Insight into the role of cholesterol in modulation of morphology and mechanical properties of CHO-K1 cells: An in situ AFM study

  • Lei Zhang
  • Lisha Zhao
  • Ping-Kai Ouyang
  • Pu ChenEmail author
Research Article


Cholesterol plays a significant role in the organization of lipids and modulation of membrane dynamics in mammalian cells. However, the effect of cholesterol depletion on the eukaryotic cell membranes seems controversial. In this study, the effects of cholesterol on the topography and mechanical behaviors of CHO-K1 cells with manipulated membrane cholesterol contents were investigated by atomic force microscopy (AFM) technique. Here, we found that the depletion of cholesterol in cell membranes could increase the membrane stiffness, reduce the cell height as well as promote cell retraction and detachment from the surface, whereas the cholesterol restoration could reverse the effect of cholesterol depletion on the membrane stiffness. Increased methyl-β-cyclodextrin levels and incubation time could significantly increase Young’s modulus and degree of stiffing on cell membrane and cytoskeleton. This research demonstratede importance of cholesterol in regulating the dynamics of cytoskeleton-mediated processes. AFM technique offers excellent advantages in the dynamic monitoring of the change in membranes mechanical properties and behaviors during the imaging process. This promising technology can be utilized in studying the membrane properties and elucidating the underlying mechanism of distinct cells in the nearnative environment.


cholesterol methyl-β-cyclodextrin atomic force microscopy Young’s modulus CHO-K1 cell 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



The authors sincerely appreciate the financial support by the Natural Sciences and Engineering Research Council of Canada Discovery Grants Program as well as the Program of Scientific Innovation Research of College Graduates in Jiangsu Province (No. CXZZ13_0455). The authors also thank Prof. Zhaobing Gao in the Shanghai Institute of Materia Medica of Chinese Academy of Science for providing the Biocatalyst AFM and related supports.

Supplementary material

11705_2018_1775_MOESM1_ESM.pdf (260 kb)
Insight into the role of cholesterol in modulation of morphology and mechanical properties of CHO-K1 cells: An in situ AFM study


