Biology subjects

Henne, W. M.

Publications and source records attributed to Henne, W. M..

10 recordsLinked to original sources

bioRxiv2024

Metabolic rewiring in fat-depleted Drosophila reveals triglyceride:glycogen crosstalk and identifies cDIP as a new regulator of energy metabolism

Tissues store nutrients as triglyceride (TG) or glycogen at specific ratios, but how these reserves are sensed and balanced remains poorly understood. Here we show that blockage of de novo lipogenesis (DNL) in the Drosophila fat body (FB) triggers a cell autonomous metabolic switch characterized by severe fat depletion and profound glycogen accumulation that supports animal development. Despite lipid loss, Drosophila develop normally but exhibit shortened lifespans and impaired female fecundity. Mechanistically, we identify SREBP-dependent metabolic rewiring that facilitates a switch from TG to glycogen storage, triggered by fatty acid deficiency when DNL is inhibited, and which is rescued by dietary fatty acids. Fat depleted FBs require glycolysis but exhibit blunted mitochondrial metabolism, and no dependence on lactate utilization. Finally, we identify histone acetyltransferases (HATs) Nej and Tip60, which support SREBP activity, as essential for this metabolic switch. Collectively, we propose that in response to DNL deficiency, the fat-depleted FB undergoes a SREBP-mediated TG-to-glycogen metabolic switch preserving organismal development at the cost of reproductive success. Key findingsO_LIFat body-specific FASN1 loss leads to fat-depleted but viable Drosophila that complete their developmental lifecycle by rewiring energy metabolism to store glycogen instead of fat C_LIO_LIFASN1-deficient larvae functionally rely on glycogen synthesis and glycolysis, but not lactate metabolism, and display blunted TCA metabolism C_LIO_LIMetabolic screening reveals a SREBP-dependent TG:glycogen metabolic switch in response to blockage of DNL fatty acid biosynthesis C_LIO_LIHistone acetyltransferases (HATs) Nej and Tip60, and acetyl-CoA synthase, are required for the TG:glycogen metabolic switch C_LI

cell biology↗

bioRxiv2024

Paraoxonase-like APMAP maintains endoplasmic reticulum-associated lipid and lipoprotein homeostasis

Oxidative stress perturbs lipid homeostasis and contributes to metabolic diseases. Though ignored compared to mitochondrial oxidation, the endoplasmic reticulum (ER) generates reactive oxygen species requiring antioxidant quality control. Using multi-organismal profiling featuring Drosophila, zebrafish, and mammalian cells, here we characterize the paraoxonase-like APMAP as an ER-localized protein that promotes redox and lipid homeostasis and lipoprotein maturation. APMAP-depleted mammalian cells exhibit defective ER morphology, elevated ER and oxidative stress, lipid droplet accumulation, and perturbed ApoB-lipoprotein homeostasis. Critically, APMAP loss is rescued with chemical antioxidant NAC. Organismal APMAP depletion in Drosophila perturbs fat and lipoprotein homeostasis, and zebrafish display increased vascular ApoB-containing lipoproteins, particles that are atherogenic in mammals. Lipidomics reveals altered polyunsaturated phospholipids and increased ceramides upon APMAP loss, which perturbs ApoB-lipoprotein maturation. These ApoB-associated defects are rescued by inhibiting ceramide synthesis. Collectively, we propose APMAP is an ER-localized antioxidant that promotes lipid and lipoprotein homeostasis. Key findings summaryO_LIAPMAP localizes primarily to the ER network in human cells and Drosophila fat body tissue, and is a type II integral membrane protein C_LIO_LILoss of APMAP or Drosophila APMAP (dAPMAP) causes ER membrane expansion, elevates CHOP-associated ER stress, promotes LD accumulation, and alters ApoB-lipoprotein secretion C_LIO_LIAPMAP-depleted cells and dAPMAP-depleted Drosophila fat tissue exhibit defective redox homeostasis; phenotypes associated with APMAP loss are rescued by antioxidant NAC C_LIO_LIZebrafish-based LipoGlo reporter reveals that loss of apmap in zebrafish causes increased vascular ApoB-containing lipoproteins C_LIO_LILipidomic profiling indicates that APMAP loss reduces PUFA-phospholipids and elevates intracellular ceramides, which perturbs ApoB maturation C_LI

cell biology↗

bioRxiv2024

Spatial proteomics of ER tubules reveals CLMN, an ER-actin tether at focal adhesions that promotes cell migration

