Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

The protocol offers a simple method for purifying active molecular motors using Sf9 baculovirus system. It also details single molecule motility and multi-motor gliding assays to study the regulatory mechanism of motor proteins. All that traditional vector expression Sf9 baculovirus expression and single step affinity purification, will lead pure and active motor protein to study the mechanistic details of Superprocessive Kinesins in single molecule and multi-motor systems.

In addition to Kinesin motors, the protocol can be applied to study biochemical and biophysical properties of other cytoskeletal based proteins such as dionine and myosin. The protocol is detailed and easy to follow. Some suggestions are to handle Sf9 cells carefully as they are prone to contamination, and to follow notes provided in the protocol.

Visual demonstration is highly effective, easy to follow and understand the critical steps, especially for those who have never performed these assays. Demonstrating the procedure will be Pushpanjali Soppina and Dipeshwari Shewale, PhD students working with me and Pradeep Naik. To begin, seed the cells in a 35 milliliter dish, at a density of 4.5 times 10 to the fifth cells per milliliter.

After 24 hours, mix one microgram of bacmid DNA and 100 microliters of unsupplemented Grace's media in a tube. Mix six microliters of transfection reagent in another tube with 100 microliters of unsupplemented Grace's media. Carefully transfer the content of the first tube into the second tube.

Mix them and incubate the mixture for 45 minutes at room temperature. Add 0.8 milliliters of unsupplemented Grace's media to the mixture. Mix and aspirate the Sf900 SFM media from the cells.

Add the prepared transfection media drop-wise on the top of the cells and incubate the plate for six hours. Then carefully remove the transfection mixture. Add two milliliters of SF900 SFM media and incubate further at 28 degrees Celsius for 48 hours.

Next, check the motor protein expression under an inverted fluorescence microscope. Harvest the media with infected cells, and spin for five minutes, at 500 G.Collect the supernatant and snap freeze the aliquots. Add 10 milliliters of SF900 SFM media with cells and one milliliter of P-zero virus stock, in a sterile, 100 milliliter, conical flask.

Incubate at 28 degrees Celsius with constant shaking at 90 rpm. After 72 hours, spin down the cells in a 15 milliliter, sterile conical tube at 500 G for five minutes and collect the supernatant. For large scale protein expression, infect 30 milliliters of the suspension culture with one milliliter of P1 stock virus.

72 hours post-infection, check the cells for protein expression and collect the cells in sterile, 50 milliliter, conical tubes. After spinning, discard the supernatant, and collect the cell pellet. To lyse the cells, add three milliliters of ice cold lysis buffer to the cell pellet.

Spin the cell lysate at 150, 000 G for 30 minutes, at four degrees Celsius. Collect the supernatant and mix it with 40 microliters of 50%ANTI-FLAG M2 affinity resin. Incubate the mixture at four degrees Celsius for three hours with end-to-end tumbling.

Now pellet the FLAG resin by spinning at 500 G for one minute at four degrees Celsius. Wash the FLAG resin pellet three times with ice cold wash buffer. And after the third wash, carefully drain the wash buffer.

Next, add 70 microliters of wash buffer containing 100 micrograms per milliliter FLAG peptide, to the resin pellet, and incubate overnight at four degrees Celsius, as demonstrated earlier. After spinning, collect the supernatant containing purified protein into a fresh tube. Prepare a polymerization mix as described in the manuscript, and add 10 microliters of tubulin to the polymerization mixture.

Transfer the tube into a heat block, pre-warmed at 37 degrees Celsius, and incubate for 30 minutes. After preparing the microtubule stabilization buffer, warm it, and add it to the polymerized microtubules. Once the motility flow cell chamber is prepared, flow 30 microliters of diluted microtubule solution through the chamber.

After 30 minutes, flow 40 to 50 microliters of blocking buffer and incubate it. Prepare a motility mixture and add one microliter of the purified motor to it. Mix well and flow the solution into the motility chamber.

Seal both ends of the motility chamber and image under TIRF illumination. Focus on the individual mCitirine tagged motors, moving processively. Mix rhodamine labeled tubulin with unlabeled tubulin in a ratio of one to 10.

After 30 minutes of polymerization, gently add 30 microliters of pre-warmed microtubule stabilization buffer and incubate further, for five minutes, at 37 degrees Celsius. Shear the microtubules by pipetting with the capillary loading tip. After preparing the motility flow chamber, flow 50 microliters of Sf9 purified GFP nanobodies and incubate for 30 minutes.

Block the cover slip surface by flowing 50 microliters of block buffer. Prepare 50 microliters of the motor mix. Flow it into the chamber after gently mixing the solution and incubate for 30 minutes.

Wash the chamber, twice, with 50 microliters of P12-casein. Prepare the gliding assay mixture and mix it gently, before flowing it into the motility chamber. Seal the ends of the motility chamber, and image microtubule gliding under TIRF illumination.

After 48 hours of transfection, the untransfected cells appear small, round and firmly attached. However, the transfected cells expressing protein, were loosely attached to the surface and showed enlarged cell diameter. After 72 hours, more than 90%of Sf9 cells expressed fluorescently tagged, kinesin-3 motor KIF1A, and cells were loosely bound to the surface.

Kinesin-3 motor protein was purified from the transfected Sf9 cells. The kymograph depicts kinesin-3 motor walking processively, along the microtubule surface. Motility events showed an average velocity and run length of approximately 2.44 micrometers per second and 10.79 micrometers respectively.

The kymograph indicates smooth microtubule gliding by the kinesin-3 motors, with an average velocity of approximately 1.37 micrometers per second. The most important thing is to add the stabilization buffer without disturbing the microtubule mix and not to shear the microtubule. The protocol would be useful for other applications, such as ATPase measurements, biophysical analysis, mechanisms, in vitro reconstitution assays, et cetera.

Using sf9 purified motors, we've demonstrated recently that kinesin-3 motors are fast and superprocessive. Interestingly, these motors exhibit high ATP turnover rates compared to well-characterized motor, Kinesin-1.