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

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Sphingolipids (SL) are ubiquitous in mammalian cell membranes, yet there is little data on the behavior of cells under SL-restriction conditions. LY-B cells derive from a CHO line in which serine palmitoyl transferase (SPT), thus <jats:italic>de novo</jats:italic> SL synthesis, is suppressed, while maintaining the capacity of taking up and metabolizing exogenous sphingoid bases from the culture medium. In the present study LY-B cells were adapted to grow in a fetal bovine serum (FBS)-deficient medium to avoid external uptake of lipids. The lowest FBS concentration that allowed LY-B cell growth, though at a slow rate, under our conditions was 0.04%, i.e. 250-fold less than the standard (10%) concentration. Cells grown under limiting SL concentrations remained viable for at least 72 h. Enriching with sphingomyelin the SL-deficient medium allowed the recovery of control LY-B cell growth rates. Studies including whole cells, plasma membrane preparations, and derived lipid vesicles were carried out. Laurdan fluorescence was recorded to measure membrane molecular order, showing a significant decrease in the rigidity of LY-B cells, not only in plasma membrane but also in whole cell lipid extract, as a result of SL limitation in the growth medium. Plasma membrane preparations and whole cell lipid extracts were also studied using atomic force microscopy in the force spectroscopy mode. Force measurements demonstrated that lower breakthrough forces were required to penetrate samples obtained from SL-poor LY-B cells than those obtained from control cells. Mass-spectroscopic analysis was also a helpful tool to understand the rearrangement undergone by the LY-B cell lipid metabolism. The most abundant SL in LY-B cells, sphingomyelin, decreased by about 85% as a result of SL limitation in the medium, the bioactive lipid ceramide and the ganglioside precursor hexosylceramide decreased similarly, together with cholesterol. Quantitative SL analysis showed that a 250-fold reduction in sphingolipid supply to LY-B cells led to a 6-fold decrease in membrane sphingolipids, underlining the resistance to changes in composition of these cells. Plasma membrane compositions exhibited similar changes, at least qualitatively, as the whole cells with SL restriction. A linear correlation was observed between the sphingomyelin concentration in the membranes, the degree of lipid order as measured by laurdan fluorescence, and membrane breakthrough forces assessed by atomic force microscopy. Concomitant changes were detected in glycerophospholipids under SL-restriction conditions.</jats:p>

Plasma Membrane Effects of Sphingolipid-Synthesis Inhibition by Myriocin in CHO Cells: A Biophysical and Lipidomic Study

“…This result is similar to the one obtained in Monasterio et al 13 when LY-B cell PM patches exhibited a large decrease, from 4.64 nN to 2.98 nN, when grown in normal or de cient medium 13 .…”

“…Myriocin-treated SM-depleted CHO cells reacted to maintain membrane order undergoing a homeostatic response, although they achieved it only partially, as their PM were more fragile when grown in SL-de cient medium. The same occurred with non-treated LYB cells 13 .…”

“…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. This was the approach in our previous study 13 in which we found that the lowest FBS concentration that allowed LY-B cell growth, though at a slow rate, under our conditions was 0.04%, i.e. 250-fold less than the standard (10%) concentration.…”

“…A novel plasma membrane (PM) preparation ('PM patches') has been used 27,28 . The study has been performed following the steps of the previous publication with genetically modi ed CHO cells (LY-B cells) 13 , so that the respective results can be compared. The LY-B study design is such that the effects of FBS removal, namely loss of sphingolipids and loss of other components in the serum, cannot be properly differentiated.…”

“…250-fold less than the standard (10%) concentration. Cells grown under those limiting SL concentrations remained viable for at least 72 h. A signi cant decrease in the rigidity of LY-B cell membranes was observed using laurdan uorescence, as well as a decrease in membrane breakthrough forces (membrane nanomechanical resistance) assessed by atomic force microscopy (AFM) 13 . Concomitantly, the SL concentration in membranes was drastically reduced, with partially compensating changes in glycerophospholipids 13 .…”