Cellular microtubule arrays contains subpopulations of microtubules with distinct dynamic properties as required for function. This protocol addresses how the collective activity of regulators bestows proximal microtubule subpopulations with distinct properties. This bottom-up restitution assay enables us to simultaneously visualize the organization and dynamics of proximal microtubule populations such as single microtubules and bundles, and to decipher the mechanisms underlying their self-organization.
Multi-component reconstitution require optimizing experimental parameters like pH, ionic strength, and protein concentration while preserving the activity of each. Systematic analysis of individual components prior to multi-protein assays is extremely helpful. To begin, pipette a mixture of 4.5 microliters of BRB ADDTT in one microliter of microtubule solution onto a microscope slide.
Cover with an 18 by 18 millimeter coverslip and seal the edges with either clear nail polish or VALAP sealant. Position the TIRF objective beneath the coverslip. Visualize the newly polymerized microtubules at a wavelength appropriate for the fluorescently labeled tubulin in the bright mix to determine what dilution of microtubules to use in the upcoming experiments.
Determine the laser intensity for the experiment empirically such that all fluorescent proteins are illuminated, but do not undergo significant photobleaching over the time course of the experiment. Set the microscope temperature to 28 degrees Celsius to view dynamic microtubules. Use lens paper to clean a 100X objective with 70%ethanol.
Use the best combination of filter cubes and emission filters depending on the fluorescent channels to be imaged. Set up the imaging sequence. For an experiment with 647 nanometer fluorophore-labeled biotinylated microtubules, 560 nanometer fluorophore-labeled non-biotinylated microtubules in soluble tubulin, and GFP-labeled protein of interest, image the 488 nanometer, 560 nanometer, and 647 nanometer channels every 10 seconds for 20 any minutes.
To capture a reference image of bundles before the addition of soluble tubulin and microtubule-associated protein, set up a sequence with one image each in 560 nanometer and 647 nanometer wavelength channels. Check the position of laser beam on the ceiling above microscope and make changes to beam using software. Ensure that the laser illuminator is aligned.
Prior to imaging, add a drop of microscope immersion oil to the objective. Prepare the soluble tubulin mixture while keeping it on ice and spin down the mixture. To immobilize microtubules via biotin-NeutrAvidin-biotin linkage, first flow in approximately 7.5 microliters of NeutrAvidin solution until the chamber is filled and incubate for five minutes.
Wash with 10 microliters of MB cold. Flow in 7.5 microliters of the blocking protein KC and incubate for two minutes. Wash with 10 microliters of MB warm to prepare the chamber for the introduction of microtubules.
Dilute the stock of biotinylated microtubules in BRB ADDTT and add one microliter of this dilution to nine microliters of MB warm. Flow the mixture into the chamber and incubate for 10 minutes. Wash away non-immobilized microtubules with 10 microliters of MB warm.
Flow 7.5 microliters of warm KC into the chamber and incubate for two minutes. During the incubation, prepare a two nanomolar solution of the crosslink or protein PRC1 in KC and spin down the solution. Flow 10 microliters of this solution into the imaging chamber and incubate for five minutes.
To make bundles, flow 10 microliters of non-biotinylated microtubules into the chamber and incubate for 10 minutes. Wash the chamber twice with 10 microliters of MB warm. During the 10-minute incubation time, prepare 10 microliters of assay mixture containing proteins of interest, soluble tubulin, nucleotides, oxygen scavengers, and antioxidants and spin it down.
Keep the mixture on ice. Load the prepared imaging chamber taped to slide holder on the 100X TIRF objective. Use the 560 nanometer and 647 nanometer channels to find a field of view that contains an optimum number and density of single microtubules and bundles.
Once a field of view is identified, take a reference image. Carefully flow in the assay mixture without disturbing the imaging chamber. Seal the open ends of the chamber with VALAP sealant.
Observe the microtubules and start the imaging sequence. After immobilizing microtubule seeds and generating bundles, fluorescence images were obtained. Single microtubules were identified by fluorescent signal in the 647 nanometer channel, whereas microtubules were identified as preformed bundles when they displayed fluorescent signals in both channels.
The dynamics of single microtubules and PRC1 crosslinked bundles were studied in the presence of the proteins KIF4A and CLASP1, which revealed that the single microtubules elongate over the course of the assay, whereas the growth of crosslinked microtubules is stalled. Be careful to keep polymerized microtubules at or above room temperature. Keep soluble tubulin on ice and protect the imaging chamber and fluorescently-labeled microtubules and proteins from light.
These assays provided mechanistic insight into how a protein module found at the mitotic spindle could differentially regulate the dynamics of two different microtubule populations that coexist in the spindle.
