Microtubules growing and shortening under constant force
Data files
Nov 01, 2023 version files 276.54 MB
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Force-clamp_viewer.zip
276.54 MB
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README.md
1.81 KB
May 27, 2024 version files 1.25 GB
Abstract
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 microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.
README: Microtubules growing and shortening under force clamp
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.
Code/Software
To run the force-clamp viewer, install the free trial of Igor Pro 9 at https://www.wavemetrics.com/software/igor-pro-9, download Force-clamp_viewer.zip, and extract all files to access Force-clamp_viewer.pxp.
When you load this file, a front panel will appear and allow you to click through recordings within a given force range with the 'Next Trace' and 'Previous Trace' buttons. Click the checkbox next to 'Mark pauses?' to highlight pauses in purple, but please note that graphs take longer to generate when this option is selected.
Force-clamp_viewer_091323.txt (available at https://doi.org/10.5281/zenodo.8433062) is an external copy of the software used to run the force-clamp viewer, but is not necessary to download to run Force-clamp_viewer.pxp.
Data available without the use of Igor Pro 9
Additionally, each recording is saved in a separate .csv file. Each .csv file contains 4 columns, the contents of which are listed below:
- Tip position of the microtubule (in nm)
- Smoothed force on the microtubule (in pN)
- 'Raw' force on the microtubule (in pN)
- Time (in s)
Force-clamp example document.dox contains an example recording and summary statistics so that users can ensure that they are downloading and interpreting these files correctly.
Finally, in Force clamp metadata.csv, each row contains the following information about a given recording:
- File name
- Average force (in pN)
- Standard deviation of force (in pN)
- Duration of the assembly period, if present (in s)
- Growth speed during the assembly period, if present (in nm/s)
- Duration of the pre-catastrophe pause period, if present (in s)
- Growth speed during the pre-catastrophe pause period, if present (in nm/s)
- Duration of the shortening period, if present (in s)
- Shortening speed during the shortening period, if present (in nm/s)
- How the event ended (detach, rescue, interrupted, or other)
Methods
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.