In animal cells, microtubules (MTs) of the mitotic apparatus (MA) communicate with the cell cortex to stimulate cytokinesis; however, the molecular nature of this stimulus remains elusive . A signal for cytokinesis likely involves the MT plus end binding family of proteins, which includes EB1, p150glued, APC, LIS1, and CLIP-170. These proteins modulate MT dynamics and facilitate interactions between growing MTs and their intracellular targets, including kinetochores, organelles, and the cell cortex . The dynein-dynactin complex mediates many of these microtubule capture events . We report that EB1 and p150glued interactions are required for stimulation of cytokinesis in dividing sea urchin eggs. Injected antibodies against EB1 or p150glued suppressed furrow ingression but did not prevent elongation of anaphase astral MTs toward the cortex, suggesting that EB1 and dynactin are both required for communication between the MA and the cortex. Targeted disruption of the interaction between EB1 and p150glued suppressed anaphase astral MT elongation and resulted in a delay of cytokinesis that could not be overcome by manipulation of the asters toward the cortex. We conclude that EB1 and dynactin participate in stimulation of the cleavage furrow, and their interaction promotes elongation of astral MTs at anaphase onset.
No 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. INTRODUCTIONCytokinesis is the process by which the cytoplasm is divided at the end of mitosis to form two daughter cells. Much has been learned about the actomyosin ring that generates the contractile force required for cytokinesis; however, many questions remain regarding mechanisms that specify the place and time for contractile ring assembly and activation. The cleavage furrow normally bisects the mitotic apparatus (MA) at the plane of the former metaphase plate (Wilson, 1928), and microtubules (MTs) of the MA are essential for communication of the contractile signals (Beams and Evans, 1940). Several models have been proposed to describe the initiation of cytokinesis, each of which attributes the source of the signal to a different MT population within the MA (Balasubramanian et al., 2004;Burgess and Chang, 2005). In one of the most well studied cell types, the cleaving echinoderm egg, positioning of the furrow is determined by equatorial stimulation from two asters and not by the bundled MTs of the spindle midzone, kinetochores, or by the polar astral MTs (Rappaport, 1996), which may play a stimulatory role in other cell types (Burgess and Chang, 2005;D'Avino et al., 2005;Glotzer, 2005). However, the nature, degree, and extent of contact between the astral MTs and the cortex required for stimulation of cytokinesis is not yet determined.Changes in MT dynamics facilitate structural reorganization of the MA during cell division. At the metaphaseanaphase transition astral MTs elongate due to decreased catastrophe or increased rescue, kinetochore MTs shorten to segregate the chromosomes, and spindle midzone MTs grow to promote separation of the spindle poles (DeBrabander et al., 1986;Snyder and Mullins, 1993;Zhai et al., 1995;Mallavarapu et al., 1999;Rusan et al., 2002;Piehl et al., 2004). Anaphase o...
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