Materials and Reagents

18 x 18 mm Square microscope cover slip (Matsunami Glass, catalog number: C218181 )
22 x 40 mm Square microscope cover slip (Matsunami Glass, catalog number: C022401 )
Double-sided tape (NICHIBAN, NICETACK^TM, catalog number: NW-5 )
Mending tape (3M, Scotch^®, catalog number: 810-3-15 )
Filter paper ϕ70 (ADVANTEC, catalog number: 00021070 )
1.5 ml microtube
M. mobile 163K strain (ATCC, catalog number: 43663 )
Polystyrene beads 1.0 µm in diameter (Polybead^® Carboxylate Microspheres 1.00 µm) (Polysciences, catalog number: 08226-15 )
PolyLink Protein Coupling Kit for COOH Microspheres (Polysciences, catalog number: 24350-1 )

PolyLink coupling buffer
PolyLink EDAC

Avidin from egg white (Sigma-Aldrich, catalog number: A9275 )
PBS with 20 mM glucose (PBS/G)
PBS with 40 mM glucose
Sulfo-NHS-LC-LC-biotin (Ez-Link^TM Sulfo-NHS-LC-LC-biotin) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 21338 )
Glucose
Nail polish
Heart infusion broth (BD, catalog number: 238400 )
Yeast extract (BD, catalog number: 212750 )
10 N NaOH
Horse serum (Thermo Fisher Scientific, Gibco^TM, catalog number: 16050122 )
Amphotericin B (Sigma-Aldrich, catalog number: A2942 )
Ampicillin Na (Nacalai Tesque, catalog number: 02739-32 )
Sodium phosphate (pH 7.3)
NaCl
Aluotto medium (see Recipes)
Phosphate-buffered saline (PBS) (see Recipes)

Equipment

Centrifuge (Sigma Laborzentrifugen, model: Sigma 1-14 )
25 cm² tissue culture flask (AS ONE, catalog number: 2-8589-01 )
25 °C incubator (Tokyo Rikakikai, model: LTI-400E )
Sonicator (Emerson Industrial Automation, BRANSON, model: 2510J-MT )
Optical microscope (Olympus, model: IX71 )
Optical tweezers system (Note 1)
High speed charge-coupled device (DigiMo, model: LRH2500XE-1 )
Power meter (Laser Power/Energy Meter) (Coherent, model: FieldMaxII-TOP, catalog number: 1098580 )
Autoclave

Software

ImageJ (http://rsbweb.nih.gov/ij/index.html)
IGOR Pro 6.33J (WaveMetrics, Portland, OR)

Procedure

Coating polystyrene beads with avidin
1. Warm polystyrene beads, coupling buffer and PBS to room temperature (RT).
2. Centrifuge 60 µl polystyrene bead suspension at 2,000 x g for 6 min at RT.
3. Discard supernatant, resuspend the pellet in 400 µl of coupling buffer and centrifuge it at 2,000 x g for 6 min at RT.
4. Discard supernatant, resuspend the pellet in 160 µl of coupling buffer.
5. Just before use, dissolve 11.7 mg of EDAC in 50 µl of coupling buffer, add 20 µl of this solution to the suspension and incubate for 5 min at RT.
6. Dissolve 0.4 mg of avidin in 10 µl of coupling buffer to be 0.13 mM as the final concentration, add the avidin solution to the suspension and incubate it with gently end-over-end mixing for 3 h at RT.
7. Centrifuge the suspension at 2,000 x g for 6 min at RT, discard the supernatant and resuspend the pellet in 450 µl of PBS. Repeat this step five times.
8. Transfer the suspension to new microtube and storage it at 4 °C until use (it can be stored for 6 months).
Biotinylation of M. mobile cell surface
1. Inoculate 1 ml of frozen stock of M. mobile into 10 ml of Aluotto medium in a 25 cm² tissue culture flask and statically cultivate it at 25 °C to reach an optical density at 600 nm of 0.06-0.08.
2. Collect 9 ml of cultured cells by centrifugation at 12,000 x g for 4 min at RT, discard supernatant, resuspend the cell pellet in 900 µl of PBS/G and centrifuge it at 12,000 x g for 4 min at RT (Note 2).
3. Just before use, prepare a 0.5 mM of Sulfo-NHS-LC-LC-biotin solution by dissolving 0.3 mg of Sulfo-NHS-LC-LC-biotin in 1 ml of PBS/G.
4. Discard supernatant of step B2, resuspend the cell pellet (Note 2) in 300 µl of Sulfo-NHS-LC-LC-biotin solution and incubate the cell suspension for 15 min at RT (Hiratsuka et al., 2006).
5. Centrifuge the suspension at 12,000 x g for 4 min at RT, discard supernatant, and resuspend the cell pellet (Note 2) in 300 µl of PBS/G. Repeat this step three times.
Force measurement by using optical tweezers
1. Prepare a tunnel chamber (5-mm interior width, 18-mm length, 60-μm wall thickness) composed of two cover glasses from box, and double-sided tapes (Figure 1).
  
