使用光镊测量运动支原体的滑行运动力

Masaki  Mizutani; Makoto  Miyata

doi:10.21769/BioProtoc.2127

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Peer-reviewed

Force Measurement on Mycoplasma mobile Gliding Using Optical Tweezers

使用光镊测量运动支原体的滑行运动力

MM Masaki Mizutani

Makoto Miyata email

发布: 2017年02月05日第7卷第3期 DOI: 10.21769/BioProtoc.2127 浏览次数: 7798

评审: Sofiane El-Kirat-ChatelAnonymous reviewer(s)

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Abstract

Dozens of Mycoplasma species, belonging to class Mollicutes form a protrusion at a pole as an organelle. They bind to solid surfaces through the organelle and glide in the direction by a unique mechanism including repeated cycles of bind, pull, and release with sialylated oligosaccharides on host animal cells. The mechanical characters are critical information to understand this unique mechanism involved in their infectious process. In this protocol, we describe a method to measure the force generated by Mycoplasma mobile, the fastest gliding species in Mycoplasma. This protocol should be useful for the studies of many kinds of gliding microorganisms.

Keywords: Mycoplasma (支原体)

Optical tweezers (光镊)

Force (力)

Avidin-biotin (抗生物素蛋白 - 生物素)

Bead (珠子)

Background

Surface motility systems are spread over many bacterial species and they are not well elucidated compared to bacterial flagella and eukaryotic motor proteins (Jarrell and McBride, 2008), although potentially they can give us critical information to understand the survival strategy of bacteria. To elucidate a motility mechanism, we need information about the structure of machinery, the flow of energy, and the mechanical characters including speed and force. Optical tweezers are a special method used for micromanipulations or force measurements in the piconewton range under microscopy, by which an object with a diffractive index different from the medium is trapped at the center of focused laser beam (Ashkin et al., 1986). This method has greatly contributed to clarifying the features of motility systems including myosin, dynein, and kinesin, and now an established method in the field of biophysics. Here, we provide a protocol on how to measure force generated by surface moving microorganisms, based on our studies (Miyata et al., 2002; Tanaka et al., 2016) for gliding mechanism of M. mobile the fastest gliding species in class Mollicutes. This is the first protocol for force measurement made using optical tweezers in bio-protocol.

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

English

中文翻译

文章信息

版权信息

如何引用

Mizutani, M. and Miyata, M. (2017). Force Measurement on Mycoplasma mobile Gliding Using Optical Tweezers. Bio-protocol 7(3): e2127. DOI: 10.21769/BioProtoc.2127.