The overall of goal of this experimental procedure is to measure the rotation of probe beads attached to individual flagellar motors in live bacterial cells. The technique that we'll describe offers the advantage of real-time tracking of motor responses and the ability to discriminate between admissible and inadmissible data in real time. The approach is helping us answer key questions in the field of mechanobiology and uncover the physics underlying molecular motor behavior.
Although we predominantly employ these techniques to study flagellar motors in E.coli, these protocols can be readily adapted to study motors in other species of flagellated bacteria. Grow overnight cultures of the desired strain carrying the sticky fliC allele in TB.The next day, inoculate 10 milliliters of fresh TB at a 1:100 dilution. Grow the culture at 33 degrees Celsius in a shaker incubator until the optical density at 600 nanometers equals 0.5.
Pellet the cells at 1500 times G for five to seven minutes. Then, redisperse the pellet vigorously in 10 milliliters of filter-sterilized motility buffer. Repeat the centrifugation and resuspension two more times.
Redisperse the final pellet in one milliliter of MB.Shear the suspension by passing it back and forth approximately 75 times between two syringes with 21 to 23 gauge adapters connected by polyethylene tubing. Limit the total time for shearing to 30 to 45 seconds. After centrifuging the sheared cells, redisperse the pellet in 100 to 500 microliters of MB.Prepare an imaging chamber by sandwiching two double-sided adhesive tapes between a cover slip and a microscope slide.
Add 0.01%poly-L-lysine solution in the chamber and after five minutes, gently rinse the surfaces with MB.Add 40 microliters of the cell suspension into the chamber and allow sufficient time for attachment to the glass surface. Flow out unstuck cells by adding 100 microliters of MB on one side of the chamber, while wicking the solution with filter paper from the other side. Next, add 10 to 15 microliters of latex beads into the chamber.
Use a range of bead sizes for the experiments that result in a good contrast. Allow the beads adequate time to settle and attach to the cells. Gently rinse the beads with 100 microliters of MB to remove unstuck beads.
Place the sample on a microscope stage and scan the surface for beads attached to motors. Employ bright-field imaging with a 60X objective as long as sufficient contrast is maintained to clearly distinguish a bright bead on a dark background. Once a bead has been selected, move the stage laterally to position the bead in a pre-determined corner.
Position beads at the same corner to ensure that the direction of rotation of the bead is correctly known. Maintain the sampling frequency higher than twice the rotational frequency of the motor to avoid errors associated with aliasing. In this work, sampling frequencies that were 10 times higher were used to observe a motor rotating at 50 hertz.
If, after tethering, the beads appear to wobble with an eccentricity greater than one to two times that of the bead radius, then it's recommended to shear the cells a greater number of times. Shown here is a visual comparison between an appropriately-tethered bead and a bead that is not tethered. The outputs from the two photomultipliers, sampled at 500 hertz, are represented over a small duration of the measurement.
The filtered outputs from the PMTs, after centering and scaling, were used to reconstruct the circular trajectories of the rotating bead, as shown here. The ideal bead trajectory is approximately circular but elliptical trajectories are admissible. The angular speeds calculated from the bead trajectories for a single motor are represented over a three-second window.
The repeated transitions of the motor between the counterclockwise and clockwise directions of rotation are indicated by switches between positive and negative values. After watching this video, you should have a good understanding of how to track beads tethered to single flagellar motors with high accuracy, using the photomultiplier-based techniques presented here. The bacterial flagellar motor presents a unique set of challenges because it is capable of rotating at speeds of several hundred hertz.
High accuracy tracking of probe beads tethered to molecular motors facilitated the characterization of several types of motors, including kinesins, myosins and F1-ATPase. The extension of the bead tethering technique to flagellar motors enabled researchers to study motor mechanics with an unprecedented accuracy. Visual demonstration of this method will help researchers wishing to utilize this technique to overcome challenges such as locating beads appropriately tethered to individual motors.
It is expected that the methods demonstrated here will enable the development of novel insights into the adaptability of these nanomachines in a variety of bacterial species.
