Induction of Cytokinesis Is Independent of Precisely Regulated Microtubule Dynamics

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...

A Global, Myosin Light Chain Kinase-dependent Increase in Myosin II Contractility Accompanies the Metaphase–Anaphase Transition in Sea Urchin Eggs

“…Mapping of contractile activity by compression revealed that myosin II activation coincides with the metaphase-anaphase transition (Figure 2), which considerably precedes the outgrowth of astral microtubules ( Figure 6). Localization studies of microtubules and Ser19-phosphorylated light chain (Figure 3) as well as live cell imaging of EB1-decorated microtubules in the same species (Strickland et al, 2005) support the notion that at the metaphase-anaphase transition, the asters have yet to contact the cortex. Once the asters contact the cell cortex, Ser19 phosphorylation becomes restricted to the cleavage plane (Figure 3), and staining of phospho-Ser19 myosin regulatory light chain (PMLC) and myosin II (Figure 4) suggests that this zone of Ser19-phosphorylated regulatory light chain reflects local regulation of myosin II activity, and not simple redistribution of the motor.…”

“…In sea urchin eggs, anaphase onset is accompanied by a dramatic elongation of microtubules toward the cortex, and active furrowing can be detected after a short latent period (Rappaport and Ebstein, 1965;Strickland et al, 2005). Although species and temperature dependent, the total time from anaphase onset to the initiation of furrowing in L. pictus has been measured to be ϳ7.5 min (Shuster and Burgess, 2002b).…”

Temporal Evaluation of Cardiac Myocyte Hypertrophy and Hyperplasia in Male Rats Secondary to Chronic Volume Overload

“…12 This adversely affects cell division because the dynamic instability of microtubules is necessary for accurate chromosome segregation and subsequent cytokinesis. 21,22 Accordingly, paclitaxel-induced stabilization of microtubules results in chromosome mis-segregation, formation of micronuclei, and multinucleated cells as a result of irregular mitosis and cytokinesis. Although rodshaped cardiomyocytes isolated from hearts at 5 days post-fistula were not positive for BrdU labeling, a small percentage of rounded cardiomyocytes did incorporate BrdU labeling in the nucleus, indicating that the extent of DNA replication and nuclear division that did occur was insufficient to produce the significant increase in the number of shorter mononucleated cells.…”

Effects of polar cortical cytoskeleton and unbalanced cortical surface tension on intercellular bridge thinning during cytokinesis

“…3d and 3e) caused by local COLC suggest that the interaction between the spindle and the polar cortex was vital for cytokinesis. Previous studies [22] have shown that astral microtubules contact the polar cortex earlier than the equatorial cortex after anaphase onset. Basu and Chang [23] reported that the interaction between the microtubule and polar cortex was a definitive signal for initiating cytokinesis and spatial regulator of cellular shape, and abnormal interaction led to termination of cell dividing and remolding of global cytoskeleton.…”