Skip to main content
SpringerLink
Log in

Modification of Cellular Cholesterol Content Affects Traction Force, Adhesion and Cell Spreading

  • Published:
Cellular and Molecular Bioengineering Aims and scope Submit manuscript

Abstract

Cellular cholesterol is a critical component of the plasma membrane, and plays a key role in determining the physical properties of the lipid bilayer, such as elasticity, viscosity, and permeability. Surprisingly, it has been shown that cholesterol depletion increases cell stiffness, not due to plasma membrane stiffening, but rather, due to the interaction between the actin cytoskeleton and the plasma membrane. This indicates that traction stresses of the acto-myosin complex likely increase during cholesterol depletion. Here we use force traction microscopy to quantify the forces individual cells are exerting on the substrate, and total internal reflection fluorescence microscopy as well as interference reflection microscopy to observe cell–substrate adhesion and spreading. We show that single cells depleted of cholesterol produce larger traction forces and have large focal adhesions compared to untreated or cholesterol-enriched cells. Cholesterol depletion also causes a decrease in adhesion area for both single cells and monolayers. Spreading experiments illustrate a decrease in spreading area for cholesterol-depleted cells, and no effect on cholesterol-enriched cells. These results demonstrate that cholesterol plays an important role in controlling and regulating the cell–substrate interactions through the actin–plasma membrane complex, cell–cell adhesion, and spreading.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Axelrod, D. Total internal reflection fluorescence microscopy in cell biology. Methods Enzymol. 361:1–33, 2003.

    Article  Google Scholar 

  2. Balaban, N. Q., U. S. Schwarz, D. Riveline, P. Goichberg, G. Tzur, et al. Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat. Cell Biol. 3:466–472, 2001.

    Article  Google Scholar 

  3. Bershadsky, A. D., N. Q. Balaban, and B. Geiger. Adhesion-dependent cell mechanosensitivity. Annu. Rev. Cell Dev. Biol. 19:677–695, 2003.

    Article  Google Scholar 

  4. Butler, J. P., I. M. Tolic-Norrelykke, B. Fabry, and J. J. Fredberg. Traction fields, moments, and strain energy that cells exert on their surroundings. Am. J. Physiol. Cell Physiol. 282:C595–C605, 2002.

    Google Scholar 

  5. Byfield, F. J., H. Aranda-Espinoza, V. G. Romanenko, G. H. Rothblat, and I. Levitan. Cholesterol depletion increases membrane stiffness of aortic endothelial cells. Biophys. J. 87:3336–3343, 2004.

    Article  Google Scholar 

  6. Byfield, F. J., S. Tikku, G. H. Rothblat, K. J. Gooch, and I. Levitan. OxLDL increases endothelial stiffness, force generation, and network formation. J. Lipid Res. 47:715–723, 2006.

    Article  Google Scholar 

  7. Cavalli, V., M. Corti, and J. Gruenberg. Endocytosis and signaling cascades: a close encounter. FEBS Lett. 498:190–196, 2001.

    Article  Google Scholar 

  8. Chen, M., R. P. Mason, and T. N. Tulenko. Atherosclerosis alters the composition, structure and function of arterial smooth-muscle cell plasma-membranes. Biochim. Biophys. Acta 1272:101–112, 1995.

    Google Scholar 

  9. Chintagari, N. R., N. Jin, P. Wang, T. A. Narasaraju, J. Chen, and L. Liu. Effect of cholesterol depletion on exocytosis of alveolar type II cells. Am. J. Respir. Cell Mol. Biol. 34:677–687, 2006.

    Article  Google Scholar 

  10. Christian, A. E., M. P. Haynes, M. C. Phillips, and G. H. Rothblat. Use of cyclodextrins for manipulating cellular cholesterol content. J. Lipid Res. 38:2264–2272, 1997.

