Drosophila embryos allocate lipid droplets to specific lineages to ensure punctual development and redox homeostasis.

Lipid droplets (LDs) are ubiquitous organelles that facilitate neutral lipid storage in cells, including energy-dense triglycerides. They are found in all investigated metazoan embryos where they are thought to provide energy for development. Intriguingly, early embryos of diverse metazoan species asymmetrically allocate LDs amongst cellular lineages, a process which can involve massive intracellular redistribution of LDs. However, the biological reason for asymmetric lineage allocation is unknown. To address this issue, we utilize the Drosophila embryo where the cytoskeletal mechanisms that drive allocation are well characterized. We disrupt allocation by two different means: Loss of the LD protein Jabba results in LDs adhering inappropriately to glycogen granules; loss of Klar alters the activities of the microtubule motors that move LDs. Both mutants cause the same dramatic change in LD tissue inheritance, shifting allocation of the majority of LDs to the yolk cell instead of the incipient epithelium. Embryos with such mislocalized LDs do not fully consume their LDs and are delayed in hatching. Through use of a dPLIN2 mutant, which appropriately localizes a smaller pool of LDs, we find that failed LD transport and a smaller LD pool affect embryogenesis in a similar manner. Embryos of all three mutants display overlapping changes in their transcriptome and proteome, suggesting that lipid deprivation results in a shared embryonic response and a widespread change in metabolism. Excitingly, we find abundant changes related to redox homeostasis, with many proteins related to glutathione metabolism upregulated. LD deprived embryos have an increase in peroxidized lipids and rely on increased utilization of glutathione-related proteins for survival. Thus, embryos are apparently able to mount a beneficial response upon lipid stress, rewiring their metabolism to survive. In summary, we demonstrate that early embryos allocate LDs into specific lineages for subsequent optimal utilization, thus protecting against oxidative stress and ensuring punctual development.

Adipose triglyceride lipase promotes prostaglandin-dependent actin remodeling by regulating substrate release from lipid droplets.

Lipid droplets (LDs), crucial regulators of lipid metabolism, accumulate during oocyte development. However, their roles in fertility remain largely unknown. During Drosophila oogenesis, LD accumulation coincides with the actin remodeling necessary for follicle development. Loss of the LD-associated Adipose Triglyceride Lipase (ATGL) disrupts both actin bundle formation and cortical actin integrity, an unusual phenotype also seen when the prostaglandin (PG) synthase Pxt is missing. Dominant genetic interactions and PG treatment of follicles indicate that ATGL acts upstream of Pxt to regulate actin remodeling. Our data suggest that ATGL releases arachidonic acid (AA) from LDs to serve as the substrate for PG synthesis. Lipidomic analysis detects AA-containing triglycerides in ovaries, and these are increased when ATGL is lost. High levels of exogenous AA block follicle development; this is enhanced by impairing LD formation and suppressed by reducing ATGL. Together, these data support the model that AA stored in LD triglycerides is released by ATGL to drive the production of PGs, which promote the actin remodeling necessary for follicle development. We speculate that this pathway is conserved across organisms to regulate oocyte development and promote fertility.

Drosophila embryos spatially sort their nutrient stores to facilitate their utilization.

Animal embryos are provided by their mothers with a diverse nutrient supply that is crucial for development. In Drosophila, the three most abundant nutrients (triglycerides, proteins and glycogen) are sequestered in distinct storage structures: lipid droplets (LDs), yolk vesicles (YVs) and glycogen granules (GGs). Using transmission electron microscopy as well as live and fixed sample fluorescence imaging, we find that all three storage structures are dispersed throughout the egg but are then spatially allocated to distinct tissues by gastrulation: LDs largely to the peripheral epithelium, YVs and GGs to the central yolk cell. To confound the embryo's ability to sort its nutrients, we employ Jabba and mauve mutants to generate LD-GG and LD-YV compound structures. In these mutants, LDs are mis-sorted to the yolk cell and their turnover is delayed. Our observations demonstrate dramatic spatial nutrient sorting in early embryos and provide the first evidence for its functional importance.

Visualizing Lipid Droplets in Drosophila Oogenesis.

