Microtubule density, dynamics, and plus end binding proteins: Understanding how astral microtubules induce ingression of the cleavage furrow in dividing sea urchin eggs

Microtubules of the mitotic apparatus (MA) are essential for spatial and temporal coordination of mitosis and cytokinesis. In the eggs of echinoderms, astral microtubules specify the position of contractile ring assembly, however much remains to be learned regarding how the MA communicates with the actin cortex. I have investigated the role of astral microtubules in stimulation of cytokinesis in sea urchin eggs. After establishing that the density of astral microtubules contacting the cortex is uniform at the time of specification of the division plane, I examined the consequences of disruption of this contact by applying the drug nocodazole to distinct regions of the cell surface. Cleavage furrows formed at sites where astral microtubules persistently contacted the cortex, arguing in favor of the prevailing model of furrow induction by equatorial stimulation. I examined the hypothesis that the dynamic behavior of astral microtubules might influence furrow induction. To test this, I exposed mitotic sea urchin eggs to a collection of compounds that affect microtubule dynamics, and then assessed the consequences for cytokinesis. I characterized the dynamic properties of the microtubules by using GFP-EB1 as a visible marker for the plus ends of elongating microtubules. I found that the cell cortex is insensitive to the dynamic state of astral microtubules, since cleavage furrows were observed under stabilizing, or destabilizing conditions. In all cases, physical contact between the asters and the cortex was required. Since the microtubule plus-end binding protein EB1 had been previously implicated in cell division, and is known to contribute to astral elongation and spindle orientation, I investigated its role in furrow initiation. I found that EB1 and its effector, p150 glued interact to promote astral elongation during anaphase, and that these proteins play a subsequent role in furrow initiation. My findings indicate that capture of astral microtubules by the cortex is mediated by the "microtubule plus-end binding" family of proteins, including EB1 and p150glued of the dynactin complex.