have used green fluorescent protein-labeled EB1 to observe MT dynamics during mitosis and cytokinesis in normal sea urchin eggs. Analysis of astral MT growth rates during anaphase shows that MTs contact the polar cortex earlier than the equatorial cortex after anaphase onset but that a normal cleavage furrow is not induced until contact with MTs has been achieved throughout the cortex. To assess the role of MT dynamics in initiation of cytokinesis, we used a collection of small molecule drugs to affect dynamics. Hexylene glycol resulted in rapid astral elongation due to decreased MT catastrophe and precocious furrowing. Taxol suppressed MT dynamics but did not inhibit furrow induction when the MA was manipulated toward the cortex. Urethane resulted in short, highly dynamic astral MTs with increased catastrophe that also stimulated furrowing upon being brought into proximity to the cortex. Our findings indicate that astral MT contact with the cortex is necessary for furrow initiation but that the dynamic state of astral MTs does not affect their competency to stimulate furrowing.

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Figures

Figure 1.
Figure 1.
Structure and dynamics of microtubules of the mitotic apparatus during completion of mitosis and cytokinesis. Cell outlines are shown in white when necessary. (A) Wide field fluorescence microscopy of microtubules (green) stained by immunofluorescence and chromosomes DNA (blue) during metaphase and anaphase in a dividing sea urchin egg. After the metaphase-anaphase transitions, the asters expand substantially, filling the egg with microtubules. Bar, 25 μm. (B) Confocal imaging of fixed eggs at different stages of mitotic progression and cytokinesis, stained for microtubules by immunofluorescence. After anaphase onset, the centrosomes expand and the astral microtubules elongate, contacting the polar cortex first, and then establishing contact at the equator before furrow ingression. Bar, 25 μm. (C) Confocal time-lapse of a GFP-EB1-microinjected egg as it proceeds through mitosis and cytokinesis. GFP-EB1 decorates the plus ends of elongating MTs and localizes abundantly to the spindle structures. Abrupt expansion of the asters is observed after anaphase onset, before furrow ingression. Bar, 25 μm. (D) Confocal time-lapse of a GFP-EB1 injected egg showing elongation of an astral microtubule during anaphase. The decoration of the MT plus ends by GFP-EB1 formed comet tails that were used to measure MT growth rates relative to their site of nucleation at the centrosome. Bar, 5 μm.
Figure 2.
Figure 2.
Geometric diagram of the MA relative to the cell cortex during anaphase. From confocal images of fixed cells stained for microtubules, we measured the distances of the polar-directed (a) and equatorially directed (b) astral MTs to the cell cortex at the time of anaphase onset, the spindle length at anaphase onset (c), and the spindle length at the time of furrow initiation (d). These measurements were used in conjunction with our analysis of anaphase astral MT growth rates to predict the time of contact between astral MTs and the polar versus equatorial cortex, relative to the time of furrow ingression.
Figure 3.
Figure 3.
Microtubule density is equal in the polar versus equatorial cortex at the time of furrow initiation. Cell outlines are shown in white. (A) Confocal immunofluorescence of microtubules shortly after anaphase onset (early), just before furrow initiation (mid), and during cytokinesis (late). To estimate MT density at different locations of the cortex, polar and equatorial regions of interest were defined (white boxes), and the Leica imaging software was used to quantify the fluorescent signal from each region. Bar, 30 μm. (B) Polar and equatorial fluorescence intensities were collected from five cells in each group and then averaged. The average value of the pole:equator ratio was then plotted to reveal that MT density is greater at the poles relative to the equator in the early group of cells, but that polar and equatorial MT densities are nearly equal before initiation of the furrow, and then remain equal during ingression.
Figure 4.
Figure 4.