Lipid droplets (LDs) are fat storage organelles highly abundant in oocytes and eggs of many vertebrates and invertebrates. They have roles both during oogenesis and in provisioning the developing embryo. In Drosophila, large numbers of LDs are generated in nurse cells during mid-oogenesis and then transferred to oocytes. Their number and spatial distribution changes developmentally and in response to various experimental manipulations. This chapter demonstrates how to visualize LDs in Drosophila follicles, both in fixed tissues and living samples. For fixed samples, the protocol explains how to prepare female flies, dissect ovaries, isolate follicles, fix, apply stains, mount the tissue, and perform imaging. For live samples, the protocol shows how to dissect ovaries, apply a fluorescent LD dye, and culture follicles such that they remain alive and healthy during imaging. Finally, a method is provided that employs in vivo centrifugation to assess colocalization of markers with LDs.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos.

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Sequestration to lipid droplets promotes histone availability by preventing turnover of excess histones.

Because both dearth and overabundance of histones result in cellular defects, histone synthesis and demand are typically tightly coupled. In Drosophila embryos, histones H2B, H2A and H2Av accumulate on lipid droplets (LDs), which are cytoplasmic fat storage organelles. Without LD binding, maternally provided H2B, H2A and H2Av are absent; however, how LDs ensure histone storage is unclear. Using quantitative imaging, we uncover when during oogenesis these histones accumulate, and which step of accumulation is LD dependent. LDs originate in nurse cells (NCs) and are transported to the oocyte. Although H2Av accumulates on LDs in NCs, the majority of the final H2Av pool is synthesized in oocytes. LDs promote intercellular transport of the histone anchor Jabba and thus its presence in the ooplasm. Ooplasmic Jabba then prevents H2Av degradation, safeguarding the H2Av stockpile. Our findings provide insight into the mechanism for establishing histone stores during Drosophila oogenesis and shed light on the function of LDs as protein-sequestration sites.

Altered sensory system activity and connectivity patterns in adductor spasmodic dysphonia.

Adductor-type spasmodic dysphonia (ADSD) manifests in effortful speech temporarily relievable by botulinum neurotoxin type A (BoNT-A). Previously, abnormal structure, phonation-related and resting-state sensorimotor abnormalities as well as peripheral tactile thresholds in ADSD were described. This study aimed at assessing abnormal central tactile processing patterns, their spatial relation with dysfunctional resting-state connectivity, and their BoNT-A responsiveness. Functional MRI in 14/12 ADSD patients before/under BoNT-A effect and 15 controls was performed (i) during automatized tactile stimulus application to face/hand, and (ii) at rest. Between-group differential stimulation-induced activation and resting-state connectivity (regional homogeneity, connectivity strength within selected sensory(motor) networks), as well as within-patient BoNT-A effects on these differences were investigated. Contralateral-to-stimulation overactivity in ADSD before BoNT-A involved primary and secondary somatosensory representations, along with abnormalities in higher-order parietal, insular, temporal or premotor cortices. Dysphonic impairment in ADSD positively associated with left-hemispheric temporal activity. Connectivity was increased within right premotor (sensorimotor network), left primary auditory cortex (auditory network), and regionally reduced at the temporoparietal junction. Activation/connectivity before/after BoNT-A within-patients did not significantly differ. Abnormal ADSD central somatosensory processing supports its significance as common pathophysiologic focal dystonia trait. Abnormal temporal cortex tactile processing and resting-state connectivity might hint at abnormal cross-modal sensory interactions.

A role for triglyceride lipase brummer in the regulation of sex differences in Drosophila fat storage and breakdown.

Triglycerides are the major form of stored fat in all animals. One important determinant of whole-body fat storage is whether an animal is male or female. Here, we use Drosophila, an established model for studies on triglyceride metabolism, to gain insight into the genes and physiological mechanisms that contribute to sex differences in fat storage. Our analysis of triglyceride storage and breakdown in both sexes identified a role for triglyceride lipase brummer (bmm) in the regulation of sex differences in triglyceride homeostasis. Normally, male flies have higher levels of bmm mRNA both under normal culture conditions and in response to starvation, a lipolytic stimulus. We find that loss of bmm largely eliminates the sex difference in triglyceride storage and abolishes the sex difference in triglyceride breakdown via strongly male-biased effects. Although we show that bmm function in the fat body affects whole-body triglyceride levels in both sexes, in males, we identify an additional role for bmm function in the somatic cells of the gonad and in neurons in the regulation of whole-body triglyceride homeostasis. Furthermore, we demonstrate that lipid droplets are normally present in both the somatic cells of the male gonad and in neurons, revealing a previously unrecognized role for bmm function, and possibly lipid droplets, in these cell types in the regulation of whole-body triglyceride homeostasis. Taken together, our data reveal a role for bmm function in the somatic cells of the gonad and in neurons in the regulation of male-female differences in fat storage and breakdown and identify bmm as a link between the regulation of triglyceride homeostasis and biological sex.

