Paired microtubules growing with a shared load

During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each chromosome. However, individual microtubules grow at intrinsically variable rates, which must be tightly regulated for a k-fiber to behave as a single unit. This exquisite coordination might be achieved biochemically, via selective binding of polymerases and depolymerases, or mechanically, because k-fiber microtubules are coupled through a shared load that influences their growth. Here, we use a novel dual laser trap assay to show that microtubule pairs growing in vitro are coordinated by mechanical coupling. Kinetic analyses show that microtubule growth is interrupted by stochastic, force-dependent pauses and indicate persistent heterogeneity in growth speed during non-pauses. A simple model incorporating both force-dependent pausing and persistent growth speed heterogeneity explains the measured coordination of microtubul..., The data here was collected using the 'dual-trap assay,' which is based on our previously developed single-trap (force-clamp) assay (Akiyoshi et al., 2010; Miller et al., 2016; Sarangapani et al., 2013), in which dynamic microtubules are grown from stabilized seeds bound to a biotinylated coverslip. Using the single laser trap, we attach an individual bead decorated with isolated yeast kinetochores to the growing plus-end of a single microtubule. A computer then continuously measures the bead position and adjusts the trap to exert a precise, constant level of tension on the microtubule via the kinetochore-decorated bead. Under this persistent feedback-controlled tension, kinetochore-beads typically track with the microtubule tips even as the tips stochastically grow and shorten.
Our new dual-trap assay uses two separate laser trapping microscopes, located adjacent to one another in the same room and connected to a single computer. On each of the two instruments, we attach a kinetochore-..., , # Paired microtubules growing with a shared load

[https://doi.org/10.5061/dryad.xksn02vnb](https://doi.org/10.5061/dryad.xksn02vnb)

Our new dual-trap assay uses two separate laser trapping microscopes, located adjacent to one another in the same room and connected to a single computer. On each of the two instruments, we attach a kinetochore-decorated bead to a dynamic microtubule plus-end. The computer then simultaneously monitors and controls the forces on both microtubules. The computer adjusts the forces dynamically to simulate an elastic coupling of both plus-ends to a single shared load. We have simulated purely elastic couplers in these recordings, with stiffnesses of *κ* = 1 or 5 pN/um.

To begin a dual-trap experiment, we first choose the spring stiffness, *κ*, and the total shared load, *FTOT*, which are kept constant. After a kinetochore-decorated bead is attached to a growing plus-end on each of the two instruments, feedback-control is initiated and the two plus-ends are ar...