Skip to main content

Journal for Biophysical Chemistry

Biointerphases Cover Image

Fluorescence microscopy investigations of ligand propagation and accessibility under adherent cells

Abstract

Fluorescence microscopy methods including total internal reflection fluorescence and confocal laser scanning microscopy have played a major role in modern cell biology research by permitting imaging of fluorescently tagged macromolecules in living cells. These methods are often used to examine the initial events in signal transduction, which involve interactions occurring between membrane receptors and ligands such as antibodies and growth factors. Most quantitative biophysical applications using these fluorescence imaging methods, including ligand binding assays, are based on the assumption that the fluorophore label of interest has equal access to all areas of the membrane on the cell. Our findings suggest that there is limited accessibility of fluorophores (25±2%)- under the basal membrane of adherent CHO-K1 cells expressing epidermal growth factor receptor plated on a bare glass in standard two-dimensional tissue cultures. The authors present a detailed study of the extent to which a small fluorescent dye molecule (Alexa 647) is able to propagate under the basal membrane of cells plated on a variety of biologically compatible substrates: fibronectin, bovine serum albumin, poly-d-lysine, collagen I, collagen IV, GeltrexTM, and fibronectin such as binding polymer. For nonspecific dye propagation the best overall accessibility was achieved using a thin layer preparation of a commercially available basement membrane matrix, GeltrexTM (67±8%). Coupling of a specific high affinity ligand (epidermal growth factor) to the dye did result in a moderate increase in propagation for most substrates examined. Despite the overall increase in propagation for most substrates (60%-80%), large areas under the central regions of the adherent cells still remained inaccessible to the fluorescently labeled ligand. More importantly, the presence of the specific ligand did not result in consistent increase in ligand propagation. Taken together these results suggest that the reduced accessibility is not exclusively due to steric effects, and the chemistry of both the ligand and the substrate may be important when working under conditions of reduced dimensionality. a) Electronic mail: jody.swift@mail.mcgill.ca b) Electronic mail: paul.wiseman@mcgill.ca

References

  1. 1

    I. Chung, R. Akita, R. Vandlen, D. Toomre, J. Schlessinger, and I. Mellman, Nature (London) 464, 783 (2010).

    Article  CAS  Google Scholar 

  2. 2

    C. Brown, B. Hebert, D. Kolin, J. Zareno, L. Whitmore, A. Horwitz, and P. Wiseman, J. Cell. Sci. 119, 5204 (2006).

    Article  CAS  Google Scholar 

  3. 3

    M. Sergeev, S. Costantino, and P. Wiseman, Biophys. J. 91, 3884 (2006).

    Article  CAS  Google Scholar 

  4. 4

    T. Gadella, Jr. and T. Jovin, J. Cell Biol. 129, 1543 (1995).

    Article  CAS  Google Scholar 

  5. 5

    S. Mukherjee, R. Ghosh, and F. Maxfield, Physiol. Rev. 77, 759 (1997).

    CAS  Google Scholar 

  6. 6

    P. Yeagle, The Structure of Biological Membranes (CRC, Boca Raton, FL, 2005), pp. 157–174.

    Google Scholar 

  7. 7

    J. B. Pawley, Handbook of Biological Confocal Microscopy, 2nd ed. (Plenum, New York, 1995), pp. 327–355.

    Google Scholar 

  8. 8

    N. Petersen, P. Höddelius, P. Wiseman, O. Seger, and K. Magnusson, Biophys. J. 65, 1135 (1993).

    Article  CAS  Google Scholar 

  9. 9

    P. Wiseman, J. Squier, M. Ellisman, and K. Wilson, J. Microsc. 200, 14 (2000).

    Article  CAS  Google Scholar 

  10. 10

    D. Huh, W. Gu, Y. Kamotani, J. Grotberg, and S. Takayama, Physiol. Meas 26, R73 (2005).

    Article  Google Scholar 

  11. 11

    11 K. Kandere-Grzybowska, C. Campbell, Y. Komarova, B. Grzybowski, and G. Borisy, Nat. Methods 2, 739 (2005).

    Article  CAS  Google Scholar 

  12. 12

    R. Ellis, Trends Biochem. Sci. 26, 597 (2001).

    Article  CAS  Google Scholar 

  13. 13

    O. Lieleg, R. Baumgärtel, and A. Bausch, Biophys. J. 97, 1569 (2009).

    Article  CAS  Google Scholar 

  14. 14

    S. Johansson, G. Svineng, K. Wennerberg, A. Armulik, and L. Lohikangas, Front. Biosci. 2, d126 (1997).

    CAS  Google Scholar 

  15. 15

    C. Chung and H. Erickson, J. Cell. Sci. 110, 1413 (1997).

    CAS  Google Scholar 

  16. 16

    16 D. F. Holmes, H. K. Graham, J. A. Trotter, and K. E. Kadler, Micron 32, 273 (2001).

    Article  CAS  Google Scholar 

  17. 17

    D. E. MacDonald, B. Markovic, M. Allen, P. Somasundaran, and A. L. Boskey, J. Biomed. Mater. Res. 41, 120 (1998).

    Article  CAS  Google Scholar 

  18. 18

    O. Mori and T. Imae, Colloids Surf., B 9, 31 (1997).

    Article  CAS  Google Scholar 

  19. 19

    J. Szymański, A. Patkowski, A. Wilk, P. Garstecki, and R. Holyst, J. Phys. Chem. B 110, 25593 (2006).

    Article  Google Scholar 

  20. 20

    B. Yari, F. Khorasheh, and A. Kheirolomoom, Chem. Phys. 321, 34 (2006).

    Article  CAS  Google Scholar 

  21. 21

    L. Groc, M. Heine, L. Cognet, K. Brickley, F. A. Stephenson, B. Lounis, and D. Choquet, Nat. Neurosci. 7, 695 (2004).

    Article  CAS  Google Scholar 

Download references

Author information

Affiliations

Authors

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Swift, J.L., Sergeev, M. & Wiseman, P.W. Fluorescence microscopy investigations of ligand propagation and accessibility under adherent cells. Biointerphases 5, 139–148 (2010). https://doi.org/10.1116/1.3523470

Download citation