Lipid droplet velocity is a microenvironmental sensor of aggressive tumors regulated by V-ATPase and PEDF.

Lipid droplets (LDs) utilize microtubules (MTs) to participate in intracellular trafficking of cargo proteins. Cancer cells accumulate LDs and acidify their tumor microenvironment (TME) by increasing the proton pump V-ATPase. However, it is not known whether these two metabolic changes are mechanistically related or influence LD movement. We postulated that LD density and velocity are progressively increased with tumor aggressiveness and are dependent on V-ATPase and the lipolysis regulator pigment epithelium-derived factor (PEDF). LD density was assessed in human prostate cancer (PCa) specimens across Gleason scores (GS) 6-8. LD distribution and velocity were analyzed in low and highly aggressive tumors using live-cell imaging and in cells exposed to low pH and/or treated with V-ATPase inhibitors. The MT network was disrupted and analyzed by α-tubulin staining. LD density positively correlated with advancing GS in human tumors. Acidification promoted peripheral localization and clustering of LDs. Highly aggressive prostate, breast, and pancreatic cell lines had significantly higher maximum LD velocity (LDVmax) than less aggressive and benign cells. LDVmax was MT-dependent and suppressed by blocking V-ATPase directly or indirectly with PEDF. Upon lowering pH, LDs moved to the cell periphery and carried metalloproteinases. These results suggest that acidification of the TME can alter intracellular LD movement and augment velocity in cancer. Restoration of PEDF or blockade of V-ATPase can normalize LD distribution and decrease velocity. This study identifies V-ATPase and PEDF as new modulators of LD trafficking in the cancer microenvironment.

Lipidomic Analysis of α-Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment.

In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for αS-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.

Developmentally regulated H2Av buffering via dynamic sequestration to lipid droplets in Drosophila embryos.

Regulating nuclear histone balance is essential for survival, yet in early Drosophila melanogaster embryos many regulatory strategies employed in somatic cells are unavailable. Previous work had suggested that lipid droplets (LDs) buffer nuclear accumulation of the histone variant H2Av. Here, we elucidate the buffering mechanism and demonstrate that it is developmentally controlled. Using live imaging, we find that H2Av continuously exchanges between LDs. Our data suggest that the major driving force for H2Av accumulation in nuclei is H2Av abundance in the cytoplasm and that LD binding slows nuclear import kinetically, by limiting this cytoplasmic pool. Nuclear H2Av accumulation is indeed inversely regulated by overall buffering capacity. Histone exchange between LDs abruptly ceases during the midblastula transition, presumably to allow canonical regulatory mechanisms to take over. These findings provide a mechanistic basis for the emerging role of LDs as regulators of protein homeostasis and demonstrate that LDs can control developmental progression.

PEDF regulates plasticity of a novel lipid-MTOC axis in prostate cancer-associated fibroblasts.