  Figure 1. Tunnel chamber (step C1)
2. To coat the glass surface with glycoproteins, inject 20 µl of 10% horse serum in PBS/G into the tunnel chamber and incubate it for 15 min at RT (Note 3).
3. Dilute the suspension of biotinylated cell with a 10 to 20-fold volume of PBS/G.
4. Wash the tunnel chamber by flowing 40 µl of PBS/G, inject 20 µl of the diluted cell suspension into the tunnel chamber and incubate it for 15 min at RT.
5. To remove floating cells, insert 20 µl of PBS/G into the tunnel chamber.
6. Add the avidin-coated beads in PBS to the same volume of PBS containing 40 mM glucose, for adjusting glucose concentration to 20 mM.
7. Dilute the bead suspension with PBS/G to get an appropriate bead density (10-20 beads per a 125.4 x 70.6 µm field of view) for the force measurements.
8. Transfer 40 µl of bead suspension to a new microtube and sonicate it for 20 sec.
9. Inject sonicated bead suspension into the tunnel chamber and seal both ends with nail polish.
10. Set and fix the tunnel chamber by mending tapes onto the stage of inverted microscope equipped with optical tweezers (Figure 2).
  
  Figure 2. Setting tunnel chamber on microscope stage (step C10)
11. Trap a floating bead near the bottom of glass surface by focused laser beam, start to record the bead movements at 200 frames per second and immediately attached the bead to the back end of the gliding cell through the avidin-biotin interaction. The cell pulls the bead from trap center of optical tweezers and finally stalls (Video 1). The recording should be continued at least 30 sec after the start of stall (Note 4). After recording, turn off the laser beam.
12. Remove the tunnel chamber and objective lens, turn on the laser beam to the same power as the step C11 and measure the applied laser power of the parallel light, which was achieved by the removal of objective lens, with using a power meter.
13. Trace the bead movement from the trap center of optical tweezers and calculate the force from the distance between centers of the bead and the laser beam, by using ImageJ and IGOR Pro, as previously described (Tanaka et al., 2016).
  
  Video 1. Trapping of bead and attachment to gliding cell (step C11). Focused point of laser beam is indicated by a red circle, marked ‘Trap center’. At 5 sec, a flowing bead came from the right side and trapped at the beam. At 18 sec, a gliding cell was moved into the field by stage handling. At 27 sec, the bead was attached to the gliding cell. The cell pulled the trapped bead and stalled from 33 sec to the end.

Data analysis

The calculation method for bead positioning is described in Thompson et al. (2002).

Notes

Construction of optical tweezers was described in Tanaka et al. (2016). Similar systems are also commercially available (Laser Optical tweezers – Mini type Basic model/without shutter, SIGMAKOKI, catalog number: LMS-M1064-2000. OTM200 Optical Tweezer Add-On, THORLABS, catalog number: OTM200).
Suspension of cell pellet
More than 100 ups and downs with the pipette is recommended to separate cells completely.
Heat treatment of horse serum
For M. mobile growth and gliding, the horse serum should be inactivated by heat treatment at 56 °C for 30 min.
Available data size for ImageJ v1.43u is limited around 1 GB. The data size of movie depends on the recording time, the field size, and the frame rate. One example is 180 sec, 4 x 4 µm square, and 200 frames per second.

Recipes

Aluotto medium
2.1% heart infusion broth
0.56% yeast extract
0.035% 10 N NaOH
Note: Dissolve the above three reagents as a mixture, autoclave, cool to lower than 37 °C, and add below three reagents in clean bench.
10% horse serum
0.025% amphotericin B
0.005% ampicillin Na
Phosphate-buffered saline (PBS)
75 mM sodium phosphate (pH 7.3)
68 mM NaCl
Sterilize by using filter or autoclave

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research on Innovative Area, ‘Harmonized Supramolecular Motility Machinery and Its Diversity’ (grant 24117002 to M. Miyata) and by a grant-in-aid for scientific research (B) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (grant 24390107 to M. Miyata).

References

Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E. and Chu, S. (1986). Observation of a single-beam gradient force optical trap for dielectric particles. Opt Lett 11(5): 288.
Hiratsuka, Y., Miyata, M., Tada, T. and Uyeda, T. Q. (2006). A microrotary motor powered by bacteria. Proc Natl Acad Sci U S A 103(37): 13618-13623.
Jarrell, K. F. and McBride, M. J. (2008). The surprisingly diverse ways that prokaryotes move. Nat Rev Microbiol 6(6): 466-476.
Miyata, M., Ryu, W. S. and Berg, H. C. (2002). Force and velocity of mycoplasma mobile gliding. J Bacteriol 184(7): 1827-1831.
Tanaka, A., Nakane, D., Mizutani, M., Nishizaka, T. and Miyata, M. (2016). Directed binding of gliding bacterium, Mycoplasma mobile, shown by detachment force and bond lifetime. MBio 7(3).
Thompson, R. E., Larson, D. R. and Webb, W. W. (2002). Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82(5): 2775-2783.