Lipidomic atlas of mammalian cell membranes reveals hierarchical variation induced by culture conditions, subcellular membranes, and cell lineages

Abstract

Lipid membranes are ubiquitous biological organizers, required for structural and functional compartmentalization of the cell and sub-cellular organelles. Membranes in living cells are compositionally complex, comprising hundreds of dynamically regulated, distinct lipid species. Cellular physiology requires tight regulation of these lipidomic profiles to achieve proper membrane functionality. While some general features of tissue- and organelle-specific lipid complements have been identified, less is known about detailed lipidomic variations caused by cell-intrinsic or extrinsic factors. Here, we use shotgun lipidomics to report detailed, comprehensive lipidomes of a variety of cultured and primary mammalian membrane preparations to identify trends and sources of variation. Unbiased principle component analysis (PCA) shows clear separation between cultured and primary cells, with primary erythrocytes, synaptic membranes, and other mammalian tissue lipidomes sharply diverging from all cultured cell lines and also from one other. Most broadly, cultured cell membrane preparations were distinguished by their paucity of polyunsaturated lipids. Cultured mammalian cell lines were comparatively similar to one another, although we detected clear, highly reproducible lipidomic signatures of individual cell lines and plasma membrane (PM) isolations thereof. These measurements begin to establish a comprehensive lipidomic atlas of mammalian cells and tissues, identifying some major sources of variation. These observations will allow investigation of the regulation and functional significance of mammalian lipidomes in various contexts.

Figure 1-. Membrane lipidomes in vivo are distinct from cultured membranes.

(A) PCA of all lipidomes reveals clustering of all cultured cell preparations, including isolated PMs, away from the five primary membrane isolations (i.e. synaptosomes, RBCs, brain, liver, and heart). (B) Loadings plot for the PCA plot. (C) Lipid unsaturation and (D) headgroup profiles of primary tissue samples and whole membrane preparations of cultured cells. Bars represent means ± SD for 2–6 samples per condition. Abbreviations: PM – Plasma Membrane; CM – Cellular Membranes; DB – double bond. All lipidomic raw data is included as a Supplemental File.

Figure 2 -. Isolated PMs have distinct lipidomes from crude membrane preparations.

(A) PCA of lipidomes from cultured cell preparations reveals that isolated PMs were consistently distinct from cellular membrane (CM) extractions. (B) Loadings plot for the PCA scores suggests that PMs are generally characterized by higher cholesterol, SM, PS, and fully saturated or monounsaturated lipids. (C) Cholesterol is generally enriched in PMs compared to cellular membranes, (D) unsaturation indices compared between PM, NF (nuclei-free crude membranes), and WC (whole cell membranes) for various cell types, (E-F) PEp and SM are enriched in PMs relative to either whole membrane preparation, (G-I) PG, PCp, and PI are enriched in whole membrane preparations, (J-L) PS, PE, and PC enrichments in PM versus CM/WC were variable between cell types. Bars represent means ± SD. Symbols represent independent preparations.

Figure 3 -. Isolated PMs have distinct lipidomes from crude membrane preparations.

(A) PCA of whole cell (WC) membrane lipidomes shows separation between cultured cell types. (B) Loadings plot for PCA. (C) Variation in SL and PS abundances between cell types. (D) Variations in lipid unsaturation levels between cell types. (E) PCA of lipidomes from PM preparations shows clear separation between various cultured cell types. PM lipids from different cultured cells vary in both (F) headgroup distribution and (G) unsaturation of lipid acyl chains. (H) PCA of lipidomes from nuclei-free (NF) preparations from various cultured cells shows significant variation between cell types but also significant variation within certain cell types. (I-J) Serum effects on HEK and RBL WC lipids are minimal as shown in little change in headgroup (I) or acyl chain unsaturation (J) distributions. Bars represent means ± SD. Symbols represent individual preparations.