Microtubules growing and shortening under constant force

Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct 'bi-oriented' kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single mi..., In these experiments, microtubules were grown with a yeast-kinetochore-decorated bead attached to the microtubule's plus end. We applied constant forces ranging from 0.5 to 17 pN to the plus end via the bead and recorded the microtubule's growth and/or shortening, as well as the stochastic switching between these two states., , # Microtubules growing and shortening under force clamp

[https://doi.org/10.5061/dryad.6djh9w16v](https://doi.org/10.5061/dryad.6djh9w16v)

In these experiments, microtubules were grown with a yeast-kinetochore-decorated bead attached to the microtubule's plus end. Using an optical trap, we applied constant forces ranging from 0.5 to 17 pN to the plus end via the bead and recorded the microtubule's growth and/or shortening, as well as the stochastic switching between these two states. A switch from growth to shortening is termed a 'catastrophe,' while a switch from shortening to growth is termed a 'rescue.'

Force-clamp viewer.pxp displays microtubule plus end position vs time and is labeled with the corresponding force under which that microtubule was grown. Growth speed vs time data is also displayed using a 2-second running velocity, as is analysis of pausing behaviors (when microtubules stochastically stop growing during periods of net growth) based on this running velocity.

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