Biochemical Membrane Lipidomics during Drosophila Development

Lipids play critical roles in energy homeostasis, membrane structure, and signaling. Using liquid chromatography and mass spectrometry, we provide a comprehensive semiquantification of lipids during the life cycle of Drosophila melanogaster (230 glycerophospholipids, 210 sphingolipids, 6 sterols and sterol esters, and 60 glycerolipids) and obtain biological insights through this biochemical resource. First, we find a high and constant triacylglycerol-to-membrane lipid ratio during pupal stage, which is nonobvious in the absence of nutrient uptake and tissue remodeling. Second, sphingolipids undergo specific changes in headgroup (glycosylation) and tail configurations (unsaturation and hydroxylation on sphingoid base and fatty acyls, respectively), which correlate with gene expression of known (GIcT/CG6437; FA2H/ CG30502) and putative (Cyt-b5-r/CG13279) enzymes. Third, we identify a gender bias in phosphoethanolamine-ceramides as a lead for future investigation into sexual maturation. Finally, we partially characterize ghiberti, required for male meiotic cytokinesis, as a homolog of mammalian serine palmitoyltransferase.

Single-cell lipidomics reveals a role for cell-to-cell sphingolipid heterogeneity in fibroblast activation

Laura Capolupo

Cell-to-cell variability plays a key role in tissue patterning by setting initial asymmetry that primes cell fate decisions. Fluctuations in the activity of regulatory molecules can commit individual cells to divergent differentiation pathways, and cell-to-cell variability can diversify the response of an otherwise homogeneous cell community to its environment. Recent advances have provided scientists with tools to study the variability associated with individual cells. However, while single-cell data are available for genomes, transcriptomes and proteomes, they are scarce for metabolic products, such as lipids. Eukaryotic cells produce thousands of lipids â each potentially contributing to specific biological functions. Furthermore, a number of metabolic switches involving lipids have been described to occur in development and cell differentiation. Few studies have directly addressed cell-to-cell lipid variations in syngeneic cell populations suggesting that lipid heterogeneity contributes to the emergence of multicellular patterns. Nonetheless, lipid biologists have so far addressed lipidomes in bulk cell extracts or selected lipids at the single-cell level. Thus, how lipidomes vary from one cell to another and which cell-to-cell lipid variations have biological meaning remains to be defined. Recent developments have provided mass spectrometry with sufficient sensitivity to reveal molecules in few hundred copies, making single-cell lipidomics possible. A rapidly emerging technique with a potential use in single-cell lipidomics is imaging mass spectrometry. Here, we devised a high-resolution MALDI imaging mass spectrometry pipeline to study cell-to-cell variability of the lipidome of hundreds of primary dermal human fibroblasts. We found that sphingolipid metabolism shows high cell-to-cell heterogeneity and that specific sphingolipids mark distinct cell sub-populations: coined herein as âlipotypesâ. Furthermore, we found that fibroblast lipotypes correspond to cell states endowed with proliferative, inflammatory or fibrogenic properties. Finally, we asked whether lipotypes participate in the specification of cell states and find that specific glycosphingolipids modulate signalling pathways (i.e., the FGF and TGF-b) to shape the transcriptional cell landscape. In summary, through the use of single-cell omics techniques, we found that: (i) specific lipid metabolic segments (i.e., the sphingolipid pathways) have cell-to-cell variation; (ii) given lipid configurations mark discrete cell states; and (iii) sphingolipids modulate dermal fibroblast activation involved in wound healing and skin homeostasis. Altogether, this study is among the first to demonstrate the potential of MALDI-IMS for single-cell lipidomics and to reveal cell-to-cell lipidome heterogeneity. It also demonstrates a role for lipids in the determination of cell states and in tissue patterning and reveals a new regulatory component to the self-organization of multicellular systems.

