The depolymerizing kinesin MCAK uses lattice diffusion to rapidly target microtubule ends

Abstract

The microtubule cytoskeleton is a dynamic structure in which the lengths of the microtubules are tightly regulated. One regulatory mechanism is the depolymerization of microtubules by motor proteins in the kinesin-13 family 1 . These proteins are crucial for the control of microtubule length in cell division [2][3][4] , neuronal development 5 and interphase microtubule dynamics 6,7 . The mechanism by which kinesin-13 proteins depolymerize microtubules is poorly understood. A central question is how these proteins target to microtubule ends at rates exceeding those of standard enzyme-substrate kinetics 8 . To address this question we developed a single-molecule microscopy assay for MCAK, the founding member of the kinesin-13 family 9 . Here we show that MCAK moves along the microtubule lattice in a one-dimensional (1D) random walk. MCAK-microtubule interactions were transient: the average MCAK molecule diffused for 0.83 s with a diffusion coefficient of 0.38 mm 2 s 21 . Although the catalytic depolymerization by MCAK requires the hydrolysis of ATP, we found that the diffusion did not. The transient transition from three-dimensional diffusion to 1D diffusion corresponds to a "reduction in dimensionality" 10 that has been proposed as the search strategy by which DNA enzymes find specific binding sites 11 . We show that MCAK uses this strategy to target to both microtubule ends more rapidly than direct binding from solution.Kinesin-13 motor proteins act at microtubule ends, where they are thought to force protofilaments into a curved conformation 12,13 , which is a likely structural intermediate in the depolymerization process 14 . Classically, kinesin motor proteins reach microtubule ends by ATP-dependent translocation along microtubules. However, LETTERSFigure 1 | MCAK-dependent microtubule depolymerization. a, Diagram of the in vitro assay depicting a microtubule (red) immobilized above the glass surface by anti-tubulin antibodies (dark blue). Excitation by total internal reflection allows the detection of single molecules (namely MCAK-GFP in green) in the evanescent field (shown in blue). b, Epifluorescence images of immobilized microtubules at the times shown in minutes. MCAK dimer (8 nM) was added at t ¼ 2 min. c, Plot of microtubule depolymerization rate against MCAK concentration. Error bars are s.d. Data fitted to Hill equations (lines plotted) yielded K m ¼ 3.9 nM and K m ¼ 6.1 nM for MCAK and MCAK-GFP, and n ¼ 2.4 and n ¼ 2.2, respectively. Red squares, MCAK-His 6 ; green circles, MCAK-His 6 -EGFP. d, Shortening of four microtubules from a mean length of 8.4 mm to 7.8 mm (black line) after the addition of 5 nM MCAK (red line). The depolymerization rate approached steady state with a time constant of 3.8 s (green fitted line). (Fig. 1a) in which microtubules were immobilized on coverslips by means of surface-adsorbed anti-tubulin antibodies. Individual rhodamine-labelled microtubules and single MCAK-GFP molecules were revealed by epifluorescence and total-internalreflection fluorescence (TIRF) illumina...

A driving and coupling “Pac-Man” mechanism for chromosome poleward translocation in anaphase A

“…2B Inset correspond to the times when the kinetochore jumps back and forth between the tip and the next tubulin subunit. Thus, the kinetochore velocity is closely coupled and controlled by the microtubule dissociation rate, which is tightly regulated in cells (1)(2)(3)(4)(28)(29)(30)(31)(32). This coupling mechanism explains the kinetochore velocity insensitivity on chromatid sizes (3,15,16), which directly correlate with its viscous drag.…”

“…If this is true, then slow microtubule dissociation would decouple the kinetochore translocation and the microtubule end. In the in vitro experiment (6), the maximum microtubule dissociation rate from one end is Ϸ1.95 m͞min (32), thus the microtubule tip should shorten at most Ϸ325 nm for 10 s. Thus, at least for some period of time, this should lead to a separation of Ͼ400 nm between the microtubule tip and the kinetochore, which is not observed in ref. 6.…”

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

“…Of particular interest are members of the kinesin superfamily that regulate the polymerization dynamics of MTs (Wordeman, 2005;Moores and Milligan, 2006;Howard and Hyman, 2007). These include members of the kinesin-13 family (Kif2A, Kif2B, and MCAK/Kif2C), which seem to be strictly MT-depolymerizing enzymes (Desai et al, 1999;Hunter et al, 2003;Helenius et al, 2006), as well as members of the Kinesin-8 family, which are MT plus end-directed motors and plus end-specific depolymerases (Gupta et al, 2006;Howard and Hyman, 2007;Mayr et al, 2007;Varga et al, 2008). MCAK has been shown to be a critical regulator of MT dynamics and organization in vitro, in Xenopus egg extracts and in mammalian cells (Walczak et al, 1996;Desai et al, 1999;Maney et al, 2001;Kline-Smith and Walczak, 2002;Cassimeris and Morabito, 2004;Zhu et al, 2005;Stout et al, 2006;Manning et al, 2007;Ohi et al, 2007;Wordeman et al, 2007;Hedrick et al, 2008).…”

Characterization of kinesin-like proteins in silkworm posterior silkgland cells

“…Previous work has revealed that members of Kinesin-13 subfamily are microtubule depolymerases. Kineins-13s form rings [9], rapidly target microtubule ends [32], form a complex at the tips, and depolymerize microtubules processively [7]. Here, since BmKinesin-13 shared a high sequence similarity with its homologues of Kinesin-13s, we speculated that BmKinesin-13 might possess similar activity to To test this idea, we overexpressed BmKinesin-13 in BmN cells and examined the integrity of the microtubule network.…”