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.

Receptor-interacting Ser/Thr kinase 1 (RIPK1) and myosin IIA–dependent ceramidosomes form membrane pores that mediate blebbing and necroptosis

“…2, C and D). Recently, ceramiderich membrane areas in the plasma membrane were shown to form pores (54). This, together with our novel data with ceramide labeling, suggests that FTY720 exposure induces the formation of membrane pores, which are enriched in endogenously generated ceramides, altering plasma membrane integrity and inducing necrotic cell death.…”

Ceramide and Exosomes: A Novel Target in Cancer Biology and Therapy

“…While it is not understood how ceramide may contribute to the inward budding of vesicles at the MVE, facilitating or inducing membrane curvature may be one of its tasks (for biophysical properties of ceramide in membrane curvature, see (Burgert et al, 2017; Goni & Alonso, 2006; Goni, Contreras, Montes, Sot, & Alonso, 2005; Pinto, Silva, Futerman, & Prieto, 2011; L. C. Silva et al, 2012).…”

“…1B). Most recently, research in our and our collaborators’ laboratories has shown that ceramide-rich platforms (CRPs) are 50–200 nm in size and contain ≥ 20 ceramide molecules (Burgert et al, 2017). CRPs are formed when sphingomyelinases convert sphingomyelin into ceramide (Fig.…”

Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy

“…Ceramide-rich membrane areas promote structural changes within the plasma membrane, which segregate membrane receptors and affect the membrane curvature and vesicle formation, fusion and trafficking. 19,20 We selected ω-N3-C6-ceramide, which is efficiently incorporated into cellular membranes and can be click-labeled with DBCO-functionalized dyes for fluorescence imaging 21,22 , for further functionalization with a primary amino group (Supporting Information and Supplementary Figures S1-S13). Therefore, we synthesized α-NH2-ω-N3-C6-ceramide from (tert-butoxycarbonyl)-Llysine ( Figure 1A).…”