Correcting Errors Aurora kinase

Error correction is thought to occur by stabilizing correct attachments while destabilizing incorrect attachments (41). Experiments in yeast showed that the inhibition of the Ipll/Aurora family of kinases prevents error correction by stabilizing incorrect attachments (38, 42), but how the active kinase corrected attachment errors was not known. This problem was particularly difficult to address because attachment errors are observed infrequently in the presence of active Aurora kinase (43). Experimental approaches that accumulated attachment errors through inhibition of Aurora kinase, for example by genetic mutation (42), did not permit subsequent kinase activation to examine error correction. Reversible small-molecule Aurora kinase inhibitors present a solution to this problem because they can be used to inhibit kinase function and subsequently removed to activate the kinase. 

To devise a strategy to address the question of how attachment errors were corrected, several issues needed to be addressed. Eirst, Aurora kinases have been implicated in multiple processes in mitosis (44). Ideally, kinase inhibition temporally would be controlled to isolate experimentally the error correction process. Second, the microtubules fibers that attach chromosomes to the spindle are highly dynamic, and the error correction likely involves some regulation of these dynamics. Live imaging would permit the analysis of microtubule dynamics with high temporal and spatial resolution. Finally, analysis of microtubule dynamics is difficult if individual fibers are obscured by other microtubules in the spindle. By creating conditions in which the improperly attached chromosomes are positioned away from the spindle body, individual fibers could be observed clearly. 

Figure 4 Correction of improper chromosome attachments by activation of Aurora kinase (45). (a) Assay schematic, (i) Treatment with the Eg5 inhibitor monastrol arrests cells in mitosis with monopolar spindles, in which sister chromosomes often are both attached to the single spindle pole, (ii) Hesperadin, an Aurora kinase inhibitor, is added as monastrol is removed. As the spindle bipolarizes with Aurora kinase inhibited, attachment errors fail to correct so that some sister chromosomes are still attached to the same pole of the bipolar spindle, (iii) Removal of hesperadin activates Aurora kinase. Incorrect attachments are destabilized by disassembling the microtubule fibers, which pulls the chromosomes to the pole, whereas correct attachments are stable, (iv) Chromosomes move from the pole to the center of the spindle as correct attachments form, (b) Structures of the Eg5 inhibitor monastrol and two Aurora kinase inhibitors, hesperadin and AKI-1. (c) Spindles were fixed after bipolarization either in the absence (i) or presence (ii) of an Aurora kinase inhibitor. Arrows indicate sister chromosomes that are both attached to the same spindle pole. Projections of multiple image planes are shown, with optical sections of boxed regions (1 and 2) to highlight attachment errors. Scale bars 5 xm. (d) After the removal of hesperadin, GFP-tubulin (top) and chromosomes (bottom) were imaged live by three-dimensional confocal fluorescence microcopy and DIC, respectively. Arrow and arrowhead show two chromosomes that move to the spindle pole (marked by circle in DIC images) as the associated kinetochore-microtubule fibers shorten and that then move to the center of the spindle. Time (minutes seconds) after the removal of hesperadin. Scale bar 5 (cm.

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