The endoplasmic reticulum (ER) is structurally and functionally diverse, yet how its functions are organized within morphological subdomains is incompletely understood. Utilizing TurboID-based proximity labeling and CRISPR knock-in technologies, here we map the proteomic landscape of the human ER and nuclear envelope. Spatial proteomics reveals enrichments of proteins into ER tubules, sheets, and nuclear envelope. We uncover an ER-enriched actin-binding protein, Calmin (CLMN), and define it as an ER-actin tether that localizes to focal adhesions adjacent to ER tubules. CLMN depletion perturbs focal adhesion disassembly, actin dynamics, and cell movement. Mechanistically, CLMN-depleted cells also exhibit defects in calcium signaling near ER-actin interfaces, suggesting CLMN promotes calcium signaling near adhesions to facilitate their disassembly. Collectively, we map the sub-organelle proteome landscape of the ER, identify CLMN as an ER-actin tether, and describe a non-canonical mechanism by which ER tubules engage actin to regulate cell migration.

cell biology↗

bioRxiv2023

Bsc2 is a novel regulator of triglyceride lipolysis that demarcates a lipid droplet subpopulation

Cells store lipids in the form of triglyceride (TG) and sterol-ester (SE) in lipid droplets (LDs). Distinct pools of LDs exist, but a pervasive question is how proteins localize to and convey functions to LD subsets. Here, we show the yeast protein YDR275W/Tld1 (for TG-associated LD protein 1) localizes to a subset of TG-containing LDs, and reveal it negatively regulates lipolysis. Mechanistically, Tld1 LD targeting requires TG, and is mediated by two distinct hydrophobic regions (HRs). Molecular dynamics simulations reveal Tld1s HRs interact with TG on LDs and adopt specific conformations on TG-rich LDs versus SE-rich LDs in yeast and human cells. Tld1-deficient yeast display no defect in LD biogenesis, but exhibit elevated TG lipolysis dependent on lipase Tgl3. Remarkably, over-expression of Tld1, but not LD protein Pln1/Pet10, promotes TG accumulation without altering SE pools. Finally, we find Tld1-deficient cells display altered LD mobilization during extended yeast starvation. We propose Tld1 senses TG-rich LDs and regulates lipolysis on LD subpopulations.

cell biology↗

bioRxiv2022

The fat body cortical actin network regulates Drosophila inter-organ nutrient trafficking and adipocyte cell size

Defective nutrient storage and adipocyte enlargement (hypertrophy) are emerging features of metabolic syndrome and type 2 diabetes. Within adipose tissues, how the cytoskeletal network contributes to adipose cell size, nutrient uptake, fat storage, and signaling remain poorly understood. Utilizing the Drosophila larval fat body (FB) as a model adipose tissue, we show that a specific actin isoform--Act5C--forms the cortical actin network necessary to expand adipocyte cell size for biomass storage in development. Additionally, we uncover a non-canonical role for the cortical actin cytoskeleton in inter-organ lipid trafficking. We find Act5C localizes to the FB cell surface and cell-cell boundaries, where it intimately contacts peripheral LDs (pLDs), forming a cortical actin network for cell architectural support. FB-specific loss of Act5C perturbs FB triglyceride (TG) storage and LD morphology, resulting in developmentally delayed larvae that fail to develop into flies. Utilizing temporal RNAi-depletion approaches, we reveal that Act5C is indispensable post-embryogenesis during larval feeding as FB cells expand and store fat. Act5C-deficient FBs fail to grow, leading to lipodystrophic larvae unable to accrue sufficient biomass for complete metamorphosis. In line with this, Act5C-deficient larvae display blunted insulin signaling and reduced feeding. Mechanistically, we also show this diminished signaling correlates with decreased lipophorin (Lpp) lipoprotein-mediated lipid trafficking, and find Act5C is required for Lpp secretion from the FB for lipid transport. Collectively, we propose that the Act5C-dependent cortical actin network of Drosophila adipose tissue is required for adipose tissue size-expansion and organismal energy homeostasis in development, and plays an essential role in inter-organ nutrient transport and signaling.

cell biology↗

bioRxiv2022

Triglyceride lipolysis driven by glucose restriction triggers liquid-crystalline phase transitions and proteome remodeling of lipid droplets

Lipid droplets (LDs) are reservoirs for triglycerides (TGs) and sterol-esters (SEs), but how these lipids are organized within LDs and influence its proteome remains unclear. Using in situ cryoelectron tomography, we show that glucose restriction triggers lipid phase transitions within LDs generating liquid-crystalline lattices inside them. Mechanistically this requires TG lipolysis, which decreases the LD TG:SE ratio, promoting SE transition to a liquid-crystalline phase. Molecular dynamics simulations reveal TG depletion promotes spontaneous TG and SE de-mixing in LDs, additionally altering the lipid packing of the phospholipid monolayer surface. Fluorescence imaging and proteomics further reveal that liquid-crystalline phases are associated with selective remodeling of the LD proteome. Some canonical LD proteins including Erg6 re-localize to the ER network, whereas others remain LD-associated. Model peptide LiveDrop also redistributes from LDs to the ER, suggesting liquid-crystalline-phases influence ER-LD inter organelle transport. Our data suggests glucose restriction drives TG mobilization, which alters the phase properties of LD lipids and selectively remodels the LD proteome.

