The interplay of the N- and C-terminal domains of MCAK control microtubule depolymerization activity and spindle assembly

Abstract

Spindle assembly and accurate chromosome segregation require the proper regulation of microtubule dynamics. MCAK, a Kinesin-13, catalytically depolymerizes microtubules, regulates physiological microtubule dynamics, and is the major catastrophe factor in egg extracts. Purified GFP-tagged MCAK domain mutants were assayed to address how the different MCAK domains contribute to in vitro microtubule depolymerization activity and physiological spindle assembly activity in egg extracts. Our biochemical results demonstrate that both the neck and the C-terminal domain are necessary for robust in vitro microtubule depolymerization activity. In particular, the neck is essential for microtubule end binding, and the C-terminal domain is essential for tight microtubule binding in the presence of excess tubulin heterodimer. Our physiological results illustrate that the N-terminal domain is essential for regulating microtubule dynamics, stimulating spindle bipolarity, and kinetochore targeting; whereas the C-terminal domain is necessary for robust microtubule depolymerization activity, limiting spindle bipolarity, and enhancing kinetochore targeting. Unexpectedly, robust MCAK microtubule (MT) depolymerization activity is not needed for sperm-induced spindle assembly. However, high activity is necessary for proper physiological MT dynamics as assayed by Ran-induced aster assembly. We propose that MCAK activity is spatially controlled by an interplay between the N- and C-terminal domains during spindle assembly.

Figure 1.

The C-terminal domain and neck of MCAK are necessary for efficient MT depolymerization activity. (A) Schematic diagram of expressed and purified MCAK proteins. The GFP tag is located on the N-terminus but was omitted from the diagrams for simplicity. (B and C) GMPCPP stabilized MTs (1 μM) were incubated with increasing concentrations of enzyme (0–1 μM) for 30 min in saturating MgATP at room temperature. MTs were sedimented, and the soluble tubulin heterodimer removed. Equal volumes of soluble tubulin and MT pellets were electrophoresed on 10% SDS polyacrylamide gels, and the gels were stained with Colloidal Coomassie Blue. The percentage of soluble tubulin was quantified from the stained gels and plotted against the log of the enzyme concentration. The four-parameter logistic equation was used to determine the EC₅₀ concentrations of each enzyme from four or more individual experiments. The curves represent the best fit curve to the data with each point represented by the mean ± SEM.

Figure 2.

The N-terminal domain of MCAK is required for efficient spindle assembly. CSF extracts were depleted of endogenous MCAK (MΔ) or mock-depleted with IgG (IgGΔ), cycled into interphase with CaCl₂, and cycled back into mitosis with additional depleted CSF extract. GFP-tagged proteins were added back to 100 nM final concentration with the second addition of CSF extract. Spindles were assembled for 90 min, fixed, and sedimented onto coverslips, and the chromatin was stained with Hoechst and mounted with anti-fade. (A) Images of representative structures from the add-back experiments are shown with MTs in magenta and chromatin in green. Scale bar, 20 μm. (B) Quantification of the spindles, large asters, and monopolar structures observed in the extracts that are represented in A. The data are graphed as the mean percentage plus the SEM from four independent extracts. An asterisk indicates a significant difference relative to GMCAK add-back.

Figure 3.

High MT depolymerization activity is not necessary for efficient sperm-induced spindle assembly. (A) The three catalytic domain mutants, GM(R522A), GM(E529A), and GM(296-308Δ), were assayed for MT depolymerization activity, quantified, and graphed as described in Figure 1, B and C. (B) The catalytic domain mutants were added back to cycled MCAK-depleted extracts (MΔ), and assayed for their ability to rescue spindle assembly as described for Figure 2. Quantification of the observed structures is graphed as the mean percentage plus the SEM from three independent experiments. An asterisk denotes a significant difference relative to add-back of GMCAK.

Figure 4.

The N-terminal domain regulates physiological MT dynamics and the C-terminal domain regulates spindle bipolarity. Asters and spindles were induced by the addition of 25 μM RanL43E to control or MCAK-depleted extracts to which 200 nM GFP, GMCAK, GM(187-731), GM(2-592), GM(187-582), GM(R522A), GM(E529A), and GM(296-308Δ) were added. Reactions were incubated for 45 min at room temperature, fixed, sedimented onto coverslips, and mounted with anti-fade. (A) Representative images are shown and were scaled identically. Scale bar, 10 μm. (B) Quantification of the structure type is graphed as the mean percentage of the total structures plus the SEM from three independent experiments. (C) Quantification of the aster types is graphed as the mean percentage from the aster and spindle total plus the SEM from three independent experiments. Small asters and spindles, which represent rescued structures, were combined and are compared with large asters. An asterisk indicates significant difference relative to GMCAK add-back.

Figure 5.

Summary of the in vitro and extract activities of GMCAK and its various mutants. The data from each experiment are summarized with plus signs to delineate the extent of activity. Four consecutive plus signs indicate the highest activity (+ + + +) and a minus sign indicates no activity (−). ND indicates activity was not determined for that assay. Each GMCAK mutant is represented schematically based on the domain(s) deleted or mutated. The domains are as depicted in Figure 1. The catalytic domain mutants are full-length GMCAK proteins with either a point mutation (R522A or E529A) or a 13-amino acid deletion (296-308Δ) within the catalytic domain.