Ecological lipidology

Dietary lipids (DLs), particularly sterols and fatty acids, are precursors for endogenous lipids that, unusually for macronutrients, shape cellular and organismal function long after ingestion. These functions - cell membrane structure, intracellular signalling, and hormonal activity - vary with the identity of DLs, and scale up to influence health, survival, and reproductive fitness, thereby affecting evolutionary change. Our Ecological Lipidology approach integrates biochemical mechanisms and molecular cell biology into evolution and nutritional ecology. It exposes our need to understand environmental impacts on lipidomes, the lipid specificity of cell functions, and predicts the evolution of lipid-based diet choices. Broad interdisciplinary implications of Ecological Lipidology include food web alterations, species responses to environmental change, as well as sex differences and lifestyle impacts on human nutrition, and opportunities for DL-based therapies

Adipose cells and tissues soften with lipid accumulation while in diabetes adipose tissue stiffens

Adipose tissue expansion involves both differentiation of new precursors and size increase of mature adipocytes. While the two processes are well balanced in healthy tissues, obesity and diabetes type II are associated with abnormally enlarged adipocytes and excess lipid accumulation. Previous studies suggested a link between cell stiffness, volume and stem cell differentiation, although in the context of preadipocytes, there have been contradictory results regarding stiffness changes with differentiation. Thus, we set out to quantitatively monitor adipocyte shape and size changes with differentiation and lipid accumulation. We quantified by optical diffraction tomography that differentiating preadipocytes increased their volumes drastically. Atomic force microscopy (AFM)-indentation and -microrheology revealed that during the early phase of differentiation, human preadipocytes became more compliant and more fluid-like, concomitant with ROCK-mediated F-actin remodelling. Adipocytes that had accumulated large lipid droplets were more compliant, and further promoting lipid accumulation led to an even more compliant phenotype. In line with that, high fat diet-induced obesity was associated with more compliant adipose tissue compared to lean animals, both for drosophila fat bodies and murine gonadal adipose tissue. In contrast, adipose tissue of diabetic mice became significantly stiffer as shown not only by AFM but also magnetic resonance elastography. Altogether, we dissect relative contributions of the cytoskeleton and lipid droplets to cell and tissue mechanical changes across different functional states, such as differentiation, nutritional state and disease. Our work therefore sets the basis for future explorations on how tissue mechanical changes influence the behaviour of mechanosensitive tissue-resident cells in metabolic disorders

Rabs on the fly: functions of Rab GTPases during development

The organisation of intracellular transport processes is adapted specifically to different cell types, developmental stages, and physiological requirements. Some protein traffic routes are universal to all cells and constitutively active, while other routes are cell-type specific, transient, and induced under particular conditions only. Small GTPases of the Rab (Ras related in brain) subfamily are conserved across eukaryotes and regulate most intracellular transit pathways. The complete sets of Rab proteins have been identified in model organisms, and molecular principles underlying Rab functions have been uncovered. Rabs provide intracellular landmarks that define intracellular transport sequences. Nevertheless, it remains a challenge to systematically map the subcellular distribution of all Rabs and their functional interrelations. This task requires novel tools to precisely describe and manipulate the Rab machinery in vivo. Here we discuss recent findings about Rab roles during development and we consider novel approaches to investigate Rab functions in vivo

Staccato/Unc-13-4 controls secretory lysosome-mediated lumen fusion during epithelial tube anastomosis

A crucial yet ill-defined step during the development of tubular networks, such as the vasculature, is the formation of connections (anastomoses) between pre-existing lumenized tubes. By studying tracheal tube anastomosis in Drosophila melanogaster, we uncovered a key role of secretory lysosome-related organelle (LRO) trafficking in lumen fusion. We identified the conserved calcium-binding protein Unc-13-4/Staccato (Stac) and the GTPase Rab39 as critical regulators of this process. Stac and Rab39 accumulate on dynamic vesicles, which form exclusively in fusion tip cells, move in a dynein-dependent manner, and contain late-endosomal, lysosomal, and SNARE components characteristic of LROs. The GTPase Arl3 is necessary and sufficient for Stac LRO formation and promotes Stac-dependent intracellular fusion of juxtaposed apical plasma membranes, thereby forming a transcellular lumen. Concomitantly, calcium is released locally from ER exit sites and apical membrane-associated calcium increases. We propose that calcium-dependent focused activation of LRO exocytosis restricts lumen fusion to appropriate domains within tip cells

DIlp7-Producing Neurons Regulate Insulin-Producing Cells in Drosophila

Cellular Insulin signaling shows a remarkable high molecular and functional conservation. Insulin-producing cells respond directly to nutritional cues in circulation and receive modulatory input from connected neuronal networks. Neuronal control integrates a wide range of variables including dietary change or environmental temperature. Although it is shown that neuronal input is sufficient to regulate Insulin-producing cells, the physiological relevance of this network remains elusive. I

