An In Vitro Model System to Test Mechano-microbiological Interactions Between Bacteria and Host Cells

  • Luís Carlos Santos
  • Emilia Laura Munteanu
  • Nicolas Biais
Part of the Methods in Molecular Biology book series (MIMB, volume 1365)


The aim of this chapter is to present an innovative technique to visualize changes of the f-actin cytoskeleton in response to locally applied force. We developed an in vitro system that combines micromanipulation of force by magnetic tweezers with simultaneous live cell fluorescence microscopy. We applied pulling forces to magnetic beads coated with the Neisseria gonorrhoeae Type IV pili in the same order of magnitude than the forces generated by live bacteria. We saw quick and robust f-actin accumulation at the sites where pulling forces were applied. Using the magnetic tweezers we were able to mimic the local response of the f-actin cytoskeleton to bacteria-generated forces. In this chapter we describe our magnetic tweezers system and show how to control it in order to study cellular responses to force.

Key words

Magnetic tweezers Neisseria f-Actin Type IV pili Magnetic beads Cytoskeleton 


  1. 1.
    McMahon HT, Boucrot E (2015) Membrane curvature at a glance. J Cell Sci 128:1065–1070PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Roux A, Cappello G, Cartaud J, Prost J, Goud B, Bassereau P (2002) A minimal system allowing tubulation with molecular motors pulling on giant liposomes. Proc Natl Acad Sci U S A 99:5394–5399PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Pollard TD (2010) Mechanics of cytokinesis in eukaryotes. Curr Opin Cell Biol 22:50–56PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Chicurel ME, Chen CS, Ingber DE (1998) Cellular control lies in the balance of forces. Curr Opin Cell Biol 10:232–239CrossRefPubMedGoogle Scholar
  5. 5.
    Mammoto T, Mammoto A, Ingber DE (2013) Mechanobiology and developmental control. Annu Rev Cell Dev Biol 29:27–61CrossRefPubMedGoogle Scholar
  6. 6.
    Roca-Cusachs P, del Rio A, Puklin-Faucher E, Gauthier NC, Biais N, Sheetz MP (2013) Integrin-dependent force transmission to the extracellular matrix by α-actinin triggers adhesion maturation. Proc Natl Acad Sci U S A 110:E1361–E1370PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Kundu AK, Putnam AJ (2006) Vitronectin and collagen I differentially regulate osteogenesis in mesenchymal stem cells. Biochem Biophys Res Commun 347:347–357CrossRefPubMedGoogle Scholar
  8. 8.
    Vogel V, Sheetz M (2006) Local force and geometry sensing regulate cell functions. Nat Rev Mol Cell Biol 7:265–275CrossRefPubMedGoogle Scholar
  9. 9.
    Vogel V, Sheetz MP (2009) Cell fate regulation by coupling mechanical cycles to biochemical signaling pathways. Curr Opin Cell Biol 21:38–46PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Farge E (2003) Mechanical induction of Twist in the Drosophila foregut/stomodeal primordium. Curr Biol 13:1365–1377CrossRefPubMedGoogle Scholar
  11. 11.
    Engler AJ, Sen S, Sweeney HL, Discher DE (2006) Matrix elasticity directs stem cell lineage specification. Cell 126:677–689CrossRefPubMedGoogle Scholar
  12. 12.
    Nisticò P, Di Modugno F, Spada S, Bissell MJ (2014) β1 and β4 integrins : from breast development to clinical practice. 1–9, Breast Cancer Research 2014, 16:459Google Scholar
  13. 13.
    Lee SW, Higashi DL, Snyder A, Merz AJ, Potter L, So M (2005) PilT is required for PI(3,4,5)P3-mediated crosstalk between Neisseria gonorrhoeae and epithelial cells. Cell Microbiol 7:1271–1284CrossRefPubMedGoogle Scholar
  14. 14.
    Bieber D, Ramer SW, Wu CY, Murray WJ, Tobe T, Fernandez R, Schoolnik GK (1998) Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli. Science 280(5372):2114–2118CrossRefPubMedGoogle Scholar
  15. 15.
    Lambrechts A, Gevaert K, Cossart P, Vandekerckhove J, Van Troys M (2008) Listeria comet tails: the actin-based motility machinery at work. Trends Cell Biol 18(April):220–227CrossRefPubMedGoogle Scholar
  16. 16.
    Cleary J, Lai L-C, Shaw RK, Straatman-Iwanowska A, Donnenberg MS, Frankel G, Knutton S (2004) Enteropathogenic Escherichia coli (EPEC) adhesion to intestinal epithelial cells: role of bundle-forming pili (BFP), EspA filaments and intimin. Microbiology 150(Pt 3):527–538CrossRefPubMedGoogle Scholar
