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Stabilizing Microtubules During Cytokinesis


By: Carol A. Rouzer, VICB Communications
Published:  March 10, 2016



Cross-talk between the contractile ring that divides a cell and central spindle microtubules is key to successful cytokinesis during mitosis.


Cell division is an exceedingly complex process requiring accurate replication of the genome (DNA synthesis) and its appropriate and equal distribution (mitosis) to the newly forming daughter cells. Importantly, the cell must also be physically divided in two, and this occurs in an equally complex series of events known as cytokinesis (Figure 1). This process, which occurs during anaphase and telophase, requires the reorganization of microtubules to create a central spindle (midzone), an interdigitating anti-parallel array of microtubules that forms between the segregated chromosomes. Concomitantly, an actin- and myosin II-containing cleavage furrow forms around the periphery of the cell. In the process of ingression, myosin II-based contraction of the furrow delineates the separation plane between the daughter cells. Eventually, the furrow pinches the microtubules of the central spindle into a tightly compacted cylindrical structure, the midbody. Soon thereafter, abscission - the separation of the daughter cells - completes the process of cytokinesis (Figure 2).



FIGURE 1. During mitosis, the chromosomes first line up along the equator of the mitotic spindle. As they begin to move apart, the microtubules of the mitotic spindle reorganize to form the central spindle while a ring of actin, myosin II, and other proteins forms around the perimeter of the cell. The contractile ring begins to ingress and eventually compresses the central spindle into a compact structure, the midbody. Finally, the process of abscission separates the two cells completely. Figure reprinted by permission from Macmillan Publishers LTD from T. D. Pollard and J.-Q. Wu (2010) Nat. Rev. Mol. Cell Biol. 11,1149. Copyright 2010.




FIGURE 2. (a) Organization of the central spindle and contractile ring. As mitosis progresses from metaphase to anaphase, the chromosomes move from their equatorial position towards the centrosomes of the mitotic spindle. During anaphase, the mitotic spindle is reorganized to form the central spindle with overlapping microtubles at the equator or midzone. The contractile ring encircles the central spindle and begins the process of ingression. When the cell reaches telophase, the contractile ring has compressed the spindle fibers into a narrow bundle termed the midbody. (b) The various stages of mitosis observed in a cell using indirect immunofluorescence to label the microtubules. Figure reprinted by permission from Macmillan Publishers LTD from M. Glotzer (2009) Nat. Rev. Mol. Cell Biol. 10, 9. Copyright 2009.



Although the events of cytokinesis appear tightly choreographed, little is known about how the activity of the central spindle is integrated with that of the contractile ring that creates the cleavage furrow. Now, Vanderbilt Institute of Chemical Biology member Puck Ohi and his graduate student Jennifer Landino show that myosin II-dependent activity in the furrow is required to stabilize central spindle microtubules. They also show that interaction of a central spindle-associated protein complex with actin in the contractile ring is required for microtubule stabilization, furrow ingression, and ultimately, cytokinesis [J. Landino and R. Ohi (2016) Curr. Biol., published online February 18, DOI:10.1016/j.cub.2016.01.018].


The investigators used a very simple assay to measure microtubule stability. They incubated growing HeLa cells at 0 oC for 10 min, and then fixed and stained the cells for tubulin. In cells that were in early anaphase, the cold treatment resulted in a disassembly of microtubules in the central spindle. However, this did not occur in cells that had reached later anaphase or telophase. In fact, the researchers found a direct correlation between the degree of ingression of the contractile ring and microtubule stability, as measured by indirect fluorescence of polymeric tubulin.


To confirm their results, Landino and Ohi used cells stably expressing green fluorescent protein- (GFP)-labeled tubulin. They treated the cells with nocodazole, a microtubule destabilizing drug, and used the GFP label to assess microtubule disassembly in the living cells. This assay confirmed their earlier results. In cells that had progressed to late anaphase with ingression of the furrow, microtubules in the central spindle remained stable in the presence of nocodazole. In contrast, cells in earlier stages of mitosis exhibited microtubule disassembly in the presence of the drug.