Hexylene glycol stabilizes microtubules and promotes precocious growth of anaphase asters. Cell outlines are shown in white when necessary. (A) Confocal immunofluorescence of microtubules in metaphase and anaphase sea urchin eggs treated with 0.75% hexylene glycol. The astral MTs seem robust and elongated relative to controls at metaphase, anaphase, and at the time of furrow ingression. Bar, 20 μm. (B) Pretreatment of eggs with 0.75% hexylene glycol protects astral microtubules from depolymerization by 1 μM nocodazole. Although 1 μM nocodazole is a sufficient dose to depolymerize the majority of the astral MTs, the addition of hexylene glycol before nocodozole permits elongation of the asters, demonstrating that hexylene glycol has a stabilizing effect on MTs. Bar, 25 μm. (C) Confocal time-lapse of a GFP-EB1-microinjected egg treated with 0.75% hexylene glycol. A robust MA, precocious anaphase astral MT elongation, and premature furrowing are apparent. Bar, 30 μm. (D) High-magnification confocal time lapse of an anaphase aster during furrow initiation in a HG-treated cell. GFP-EB1 is seen to decorate many MTs along the length of their lattice, rather than just at the tips, suggesting prolonged periods of growth and decreased catastrophe. Bar, 15 μm. (E) Confocal time lapse showing elongation of an astral microtubule during anaphase, in a cell treated with 0.75% HG. Bar, 5 μm.
Figure 5.
Figure 5.
Taxol treatment results in diminished MT dynamics but does not prevent furrow formation. Cell outlines are shown in white when necessary. (A) Confocal immunofluorescence of microtubules in a metaphase cell treated with 100 μM taxol. The asters are extremely dense and slightly longer than the metaphase asters of control cells. The centrosomes are abnormally shaped ellipses. Many cytosolic MTs are also present. Bar, 40 μm. (B) Confocal time-lapse of a GFP-EB1-microinjected cell, treated with 50 μM taxol at metaphase. The density and length of astral MTs was increased at metaphase, but as anaphase progressed, GFP-EB1 binding diminished, suggesting suppression of dynamics. Astral MTs did not seem to substantially elongate beyond their metaphase length. The cytoplasmic clusters of MTs were observed in high abundance near the equatorial cortex. The cell elongated, but furrowing was never observed despite the fact that mitosis completed. Bar, 50 μm. (C) High-magnification confocal time-lapse of GFP-EB1 during metaphase/anaphase transition in a 50 μM taxol-treated cell. The cell did not initiate a conventional cleavage furrow, but cortical contractility occurred at sites associated with cytasters (arrow). Cell outline is shown in white. Bar, 20 μm. (D) Time-lapse of Hoechst-stained chromosomes in a cell that was microinjected with 100 μM taxol on one side of the anaphase MA (arrow indicates injection site). Microinjection blocked furrow formation on the side of injection but a normal cleavage furrow formed on the opposite side, indicating local suppression of MT dynamics on the injected side. (E) Localized microinjection of 100 μM taxol followed by micromanipulation of the MA into contact with the cortex permits initiation of a furrow. Bar, 20 μm. (White line indicates position of glass manipulator. Arrow indicates injection site.)
Figure 6.
Figure 6.
Urethane treatment results in short, dense asters undergoing increased catastrophe, but does not inhibit furrowing if the MA is micromanipulated toward the cortex. (A) Confocal immunofluorescence of microtubules during metaphase, anaphase, and cytokinesis in cells treated with 60 mM urethane. The asters are reduced in size and never elongate during anaphase. They do seem to become more dense during anaphase, consistent with increased occurrence of catastrophe, followed by brief periods of elongation. The spindle is reduced in length relative to controls, and never elongates before telophase, suggesting an absence of anaphase b. Bar, 20 μm. (B) Nomarski DIC image showing that micromanipulation of the MA toward the cortex in a urethane treated cell results in furrowing. Bar, 20 μm. (C) Confocal time lapse of a GFP-EB1-microinjected cell, treated with 60 mM urethane at metaphase. The asters are diminished in length but highly dynamic. The centrosomes do not substantially expand and the spindle poles do not separate during anaphase. Bar, 15 μm.
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