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, 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. 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.

‘Dual-trap_viewer_103124.pxp’ displays microtubule plus end position vs time of each microtubule in a growing pair, labeled with the stiffness used to couple that pair. ‘Raw’ and re-smoothed force vs time data is also displayed for each microtubule (see details about re-smoothing under ‘Data available without the use of Igor Pro 9’). In our most recent update, the user can now view either measured tip position or the tip position adjusted for system stiffness. Please see ‘Update to dual-trap data and simulations.pdf’ for details about how and why wanted to use tip position adjusted for system stiffness. This document also describes the correction of a minor bug affecting the tip separations of our simulated data, although this bug does not affect any of the files uploaded here and does not alter any conclusions of our publication.

Code/Software

To run the dual-trap data viewer, install the free trial of Igor Pro 9 at https://www.wavemetrics.com/software/igor-pro-9 and download ‘Dual-trap_viewer_103124.pxp.’

When you load this file, a front panel will appear and allow you to click through recordings of growing microtubule pairs using the ‘Next Trace’ and ‘Previous Trace’ buttons. Look at traces recorded with either soft or stiff couplers (κ = 1 or 5 pN/um) by clicking the up or down arrows to the right of ‘Spring stiffness.’ Use the checkboxes to look at measured tip position and/or tip position adjusted for system stiffness. See ’Dual-trap example document.docx’ for the description of an example recording using this viewer.

The Dual-trap_analysis.txt (available at https://doi.org/10.5281/zenodo.8433109) contains functions we used to collect and organize dual-trap data, as well as to gather statistics about the time-dependent tip separation between microtubules in a pair.

Data available without the use of Igor Pro 9

Additionally, each recording is saved in two separate .csv files, one with the measured tip positions reported in our original publication, the other with tip positions adjusted for system stiffness (please see ‘Update to dual-trap data and simulations.pdf’ for details about this adjustment). These files are available in folders within ‘Individual_trace_data.zip.’

The .csv files with raw tip positions contain 7 columns, the contents of which are listed below:

1. Measured tip position of microtubule 1 (in nm)
2. Measured tip position of microtubule 2 (in nm)
3. Smoothed force on microtubule 1 (in pN) – smoothed from original data
4. Smoothed force on microtubule 2 (in pN) – smoothed from original data
5. ‘Raw’ force on microtubule 1 (in pN)
6. ‘Raw’ force on microtubule 2 (in pN)
7. Time (in s)

The smoothed forces in columns 3 and 4 are smoothed from the original data using a 0.5s smoothing window, so the first and last 0.25s of smoothed forces are affected by forces 0.25s before and after (respectively) the ‘raw’ forces in columns 5 and 6. This occasionally results in a small jump in force in the first or last 0.25s of the smoothed forces. Since this jump can affect our estimates of the initial force and thus the correction we need to apply to adjust for system stiffness, we performed the same smoothing using only the ‘raw’ forces in columns 5 and 6 and report these resmoothed forces in the .csv files described below.

The .csv files with tip positions adjusted for system stiffness contain 4 columns, the contents of which are listed below:

1. Tip position of microtubule 1, adjusted for system stiffness (in nm)
2. Tip position of microtubule 2, adjusted for system stiffness (in nm)
3. Re-smoothed force on microtubule 1 (in pN)
4. Re-smoothed force on microtubule 2 (in pN)

‘Dual-trap example document.docx’ contains an example recording and summary statistics so that users can ensure that they are downloading and interpreting these files correctly.

Finally, in Dual-trap assembly metadata.xslx, each row contains the following information about a given recording:

1. File name
2. Duration (in s)
3. How the event ended (detach, catastrophe, or other)
4. Average growth speed of microtubule 1 (in nm/s)
5. Average growth speed of microtubule 2 (in nm/s)
6. Estimated simulated spring constant (in pN/um)
7. How far ahead the microtubule tip was that started to disassembly (labeled ‘catastrophe’ and measured in nm, if applicable). Positive values indicate that the leading tip underwent catastrophe, while negative values indicate that the lagging tip underwent catastrophe.

For recording 21_0906_044016, this pair probably used a stiff coupler because of the previous entry but the pair stayed too close together for the algorithm to tell.

Other microtubule dynamics, such as average growth speed and catastrophe rate, were also measured for the entire population of soft or stiff couplers and are shown in this excel file.

Change log

Update on 11/13/24: We adjust our tip position recordings to account for series stiffness of our laser trap setup. Both raw and adjusted tip positions can now be downloaded or viewed using ‘Dual-trap_viewer_103124.pxp.’ Dual-trap_assembly_metadata.xlsx’ does NOT use tip positions adjusted for series stiffness. We describe this update in detail in ‘Update_to_dual-trap-data_and_simulations.pdf.’ This document also describes the correction of a minor bug affecting the tip separations of our simulated data, although this bug does not affect any of the files uploaded here and does not alter any conclusions of our publication.