Measuring Pushing and Braking Forces Generated by Ensembles of Kinesin-5 Crosslinking Two Microtubules.

Proteins Full-length Xenopus laevis kinesin-5 tagged at the C-terminus with EGFP was expressed and purified as previously described ( Kwok et al., 2006 ). Microtubules were polymerized from a mixture of X-rhodamine tubulin, biotinylated tubulin and unmodified tubulin with 1 mM GMPCPP and stabilized by Taxol. A rigor kinesin mutant, used for bead-microtubule attachment, was expressed and purified as previously described ( Rice et al., 1999 ).

Assays

Measurement of kinesin-5 force development was performed on an inverted microscope equipped with force-calibrated optical tweezers and dual-mode TIRF and Epifluorescence imaging optics ( Forth et al., 2014 ). To generate microtubule ‘sandwiches’ crosslinked by kinesin-5, a flow chamber assembled with a PEG-coated coverslip ( Subramanian et al., 2013 ) was incubated with reagents in the following order: 0.5 mg/ml α-casein for 3 min, 0.2 mg/ml neutravidin for 5 min, biotinylated microtubules for 10 min, and motility sample (non-biotinylated microtubules, 1 nM kinesin-5-GFP, 2 mM ATP, 3 mM MgCl 2 , 1 mM EGTA, 60 mM PIPES (pH 6.8), 4.5 mg/ml glucose, 350U/ml glucose oxidase, 34U/ml catalase, 0.5 mg/ml α-casein, 1 mM DTT, 20 μM Taxol and ~0.1 pM beads). After sealing the chamber with VaLaP, a microtubule pair was selected and an optically-trapped bead was attached to the non-biotinylated microtubule within 2-5 microns of the region of microtubule overlap to prevent filament buckling. The bead position was recorded using a quadrant photodiode (0.1-0.5 kHz sampling rate). Microtubules and kinesin-5-GFP were imaged (interval: 1-5 s; exposure time: 200-300 ms), in parallel with the force measurement. For measurements within sliding microtubule pairs, the piezo stage was moved along the microtubule axis at constant velocity once the kinesin-5 dependent force reached a plateau. The trapped bead was held ~1 μm above the coverslip surface, where the trap stiffness was determined to be 0.065 pN/nm. For all experiments, beads were held within ±250 nm of the trap center to maintain a linear trap stiffness. The rupture-based counting method was performed by moving the sample stage orthogonal to the surface microtubule at a speed of 0.3 μm/s while holding the trap center at a fixed position. The motion of the trapped bead was recorded at 0.5 kHz and processed as described to estimate the rupture number. For microtubule flipping experiments, the bead-held microtubule was detached from the surface microtubule and then flipped by 180 ° using fluid flow induced by motion of the sample stage. The flipped filament was then brought into contact with the same surface microtubule.

Data analysis

The maximum force developed within antiparallel microtubule pairs was determined at the plateau of the force development record, defined as the region where the force signal persisted for more than 1 s without significant fluctuation or sudden drop (