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Reply: reducing background fluorescence reveals adhesions in 3D matrices

Nature Cell Biology volume 13, pages 5–7 (2011)Cite this article

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Fraley et al. reply:

Kubow and Horwitz present evidence supporting the presence of focal adhesion complexes in the mesenchymal cell lines U2OS and HT-1080 when these cells are embedded in a three-dimensional (3D) collagen matrix. The initial adhesions were approximately 0.4 μm in diameter and achieved a median length of 1 μm. They also report that the formation and rearward movement of these adhesions were similar to that observed for these cells in two-dimensional (2D) systems. In contrast, in our recent article1, we reported that we did not observe any structured organization of focal adhesion proteins of this size in migrating HT-1080 cells or non-motile U2OS cells that were completely embedded in a similar 3D collagen gel. The conditions of our experiments and the imaging techniques employed were similar to those used by Kubow and Horwitz, but for one technical difference (see below), and thus should be able to detect focal adhesions of 0.4 μm or greater (our resolution is 0.21 μm or greater). Kubow and Horwitz suggest that the discrepancy between their results and ours was because of the high background fluorescence resulting from overexpression of GFP (green fluorescent protein)-tagged FA proteins in our experiments, which obscures the observation of focal complexes. To circumvent this problem, Kubow and Horwitz used a 'crippled' CMV (cygtomegalovirus) promoter that expresses less protein in cells2.

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Figure 1: Focal adhesion visualization in live HT-1080 cells.
Figure 2: Properties of cells on or near the bottom versus fully embedded in 3D collagen gel.

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Acknowledgements

This research was supported by NIH grants CA143868, GM084204, GM080673 and CA85839 to G.D.L. and D.W. S.I.F. was supported by ARCS and NSF-GRFP.

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Authors and Affiliations

  1. Departments of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, 21218, MD, USA

    Stephanie I. Fraley & Denis Wirtz

  2. Johns Hopkins Physical Sciences in Oncology Centre, Johns Hopkins University, Baltimore, 21218, MD, USA

    Stephanie I. Fraley, Yunfeng Feng, Denis Wirtz & Gregory D. Longmore

  3. Departments of Medicine and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, 63110, MO, USA

    Yunfeng Feng & Gregory D. Longmore

  4. BRIGHT Institute, Washington University School of Medicine, St. Louis, 63110, MO, USA

    Yunfeng Feng & Gregory D. Longmore

Authors
  1. Stephanie I. Fraley
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  2. Yunfeng Feng
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Contributions

Y. F. generated YFP-paxillin and GFP-zyxin constructs. S. F. performed all experiments and analysis and co-wrote the manuscript; G. L. and D. W. co-supervised the project and co-wrote the manuscript.

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Correspondence to Denis Wirtz or Gregory D. Longmore.

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Fraley, S., Feng, Y., Wirtz, D. et al. Reply: reducing background fluorescence reveals adhesions in 3D matrices. Nat Cell Biol 13, 5–7 (2011). https://doi.org/10.1038/ncb0111-5

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