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DNA-Based Probes for Measuring Mechanical Forces in Cell-Cell Contacts: Application to B Cell Antigen Extraction from Immune Synapses

Part of the Methods in Molecular Biology book series (MIMB,volume 1707)

Abstract

The production of antibodies requires the expansion and selection of high-affinity B cell clones. This process is initiated by antigen uptake through the B cell receptor (BCR), which recognizes and binds antigen displayed on the surface of an antigen-presenting cell (APC). To acquire the antigen, B cells use myosin contractility to physically pull BCR-antigen clusters from the APC membrane. These mechanical forces influence association and dissociation rates of BCR-antigen bonds, resulting in affinity-dependent acquisition of antigen by B cells. Mechanical regulation of B cell antigen acquisition from APCs remains poorly understood, although the recent development of DNA-based force sensors has enabled the measurement of mechanical forces generated in B cell-APC contacts. In this chapter, we describe a protocol to design, synthesize, and purify DNA-based force sensors to measure B cell antigen extraction forces using fluorescence microscopy.

Key words

  • B cells
  • B cell receptor
  • Immune synapse
  • Antigen internalization
  • DNA force sensor
  • DNA-protein conjugation
  • Cell mechanics
  • Fluorescence microscopy

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  • DOI: 10.1007/978-1-4939-7474-0_5
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References

  1. Batista FD, Iber D, Neuberger MS (2001) B cells acquire antigen from target cells after synapse formation. Nature 411:489–494

    CAS  CrossRef  PubMed  Google Scholar 

  2. Fleire SJ, Goldman JP, Carrasco YR, Weber M, Batista FD (2006) B cell ligand discrimination through a spreading and contraction response. Science 312:738–741

    CAS  CrossRef  PubMed  Google Scholar 

  3. Natkanski E, Lee W-Y, Mistry B, Casal A, Molloy JE, Tolar P (2013) B cells use mechanical energy to discriminate antigen affinities. Science 340:1587–1590

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  4. Nowosad CR, Spillane KM, Tolar P (2016) Germinal center B cells recognize antigen through a specialized immune synapse architecture. Nat Immunol 17:870–877

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  5. Tolar P, Spillane KM (2014) Force generation in B-cell synapses: mechanisms coupling B-cell receptor binding to antigen internalization and affinity discrimination. Adv Immunol 123:69–100

    CAS  CrossRef  PubMed  Google Scholar 

  6. Neuman KC, Nagy A (2008) Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods 5:491–505

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  7. Morimatsu M, Mekhdjian AH, Adhikari AS, Dunn AR (2013) Molecular tension sensors report forces generated by single integrin molecules in living cells. Nano Lett 13:3985–3989

    CAS  CrossRef  PubMed  Google Scholar 

  8. Grashoff C, Hoffman BC, Brenner MD, Zhou R, Parsons M, Yang MT, McLean MA, Sligar SG, Chen CS, Ha T, Schwartz MA (2010) Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature 466:263–266

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  9. Blakely BL, Dumelin CE, Trappmann B, McGregor LM, Choi CK, Anthony PC, Duesterberg VK, Baker BM, Block SM, Liu DR, Chen CS (2014) A DNA-based molecular probe for optically reporting cellular traction forces. Nat Methods 11:1229–1232

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  10. Zhang Y, Ge C, Zhu C, Salaita K (2014) DNA-based digital tension probes reveal integrin forces during early cell adnhesion. Nat Commun 5:5167

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  11. Spillane KM, Tolar P (2017) B cell antigen extraction is regulated by physical properties of antigen-presenting cells. J Cell Biol 216:217–230

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  12. Wang X, Ha T (2013) Defining single molecular forces required to activate integrin and Notch signalling. Science 340:991–994

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

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Correspondence to Pavel Tolar .

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Spillane, K.M., Tolar, P. (2018). DNA-Based Probes for Measuring Mechanical Forces in Cell-Cell Contacts: Application to B Cell Antigen Extraction from Immune Synapses. In: Liu, C. (eds) B Cell Receptor Signaling. Methods in Molecular Biology, vol 1707. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7474-0_5

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  • DOI: https://doi.org/10.1007/978-1-4939-7474-0_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7473-3

  • Online ISBN: 978-1-4939-7474-0

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