The ceramide synthase 2b gene mediates genomic sensing and regulation of sphingosine Levels during zebrafish embryogenesis
Abstract
Sphingosine-1-phosphate (S1P) is generated through phosphorylation of sphingosine by sphingosine kinases (Sphk1 and Sphk2). We show that sphk2 maternal-zygotic mutant zebrafish embryos (sphk2MZ) display early developmental phenotypes, including a delay in epiboly, depleted S1P levels, elevated levels of sphingosine, and resistance to sphingosine toxicity. The sphk2MZ embryos also have strikingly increased levels of maternal transcripts encoding ceramide synthase 2b (Cers2b), and loss of Cers2b in sphk2MZ embryos phenocopies sphingosine toxicity. An upstream region of the cers2b promoter supports enhanced expression of a reporter gene in sphk2MZ embryos compared to wildtype embryos. Furthermore, ectopic expression of Cers2b protein itself reduces activity of the promoter, and this repression is relieved by exogenous sphingosine. Therefore, the sphk2MZ genome recognizes the lack of sphingosine kinase activity and up-regulates cers2b as a salvage pathway for sphingosine turnover. Cers2b can also function as a sphingolipid-responsive factor to mediate at least part of a feedback regulatory mechanism.
Article and author information
Author details
Funding
National Institutes of Health (HL089934)
- Timothy Hla
National Institutes of Health (CA077839)
- Timothy Hla
National Institutes of Health (HL111400)
- Todd Evans
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (2011-100) of the Weill Cornell Medical College.
Copyright
© 2017, Mendelson 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
-
- 1,376
- views
-
- 277
- downloads
-
- 19
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
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)
Further reading
-
- Biochemistry and Chemical Biology
- Chromosomes and Gene Expression
The mRNA 5'-cap structure removal by the decapping enzyme DCP2 is a critical step in gene regulation. While DCP2 is the catalytic subunit in the decapping complex, its activity is strongly enhanced by multiple factors, particularly DCP1, which is the major activator in yeast. However, the precise role of DCP1 in metazoans has yet to be fully elucidated. Moreover, in humans, the specific biological functions of the two DCP1 paralogs, DCP1a and DCP1b, remain largely unknown. To investigate the role of human DCP1, we generated cell lines that were deficient in DCP1a, DCP1b, or both to evaluate the importance of DCP1 in the decapping machinery. Our results highlight the importance of human DCP1 in decapping process and show that the EVH1 domain of DCP1 enhances the mRNA-binding affinity of DCP2. Transcriptome and metabolome analyses outline the distinct functions of DCP1a and DCP1b in human cells, regulating specific endogenous mRNA targets and biological processes. Overall, our findings provide insights into the molecular mechanism of human DCP1 in mRNA decapping and shed light on the distinct functions of its paralogs.
-
- Biochemistry and Chemical Biology
- Cell Biology
Myosin 10 (Myo10) is a motor protein known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the absolute number of Myo10 molecules during the filopodial lifecycle. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. We combined SDS-PAGE densitometry with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it enriches at opposite cellular ends. Hundreds of Myo10s are in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Live-cell movies reveal a dense cluster of over a hundred Myo10 molecules that initiates filopodial elongation. Hundreds of Myo10 molecules continue to accumulate during filopodial growth, but accumulation ceases when retraction begins. Rates of filopodial elongation, second-phase elongation, and retraction are inversely related to Myo10 quantities. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane compartments. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation.