Chen Y and Hancock WO (2015), Kinesin-5 is a microtubule polymerase.

XB-ART-52074

Nat Commun 2015 Oct 06;6:8160. doi: 10.1038/ncomms9160.

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Kinesin-5 is a microtubule polymerase.

Chen Y , Hancock WO .

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Kinesin-5 slides antiparallel microtubules during spindle assembly, and regulates the branching of growing axons. Besides the mechanical activities enabled by its tetrameric configuration, the specific motor properties of kinesin-5 that underlie its cellular function remain unclear. Here by engineering a stable kinesin-5 dimer and reconstituting microtubule dynamics in vitro, we demonstrate that kinesin-5 promotes microtubule polymerization by increasing the growth rate and decreasing the catastrophe frequency. Strikingly, microtubules growing in the presence of kinesin-5 have curved plus ends, suggesting that the motor stabilizes growing protofilaments. Single-molecule fluorescence experiments reveal that kinesin-5 remains bound to the plus ends of static microtubules for 7 s, and tracks growing microtubule plus ends in a manner dependent on its processivity. We propose that kinesin-5 pauses at microtubule plus ends and enhances polymerization by stabilizing longitudinal tubulin-tubulin interactions, and that these activities underlie the ability kinesin-5 to slide and stabilize microtubule bundles in cells.

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Species referenced: Xenopus laevis

