Techniques to Measure Pilus Retraction Forces
The importance of physical forces in biology is becoming more appreciated. Neisseria gonorrhoeaehas become a paradigm for the study of physical forces in the bacterial world. Cycles of elongations and retractions of Type IV pili enables N. gonorrhoeaebact
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1. Introduction Type IV pili (Tfp) play a critical role in the life cycles of both Neisseria meningitidis and Neisseria gonorrhoeae, the two main pathogens of the Neisseria genus. These appendages are thin (6 nm in diameter) and long (up to 30 Pm in length) dynamic polymers emanating from the surface of the bacteria. They are associated with a wide range of functions including DNA uptake, twitching motility, cell adhesion, and biofilm formation (1). An explanation for this wide spectrum of functions stems from the extraordinary diversity of Tfp filaments: the main subunit, pilin, can undergo high frequency antigenic variation, (2) harbors various post-translational modifications (3) and the filaments themselves possess different minor pilins (4). Alongside these chemical and genetic variabilities, force generation has also been implicated as another important function of Tfp biology.
Myron Christodoulides (ed.), Neisseria meningitidis: Advanced Methods and Protocols, Methods in Molecular Biology, vol. 799, DOI 10.1007/978-1-61779-346-2_13, © Springer Science+Business Media, LLC 2012
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During the last decade, the role of physical forces in biology has emerged as a central theme. From development to differentiation, from motility to signaling, physical forces modulate biological fates and the bacterial world is no exception (5–8). N. gonorrhoeae Tfp can undergo cycles of extension and retractions and they can generate force during retraction (9). A single Tfp fiber can exert forces of 100 picoNewtons (pN) (10) and bundles of 8–10 Tfp can be retracted in unison exerting forces of up to 1 nanoNewtons (nN) which can be maintained for hours (11). In the context of cellular infection, these forces elicit dramatic rearrangements in the cortex of eukaryotic cells (12) and have been shown to trigger cytoprotective pathways (13). The techniques designed to measure those forces in N. gonorrhoeae can readily be adapted to measure the force generation in N. meningitidis or other pili-bearing bacteria. For this chapter, we present two complementary techniques to measure the forces generated by the retraction of pili. The first one utilizes optical tweezers and is geared toward the measurement of forces from a single bacterium. The second utilizes arrays of Polyacrylamide MicroPillars (PoMPs) and is geared toward the measurement of forces from multiple bacteria in “infection-like” conditions. The use of these techniques will increase our understanding of the role of physical forces in the biology of the pathogenic Neisseriae and other pilus-bearing bacteria.
2. Materials 2.1. Calibration of Optical Tweezers
1. A basic laser tweezers set-up with a fixed laser trap obtained from a 2 W neodymium-doped yttrium aluminum garnet neodymium (Nd:YAG) laser (14). 2. Carboxylated silica or latex beads (Polysciences or Bangs laboratories). 3. Eppendorf tubes. 4. Piezoelectric stage P-517 (Physik Instrumente). 5. #1 coverglass (Fisher Scientific). 6. Observation chamber: a metallic slide with a 20 × 20 mm opening (15) (see F
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