Characterization of Plasma Membrane Ceramides by Super‐Resolution Microscopy

Abstract

The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane which segregates membrane receptors and affect membrane curvature and vesicle formation, fusion and trafficking. Here, we label ceramides by immunocytochemistry and visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super-resolution images show that independent of labeling conditions and cell type 50-60% of all membrane ceramides are located in ceramide-rich platforms (CRPs) with a size of ~ 75 nm which are composed of at least ~ 20 ceramides. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold. Graphical Abstractd STORM reveals that independent of the cell type membrane ceramides form nanodomains (CRPs) with a diameter of ~ 75 nm containing 50-60% of all ceramides. Treatment with sphingomyelinase increases the quantity of ceramides, the size and density of CRPs as well as the ceramide concentration in CRPs.Correspondence to: Markus Sauer. Supporting information for this article is given via a link at the end of the document. The plasma membrane is mainly composed of glycerophospholipids, sphingolipids and cholesterol. Sphingolipids are natural lipids comprised of the sphingoid base backbone sphingosine, which when N-acylated with fatty acids forms ceramide, a central molecule in sphingolipid biology. De-novo synthesis of ceramide occurs in the endoplasmatic reticulum followed by conversion into complex sphingolipids in the Golgi apparatus. In addition, sphingomyelinases (SMases) can generate ceramide from sphingomyelin in the plasma membrane. [1,2] Interactions of sphingolipids with one another and with cholesterol typically result in membrane microdomains or 'rafts' which segregate membrane-associated proteins and compartmentalize signaling components within the plasma membrane. [3][4][5][6] Activation of SMases by cellular stress or ligation of receptors results in ceramide synthesis and subsequent self-association within the plasma membrane resulting in ceramide-rich platforms (CRPs). [7,8] The formation of CRPs rearranges the organization of the plasma membrane including clustering of diverse receptors and facilitates vesicle formation and fusion. [8,9] These changes induce differentiation, proliferation, growth arrest, and cell death. [1,2] Moreover, SMases and ceramides have been shown to be critically involved in the internalization of pathogens. [10][11][12][13][14][15] The recent observation, that CRP formation is essential in controlling the metabolic activity of regulatory T cells demonstrates that ceramides play also an essential role in regulating immune functions and i...

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