Prostate tumors make metabolic adaptations to ensure adequate energy and amplify cell cycle regulators, such as centrosomes, to sustain their proliferative capacity. It is not known whether cancer-associated fibroblasts (CAFs) undergo metabolic re-programming. We postulated that CAFs augment lipid storage and amplify centrosomal or non-centrosomal microtubule-organizing centers (MTOCs) through a pigment epithelium-derived factor (PEDF)-dependent lipid-MTOC signaling axis. Primary human normal prostate fibroblasts (NFs) and CAFs were evaluated for lipid content, triacylglycerol-regulating proteins, MTOC number and distribution. CAFs were found to store more neutral lipids than NFs. Adipose triglyceride lipase (ATGL) and PEDF were strongly expressed in NFs, whereas CAFs had minimal to undetectable levels of PEDF or ATGL protein. At baseline, CAFs demonstrated MTOC amplification when compared to 1-2 perinuclear MTOCs consistently observed in NFs. Treatment with PEDF or blockade of lipogenesis suppressed lipid content and MTOC number. In summary, our data support that CAFs have acquired a tumor-like phenotype by re-programming lipid metabolism and amplifying MTOCs. Normalization of MTOCs by restoring PEDF or by blocking lipogenesis highlights a previously unrecognized plasticity in centrosomes, which is regulated through a new lipid-MTOC axis.This article has an associated First Person interview with the first author of the paper.

Emerging Links between Lipid Droplets and Motor Neuron Diseases.

Lipid droplets (LDs) are ubiquitous fat storage organelles and play key roles in lipid metabolism and energy homeostasis; in addition, they contribute to protein storage, folding, and degradation. However, a role for LDs in the nervous system remains largely unexplored. We discuss evidence supporting an intimate functional connection between LDs and motor neuron disease (MND) pathophysiology, examining how LD functions in systemic energy homeostasis, in neuron-glia metabolic coupling, and in protein folding and clearance may affect or contribute to disease pathology. An integrated understanding of LD biology and neurodegeneration may open the way for new therapeutic interventions.

Combined Ceria Reduction and Methane Reforming in a Solar-Driven Particle-Transport Reactor.

We report on the experimental performance of a solar aerosol reactor for carrying out the combined thermochemical reduction of CeO2 and reforming of CH4 using concentrated radiation as the source of process heat. The 2 kWth solar reactor prototype utilizes a cavity receiver enclosing a vertical Al2O3 tube which contains a downward gravity-driven particle flow of ceria particles, either co-current or counter-current to a CH4 flow. Experimentation under a peak radiative flux of 2264 suns yielded methane conversions up to 89% at 1300 °C for residence times under 1 s. The maximum extent of ceria reduction, given by the nonstoichiometry δ (CeO2-δ), was 0.25. The solar-to-fuel energy conversion efficiency reached 12%. The syngas produced had a H2:CO molar ratio of 2, and its calorific value was solar-upgraded by 24% over that of the CH4 reformed.

Lipid droplet functions beyond energy storage.

Lipid droplets are cytoplasmic organelles that store neutral lipids and are critically important for energy metabolism. Their function in energy storage is firmly established and increasingly well characterized. However, emerging evidence indicates that lipid droplets also play important and diverse roles in the cellular handling of lipids and proteins that may not be directly related to energy homeostasis. Lipid handling roles of droplets include the storage of hydrophobic vitamin and signaling precursors, and the management of endoplasmic reticulum and oxidative stress. Roles of lipid droplets in protein handling encompass functions in the maturation, storage, and turnover of cellular and viral polypeptides. Other potential roles of lipid droplets may be connected with their intracellular motility and, in some cases, their nuclear localization. This diversity highlights that lipid droplets are very adaptable organelles, performing different functions in different biological contexts. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.

A Luciferase-fragment Complementation Assay to Detect Lipid Droplet-associated Protein-Protein Interactions.

A critical challenge for all organisms is to carefully control the amount of lipids they store. An important node for this regulation is the protein coat present at the surface of lipid droplets (LDs), the intracellular organelles dedicated to lipid storage. Only limited aspects of this regulation are understood so far. For the probably best characterized case, the regulation of lipolysis in mammals, some of the major protein players have been identified, and it has been established that this process crucially depends on an orchestrated set of protein-protein interactions. Proteomic analysis has revealed that LDs are associated with dozens, if not hundreds, of different proteins, most of them poorly characterized, with even fewer data regarding which of them might physically interact. To comprehensively understand the mechanism of lipid storage regulation, it will likely be essential to define the interactome of LD-associated proteins.Previous studies of such interactions were hampered by technical limitations. Therefore, we have developed a split-luciferase based protein-protein interaction assay and test for interactions among 47 proteins from Drosophila and from mouse. We confirmed previously described interactions and identified many new ones. In 1561 complementation tests, we assayed for interactions among 487 protein pairs of which 92 (19%) resulted in a successful luciferase complementation. These results suggest that a prominent fraction of the LD-associated proteome participates in protein-protein interactions.In targeted experiments, we analyzed the two proteins Jabba and CG9186 in greater detail. Jabba mediates the sequestration of histones to LDs. We successfully applied our split luciferase complementation assay to learn more about this function as we were e.g. able to map the interaction between Jabba and histones. For CG9186, expression levels affect the positioning of LDs. Here, we reveal the ubiquitination of CG9186, and link this posttranslational modification to LD cluster induction.

