The overall goal of this procedure is to use a gliding assay to measure the UAL rigidity of microtubules. This is accomplished by first specifically attaching the motor protein kinesin to a microscope slide with motor domains free to bind microtubules. The second step is to add fluorescently labeled microtubules to the sample, which are translated by the kinesin.
Next videos of gliding microtubules are collected using fluorescence microscopy. The final step is analyzing the gliding assay videos to calculate the micro tubule flexural rigidity. Ultimately, the gliding assays are used to determine the flexural rigidity of single microtubules.
This method can help answer key questions in the field of cytoskeletal mechanics, such as the bending stiffness of individual cytoskeletal polymers. Demonstrating this procedure will be Anna Ratliff, a student from my laboratory. To begin this procedure, prepare the working assay buffer by adding 10 microliters of 200 milli mole DIO three atol or DTT to one milliliter of the stock assay buffer solution and mix.
Keep this and all other working solutions at room temperature during the procedure. Next, using the working assay buffer, prepare 40 microliters of biotin BSA buffer by combining sterile filtered biotinylated bovine serum albumin at two milligrams per milliliter with the working assay buffer in a one-to-one ratio and mixing the solution. Then prepare the BSA buffer by combining 645 microliters of assay buffer with 5.5 microliters of 117 milligrams per milliliter BSA, and mixing the solution.
Also prepare the tritin buffer by mixing 57 microliters of assay buffer with three microliters of sterile filtered strep din at 10 milligrams per milliliter in assay buffer. Next, construct the flow lanes on a 24 by 60 millimeter cover slip extrude five thin lines of vacuum grease from a 10 milliliter syringe. Using a 200 microliter pipette tip to regulate the flow, the lines should be approximately four millimeters apart.
Place a 22 by 22 millimeter cover slip on top of the vacuum grease and lightly press down to form the channels. With an approximate volume of 10 microliters, the vacuum grease lines should spread out to approximately twice the original width. The next step is to preco the lanes with biotinylated bovine serum albumin by pipetting 10 microliters of the bioTE BSA buffer into each flow lane using capillary force to draw in the buffer incubate for five minutes at room temperature.
Then rinse each lane three times by injecting 15 microliters of the BSA buffer into one side of each lane while drawing the solution out the other side using a Kim wipe or filter paper. Take care not to introduce air bubbles into the lanes. Next flow, 15 microliters of streptavidin buffer into each lane and incubate for up to two hours with a minimum of 10 minutes.
During the incubation, prepare the alpha KC in buffer by mixing 200 microliters of BSA buffer 50 microliters of sterile filtered alpha KC in at five milligrams per milliliter in assay buffer and 0.8 microliters of 15 micromolar a TP at pH seven. Also prepare the kinesin solution by pipetting the alpha KCM buffer into a new tube and adding the biotin kinesin to 10 nano molar and then mixing following incubation with the streptavidin buffer. Rinse each lane three times with 15 microliters of BSA buffer.
Then rinse each lane with 15 microliters of the alpha kcn buffer in order to reduce non-specific binding of kinesin and microtubules to the glass. Next, add 15 microliters of kinesin solution to each lane and incubate for 15 minutes to an hour. During this time, the biotin kinesin will form a strong bond with the surface bound streptavidin and allow the kinesin motor domains to be accessible for binding microtubules.
In the meantime, prepare a solution of 40 micromolar paclitaxel and alpha KC and buffer. The Paclitaxel is important for preventing microtubules from depolymerizing. In a fume hood, prepare the fluorescence anti bleaching buffer by mixing together 95 microliters of alpha KCM buffer, 2.5 microliters of glucose stock, one microliter of 100 x oxygen scavenging.
Mix one microliter of Paclitaxel and DMSO at four millimolar. And finally, one microliter of beta me capto ethanol. Following incubation, rinse away any free biotin kinesin by washing each lane with 15 microliters of room temperature.
Paclitaxel in alpha Kian solution using cold buffer may depolymerize microtubules. Finally pipee the fluorescence anti bleach buffer to a new tube and add the fluorescently labeled microtubules to a dilution of one to 100 to one to 1000. Then add one millimolar a TP and mix.
Inject 15 microliters of the micro tubule solution into one lane and observe using fluorescence microscopy within 30 minutes. The most critical aspects of this procedure is to have an optimal concentration of microtubules to ensure they don't overlap and to have a low background. The remaining lanes are used to optimize the micro tubule concentration, and we use the microscope itself to align and focus them.
The imaging of gliding microtubules is done using a homemade total internal reflection fluorescence microscope. A 100 x high numerical aperture objective displays a field of approximately 50 microns by 50 microns, which is enough space to view movement for approximately 100 seconds at a rate of 0.5 microns per second set. The laser used for excitation to a power setting of three to five milliwatts.
This power is low enough to prevent photobleaching of the floor of fours more quickly than 100 seconds to allow for adequate imaging time. Using image capture software, collect sequences of images at five frames per second for two minutes. Sequences should be long enough for the microtubules to traverse the entire field of view.
Analyze the image data using an IDL routine such as the file, get LP Pro, which can be obtained from the supplementary material. This DL routine returns a persistence length value that is based on all microtubules gliding in a given image sequence. Modify the intensity parameters depending on fluoro four intensity and microscope setup.
Shown here is a gliding assay taken with total internal reflection fluorescence microscopy. The Fluor fours can be seen sparsely decorated along the length of the microtubules. The single fluoro four trajectories just shown are merged, combining an average of 10 Fluor fours into a single point per 100 nanometers to improve data analysis.
When looking at a single microtubule, the tangent angle between each point can be easily calculated and mapped as a function of length. Using tangent angles, the persistence length can be calculated and the average cosign per segment length can be plotted using the equation shown here. A best fit line can be determined Once mastered.
This technique can be used to measure the bending stiffness of several dozen microtubules in three or four hours if it is performed properly.