The results suggested a correlation between ring contraction and microtubule stability. This led the researchers to hypothesize that myosin II activity in the contractile ring was required to promote the observed increased stability of spindle microtubules. To test this hypothesis, they treated cells with blebbistatin, an inhibitor of non-muscle myosin that prevented ingression but did not impair assembly of the contractile ring. Consistent with their hypothesis, they discovered that microtubules in blebbistatin-treated cells exhibited reduced stability in the presence of either cold treatment or nocodazole.


The chromosome passenger complex (CPC) is a heterotetramer comprising aurora B kinase, borealin, survivin, and the inner centromere protein (INCENP) (Figure 3). Known to play a role in multiple aspects of cytokinesis, the CPC promotes central spindle assembly and helps to define the site of the cleavage plane. Prior work had shown that the CPC is located at the intersection of actin and microtubules during cytokinesis, leading Landino and Ohi to hypothesize that it may mediate cross-talk between the spindle microtubules and the contractile ring. To test this hypothesis, they treated cells with an inhibitor of aurora B kinase and then exposed the cells to either cold or nocodazole. They discovered that the inhibitor had no effect on localization of aurora B kinase in the cells, but it reduced microtubule stability in the presence of both of the stresses.



FIGURE 3. Diagrammatic representation of the four proteins that form the chromosome passenger complex (CPC). The arrows indicate sites of protein-protein interactions. The inset depicts the structure of the survivin, borealin, INCENP complex, highlighting the three-helical bundle formed by the N-termini of the three proteins. Figure reprinted by permission from Macmillan Publishers LTD from M. Glotzer (2009) Nat. Rev. Mol. Cell Biol. 10, 9. Copyright 2009.



Prior studies in Dictyostelium had shown that INCENP interacts directly with actin, suggesting that the CPC may provide a link between the contractile ring and the spindle microtubules. By expressing GFP-labeled constructs derived from INCENP in HeLa cells, the investigators found that, indeed, an alpha helical domain comprising amino acids 500 through 680 of INCENP bound directly to actin. Because this region of the protein is highly positively charged, the investigators hypothesized that the INCENP-actin interaction occurred through electrostatic attractions. They supported this hypothesis by mutating the ten positively charged amino acids in INCENP500-680 to glutamic acid. Expressing this charge-reversal mutant (INCENP500-680 CR) in cells demonstrated that the altered protein exhibited no ability to bind to actin. Further work showed that wild type His-tagged INCENP500-680 but not His-tagged INCENP500-680 CR sedimented with actin in vitro.


To further test the role of an interaction between INCENP and actin, the investigators depleted cells of endogenous INCENP and then replaced it by expressing GFP-labeled wild-type INCENP or GFP-labeled INCENP CR, a full length protein carrying the charge reversal mutations. In both cases, the expressed proteins localized normally to the central spindle and the cell cortex in the region of the furrow. Cells expressing the wild-type protein exhibited normal stabilization of midzone microtubules in late anaphase and proceeded through cytokinesis with no apparent defects. In contrast, cells expressing the INCENP CR mutant exhibited reduced midzone microtubule stability in response to cold and a marked reduction in their ability to ingress and form a midbody. Treatment of these cells with taxol, a microtubule stabilizing drug both increased nocodazole resistance and enabled them to complete normal furrow ingression.


The results demonstrate a previously unappreciated cross-talk between myosin II activity in the contractile ring and microtubule stability in the central spindle midzone. This cross-talk appears to be mediated, at least in part, by aurora B kinase through an INCENP-dependent association with actin. The exact mechanism by which aurora B kinase translates myosin II-dependent ingression to midzone microtubule stability will be the subject of further experiments. However, these findings provide new insight into the process of cytokinesis, without which normal cell growth and development cannot occur.


View Curr Biol. article: The Timing of Midzone Stabilization during Cytokinesis Depends on Myosin II Activity and an Interaction between INCENP and Actin










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