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

doi:10.1007/s11705-018-1775-y

Front. Chem. Sci. Eng.

2019, Vol. 13

Issue (1) : 98-107 https://doi.org/10.1007/s11705-018-1775-y

RESEARCH ARTICLE

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

Lei Zhang^1,², Lisha Zhao³, Ping-Kai Ouyang², Pu Chen^1,²(

)

¹. Department of Chemical Engineering and Waterloo Institute for Nanotechnology, University of Waterloo, Ontario N2L 3G1, Canada
². College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China
³. Department of Agricultural, Food and Nutritional Science, University of Alberta, Alberta T6G 2P5, Canada

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Abstract

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 near-native environment.

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

Corresponding Author(s): Pu Chen

Just Accepted Date: 06 September 2018 Online First Date: 25 January 2019 Issue Date: 25 February 2019

Cite this article:

Lei Zhang,Lisha Zhao,Ping-Kai Ouyang, et al. Insight into the role of cholesterol in modulation of morphology and mechanical properties of CHO-K1 cells: An in situ AFM study[J]. Front. Chem. Sci. Eng., 2019, 13(1): 98-107.

URL:

https://academic.hep.com.cn/fcse/EN/10.1007/s11705-018-1775-y
https://academic.hep.com.cn/fcse/EN/Y2019/V13/I1/98

Fig.1 Representative AFM images from both untreated (control) and treated CHO-K1 cells with 10 mol/L MβCD incubation for different time intervals. From the left side, the first (a_i?d_i) and second (a_ii?d_ii) columns showed deflection-error images and height images (100 mm²) of the cells, respectively. The third column (a_iii?d_iii) showed three-dimensional (3D) images of the cells and the last column (a_iv?d_iv) presented the cell height profiles corresponding to individual cell position selected in each figure in the second column. A white line in each figure denoted the position selected on the cell surface. The colors in the images indicated different heights with dark and light regions corresponding to lower and higher positions of the topography, respectively. The red arrows indicated the morphology diversification

Fig.2 The effect of incubation time of 10 mol/L MβCD on cell heights of CHO-K1 cells. (A) The cell heights of five cells (A?E) untreated (control) and treated with the 10 mol/L MβCD solution for 30, 150 and 270 min. Five cells were presented as A?E in Fig. 1(a_i); (B) The effect of incubation time on the mean heights of cells (mean±SD, n = 16, three cultures, P₁, P₂, P₃<0.001 (P₁: 30 min versus control, P₂: 270 min versus control, P₃: 270 minversus 150 min); (C) Mean cell height of untreated cells incubated for 0 and 270 min. There was no significant difference in intensity in control cells between 0 and 270 min (mean±SD, n = 10, P>0.05 versus control). In (B) and (C), the height of cell at 0 min (control) was normalized to 3.5

Fig.3 The effects of MβCD concentration and incubation time on Young’s modulus of CHO-K1 cells. (A) Young’s modulus of a single cell as a function of incubation time at different MβCD concentrations (Each point includes threes measurements of one individual cell); (B) Mean values of Young’s modulus of treated cells as a function of MβCD concentration for 30 min (Results were described as mean±SD (n = 3, three cultures), with a significant difference in intensity between treated and control cells. P₁, P₂<0.01 (P₁: 5 mol/L MβCD versus control, P₂: 10 mol/L MβCD versus control)); (C) Young’s modulus of control cells without MβCD treatment as a function of incubation time; (D) Mean value of Young’s modulus of control cells without MβCD treatment for up to 30 min. There was no significant difference (P>0.05) in the intensity among all the control cells at different time intervals. In (B) and (D), the height of cell at 0 min (control) was normalized to 2.4

Fig.4 The effect of cholesterol restoration on Young’s modulus of CHO-K1 cells using the 5 mol/L MβCD-cholesterol agent. (A) Normalized Young’s modulus of treated cells as a function of incubation time during the dynamic characterization process: untreated (5 min) and treated with 5 mol/L MβCD (15 min) and followed by 5 mol/L MβCD-cholesterol (15 min) (Each point includes threes measurements of one individual cell); (B) Mean value of Young’s modulus of cells (untreated, treated with 5 mol/L MβCD and 5 mol/L MβCD-cholesterol) (Results were described as mean±SD, n = 3, three cultures, P₁<0.01, P₂>0.05 (P₁: 5 mol/L MβCD versus control, P₂: 5 mol/L MβCD-cholesterol versus control); (C) Young’s modulus of control cells for 5 min and then incubated with 5 mol/L MβCD-cholesterol for up to 30 min; (D) Mean value of Young’s modulus of control cells without treatment and subject to 5 mol/L MβCD-cholesterol treatments for up to 30 min. There was no significant difference in intensity among all the control cells at different time intervals (P>0.05). In (B) and (D), the height of cell at 0 min (control) was normalized to 2.4

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