MCAK and Paclitaxel Have Differential Effects on Spindle Microtubule Organization and Dynamics

Abstract

Within the mitotic spindle, there are multiple populations of microtubules with different turnover dynamics, but how these different dynamics are maintained is not fully understood. MCAK is a member of the kinesin-13 family of microtubule-destabilizing enzymes that is required for proper establishment and maintenance of the spindle. Using quantitative immunofluorescence and fluorescence recovery after photobleaching, we compared the differences in spindle organization caused by global suppression of microtubule dynamics, by treating cells with low levels of paclitaxel, versus specific perturbation of spindle microtubule subsets by MCAK inhibition. Paclitaxel treatment caused a disruption in spindle microtubule organization marked by a significant increase in microtubules near the poles and a reduction in K-fiber fluorescence intensity. This was correlated with a faster t 1/2 of both spindle and K-fiber microtubules. In contrast, MCAK inhibition caused a dramatic reorganization of spindle microtubules with a significant increase in astral microtubules and reduction in K-fiber fluorescence intensity, which correlated with a slower t 1/2 of K-fibers but no change in the t 1/2 of spindle microtubules. Our data support the model that MCAK perturbs spindle organization by acting preferentially on a subset of microtubules, and they support the overall hypothesis that microtubule dynamics is differentially regulated in the spindle. INTRODUCTIONThe cytoskeleton must undergo dramatic reorganization as cells transition from interphase to mitosis to assemble the mitotic spindle. The spindle is a macromolecular structure composed of a dynamic array of microtubules (MTs) and their associated proteins that is necessary for proper chromosome segregation (Compton, 2000;Walczak and Heald, 2008). At the onset of mitosis, there is an increase in MT turnover that allows for breakdown of the interphase array and assembly of the mitotic spindle (Saxton et al., 1984;. This change is correlated with an increased catastrophe frequency (transition from growth to shrinkage) and a decreased rescue frequency (transition from shrinkage to growth) (Belmont et al., 1990;Gliksman et al., 1992;Verde et al., 1992;Rusan et al., 2001). The changes in MT dynamics are also correlated with a change in MT polymer levels during mitosis (Zhai et al., 1996). At nuclear envelope breakdown, there is a dramatic decrease in MT polymer levels, which may allow the cell to rapidly assemble a mitotic spindle because the released tubulin dimers can polymerize into new spindle MTs. As the cell progresses from mitosis to the next interphase, there is no change in the levels of MT polymer (Zhai et al., 1996), suggesting that MT polymer gets reorganized to reform the interphase MT cytoskeleton, without a significant change in the dynamics of the MTs. These studies highlight the need for the cell to possess a mechanism to temporally regulate MT dynamics.In addition to the temporal changes in dynamics throughout the cell cycle, the dynamics of MTs within mitosis are also...

Microtubule assembly during mitosis – from distinct origins to distinct functions?

“…These data provide a molecular link between the chromosomal MTs and the K-fibers that goes beyond previous observations of the formation of acentrosomal MTs at the kinetochores (Khodjakov et al, 2003;Maiato et al, 2004). Interestingly, the distinct spindle MT subclasses have distinct dynamic properties, (Rizk et al, 2009), and functional studies have shown that MCRS1 could have a role in the regulation of Kfiber minus-end stability (Meunier and Vernos, 2011).…”

Welcome to a new kind of tension: translating kinetochore mechanics into a wait-anaphase signal

“…First, extended treatment (24 hours) of PtK cells with nanomolar concentrations of taxol was recently reported to reduce kinetochore fiber fluorescence intensity suggesting that taxol can compromise microtubule attachment (Rizk et al, 2009). A second drawback is that the three cited observations that support this theory were made in different cell types (HeLa or PtK cells) with variable concentrations of taxol (1 mM versus 10 mM).…”

Regulation of microtubule dynamic instability

“…MTs in kif2a −/− cells frequently fail to stop growing after reaching the cell edge, resulting in bent and overextended MTs; this suggests that Kif2A promotes catastrophes at the cell cortex. During mitosis, different kinesin-13 family members share the workload: whereas Kif2A and Kif2B are primarily associated with the centrosome, Kif2C/MCAK predominates at the kinetochores [64,65]; therefore these proteins are likely to affect diverse MT subpopulations differently ( [5] and references therein).…”

“…Free MT ends can elongate or shorten, and even mild suppression of MT dynamics by low doses of MT-stabilizing or -destabilizing drugs has a profound effect on the organization of the mitotic apparatus, directional migration and even synaptogenesis in neuronal cells [5][6][7]. Cells express an arsenal of MT-modulating factors, some of which promote assembly [such as XMAP215 (Xenopus microtubule-associated protein 215)] or disassembly [kinesin-13s and stathmin/SCG10 (superior cervical ganglion-10 protein) proteins], while others have more specific roles only on a subset of MTs.…”