Our protocol allows researchers to build cytoskeletal motifs out of purified components and directly measure the forces these networks generate in order to understand how these mechanical components of the cell function in both healthy and disease states. This method allows us to quantify forces and correlate these numbers directly to keep parameters of the proteins involved, including protein concentration and density. Parameters that are generally impossible to acquire within the cell.
This method can provide insights into any type of cytoskeleton network, such as those involved in mitosis or neural development. It can readily be adapted to study networks involved in muscle contraction or cell migration by simply swapping out this specific proteins being used. The trickiest aspect of this method is the coordination of the different technologies, which include a single-beam optical trap, and a TIRF microscope.
Additionally, single-molecule experiments require careful preparation of surfaces, such as the cover slip, so preparing high-quality samples is paramount. Start with constructing a humidity chamber by placing a folded-up, lint-free tissue dampened with ultrapure water at the bottom of an empty pipette tip box. Introduce all the reagents by pipetting the liquid at one end of the channel and wicking the liquid out at the opposite end.
Using a 20-microliter-angled pipette tip, slowly flow in 10 microliters of 0.5 milligrams per milliliter streptavidin or NeutrAvidin. Incubate the slide in the humidity chamber for 4 minutes with the cover slip facing down. Flush out the chamber using 20 microliters of a 1x BRB80 using a lint-free tissue to draw out liquids.
After repeating flowing reagents and incubation one more time, flush step with 10 microliters of 0.5 milligrams per milliliter casein blocking solution. Flow 10 microliters of diluted biotinylated-far-red microtubules into the chamber, then flush the chamber with 20 microliters of 1x BRB80. After flowing the reagent and incubating as demonstrated previously, flush the step with 10 microliters of an appropriate concentration of non-motor protein to be studied, then flow in 10 microliters of rhodamine microtubules and repeat the incubation and flushing.
Next, combine 1 microliter of rigor kinesin-coated beads and 19 microliters of reaction buffer. Seal both edges of the chamber with clear nail polish and wait until the polish dries. Employ an inverted microscope instrument that has total internal reflection fluorescence imaging capabilities, and into which a single-beam optical trap has been constructed.
Add 1 drop of immersion oil onto the cover slip of the sample chamber. With the cover slip side of the sample chamber facing downward, place the sample to be imaged on the microscope platform. Slowly bring the objective up so that there is contact between both the cover slip and the objective through the oil.
Adjust the objective height so that the cover slip surface of the sample chamber is in focus. Record single-frame images of the samples in all three channels. For the experiments here, use 100 to 200 milliseconds of exposure.
Adjust the laser power to achieve a good signal-to-noise ratio from the samples while minimizing photo bleaching over 2 to 5 minutes when the individual bundle is assayed. The frequency of microtubule bundles per field of view should be approximately three to four microtubule bundles per 100 microliters by 100 microliters field of view per chamber. Observe the density of beads in the sample using a bright field and the 100x objective on the microscope.
Employ a concentration of beads such that the beads are a minimum of 10 micrometers from one another on average, and ensure that they are free from any kind of surface confinement or clustering. Ensure that the biotinylated far-red microtubules are firmly attached to the surface of the cover slip and that the rhodamine with no visible Brownian motion. Ensure that the rhodamine microtubules are attached to biotinylated microtubules via the cross-linking map and are visibly fluctuating at their free ends.
Identify a suitable microtubule bundle that contains only two microtubules, one biotinylated with full surface attachment and one non-biotinylated microtubule that is partly free in solution and is aligned on either the x or y-axis. Use the optical trap to capture a free bead demonstrating Brownian motion. Once the bead has been trapped, carefully move the bead over to the selected microtubule bundle ensuring no other beads are pulled into the trap in the process.
Ensure that the bead is several microns away from the overlap region containing cross-linking proteins. Carefully lower the bead in the z-axis until it makes contact with the edge of the free segment of the rhodamine microtubule. Pull up gently and move perpendicular to the microtubule axis to check for attachment by observing the rhodamine microtubule bending and following the bead position.
Carefully realign the rhodamine microtubule along the microtubule axis of the surface-attached microtubule by moving the nano-positioning stage and set up the parameters for automated pulling of the microtubule bundle. Set the direction along the parallel axis of the microtubules. Set the desired pull speed and desired pull time depending on overlap length.
Begin recording fluorescence data in the three channels at a rate of 1 to 2 frames per second. Use the optimized the laser powers and exposure times. Initiate automated stage motion and record trapping data from the position-sensitive detector stage and total internal reflection fluorescence camera stage.
Deactivate the traps to release the bead and repeat the experiment as desired. Ensembles of the essential mitotic motor protein kinesin-5 can regulate microtubule sliding by generating both pushing and braking forces that scale linearly with overlap length. It was found that the C-terminal tail domain is required for efficient cross-linking and force generation.
The full-length protein is able to generate sustained forces that plateau when all motor proteins reach their individual stall force. However, the kinesin-5 motor lacking its C-terminal tail generates maximum forces that are nearly fivefold smaller in magnitude. Ensembles of the non-motor mitotic protein PRC1 operate as a mechanical dash pod to resist microtubule sliding in anaphase spindle mid-zone.
The resistive forces do not depend on the length of overlap regions between microtubule pairs or the local crosslinker density, but they do strongly depend on the total concentration of engaged PRC1 molecules. This method can be adapted to study diverse biological systems such as microbial organization and neurons by using alternate proteins. It can also readily be expanded to analyze actin-based network geometries to study muscle contraction or cell motility.
This technique lets one study unique cell total geometries, where filaments are both track and cargo and are organized by small ensembles of proteins. These geometries better mimic what's happening in cells during numerous biological processes.