Endogenously Tagged Rab Proteins: A Resource to Study Membrane Trafficking in Drosophila

SummaryMembrane trafficking is key to the cell biological mechanisms underlying development. Rab GTPases control specific membrane compartments, from core secretory and endocytic machinery to less-well-understood compartments. We tagged all 27 Drosophila Rabs with YFPMYC at their endogenous chromosomal loci, determined their expression and subcellular localization in six tissues comprising 23 cell types, and provide this data in an annotated, searchable image database. We demonstrate the utility of these lines for controlled knockdown and show that similar subcellular localization can predict redundant functions. We exploit this comprehensive resource to ask whether a common Rab compartment architecture underlies epithelial polarity. Strikingly, no single arrangement of Rabs characterizes the five epithelia we examine. Rather, epithelia flexibly polarize Rab distribution, producing membrane trafficking architectures that are tissue- and stage-specific. Thus, the core machinery responsible for epithelial polarization is unlikely to rely on polarized positioning of specific Rab compartments

Lipoproteins in <em>Drosophila melanogaster</em>—Assembly, Function, and Influence on Tissue Lipid Composition

<div><p>Interorgan lipid transport occurs via lipoproteins, and altered lipoprotein levels correlate with metabolic disease. However, precisely how lipoproteins affect tissue lipid composition has not been comprehensively analyzed. Here, we identify the major lipoproteins of <em>Drosophila melanogaster</em> and use genetics and mass spectrometry to study their assembly, interorgan trafficking, and influence on tissue lipids. The apoB-family lipoprotein Lipophorin (Lpp) is the major hemolymph lipid carrier. It is produced as a phospholipid-rich particle by the fat body, and its secretion requires Microsomal Triglyceride Transfer Protein (MTP). Lpp acquires sterols and most diacylglycerol (DAG) at the gut via Lipid Transfer Particle (LTP), another fat body-derived apoB-family lipoprotein. The gut, like the fat body, is a lipogenic organ, incorporating both <em>de novo</em>–synthesized and dietary fatty acids into DAG for export. We identify distinct requirements for LTP and Lpp-dependent lipid mobilization in contributing to the neutral and polar lipid composition of the brain and wing imaginal disc. These studies define major routes of interorgan lipid transport in <em>Drosophila</em> and uncover surprising tissue-specific differences in lipoprotein lipid utilization.</p> </div

Lpp and LTP function together to mobilize lipids from the gut.

<p>(A) Immunofluorescence of a second instar larval gut, stained for Lpp, LTP and neutral lipid droplets. Lpp, LTP and lipid droplets are enriched in the same subsets of the anterior and posterior midgut. (*) indicates a nearby piece of fat body. GC: gastric caeca; AM: anterior midgut; PM: posterior midgut; HG: hindgut; MT: malpighian tubule. Scale bars = 200 µm. (B) Lipid droplets of guts from first instar wild-type and <i>mtp^Δex1</i> larvae, visualized with Nile red. Loss of MTP causes strong neutral lipid accumulation. Yellow: neutral lipids; red: phospholipids. GC: gastric caeca; AM: anterior midgut; PM: posterior midgut; HG: hindgut. Scale bars = 200 µm. Scale bars blow ups = 20 µm. (C) Lipid droplets of posterior midguts upon MTP, LTP or Lpp RNAi, visualized with Nile red. Knock-down of either protein causes similar accumulation of neutral lipid. Scale bars = 50 µm.</p

LTP promotes the export of lipids from the gut to Lpp.

<p>(A) Immunoblot of isopycnic KBr gradients from LTP or Lpp RNAi hemolymph. Knock-down of LTP increases Lpp density. Knock-down of Lpp decreases LTP density. (B) Immunoblot of isopycnic KBr gradients from gut lipid transfer experiments, showing that LTP facilitates lipid export from the gut to Lpp. (C) Cartoon of the posterior midgut. Optical section planes of (D), (F) are indicated in red. (D) Immunofluorescence of a second larval instar posterior midgut, showing the subcellular localization of Lpp, LTP and neutral lipid droplets. Scale bars = 10 µm. (E) Lipid droplets in the gut of second instar larvae at different time points after the induction of dominant negative (DN) dynamin (shibire), visualized with Nile red. Within a few hours after induction, neutral lipid droplets accumulate to a similar extent as in the gut of lipoprotein-deficient larvae. Yellow: neutral lipids; red: phospholipids. Scale bars = 50 µm. (F) Immunofluorescence of second larval instar posterior midguts at different time points after the induction of dominant negative dynamin, stained for Lpp and LTP. Scale bars = 10 µm.</p