    Google Scholar 

  11. Corvera, S., C. DiBonaventura, and H. S. Shpetner. Cell confluence-dependent remodeling of endothelial membranes mediated by cholesterol. J. Biol. Chem. 275:31414–31421, 2000.

    Article  Google Scholar 

  12. Curtis, A. S. G. Mechanism of adhesion of cells to glass—study by interference reflection microscopy. J. Cell Biol. 20:199, 1964.

    Article  Google Scholar 

  13. Dembo, M., and Y. L. Wang. Stresses at the cell-to-substrate interface during locomotion of fibroblasts. Biophys. J. 76:2307–2316, 1999.

    Article  Google Scholar 

  14. Dubin-Thaler, B. J., G. Giannone, H. G. Dobereiner, and M. P. Sheetz. Nanometer analysis of cell spreading on matrix-coated surfaces reveals two distinct cell states and STEPs. Biophys. J. 86:1794–1806, 2004.

    Article  Google Scholar 

  15. Engler, A., L. Bacakova, C. Newman, A. Hategan, M. Griffin, and D. Discher. Substrate compliance versus ligand density in cell on gel responses. Biophys. J. 86:617–628, 2004.

    Article  Google Scholar 

  16. Galbraith, C. G., and M. P. Sheetz. A micromachined device provides a new bend on fibroblast traction forces. Proc. Natl Acad. Sci. USA 94:9114–9118, 1997.

    Article  Google Scholar 

  17. Gauthier, N. C., O. M. Rossier, A. Mathur, J. C. Hone, and M. P. Sheetz. Plasma membrane area increases with spread area by exocytosis of a GPI-anchored protein compartment. Mol. Biol. Cell 20:3261–3272, 2009.

    Article  Google Scholar 

  18. Guo, W. H., M. T. Frey, N. A. Burnham, and Y. L. Wang. Substrate rigidity regulates the formation and maintenance of tissues. Biophys. J. 90:2213–2220, 2006.

    Article  Google Scholar 

  19. Ilangumaran, S., and D. C. Hoessli. Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane. Biochem. J. 335:433–440, 1998.

    Google Scholar 

  20. Joos, U., T. Biskup, O. Ernst, I. Westphal, C. Gherasim, et al. Investigation of cell adhesion to structured surfaces using total internal reflection fluorescence and confocal laser scanning microscopy. Eur. J. Cell Biol. 85:225–228, 2006.

    Article  Google Scholar 

  21. Klausen, T. K., C. Hougaard, E. K. Hoffmann, and S. F. Pedersen. Cholesterol modulates the volume-regulated anion current in Ehrlich-Lettre ascites cells via effects on Rho and F-actin. Am. J. Physiol. Cell Physiol. 291:C757–C771, 2006.

    Article  Google Scholar 

  22. Kong, H. J., T. R. Polte, E. Alsberg, and D. J. Mooney. FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness. Proc. Natl Acad. Sci. USA 102:4300–4305, 2005.

    Article  Google Scholar 

  23. Kowalsky, G. B., F. J. Byfield, and I. Levitan. oxLDL facilitates flow-induced realignment of aortic endothelial cells. Am. J. Physiol. Cell Physiol. 295:C332–C340, 2008.

    Article  Google Scholar 

  24. Kwik, J., S. Boyle, D. Fooksman, L. Margolis, M. P. Sheetz, and M. Edidin. Membrane cholesterol, lateral mobility, and the phosphatidylinositol 4,5-bisphosphate-dependent organization of cell actin. Proc. Natl Acad. Sci. USA 100:13964–13969, 2003.

    Article  Google Scholar 

  25. Levitan, I., A. E. Christian, T. N. Tulenko, and G. H. Rothblat. Membrane cholesterol content modulates activation of volume-regulated anion current in bovine endothelial cells. J. Gen. Physiol. 115:405–416, 2000.

    Article  Google Scholar 

  26. Levitan, I., and K. J. Gooch. Lipid rafts in membrane–cytoskeleton interactions and control of cellular biomechanics: actions of oxLDL. Antioxid. Redox Signal. 9:1519–1534, 2007.