  1. 1.
    de Oliveira Andrade L. Understanding the role of cholesterol in cellular biomechanics and regulation of vesicular trafficking: The power of imaging. Biomedical Spectroscopy and Imaging, 2016, 5 (s1): S101–S117Google Scholar
  2. 2.
    Evangelisti E, Cecchi C, Cascella R, Sgromo C, Becatti M, Dobson C M, Chiti F, Stefani M. Membrane lipid composition and its physicochemical properties define cell vulnerability to aberrant protein oligomers. Journal of Cell Science, 2012, 125(10): 2416–2427Google Scholar
  3. 3.
    Redondo-Morata L, Giannotti M I, Sanz F. Influence of cholesterol on the phase transition of lipid bilayers: A temperature-controlled force spectroscopy study. Langmuir, 2012, 28(35): 12851–12860Google Scholar
  4. 4.
    Zhao L, Temelli F. Preparation of liposomes using supercritical carbon dioxide via depressurization of the supercritical phase. Journal of Food Engineering, 2015, 158: 104–112Google Scholar
  5. 5.
    Magarkar A, Dhawan V, Kallinteri P, Viitala T, Elmowafy M, Rog T, Bunker A. Cholesterol level affects surface charge of lipid membranes in saline solution. Scientific Reports, 2014, 4: 2045–2322Google Scholar
  6. 6.
    Zhao L, Temelli F, Curtis J M, Chen L. Encapsulation of lutein in liposomes using supercritical carbon dioxide. Food Research International, 2017, 100: 168–179Google Scholar
  7. 7.
    de Meyer F, Smit B. Effect of cholesterol on the structure of a phospholipid bilayer. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(10): 3654–3658Google Scholar
  8. 8.
    Sułkowski W, Pentak D, Nowak K, Sułkowska A. The influence of temperature, cholesterol content and pH on liposome stability. Journal of Molecular Structure, 2005, 744: 737–747Google Scholar
  9. 9.
    Zhao L, Temelli F, Curtis J M, Chen L. Preparation of liposomes using supercritical carbon dioxide technology: Effects of phospholipids and sterols. Food Research International, 2015, 77: 63–72Google Scholar
  10. 10.
    Zhao L, Temelli F. Preparation of liposomes using a modified supercritical process via depressurization of liquid phase. Journal of Supercritical Fluids, 2015, 100: 110–120Google Scholar
  11. 11.
    Khatibzadeh N, Spector A A, Brownell W E, Anvari B. Effects of plasma membrane cholesterol level and cytoskeleton F-actin on cell protrusion mechanics. PLoS One, 2013, 8(2): e57147Google Scholar
  12. 12.
    Mañes S, Martínez-A C. Cholesterol domains regulate the actin cytoskeleton at the leading edge of moving cells. Trends in Cell Biology, 2004, 14(6): 275–278Google Scholar
  13. 13.
    Sun M, Northup N, Marga F, Huber T, Byfield F J, Levitan I, Forgacs G. The effect of cellular cholesterol on membranecytoskeleton adhesion. Journal of Cell Science, 2007, 120(13): 2223–2231Google Scholar
  14. 14.
    Norman L L, Oetama R J, Dembo M, Byfield F, Hammer D A, Levitan I, Aranda-Espinoza H. Modification of cellular cholesterol content affects traction force, adhesion and cell spreading. Cellular and Molecular Bioengineering, 2010, 3(2): 151–162Google Scholar
  15. 15.
    Yang Y T, Liao J D, Lin C C K, Chang C T, Wang S H, Ju M S. Characterization of cholesterol-depleted or-restored cell membranes by depth-sensing nano-indentation. Soft Matter, 2012, 8(3): 682–687Google Scholar
  16. 16.
    Kilbride P, Woodward H J, Tan K B, Thanh N T, Chu K E, Minogue S, Waugh MG. Modeling the effects of cyclodextrin on intracellular membrane vesicles from Cos-7 cells prepared by sonication and carbonate treatment. PeerJ, 2015, 3: e1351Google Scholar
  17. 17.
    Zidovetzki R, Levitan I. Use of cyclodextrins to manipulate plasma membrane cholesterol content: Evidence, misconceptions and control strategies. Biochimica et Biophysica Acta (BBA)—Biomembranes, 2007, 1768(6): 1311–1324Google Scholar
  18. 18.
    Christian A, Haynes M, Phillips M, Rothblat G. Use of cyclodextrins for manipulating cellular cholesterol content. Journal of Lipid Research, 1997, 38(11): 2264–2272Google Scholar
