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High-throughput optical screening of cellular mechanotransduction

Abstract

We introduce an optical platform for rapid, high-throughput screening of exogenous molecules that affect cellular mechanotransduction. Our method initiates mechanotransduction in adherent cells using single laser-microbeam generated microcavitation bubbles without requiring flow chambers or microfluidics. These microcavitation bubbles expose adherent cells to a microtsunami, a transient microscale burst of hydrodynamic shear stress, which stimulates cells over areas approaching 1 mm2. We demonstrate microtsunami-initiated mechanosignalling in primary human endothelial cells. This observed signalling is consistent with G-protein-coupled receptor stimulation, resulting in Ca2+ release by the endoplasmic reticulum. Moreover, we demonstrate the dose-dependent modulation of microtsunami-induced Ca2+ signalling by introducing a known inhibitor to this pathway. The imaging of Ca2+ signalling and its modulation by exogenous molecules demonstrates the capacity to initiate and assess cellular mechanosignalling in real time. We utilize this capability to screen the effects of a set of small molecules on cellular mechanotransduction in 96-well plates using standard imaging cytometry.

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Figure 1: Pulsed laser microbeam exposes adherent cells to a transient μCB.
Figure 2: Radial dependence of a μtsunami-generated hydrodynamic shear stress impulse.
Figure 3: Ca2+ signalling following μtsunami exposure.
Figure 4: Dose-dependent effect of IP3 inhibitor on μtsumani-induced Ca2+ signalling.
Figure 5: Use of μtsunamis for HTS.

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Acknowledgements

The authors thank A. Hellman for her early contributions to this research, M. Khine for assistance with microfabrication and C. Hughes for supplying HUVECs. This research was funded through the Laser Microbeam and Medical Program, a National Biomedical Technology Resource (P41-EB015890) supported by the National Institutes of Health, the National Science Foundation through the Integrative Graduate Education and Research Traineeship (IGERT) Program (DGE-1144901), and UC Irvine through the I3 Award Program.

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Authors

Contributions

E.B. and V.V. conceived the project. J.L.C., J.C.L., E.B. and V.V. designed the experiments. J.L.C., J.C.L. and H.M. performed the experiments. J.L.C. and V.V. developed the hydrodynamic model. J.L.C., J.C.L., E.B. and V.V. performed the data analysis. J.L.C., J.C.L., E.B. and V.V. wrote the manuscript.

Corresponding authors

Correspondence to Elliot Botvinick or Vasan Venugopalan.

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Competing interests

Authors Compton, Botvinick and Venugopalan filed a non-provisional US patent application “Mechanical Stress Response Analysis of Cells and Tissues” Serial No. 14/046,804 on 4 October 2013 based on the findings of this study.

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Compton, J., Luo, J., Ma, H. et al. High-throughput optical screening of cellular mechanotransduction. Nature Photon 8, 710–715 (2014). https://doi.org/10.1038/nphoton.2014.165

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