Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides
- University of Osnabrück, Germany
- Ludwig Maximilians University Munich, Germany
- Max Planck Institute of Biochemistry, Germany
- New York University, United States
- University of Osnabrück, Germany
- Oregon Health and Science University, United States
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.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting source data file. Source data files have been provided for Figures 4-6.
Article and author information
Author details
Henri G Franquelim
Funding
Deutsche Forschungsgemeinschaft (SFB1032)
- Henri G Franquelim
- Petra Schwille
- Dirk Trauner
- James A Frank
National Sciences and Engineering Research Council of Canada
- Ben Williams
Deutsche Forschungsgemeinschaft (SFB944)
- Matthijs Kol
- Joost CM Holthuis
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Arun Radhakrishnan, University of Texas Southwestern Medical Center, United States
Version history
- Received: October 30, 2018
- Accepted: February 2, 2019
- Accepted Manuscript published: February 5, 2019 (version 1)
- Version of Record published: February 22, 2019 (version 2)
Copyright
© 2019, Kol et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,145
- views
-
- 540
- downloads
-
- 27
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides
eLife 8:e43230.
https://doi.org/10.7554/eLife.43230
Further reading
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
To date, all major modes of monoclonal antibody therapy targeting SARS-CoV-2 have lost significant efficacy against the latest circulating variants. As SARS-CoV-2 omicron sublineages account for over 90% of COVID-19 infections, evasion of immune responses generated by vaccination or exposure to previous variants poses a significant challenge. A compelling new therapeutic strategy against SARS-CoV-2 is that of single-domain antibodies, termed nanobodies, which address certain limitations of monoclonal antibodies. Here, we demonstrate that our high-affinity nanobody repertoire, generated against wild-type SARS-CoV-2 spike protein (Mast et al., 2021), remains effective against variants of concern, including omicron BA.4/BA.5; a subset is predicted to counter resistance in emerging XBB and BQ.1.1 sublineages. Furthermore, we reveal the synergistic potential of nanobody cocktails in neutralizing emerging variants. Our study highlights the power of nanobody technology as a versatile therapeutic and diagnostic tool to combat rapidly evolving infectious diseases such as SARS-CoV-2.
-
- Biochemistry and Chemical Biology
Phosphoinositide 3-kinase (PI3K) beta (PI3Kβ) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kβ prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kβ localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kβ when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kβ membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kβ prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GβGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kβ to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GβGγ or pY/Rac1(GTP), PI3Kβ activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kβ is synergistically activated by pY/GβGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.