Experimental Demonstration of the Thermochemical Reduction of Ceria in a Solar Aerosol Reactor.

We report on the experimental demonstration of an aerosol solar reactor for the thermal reduction of ceria, as part of a thermochemical redox cycle for splitting H2O and CO2. The concept utilizes a cavity-receiver enclosing an array of alumina tubes, each containing a downward gravity-driven aerosol flow of ceria particles countercurrent to an inert sweep gas flow for intrinsic separation of reduced ceria and oxygen. A 2 kWth lab-scale prototype with a single tube was tested under radiative fluxes approaching 4000 suns, yielding reaction extents of up to 53% of the thermodynamic equilibrium at 1919 K within residence times below 1 s. Upon thermal redox cycling, fresh primary particles of 2.44 µm mean size initially formed large agglomerates of 1000 µm mean size, then sintered into stable particles of 150 µm mean size. The reaction extent was primarily limited by heat transfer for large particles/agglomerates (mean size > 200 µm) and by the gas phase advection of product O2 for smaller particles.

Temporal control of bidirectional lipid-droplet motion in Drosophila depends on the ratio of kinesin-1 and its co-factor Halo.

During bidirectional transport, individual cargoes move continuously back and forth along microtubule tracks, yet the cargo population overall displays directed net transport. How such transport is controlled temporally is not well understood. We analyzed this issue for bidirectionally moving lipid droplets in Drosophila embryos, a system in which net transport direction is developmentally controlled. By quantifying how the droplet distribution changes as embryos develop, we characterize temporal transitions in net droplet transport and identify the crucial contribution of the previously identified, but poorly characterized, transacting regulator Halo. In particular, we find that Halo is transiently expressed; rising and falling Halo levels control the switches in global distribution. Rising Halo levels have to pass a threshold before net plus-end transport is initiated. This threshold level depends on the amount of the motor kinesin-1: the more kinesin-1 is present, the more Halo is needed before net plus-end transport commences. Because Halo and kinesin-1 are present in common protein complexes, we propose that Halo acts as a rate-limiting co-factor of kinesin-1.

How Brain Fat Conquers Stress.

A new paper by Bailey et al. reveals that lipid droplets, crucial organelles for energy storage, can also protect against oxidative stress. In Drosophila larvae, lipid droplets in glia allow neuronal stem cells to keep proliferating under hypoxic conditions. Protection likely involves sequestering vulnerable membrane lipids away from reactive oxygen species.

Expanding roles for lipid droplets.

Lipid droplets are the intracellular sites for neutral lipid storage. They are critical for lipid metabolism and energy homeostasis, and their dysfunction has been linked to many diseases. Accumulating evidence suggests that the roles lipid droplets play in biology are significantly broader than previously anticipated. Lipid droplets are the source of molecules important in the nucleus: they can sequester transcription factors and chromatin components and generate the lipid ligands for certain nuclear receptors. Lipid droplets have also emerged as important nodes for fatty acid trafficking, both inside the cell and between cells. In immunity, new roles for droplets, not directly linked to lipid metabolism, have been uncovered, with evidence that they act as assembly platforms for specific viruses and as reservoirs for proteins that fight intracellular pathogens. Until recently, knowledge about droplets in the nervous system has been minimal, but now there are multiple links between lipid droplets and neurodegeneration: many candidate genes for hereditary spastic paraplegia also have central roles in lipid-droplet formation and maintenance, and mitochondrial dysfunction in neurons can lead to transient accumulation of lipid droplets in neighboring glial cells, an event that may, in turn, contribute to neuronal damage. As the cell biology and biochemistry of lipid droplets become increasingly well understood, the next few years should yield many new mechanistic insights into these novel functions of lipid droplets.