    Article  Google Scholar 

  27. Lundbaek, J. A., P. Birn, J. Girshman, A. J. Hansen, and O. S. Andersen. Membrane stiffness and channel function. Biochemistry 35:3825–3830, 1996.

    Article  Google Scholar 

  28. Maxfield, F. R., and I. Tabas. Role of cholesterol and lipid organization in disease. Nature 438:612–621, 2005.

    Article  Google Scholar 

  29. Michiels, C. Endothelial cell functions. J. Cell. Physiol. 196:430–443, 2003.

    Article  Google Scholar 

  30. Needham, D., and R. S. Nunn. Elastic-deformation and failure of lipid bilayer-membranes containing cholesterol. Biophys. J. 58:997–1009, 1990.

    Article  Google Scholar 

  31. Pourati, J., A. Maniotis, D. Spiegel, J. L. Schaffer, J. P. Butler, et al. Is cytoskeletal tension a major determinant of cell deformability in adherent endothelial cells? Am. J. Physiol. Cell Physiol. 43:C1283–C1289, 1998.

    Google Scholar 

  32. Ramprasad, O. G., G. Srinivas, K. S. Rao, P. Joshi, J. P. Thiery, et al. Changes in cholesterol levels in the plasma membrane modulate cell signaling and regulate cell adhesion and migration on fibronectin. Cell Motil. Cytoskeleton 64:199–216, 2007.

    Article  Google Scholar 

  33. Raucher, D., and M. P. Sheetz. Cell spreading and lamellipodial extension rate is regulated by membrane tension. J. Cell Biol. 148:127–136, 2000.

    Article  Google Scholar 

  34. Reinhart-King, C. A., M. Dembo, and D. A. Hammer. Endothelial cell traction forces on RGD-derivatized polyacrylamide substrata. Langmuir 19:1573–1579, 2003.

    Article  Google Scholar 

  35. Riveline, D., E. Zamir, N. Q. Balaban, U. S. Schwarz, T. Ishizaki, et al. Focal contacts as mechanosensors: Externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism. J. Cell Biol. 153:1175–1185, 2001.

    Article  Google Scholar 

  36. Romanenko, V. G., G. H. Rothblat, and I. Levitan. Modulation of endothelial inward-rectifier K+ current by optical isomers of cholesterol. Biophys. J. 83:3211–3222, 2002.

    Article  Google Scholar 

  37. Romanenko, V. G., G. H. Rothblat, and I. Levitan. Sensitivity of volume-regulated anion current to cholesterol structural analogues. J. Gen. Physiol. 123:77–87, 2004.

    Article  Google Scholar 

  38. Saez, A., A. Buguin, P. Silberzan, and B. Ladoux. Is the mechanical activity of epithelial cells controlled by deformations or forces? Biophys. J. 89:L52–L54, 2005.

    Article  Google Scholar 

  39. Sato, M., D. P. Theret, L. T. Wheeler, N. Ohshima, and R. M. Nerem. Application of the micropipette technique to the measurement of cultured porcine aortic endothelial-cell viscoelastic properties. J. Biomech. Eng. 112:263–268, 1990.

    Article  Google Scholar 

  40. Schwarz, U. S., N. Q. Balaban, D. Riveline, A. Bershadsky, B. Geiger, and S. A. Safran. Calculation of forces at focal adhesions from elastic substrate data: the effect of localized force and the need for regularization. Biophys. J. 83:1380–1394, 2002.

    Article  Google Scholar 

  41. Sengupta, K., H. Aranda-Espinoza, L. Smith, P. Janmey, and D. Hammer. Spreading of neutrophils: from activation to migration. Biophys. J. 91:4638–4648, 2006.

    Article  Google Scholar 

  42. Sheetz, M. P., J. E. Sable, and H. G. Dobereiner. Continuous membrane–cytoskeleton adhesion requires continuous accommodation to lipid and cytoskeleton dynamics. Annu. Rev. Biophys. Biomol. Struct. 35:417–434, 2006.