  17. 17.
    Caron E, Crepin VF, Simpson N, Knutton S, Garmendia J, Frankel G (2006) Subversion of actin dynamics by EPEC and EHEC. Curr Opin Microbiol 9:40–45CrossRefPubMedGoogle Scholar
  18. 18.
    Merz AJ, So M (1997) Attachment of piliated, Opa- and Opc- gonococci and meningococci to epithelial cells elicits cortical actin rearrangements and clustering of tyrosine-phosphorylated proteins. Infect Immun 65:4341–4349PubMedCentralPubMedGoogle Scholar
  19. 19.
    Merz AJ, Enns CA, So M (1999) Type IV pili of pathogenic Neisseriae elicit cortical plaque formation in epithelial cells. Mol Microbiol 32:1316–1332CrossRefPubMedGoogle Scholar
  20. 20.
    Merz AJ, So M (2000) Interactions of pathogenic neisseriae with epithelial cell membranes. Annu Rev Cell Dev Biol 16:423–457CrossRefPubMedGoogle Scholar
  21. 21.
    Howie HL, Glogauer M, So M (2005) The N. gonorrhoeae type IV pilus stimulates mechanosensitive pathways and cytoprotection through a pilT-dependent mechanism. PLoS Biol 3:e100PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Higashi DL, Zhang GH, Biais N, Myers LR, Weyand NJ, Elliott DA, So M (2009) Influence of type IV pilus retraction on the architecture of the Neisseria gonorrhoeae-infected cell cortex. Microbiology 155(Pt 12):4084–4092PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Tanase M, Biais N, Sheetz M (2007) Magnetic tweezers in cell biology. Trends Cell Biol 2:116–118Google Scholar
  24. 24.
    Kuo SC, Sheetz MP (1992) Optical tweezers in cell biology. Trends Cell Biol 2:116–118CrossRefPubMedGoogle Scholar
  25. 25.
    Kollmannsberger P, Fabry B (2007) High-force magnetic tweezers with force feedback for biological applications. Rev Sci Instrum 78:114301CrossRefPubMedGoogle Scholar
  26. 26.
    Webster KD, Crow A, Fletcher DA (2011) An AFM-based stiffness clamp for dynamic control of rigidity. PLoS One 6:1–7Google Scholar
  27. 27.
    Desprat N, Richert A, Simeon J, Asnacios A (2005) Creep function of a single living cell. Biophys J 88:2224–2233PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Fouchard J, Bimbard C, Bufi N, Durand-Smet P, Proag A, Richert A, Cardoso O, Asnacios A (2014) Three-dimensional cell body shape dictates the onset of traction force generation and growth of focal adhesions. Proc Natl Acad Sci U S A 111(36):13075–13080PubMedCentralCrossRefPubMedGoogle Scholar
  29. 29.
    Jiang G, Giannone G, Critchley DR, Kukomoto E, Sheetz MP (2003) Two-piconewton slip bond between fibronectin and the cytoskeleton depends on talin. Nature 424:334-337Google Scholar
  30. 30.
    Dai J, Sheetz MP (1999) Membrane tether formation from blebbing cells. Biophys J 77(6):3363–3370Google Scholar
  31. 31.
    Jiang G, Huang AH, Cai Y, Tanase M, Sheetz MP (2006) Rigidity sensing at the leading edge through alphavbeta3 integrins and RPTPalpha. Biophys J 90:1804–1809PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Biais N, Ladoux B, Higashi D, So M, Sheetz M. (2008) Cooperative retraction of bundled type IV pili enables nanonewton force generation. PLoS Biology 15;6(4):e87Google Scholar
  33. 33.
    Opitz D, Maier B (2011) Rapid cytoskeletal response of epithelial cells to force generation by type IV pili. PLoS One 6:8Google Scholar
  34. 34.
    Johnson HW, Schell MJ (2010) Neuronal IP3 3-kinase is an F-actin-bundling protein: role in dendritic targeting and regulation of spine morphology. Mol Biol Cell 20:5166–5180Google Scholar
  35. 35.
    Biais N, Higashi D, So M, Ladoux B (2012) Techniques to measure pilus retraction forces. Methods Mol Biol 799:197–216CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Luís Carlos Santos
    • 1
    • 4
  • Emilia Laura Munteanu
    • 1
    • 2
  • Nicolas Biais
    • 1
    • 3
  1. 1.Department of BiologyBrooklyn College of the City University of New YorkBrooklynUSA
  2. 2.Herbert Irving Comprehensive Cancer CenterColumbia UniversityNew YorkUSA
  3. 3.The Graduate Center of the City University of New YorkNew YorkUSA
  4. 4.Icahn School of Medicine at Mount SinaiNew YorkUSA

Personalised recommendations