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	Figure 1. Kinesin-5 stabilizes GMPCPP microtubules.(a) Diagram of dimeric kinesin-5 constructs Kin5_18 and Kin5_14 (ref. 33). (b,c) Kinesin-5 slows depolymerization of GMPCPP microtubules. Surface-immobilized GMPCPP microtubules were incubated in the presence or absence of motors, as indicated. (b) Superimposed before/after images with initial microtubule image in red and microtubule after 20 min in green. Scale bar, 1 μm for all. (c) Mean depolymerization rate in the absence and presence of 20 nM Kin5_14 or 25 nM Kin5_18 over a 20-min interval. P values are from two sample t-tests.
	Figure 2. Kinesin-5 acts as a microtubule polymerase.(a) Sample images of microtubules extended from GMPCPP seeds for 5 min in 10 μM free tubulin in the presence and absence of 20 nM Kin5_18. Scale bar, 2 μm (for all). (b,c) Microtubule growth rate and catastrophe frequency in the absence of motor (control), or in the presence of 100 nM kinesin-1 or 20 nM Kin5_18, showing that kinesin-5 causes a significant increase in the growth rate and significant decrease in the catastrophe frequency. (d) Diagram of proposed plus-end structures for microtubules growing under different conditions. (e) Fluorescence intensity profile of growing microtubule plus ends. Line scans (points) of fluorescence intensity along microtubules were each normalized relative to background, and then fit by error functions (lines). (f) Tip s.d. values obtained from error function fits in e, showing that Kin5_18 causes significant increase plus-end tapering. (g) Diagram of simulation used to predict fluorescence profile of tapered microtubule plus ends. To simulate the end tapering of a 13 protofilament microtubule, the longest protofilament was fixed at Nmax tubulin subunits longer than the shortest protofilament, and the lengths of the remaining 11 protofilaments were randomly selected from 0 to Nmax. The dye density (1 dye per 20 tubulin dimers) matched the experimental conditions. (h) An exemplary intensity profile and corresponding fit from a simulation with Nmax=150 tubulin. (i) Plot of tip s.d. versus Nmax, showing that s.d.tip of 222 for the control corresponds to tapering over ∼60 tubulin subunits and s.d.tip of 418 nm for the motor case corresponds to tapering over 140 tubulin. All error bars in figure represent s.e.m.
	Figure 3. Diverse plus-end structures of microtubules grown in the presence of kinesin-5.(a–e) Images of microtubules growing in the presence of 10 μM tubulin and 30 nM Kin5_18GFP, showing straight (a), bifurcated (b), curved (c,d) and looped (e) plus ends. Tubulin was unlabelled and fluorescence was solely due to the Kin5_18GFP. (f) Annealing of a ‘banana peel'. In this case, the microtubule initially grows with a bifurcated plus end, one side straightens at 134 s, and the second side straightens and anneals between 135.5 and 140 s. For full sequence, see Supplementary Movie 2. (g,h) Images and diagram of protofilaments curling to make a loop and then breaking. The curled plus end at 0 s grows to form a closed loop at 91 s, which breaks at 98 s. For full sequence, see Supplementary Movie 3.
	Figure 4. Kinesin-5 pauses at plus ends of taxol-stabilized microtubules.(a,b) Kin5_18GFP and Kin5_14GFP (5 and 3 nM, respectively, in 1 mM ATP) walking on and accumulating at plus ends of taxol-stabilized microtubules. (c,d) Sequence of images (c) and kymograph (d) at 25 pM motor concentration, showing a single Kin5_14GFP molecule walking along a taxol-stabilized microtubule and pausing at the plus end before dissociating. (e) Distribution of motor residence times at plus ends of taxol-stabilized microtubules, along with mean and s.e. of exponential fit. N=137 and 98 for Kin5_18 and Kin5_14, respectively. (f) Simplified hydrolysis cycle of a motor waiting at a microtubule plus end. Three possible explanations for the end-binding duration are that the motor binds in a two-head-bound configuration, the motor undergoing futile hydrolysis cycles, or ATP hydrolysis requires binding of the tethered head to the next tubulin on the lattice (see text for further details). (g) Diagram and kymograph of Kin5_18GFP landing on and dissociating from an immobilized microtubule in 1 mM ADP. (h) Distribution of motor binding durations in 1 mM ADP, along with mean and s.e. of exponential fit to the data, giving an average residence time that is considerably shorter than the dwell time at plus ends in ATP.
	Figure 5. Plus-end pausing under crowded conditions.(a) Diagram of spiking experiments. (b) Kymograph of Kin5_14GFP walking to plus end of a taxol-stabilized microtubule in the presence of excess unlabelled motors. (c) Distribution of dwell times of motors at plus ends under crowded conditions, with exponential fits to data, resulting mean durations of 2.26s and 2.61 s for Kin5_18 and Kin5_14, respectively. Experimental conditions: 50 pM Kin5_14GFP plus 15 nM unlabelled Kin5_14, and 70 pM Kin5_18GFP plus 25 nM unlabelled Kin5_18.
	Figure 6. Kinesin-5 tracks growing microtubule plus ends.(a,b) Images and kymographs of Kin5_18GFP and Kin5_14GFP labelling plus ends of growing microtubules. Conditions were 20 nM Kin5_14GFP or 30 nM Kin5_18GFP, 20 μM tubulin and 1 mM ATP. (c) Single-molecule kymograph. A total of 50 pM Kin5_14GFP and 20 μM tubulin were used. (d) Mean duration of kinesin-5 residence at plus ends, compared with motor step duration on microtubule lattice. (e) Gel filtration of 3 μM Kin5_14 and 20 μM tubulin in 1 mM ATP shows distinct peaks of tubulin and motors and no evidence of motor–tubulin complex.
	Figure 7. Proposed kinesin-5 polymerase mechanism.Motors walk to the plus end of protofilaments where tubulin subunits are being rapidly and reversibly exchanged (state 1). If the motor steps before a newly added tubulin subunit dissociates, then it stabilizes the longitudinal tubulin–tubulin interactions and slows the tubulin off-rate (state 3). This stabilization enhances the microtubule growth rate and suppresses the catastrophe frequency. Over time, growing protofilaments will be stabilized by lateral interactions with adjacent protofilaments (state 4), leading to long and stable protofilament bundles. Following catastrophe, the rapid depolymerization rates preclude plus-end motor accumulation and hence are expected to have little effect on the depolymerization rate.

References [+] :

Ayaz, A tethered delivery mechanism explains the catalytic action of a microtubule polymerase. 2014, Pubmed, Xenbase