Nanoscale 3D Dynamics of Optically Trapped Spheres Interacting with Adherent Cell Lamellae

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We demonstrate an optical trap instrument and experimental methodology for reporting the nanoscale dynamics of binding individual cell-surface receptors and the cyto-mechanical responses initiated by bi-directional outside-in and inside-out signals. The probe, a 1-μm diameter chemically labeled glass sphere held in the trap, is positioned above the lamella of an adherent motile cell (cf. figs. A-B), and tracked in 3D at a framing rate of ∼100/s. In addition to a lateral precision of 2nm in 2D tracking, we have developed a practical approach to incorporate a comparable 4-6 nm precision in vertical tracking of the trapped sphere. We apply a novel Brownian analysis of vertical probe fluctuations in soft contact with the cell lamella to determine the apparent rigid boundary of the cell surface and elastic compliance of the glycocalyx. The adherent motile leukocytic (Jurkat) cells were spread on coverglass coated with the α4β1-integrin ligand VCAM-1 in isotonic Ca2+ and/or Mg2+ buffers. Spheres were conjugated with fibronectin fragments to demonstrate the nanoscale dynamics of integrin ligation and release (cf. figs. C-D), the measurement of bond-surface separation and extensional compliance, plus intriguing transient subsurface movements triggered by bond formation.View Large Image | View Hi-Res Image | Download PowerPoint Slide