Abstract
Ceramides are central intermediates of sphingolipid metabolism that also function as potent messengers in stress signaling and apoptosis. Progress in understanding how ceramides execute their biological roles is hampered by a lack of methods to manipulate their cellular levels and metabolic fate with appropriate spatiotemporal precision. Here, we report on clickable, azobenzene-containing ceramides, caCers, as photoswitchable metabolic substrates to exert optical control over sphingolipid production in cells. Combining atomic force microscopy on model bilayers with metabolic tracing studies in cells, we demonstrate that light-induced alterations in the lateral packing of caCers lead to marked differences in their metabolic conversion by sphingomyelin synthase and glucosylceramide synthase. These changes in metabolic rates are instant and reversible over several cycles of photoswitching. Our findings disclose new opportunities to probe the causal roles of ceramides and their metabolic derivatives in a wide array of sphingolipid-dependent cellular processes with the spatiotemporal precision of light.
Highlights
Sphingolipids are unusually versatile membrane components in eukaryotic cells that contribute to mechanical stability, cell signaling and molecular sorting (Holthuis et al, 2001)
Each caCer has an N-acyl chain possessing a terminal alkyne for click-derivatization with a fluorophore reporter. caCer-1 is a clickable variant of azobenzene-containing ceramides (ACes)-1 on which we reported earlier (Frank et al, 2016b) (Figure 1c), and carries the azobenzene photoswitch in its N-acyl chain with the diazene group mimicking a 9-double bond. caCer-2 has a shorter N-acyl
We examined whether metabolic conversion of caCers by SMS2 can be controlled in a reversible manner using the temporal precision of light
Summary
Sphingolipids are unusually versatile membrane components in eukaryotic cells that contribute to mechanical stability, cell signaling and molecular sorting (Holthuis et al, 2001). They derive from the addition of various polar head groups to ceramide, a hydrophobic molecule containing saturated or trans-unsaturated acyl chains linked to a serine backbone (Wegner et al, 2016). The enzymes responsible for sphingolipid production and turnover comprise a complex metabolic network that gives rise to numerous bioactive molecules. Intermediates of sphingolipid metabolism, notably sphingosine, ceramide and their phosphorylated derivatives, influence a multitude of physiological processes, including cell proliferation, cell death, migration, stress adaptation, immune responses and angiogenesis (Hannun and Obeid, 2018). The hydrolysis of plasma membrane SM by sphingomyelinases generates ceramide, which mediates several stress responses
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