EPFL2021

Elucidating regulatory mechanisms shaping the transcriptome and proteome of the gut immune response in Drosophila melanogaster

Michael Vincent Frochaux

Work on Drosophila melanogaster paved the way to our current understanding of modern genetics. Since then, this model organism has contributed greatly to various fields such as neurobiology, development, and immunology. The discovery and analysis of the various pathways of the Drosophila immune response led the way to the in-depth characterization of the gut immune response. Furthermore, a genome-wide association study on a population of Drosophila identified resistance to infection by the entomopathogenic bacteria Pseudomonas entomophila (P.e.) as a complex phenotype, with highly resistant and susceptible lines, and highlighted genes and pathways mediating inter-individual differences. However, the molecular mechanisms mediating the resistance to enteric infection remain largely uncharacterized. This study uses the same Drosophila population to analyze what are the molecular mechanisms mediating the resistance to enteric infection. We analyzed the genetic determinants of gene expression variation among the most resistant and susceptible lines in response to infection using expression quantitative trait loci (eQTL) analysis. We also characterized the mode of action of these eQTLs into cis- and trans-acting. Furthermore, we identified the gene nutcracker (ntc) as the only differentially-expressed gene between resistance classes. We then demonstrate that loss of function ntc mutants are significantly more susceptible to infection and then identify a single nucleotide polymorphism (SNP) located in a transcription factor binding site (TFBS) which affects the binding affinity of the transcription factor Broad leading to allele-specific expression variation of the gene ntc. If the transcriptomic response to infection has been thoroughly characterized, it is not the case for the proteomic response. We thus sought to characterize for the first time the proteomic response of the Drosophila to enteric infection. We performed mass spectrometry and RNA sequencing on dissected guts from flies infected by two Gram-negative bacteria, Erwinia carotovora carotovora 15 (Ecc15) or Pseudomonas entomophila, 4h and 16h after infection. We found that a large portion of the measurable proteome (12%) varies after infection and that protein changes are strongly time- and infection-dependent. We showed the relatively poor correlation between gene expression and protein abundance in the Drosophila enteric immune response. Finally, we performed a screen of several proteins that were identified in our proteomics work but had previously not been found when assessing the gene expression response to infection using loss-of-function mutants and identifying 7 genes that modulate overall susceptibility to P.e. infection. In summary, this work analyze the genetic determinants of gene expression variation in the DGRP population upon infection and characterize them according to their mode of action. Furthermore, we describe how a non-coding variant lowers resistance to infection by modulating ntc gene expression through altered Broad repressor binding. Finally, it provides the first comprehensive characterization of the Drosophila gut proteome upon infection by Gram-negative bacteria which can be the basis of future proteomic analysis of the enteric immune response

EPFL2019

Niacin: an old lipid drug in a new NAD(+) dress

Johan Auwerx, Dina Hofer, Elena Katsyuba, Mario Romani

Niacin, the first antidyslipidemic drug, has been at the center stage of lipid research for many decades before the discovery of statins. However, to date, despite its remarkable effects on lipid profiles, the clinical outcomes of niacin treatment on cardiac events is still debated. In addition to its historically well-defined interactions with central players of lipid metabolism, niacin can be processed by eukaryotic cells to synthesize a crucial cofactor, NAD(+). NAD(+) acts as a cofactor in key cellular processes, including oxidative phosphorylation, glycolysis, and DNA repair. More recently, evidence has emerged that NAD(+) also is an essential cosubstrate for the sirtuin family of protein deacylases and thereby has an impact on a wide range of cellular processes, most notably mitochondrial homeostasis, energy homeostasis, and lipid metabolism. NAD(+) achieves these remarkable effects through sirtuin-mediated deacetylation of key transcriptional regulators, such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha, LXR, and SREBPs, that control these cellular processes. Here, we present an alternative point of view to explain niacin's mechanism of action, with a strong focus on the importance of how this old drug acts as a control switch of NAD(+)/sirtuin-mediated control of metabolism.