Plasma membrane effects of sphingolipid-synthesis inhibition by myriocin in CHO cells: a biophysical and lipidomic study

Abstract

Suppression of a specific gene effect can be achieved by genetic as well as chemical methods. Each approach may hide unexpected drawbacks, usually in the form of side effects. In the present study, the specific inhibitor myriocin was used to block serine palmitoyltransferase (SPT), the first enzyme in the sphingolipid synthetic pathway, in CHO cells. The subsequent biophysical changes in plasma membranes were measured and compared with results obtained with a genetically modified CHO cell line containing a defective SPT (the LY-B cell line). Similar effects were observed with both approaches: sphingomyelin values were markedly decreased in myriocin-treated CHO cells and, in consequence, their membrane molecular order (measured as laurdan general polarization) and mechanical resistance (AFM-measured breakthrough force values) became lower than in the native, non-treated cells. Cells treated with myriocin reacted homeostatically to maintain membrane order, synthesizing more fully saturated and less polyunsaturated GPL than the non-treated ones, although they achieved it only partially, their plasma membranes remaining slightly more fluid and more penetrable than those from the control cells. The good agreement between results obtained with very different tools, such as genetically modified and chemically treated cells, reinforces the use of both methods and demonstrates that both are adequate for their intended use, i.e. the complete and specific inhibition of sphingolipid synthesis in CHO cells, without apparent unexpected effects.

Figure 1

Myriocin-treated CHO and LY-B cell growth. (A) LY-B and CHO cell growth as a function of time in sphingolipid-deficient (0.04% FBS) medium, plus/minus 2.5 µM myriocin. Statistical symbols correspond to differences between green circles (CHO 0.04) and the other three samples; Student’s t-test ***P < 0.001. (B) Myriocin-treated and non-treated CHO cell growth after 72 h in sphingolipid-deficient medium supplemented with sphinganine (seeded cells: 0.25 × 10⁶). Values are given as average values ± S.D. (n = 3). Significance, according to Student’s t-test, myriocin vs. control: *P < 0.05; ***P < 0.001. (C) A representative FACS assessment of cell viability of CHO cells after 72 h in sphingolipid-deficient medium plus 2.5 µM myriocin. Three independent experiments were performed counting 10,000 cells in each case.

Figure 2

mCherry-Lysenin-stained myriocin-treated or non-treated cells after 72 h growth. Non-treated (A) and myriocin-treated (B) CHO cells in standard medium. Non-treated (C) and myriocin-treated (D) CHO cells in deficient medium. Non-treated (E) and myriocin-treated (F) LY-B cells in standard medium. Non-treated (G) and myriocin-treated (H) LY-B cells in deficient medium. Bar = 75 µm.

Figure 3

FACS-mediated, quantification of cell staining with SM specific mCherry-lysenin as a function of time. CHO cells grown in standard medium vs. myriocin-treated or non-treated CHO cells grown in deficient medium for 24 h (A) or 72 h (B). LY-B cells grown in standard medium vs. treated or non-treated LY-B cells grown in deficient medium for 24 h (C) or 72 h (D). Total CHO and LY-B lysenin-mCherry signals and CHO/LY-B lysenin-mCherry signal ratios (E). Histograms in red correspond to control cells (CHO or LY-B grown in standard-medium); histograms in black correspond to the samples of interest, as indicated in each case. Geometric mean ± S.D. (n = 3). Representative histograms are shown; three independent experiments were performed counting 10,000 cells in each case. Statistical significance was calculated with the Student’s t-test, ***P < 0.001.

Figure 4

Two-photon microscopy images of PM patches stained with laurdan for GP measurements. PM patches of non-treated (A) and myriocin-treated (B) CHO cells grown in deficient medium. (C) Box plot graph of GP values of non-treated (black) and myriocin-treated (blue) CHO cell PM patches. GP are given as average values ± S.D. Three independent experiments were performed; n = 150 (50 PM patches × 3 replicas). Statistical significance (GP of control vs. myriocin-treated cells) was calculated with the Student’s t-test, P = 0.03. Bar = 30 µm.

Figure 5

Laurdan staining and GP processing. A representative CHO cell grown in deficient medium (A), and the PM selection of the cell in panel A (B). A representative LY-B cell grown in deficient medium (C), and the PM selection of the cell in panel C (D). A representative 2.5 µM myriocin-treated CHO cell grown in deficient medium (E), and the PM selection of the cell in panel E (F). A representative 2.5 µM myriocin-treated LY-B cell grown in deficient medium (G), and the PM selection of the cell in panel E (H). Box plot graph of GP values of non-treated and myriocin-treated CHO (black and blue dots) and LY-B (red and green dots) PM selection values, including average values ± S.D. Three independent experiments were performed; n = 75 (25 PM selections × 3 replicas). Treated vs non-treated CHO cell P = 0.027 (I). Statistical significance was calculated with the Student´s t-test.

Figure 6

Topographic image and breakthrough force distributions of PM patches. PM patches from non-treated (A) and myriocin-treated (B) CHO cells grown in deficient medium. Three independent experiments were performed; n = 150–170 (50–60 breakthrough forces × 3 replicas. Statistical significance was calculated with the Student´s t-test, P = 0.0008.

Figure 7

Myriocin treatment effects on the lipid composition of CHO ad LY-B PM patches. Total SM (A), Cer (B) and HexCer (C). Fully saturated (DB = double bond) (D) and polyunsaturated (E) GPL. Short-chain (30-32C) (F), long-chain (34-40C) (G) and very-long chain (42-44C) GPL (H). Only selected lipids are included in the figure, a comprehensive description of the various lipid compositions can be seen in the Supplementary Material Table S1. n = 3. Statistical significance (control vs. myriocin-treated) was calculated with the Student´s t-test: *P < 0.05; ***P < 0.001.

Figure 8

A correlative presentation of myriocin effects on the plasma membranes of CHO cells grown on SL-deficient medium. Black bars: control, non-myriocin treated cells; grey bars: myriocin-treated cells. (A) Laurdan general polarization in PM patches (data from Fig. 4). (B) Bilayer breakthrough forces obtained by AFM in the force-spectroscopy mode (data from Fig. 6). (C) Mole percent SM contents in PM patches (data from Table S1 and Fig. 7A). (D) Saturated/unsaturated fatty-acyl mole ratio (data from Table S1 and Fig. 7D, E). (E) Short-chain/very-long-chain fatty-acyl mole ratio (data from Table S1 and Fig. 7 F, H). Significance (control vs. myriocin-treated cells) according to Student’s t-test: *P < 0.05; **P < 0.01, ***P < 0.001.