  19. 19.
    Romanenko V G, Fang Y, Byfield F, Travis A J, Vandenberg C A, Rothblat G H, Levitan I. Cholesterol sensitivity and lipid raft targeting of Kir2. 1 channels. Biophysical Journal, 2004, 87(6): 3850–3861Google Scholar
  20. 20.
    Mahammad S, Parmryd I. Cholesterol depletion using methylcyclodextrin. Methods in Membrane Lipids, 2015: 91–102Google Scholar
  21. 21.
    Romanenko V G, Rothblat G H, Levitan I. Modulation of endothelial inward-rectifier K+ current by optical isomers of cholesterol. Biophysical Journal, 2002, 83(6): 3211–3222Google Scholar
  22. 22.
    Levitan I, Christian A E, Tulenko T N, Rothblat G H. Membrane cholesterol content modulates activation of volume-regulated anion current in bovine endothelial cells. Journal of General Physiology, 2000, 115(4): 405–416Google Scholar
  23. 23.
    Niu S L, Mitchell D C, Litman B J. Manipulation of cholesterol levels in rod disk membranes by methyl-β-cyclodextrin effects on receptor activation. Journal of Biological Chemistry, 2002, 277(23): 20139–20145Google Scholar
  24. 24.
    Klein U, Gimpl G, Fahrenholz F. Alteration of the myometrial plasma membrane cholesterol content with β-cyclodextrin modulates the binding affinity of the oxytocin receptor. Biochemistry, 1995, 34(42): 13784–13793Google Scholar
  25. 25.
    Roduit C, van der Goot F G, De Los Rios P, Yersin A, Steiner P, Dietler G, Catsicas S, Lafont F, Kasas S. Elastic membrane heterogeneity of living cells revealed by stiff nanoscale membrane domains. Biophysical Journal, 2008, 94(4): 1521–1532Google Scholar
  26. 26.
    Bronder A M, Bieker A, Elter S, Etzkorn M, Häussinger D, Oesterhelt F. Oriented membrane protein reconstitution into tethered lipid membranes for AFM force spectroscopy. Biophysical Journal, 2016, 111(9): 1925–1934Google Scholar
  27. 27.
    Casuso I, Khao J, Chami M, Paul-Gilloteaux P, Husain M, Duneau J P, Stahlberg H, Sturgis J N, Scheuring S. Characterization of the motion of membrane proteins using high-speed atomic force microscopy. Nature Nanotechnology, 2012, 7(8): 525–529Google Scholar
  28. 28.
    Hutter J L, Bechhoefer J. Calibration of atomic-force microscope tips. Review of Scientific Instruments, 1993, 64(7): 1868–1873Google Scholar
  29. 29.
    Sneddon I N. The relation between load and penetration in the axisymmetric Boussinesq problem for a punch of arbitrary profile. International Journal of Engineering Science, 1965, 3(1): 47–57Google Scholar
  30. 30.
    Matzke R, Jacobson K, Radmacher M. Direct, high-resolution measurement of furrow stiffening during division of adherent cells. Nature Cell Biology, 2001, 3(6): 607–610Google Scholar
  31. 31.
    Emad A, Heinz W F, Antonik M D, D’Costa N P, Nageswaran S, Schoenenberger C A, Hoh J H. Relative microelastic mapping of living cells by atomic force microscopy. Biophysical Journal, 1998, 74(3): 1564–1578Google Scholar
  32. 32.
    Lam R S, Shaw A R, Duszyk M. Membrane cholesterol content modulates activation of BK channels in colonic epithelia. Biochimica et Biophysica Acta (BBA)—Biomembranes, 2004, 1667(2): 241–248Google Scholar
  33. 33.
    Toselli M, Biella G, Taglietti V, Cazzaniga E, Parenti M. Caveolin-1 expression and membrane cholesterol content modulate N-type calcium channel activity in NG108-15 cells. Biophysical Journal, 2005, 89(4): 2443–2457Google Scholar
  34. 34.
    Oh H, Mohler E R III, Tian A, Baumgart T, Diamond S L. Membrane cholesterol is a biomechanical regulator of neutrophil adhesion. Arteriosclerosis, Thrombosis, and Vascular Biology, 2009, 29(9): 1290–1297Google Scholar
  35. 35.
    Corvera S, DiBonaventura C, Shpetner H S. Cell confluencedependent remodeling of endothelial membranes mediated by cholesterol. Journal of Biological Chemistry, 2000, 275(40): 31414–31421Google Scholar
  36. 36.
