Paired microtubules growing with a shared load

Description

Other

Funding provided by: National Institutes of Health
Crossref Funder Registry ID: https://ror.org/01cwqze88
Award Number:

Funding provided by: Howard Hughes Medical Institute
Crossref Funder Registry ID: https://ror.org/006w34k90
Award Number:

Methods

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-decorated bead to a dynamic microtubule plus-end. The computer then simultaneously monitors and controls the forces on both microtubules. Rather than keeping the force constant on each microtubule, the computer adjusts the forces dynamically to simulate an elastic coupling of both plus-ends to a single shared load. Thus far, we have simulated only purely elastic couplers, where both coupling springs are linear (Hookean) with stiffness, κ.

To begin a dual-trap experiment, we first choose the spring stiffness, κ, and the total shared load, F_TOT, 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 arbitrarily considered to be parallel, with tips side-by-side (i.e., both at x₁ = x₂ = 0) and sharing the load equally (F₁ = F₂ = ½·F_TOT). Because microtubule growth is intrinsically variable, the two microtubules subsequently grow at different speeds. The computer then dynamically monitors the bead positions and adjusts the forces, F₁ and F₂, according to the elastic coupling model. For purely elastic couplers, the force difference across the two microtubules equals the tip separation, (x₂ – x₁), multiplied by the coupling stiffness (κ). When one microtubule grows more quickly than the other, tension on the leading (faster-growing) microtubule decreases, and tension on the lagging (slower-growing) microtubule increases to maintain a constant total force on the pair, F_TOT. For all experiments, F_TOT = 8 pN.