The Saccharomyces cerevisiae kinesin-5 Cin8 is a bidirectional motor protein. Cin8 moves in the minus-end direction of the microtubules as a single molecule, and changes directionality under a number of different experimental conditions. The overall goal of our research is to understand the mechanism of bidirectional motility by studying factors and structural elements that regulate the bidirectional motility of Cin8.
This protocol comprehensively explains the purification of GFP-type full-length Cin8 overexpressed in yeast cells, the single-molecule motility assay, and the subsequent analysis of motile properties of single molecules and clusters of Cin8. This separation is important, since the directionality of Cin8 is affected by its accumulation in clusters on the microtubule. This technique can be easily adapted to purify other nuclear proteins from the yeast.
Additionally, it can be applied to any fluorescent particles that need to be separated into size groups based on their fluorescence intensity. Demonstrating the procedure will be Dr.Mary Popov, our lab manager, Dr.Alina Goldstein-Levitin, and Dr.Nurit Siegler, postdoctoral fellows;Tatiana Zvagelsky, Mayan Sadan, and Shira Hershfinkel, graduate students;and Neta Yanir, Yahel Abraham, Roy Avraham, and Meital Haberman, undergraduate students from our laboratory. To begin, grow Saccharomyces cerevisiae cells containing the plasmid for overexpression of Cin8-GFP-6His to the exponential growth phase in one liter of yeast-selective medium, supplemented with 2%raffinose at 28 degrees Celsius.
Then induce Cin8-GFP-6His overexpression by adding 2%galactose, and monitor the yeast culture growth by measuring absorbance at 600 nanometers. Five hours after galactose addition, harvest the cells by centrifugation. Re-suspend the cells in lysis buffer, and freeze them in liquid nitrogen.
Next, using a chilled mortar and pestle, grind the frozen cells in liquid nitrogen. Keep adding liquid nitrogen during the grinding, so that the extracts remain frozen. Monitor the cell lysis under a phase or differential interference contrast microscope.
Then thaw the ground cells, and centrifuge them. Load the supernatant onto a gravity-flow column filled with two milliliters of Nickel-NTA, and pre-equilibrate it with lysis buffer. Let the supernatant flow out through the column.
Wash the column with five column volumes of lysis buffer, followed by five column volumes of lysis buffer supplemented with 25-millimolar imidazole. Then elute the Cin8-GFP-6His with elution buffer. Analyze the eluted samples by SDS-PAGE fractionation, followed by Coomassie blue staining and Western blot analysis probed with anti-GFP antibody.
After pooling the fractions containing Cin8-GFP-6His, purify them by size-exclusion chromatography at a flow rate of 0.5 milliliters per minute, and a column pressure limit of 1.5 megapascals. Simultaneously monitor the absorbance at 280 nanometers. Collect the fractions corresponding to the Cin8-GFP tetramer and analyze them by SDS-PAGE and Western blotting.
Then aliquot the selected fractions, snap-freeze, and store until use at minus 80 degrees Celsius. To polymerize and stabilize the biotin and rhodamine labeled microtubules, mix the reaction components in a 1.5-milliliter tube, and incubate the mixture for one hour at 37 degrees Celsius. Then add 80 microliters of warm general tubulin buffer, mix carefully, and centrifuge.
Discard the supernatant, and re-suspend the pellet carefully by pipetting up and down with 50 microliters of warm general tubulin buffer. Then store the suspension at 28 or 37 degrees Celsius. Examine the microtubules with a fluorescence microscope, using the 647-nanometer rhodamine channel.
Next, remove the protective paper from the tape strips and place a silanized coverslip on the strips to create three 10-microliter volume flow chambers. Then perform the avidin coating of the coverslip by sequential addition of the indicated reagents. Incubate the coverslip for three to five minutes before washing it with 80 microliters of general tubulin buffer.
Next, attach the biotinylated microtubules to the b-BSA/avidin-coated coverslips by inserting 20 microliters of microtubules into the flow chamber. Incubate the slides with the coverslip facing downwards in a dark humidity chamber for five minutes at room temperature. Then wash the slides with 200 microliters of general tubulin buffer.
Next, apply 30 microliters of motility reaction mix followed by Cin8-GFP motors diluted in 20 microliters of the motility reaction mix into the flow chamber, and immediately proceed to image the movement of the motors along the microtubules. For motor motility imaging, place a drop of immersion oil on the microscope objective, then place the flow chamber on the fluorescent microscope stage, with the coverslip facing the objective. Turn on the rhodamine channel to focus on the microtubules attached to the coverslip surface.
Then, using ImageJ Micro-Manager software, acquire the image with a 20-millisecond exposure. Next, turn on the GFP channel and acquire 90 time-lapse images with a one-second interval and 800-millisecond exposure. In the ImageJ Fiji software, open the time-lapse movie and the corresponding microtubule field image.
Synchronize these two windows by choosing Analyze, followed by Tools and Synchronize Windows. To obtain a kymograph, highlight one microtubule using the Segmented Line option. Then, from Analyze, select the Multi Kymograph tab.
To determine the cluster size of the Cin8-GFP molecules, perform the background subtraction and correction for uneven illumination by clicking on Process, followed by Subtract Background. Set the Rolling ball radius to 100 pixels, and check the Sliding paraboloid option. Then track a specific non-motile Cin8-GFP motor using the TrackMate plugin of the ImageJ Fiji software.
Choose Plugins, followed by Tracking, TrackMate, LoG detector, and Simple LAP tracker to follow the mean fluorescence intensity of the Cin8-GFP motor as a function of time within a circle of four pixels radius. After repeating the procedure for different Cin8-GFP motors, plot the fluorescence intensities as a function of time. Next, for tracking the motility of the Cin8-GFP molecules along the microtubule tracks, crop the microtubules to be analyzed in the time-lapse sequence of recorded frames by highlighting it with the rectangle tool and clicking Image, followed by Crop.
Choose a fluorescent Cin8-GFP particle for the analysis. Record the particle coordinates in each frame of the time-lapse sequence using the point tool and Measure options. Similarly, record coordinates for other fluorescent particles in the time-lapse sequence.
Then assign cluster size to all the examined Cin8-GFP particles in the first frame of their appearance, as demonstrated earlier. Next, using the indicated formula and the determined Cin8-GFP movement coordinates, calculate the displacements of Cin8-GFP at each time point with respect to the initial coordinates. From these displacement values, calculate the displacement for all possible time intervals for a specified Cin8-GFP particle.
Repeat the procedure for all the examined Cin8-GFP particles. Finally, plot the mean displacement of all examined Cin8-GFP particles versus time interval, and subject it to a linear fit. The slope of this fit represents the mean velocity of motile Cin8-GFP particles.
The motility characteristics of bidirectional motor protein Cin8 of different cluster sizes on single microtubules are shown here. For each cluster size category, more than 40 trajectories of individual Cin8-GFP were extracted from the recordings. Plotting the mean displacements of single molecules and clusters of Cin8 motors as a function of the time interval demonstrated that single Cin8-GFP molecules move in a unidirectional minus-end-directed manner with high velocity.
In contrast, Cin8 clusters exhibit lower velocity and higher bidirectional motility. While performing this procedure, high-purity motor protein must be used to avoid any contaminations that can affect motor clustering. In analyzing the motor fluorescence intensity, it is important to examine the motors in the first frame they appear, to avoid the effect of photobleaching.
When studying the motility of bidirectional motors, it is extremely important to separately analyze single molecules and clusters, as motor clustering is one of the key factors that affect motor directionality.