cell biology↗

bioRxiv2021

Experimental and in silico Alphafold2 derived structures of the SNX-RGS proteins suggest a new class of lipid transfer protein

Recent advances in protein structure prediction using machine learning such as AlphaFold2 and RosettaFold presage a revolution in structural biology. Genome-wide predictions of protein structures are providing unprecedented insights into their architecture and intradomain interactions, and applications have already progressed towards assessing protein complex formation. Here we present detailed analyses of the sorting nexin proteins that contain regulator of G-protein signalling domains (SNX-RGS proteins), providing a key example of the ability of AlphaFold2 to reveal novel structures with previously unsuspected biological functions. These large proteins are conserved in most eukaryotes and are known to associate with lipid droplets (LDs) and sites of LD-membrane contacts, with key roles in regulating lipid metabolism. They possess five domains, including an N-terminal transmembrane domain that anchors them to the endoplasmic reticulum, an RGS domain, a lipid interacting phox homology (PX) domain and two additional domains named the PXA and PXC domains of unknown structure and function. Here we report the crystal structure of the RGS domain of sorting nexin 25 (SNX25) and show that the AlphaFold2 prediction closely matches the experimental structure. Analysing the full-length SNX-RGS proteins across multiple homologues and species we find that the distant PXA and PXC domains in fact fold into a single unique structure that notably features a large and conserved hydrophobic pocket. The nature of this pocket strongly suggests a role in lipid or fatty acid binding, and we propose that these molecules represent a new class of conserved lipid transfer proteins.

biochemistry↗

bioRxiv2021

Liquid-crystalline lipid phase transitions in lipid droplets selectively remodel the LD proteome

Lipid droplets (LDs) are reservoirs for triglycerides (TGs) and sterol-esters (SEs). How lipids are organized within LDs and influence the LD proteome remains unclear. Using in situ cryo-electron tomography, we show that glucose restriction triggers lipid phase transitions within LDs generating liquid-crystalline lattices inside them. Mechanistically, this requires TG lipolysis, which alters LD neutral lipid composition and promotes SE transition to a liquid-crystalline phase. Fluorescence imaging and proteomics further reveal that LD liquid-crystalline lattices selectively remodel the LD proteome. Some canonical LD proteins including Erg6 re-localize to the ER network, whereas others remain on LDs. Model peptide LiveDrop also redistributes from LDs to the ER, suggesting liquid-crystalline-phases influence ER-LD inter-organelle transport. Proteomics also indicates glucose restriction elevates peroxisome lipid oxidation, suggesting TG mobilization provides fatty acids for cellular energetics. This suggests glucose restriction drives TG mobilization, which alters the phase properties of LD lipids and selectively remodels the LD proteome.

cell biology↗

bioRxiv2021

ER-localized phosphatidylethanolamine synthase plays a conserved role in lipid droplet formation

The asymmetric distribution of phospholipids in membranes is a fundamental principle of cellular compartmentalization and organization. Phosphatidylethanolamine (PE), a nonbilayer phospholipid that contributes to organelle shape and function, is synthesized at several subcellular localizations via semi-redundant pathways. Previously, we demonstrated in the yeast Saccharomyces cerevisiae that the PE synthase Psd1, which primarily operates on the mitochondrial inner membrane, is additionally targeted to the endoplasmic reticulum (ER). While ER-localized Psd1 is required to support cellular growth in the absence of redundant pathways, its physiological function at the ER is unclear. We now demonstrate that ER-localized Psd1 sub-localizes on the ER to lipid droplet (LD) attachment sites and further show it is specifically required for normal LD formation. We also find that the role of PSD enzymes in LD formation is conserved in other organisms. Thus, we have identified PSD enzymes as novel regulators of LDs and demonstrate that both mitochondria and LDs in yeast are organized and shaped by the spatial positioning of a single PE synthesis enzyme.

cell biology↗

bioRxiv2021

Vacuole-specific lipid release for tracking intracellular lipid metabolism and transport in Saccharomyces cerevisiae

Lipid metabolism is spatiotemporally regulated within cells, yet intervention into lipid functions at subcellular resolution remains difficult. Here we report a method that enables site-specific release of sphingolipids and cholesterol inside the vacuole in Saccharomyces cerevisiae. Using this approach, we monitored real-time sphingolipid metabolic flux out of the vacuole by mass spectrometry and found that the ER-vacuole tethering protein Mdm1 facilitated the metabolism of sphingoid bases into ceramides. In addition, we showed that cholesterol, once delivered into yeast using our method, could restore cell proliferation induced by ergosterol deprivation, overcoming the previously described sterol-uptake barrier under aerobic conditions. Together, these data define a new way to study intracellular lipid metabolism and transport from the vacuole in yeast.

cell biology↗