    Frankel D, Pfeiffer J, Surviladze Z, Johnson A, Oliver J, Wilson B, Burns A. Revealing the topography of cellular membrane domains by combined atomic force microscopy/fluorescence imaging. Biophysical Journal, 2006, 90(7): 2404–2413Google Scholar
  37. 37.
    Kwik J, Boyle S, Fooksman D, Margolis L, Sheetz M P, Edidin M. Membrane cholesterol, lateral mobility, and the phosphatidylinositol 4,5-bisphosphate-dependent organization of cell actin. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(24): 13964–13969Google Scholar
  38. 38.
    Byfield F J, Tikku S, Rothblat G H, Gooch K J, Levitan I. OxLDL increases endothelial stiffness, force generation, and network formation. Journal of Lipid Research, 2006, 47(4): 715–723Google Scholar
  39. 39.
    Zhang X, Hurng J, Rateri D L, Daugherty A, Schmid-Schönbein G W, Shin H Y. Membrane cholesterol modulates the fluid shear stress response of polymorphonuclear leukocytes via its effects on membrane fluidity. American Journal of Physiology. Cell Physiology, 2011, 301(2): C451–C460Google Scholar
  40. 40.
    Borbiev T, Radel C, Rizzo V. Participation of caveolae in β1 integrin-mediated mechanotransduction. FASEB Journal, 2007, 21 (6): A752–A752Google Scholar
  41. 41.
    Qi Y, Andolfi L, Frattini F, Mayer F, Lazzarino M, Hu J. Membrane stiffening by STOML3 facilitates mechanosensation in sensory neurons. Nature Communications, 2015, 6(1): 8512Google Scholar
  42. 42.
    Byfield F J, Aranda-Espinoza H, Romanenko V G, Rothblat G H, Levitan I. Cholesterol depletion increases membrane stiffness of aortic endothelial cells. Biophysical Journal, 2004, 87(5): 3336–3343Google Scholar
  43. 43.
    Hissa B, Pontes B, Roma P M S, Alves A P, Rocha C D, Valverde T M, Aguiar P H N, Almeida F P, Guimaraes A J, Guatimosim C, et al. Membrane cholesterol removal changes mechanical properties of cells and induces secretion of a specific pool of lysosomes. PLoS One, 2013, 8(12): e82988Google Scholar
  44. 44.
    Brown R E. Sphingolipid organization in biomembranes: What physical studies of model membranes reveal. Journal of Cell Science, 1998, 111(1): 1–9Google Scholar
  45. 45.
    Pralle A, Keller P, Florin E L, Simons K, Hörber J H. Sphingolipidcholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. Journal of Cell Biology, 2000, 148(5): 997–1008Google Scholar
  46. 46.
    Wakatsuki T, Schwab B, Thompson N C, Elson E L. Effects of cytochalasin D and latrunculin B on mechanical properties of cells. Journal of Cell Science, 2001, 114(5): 1025–1036Google Scholar
  47. 47.
    Khatibzadeh N, Gupta S, Farrell B, Brownell WE, Anvari B. Effects of cholesterol on nano-mechanical properties of the living cell plasma membrane. Soft Matter, 2012, 8(32): 8350–8360Google Scholar
  48. 48.
    Zhang L, Bennett W F D, Zheng T, Ouyang P K, Ouyang X P, Qiu X Q, Luo A Q, Karttunen M, Chen P. Effect of cholesterol on cellular uptake of cancer drugs pirarubicin and ellipticine. Journal of Physical Chemistry B, 2016, 120(12): 3148–3156Google Scholar
  49. 49.
    Ramprasad O, Srinivas G, Rao K S, Joshi P, Thiery J P, Dufour S, Pande G. Changes in cholesterol levels in the plasma membrane modulate cell signaling and regulate cell adhesion and migration on fibronectin. Cytoskeleton, 2007, 64(3): 199–216Google Scholar
  50. 50.
    López C A, de Vries A H, Marrink S J. Molecular mechanism of cyclodextrin mediated cholesterol extraction. PLoS Computational Biology, 2011, 7(3): e1002020Google Scholar

Copyright information

© Higher Education Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Lei Zhang
    • 1
    • 2
  • Lisha Zhao
    • 3
  • Ping-Kai Ouyang
    • 2
  • Pu Chen
    • 1
    • 2
    Email author
  1. 1.Department of Chemical Engineering and Waterloo Institute for NanotechnologyUniversity of WaterlooWaterlooCanada
  2. 2.College of Biotechnology and Pharmaceutical EngineeringNanjing Tech UniversityNanjingChina
  3. 3.Department of Agricultural, Food and Nutritional ScienceUniversity of AlbertaEdmontonCanada

Personalised recommendations