CHO/LY‐B cell growth under limiting sphingolipid supply: Correlation between lipid composition and biophysical properties of sphingolipid‐restricted cell membranes

Monasterio, Bingen G.; Jiménez‐Rojo, Noemi; García-Arribas, Aritz B.; Riezman, Howard; Goñi, Félix M.; Alonso, Alicia

doi:10.1096/fj.202001879rr

Cited by 6 publications

(18 citation statements)

References 85 publications

(183 reference statements)

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“…Flow-cytometry analyses demonstrated that 84% of CHO cells grown in SL-deficient medium for 72 h and treated with 2.5 µM myriocin remained viable (Fig. 1 C), similar to the 86% value obtained with non-treated LY-B cells grown in SL-deficient medium 13 . Values for Myr-treated LY-B cells were the same as for the non-treated ones.…”

Section: Resultssupporting

confidence: 79%

“…Results are occasionally shown of cells grown in myriocin-free, SL-deficient medium. The latter are only for comparative purposes, they have been published in 13 . Cell count measurements were performed using a BioRad TC20 hemocytometer to assess myriocin treatment effects on CHO (wild type) and LY-B (SPT-defective) cell division rate and integrity.…”

Section: Resultsmentioning

confidence: 99%

“…S1 C), as seen in Monasterio et al . 13 . It was concluded that myriocin-mediated lack of SPT activity could be masked when appropriate SL supplementation was added in the deficient-medium.…”

Section: Resultsmentioning

confidence: 99%

“…Mutant LY-B and wild type CHO cells could be comparatively studied to determine the effect of SL depletion on the cell membrane biophysical properties. In our previous study 13 , with cells grown under limiting SL concentrations, a significant decrease in the rigidity of LY-B cell membranes was observed using laurdan fluorescence, as well as a decrease in membrane breakthrough forces (membrane nanomechanical resistance) assessed by atomic force microscopy (AFM) 13 . Concomitantly, SL concentration in membranes was drastically reduced, with partially compensating changes in glycerophospholipids 13 .…”

Section: Introductionmentioning

confidence: 97%

“…A novel plasma membrane (PM) preparation (‘PM patches’) has been used 20 , 21 . The study has been performed following the steps of the previous publication with genetically modified CHO cells (LY-B cells) 13 , so that the respective results can be compared. The LY-B study design was such that the effects of FBS removal, namely loss of sphingolipids and loss of other components in the serum, could not be properly differentiated.…”

Section: Introductionmentioning

confidence: 99%

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

Monasterio

Jiménez‐Rojo

García-Arribas

et al. 2022

Sci Rep

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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.

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Section: Resultssupporting

confidence: 79%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%