    Article  Google Scholar 

  43. Shibamoto, S., M. Hayakawa, K. Takeuchi, T. Hori, N. Oku, et al. Tyrosine phosphorylation of beta-catenin and plakoglobin enhanced by hepatocyte growth-factor and epidermal growth-factor in human carcinoma-cells. Cell Adhes. Commun. 1:295–305, 1994.

    Article  Google Scholar 

  44. Smith, L. A., H. Aranda-Espinoza, J. B. Haun, M. Dembo, and D. A. Hammer. Neutrophil traction stresses are concentrated in the uropod during migration. Biophys. J. 92:L58–L60, 2007.

    Article  Google Scholar 

  45. Solon, J., I. Levental, K. Sengupta, P. C. Georges, and P. A. Janmey. Fibroblast adaptation and stiffness matching to soft elastic substrates. Biophys. J. 93:4453–4461, 2007.

    Article  Google Scholar 

  46. Sun, M., N. Northup, F. Marga, T. Huber, F. J. Byfield, et al. The effect of cellular cholesterol on membrane–cytoskeleton adhesion. J. Cell Sci. 120:2223–2231, 2007.

    Article  Google Scholar 

  47. Tan, J. L., J. Tien, D. M. Pirone, D. S. Gray, K. Bhadriraju, and C. S. Chen. Cells lying on a bed of microneedles: an approach to isolate mechanical force. Proc. Natl Acad. Sci. USA 100:1484–1489, 2003.

    Article  Google Scholar 

  48. Ukropec, J. A., M. K. Hollinger, S. M. Salva, and M. J. Woolkalis. SHP2 association with VE-cadherin complexes in human endothelial cells is regulated by thrombin. J. Biol. Chem. 275:5983–5986, 2000.

    Article  Google Scholar 

  49. Wang, Y. L., and R. J. Pelham. Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of culture cells. Methods Enzymol. 298:489–496, 1998.

    Article  Google Scholar 

  50. Yeung, T., P. C. Georges, L. A. Flanagan, B. Marg, M. Ortiz, et al. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil. Cytoskeleton 60:24–34, 2005.

    Article  Google Scholar 

Download references

Acknowledgments

The authors thank Elena Tous for assistance with monolayer staining and preparations. This work was supported by NSF Grant CMMI-0643783 to HAE, National Institutes of Health Grants HL083298 and HL073965 to IL, and a Department of Defense CREST Graduate Fellowship to LLN.

Author information

Authors and Affiliations

  1. Fischell Department of Bioengineering, University of Maryland, Room 3138 Jeong H. Kim Engineering Building (Bldg. #225), College Park, MD, 20742, USA

    Leann L. Norman, Ratna J. Oetama & Helim Aranda-Espinoza

  2. Department of Biomedical Engineering, Boston University, Boston, MA, 02215, USA

    Micah Dembo

  3. Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, 19104, USA

    F. Byfield & Daniel A. Hammer

  4. Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Daniel A. Hammer

  5. Pulmonary, Critical Care and Sleep Medicine, University of Illinois at Chicago, Chicago, IL, 60612, USA

    Irena Levitan

Authors
  1. Leann L. Norman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ratna J. Oetama
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Micah Dembo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. F. Byfield
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Daniel A. Hammer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Irena Levitan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Helim Aranda-Espinoza
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Helim Aranda-Espinoza.

Additional information

Associate Editor Yu-Li Wang oversaw the review of this article.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Norman, L.L., Oetama, R.J., Dembo, M. et al. Modification of Cellular Cholesterol Content Affects Traction Force, Adhesion and Cell Spreading. Cel. Mol. Bioeng. 3, 151–162 (2010). https://doi.org/10.1007/s12195-010-0119-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12195-010-0119-x

Keywords

Search

Navigation