The fat body cortical actin network regulates Drosophila inter-organ nutrient trafficking, signaling, and adipose 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.

Snx14 proximity labeling reveals a role in saturated fatty acid metabolism and ER homeostasis defective in SCAR20 disease.

Fatty acids (FAs) are central cellular metabolites that contribute to lipid synthesis, and can be stored or harvested for metabolic energy. Dysregulation in FA processing and storage causes toxic FA accumulation or altered membrane compositions and contributes to metabolic and neurological disorders. Saturated lipids are particularly detrimental to cells, but how lipid saturation levels are maintained remains poorly understood. Here, we identify the cerebellar ataxia spinocerebellar ataxia, autosomal recessive 20 (SCAR20)-associated protein Snx14, an endoplasmic reticulum (ER)-lipid droplet (LD) tethering protein, as a factor required to maintain the lipid saturation balance of cell membranes. We show that following saturated FA (SFA) treatment, the ER integrity of SNX14KO cells is compromised, and both SNX14KO cells and SCAR20 disease patient-derived cells are hypersensitive to SFA-mediated lipotoxic cell death. Using APEX2-based proximity labeling, we reveal the protein composition of Snx14-associated ER-LD contacts and define a functional interaction between Snx14 and Δ-9 FA desaturase SCD1. Lipidomic profiling reveals that SNX14KO cells increase membrane lipid saturation following exposure to palmitate, phenocopying cells with perturbed SCD1 activity. In line with this, SNX14KO cells manifest delayed FA processing and lipotoxicity, which can be rescued by SCD1 overexpression. Altogether, these mechanistic insights reveal a role for Snx14 in FA and ER homeostasis, defects in which may underlie the neuropathology of SCAR20.

Drosophila Snazarus Regulates a Lipid Droplet Population at Plasma Membrane-Droplet Contacts in Adipocytes.

Adipocytes store nutrients as lipid droplets (LDs), but how they organize their LD stores to balance lipid uptake, storage, and mobilization remains poorly understood. Here, using Drosophila fat body (FB) adipocytes, we characterize spatially distinct LD populations that are maintained by different lipid pools. We identify peripheral LDs (pLDs) that make close contact with the plasma membrane (PM) and are maintained by lipophorin-dependent lipid trafficking. pLDs are distinct from larger cytoplasmic medial LDs (mLDs), which are maintained by FASN1-dependent de novo lipogenesis. We find that sorting nexin CG1514 or Snazarus (Snz) associates with pLDs and regulates LD homeostasis at ER-PM contact sites. Loss of SNZ perturbs pLD organization, whereas Snz over-expression drives LD expansion, triacylglyceride production, starvation resistance, and lifespan extension through a DESAT1-dependent pathway. We propose that Drosophila adipocytes maintain spatially distinct LD populations and identify Snz as a regulator of LD organization and inter-organelle crosstalk.

Cerebellar ataxia disease-associated Snx14 promotes lipid droplet growth at ER-droplet contacts.

Lipid droplets (LDs) are nutrient reservoirs used by cells to maintain homeostasis. Nascent droplets form on the endoplasmic reticulum (ER) and grow following an influx of exogenous fatty acids (FAs). The budding of LDs requires extensive ER-LD crosstalk, but how this is regulated remains poorly understood. Here, we show that sorting nexin protein Snx14, an ER-resident protein associated with the cerebellar ataxia SCAR20, localizes to ER-LD contacts following FA treatment, where it promotes LD maturation. Using proximity-based APEX technology and topological dissection, we show that Snx14 accumulates specifically at ER-LD contacts independently of Seipin, where it remains ER-anchored and binds LDs in trans. SNX14KO cells exhibit perturbed LD morphology, whereas Snx14 overexpression promotes LD biogenesis and extends ER-LD contacts. Multi-time point imaging reveals that Snx14 is recruited to ER microdomains containing the fatty acyl-CoA ligase ACSL3, where nascent LDs bud. We propose that Snx14 is a novel marker for ER-LD contacts and regulates FA-stimulated LD growth.

Mdm1 maintains endoplasmic reticulum homeostasis by spatially regulating lipid droplet biogenesis.

Lipid droplets (LDs) serve as cytoplasmic reservoirs for energy-rich fatty acids (FAs) stored in the form of triacylglycerides (TAGs). During nutrient stress, yeast LDs cluster adjacent to the vacuole/lysosome, but how this LD accumulation is coordinated remains poorly understood. The ER protein Mdm1 is a molecular tether that plays a role in clustering LDs during nutrient depletion, but its mechanism of function remains unknown. Here, we show that Mdm1 associates with LDs through its hydrophobic N-terminal region, which is sufficient to demarcate sites for LD budding. Mdm1 binds FAs via its Phox-associated domain and coenriches with fatty acyl-coenzyme A ligase Faa1 at LD bud sites. Consistent with this, loss of MDM1 perturbs free FA activation and Dga1-dependent synthesis of TAGs, elevating the cellular FA level, which perturbs ER morphology and sensitizes yeast to FA-induced lipotoxicity. We propose that Mdm1 coordinates FA activation adjacent to the vacuole to promote LD production in response to stress, thus maintaining ER homeostasis.