Biology subjects

Canals, D.

Publications and source records attributed to Canals, D..

7 recordsLinked to original sources

bioRxiv2026

Dithionite quenching of NBD-labeled lipids reveals artificial lipid droplet purity and neutral lipid surface accessibility

Artificial lipid droplets (aLDs) provide a controllable platform for studying lipid biochemistry, but their use is limited by contamination with other membrane structures and the lack of quantitative methods to assess sample purity. Here, we establish dithionite quenching of NBD-labeled lipids as a simple approach to evaluate aLD purity. The approach relies on dithionites ability to selectively quench NBD fluorophores exposed in the phospholipid monolayer of aLDs and in the outer leaflet of liposome bilayers, but not those protected within the inner leaflet of liposome bilayers. Consistent with liposome contamination, bulk aLD preparations exhibit incomplete quenching, which can be separated by sucrose gradient centrifugation into liposome-like and droplet-enriched populations based on quenching behavior. Guided by this assay, sonication conditions were optimized to increase aLD purity and reduce liposome contamination. A biotin-streptavidin immobilization strategy further enabled stable imaging of individual aLDs. Finally, we applied dithionite quenching to probe the accessibility of neutral lipids within aLDs. This revealed hydrophobicity-dependent quenching kinetics of neutral lipids, with less hydrophobic diacylglycerols showing greater surface exposure within aLDs than more hydrophobic triacylglycerols and cholesterol esters. Taken together, these establish dithionite quenching of NBD-labeled lipids as a simple quantitative method for assessing aLD purity and demonstrate its utility for studying lipid accessibility.

biochemistry↗

bioRxiv2026

Sphingolipid regulation by yeast Mdm1 supports adaptive remodeling of the methionine transporter Mup1

Membrane lipid composition influences endocytic remodeling of nutrient transporters, yet how lipid metabolism is spatially coordinated to support sustained adaptation to nutrient limitations remains unclear. Here, we investigated whether the ER-vacuole tether Mdm1 links sphingolipid homeostasis to regulation of the high-affinity methionine permease Mup1 in budding yeast. To test this, we examined Mup1 trafficking, amino acid homeostasis, and sphingolipid composition in mdm1{Delta} cells during starvation. We found that loss of Mdm1 causes persistent retention of Mup1 at the plasma membrane, accompanied by reduced intracellular methionine and broad amino acid depletion. Lipidomic analyses revealed decreased sphingoid bases and altered ceramide composition in mdm1{Delta} cells. Importantly, supplementation with the sphingolipid precursor phytosphingosine restored sphingolipid pools, rescued Mup1 endocytosis, and improved amino acid homeostasis. Consistent with a chronic amino acid restriction-like state, mdm1{Delta} cells exhibited extended chronological lifespan. Together, these findings identify Mdm1 as a spatial organizer of sphingolipid metabolism required for adaptive endocytic remodeling of Mup1, thereby linking ER-vacuole contact site function to plasma membrane proteostasis and metabolic adaptation.

cell biology↗

bioRxiv2026

Metabolic plasticity of sphingolipids governs cancer cell fitness in acidic tumor ecosystems

Cell state plasticity enables cancer cells to rapidly adapt to fluctuating microenvironments without requiring genetic alteration, shaping tumor evolution under stress. Extracellular acidosis is a persistent feature of solid tumors that impose strong selective pressure, yet how cancer cells maintain fitness under acute and chronic acidic conditions remains unclear. Here, we show that adaptation to acidosis is mediated by plastic rewiring of sphingolipid metabolism centered on ceramide turnover. Spatial multi-omics analysis of three-dimensional tumor models revealed enrichment of ceramides within acidic niches, consistent with a stress-induced phenotype. While acute acidosis promoted ceramide accumulation and reduced fitness, chronic exposure selected for cells capable of dynamically redistributing sphingolipid flux across multiple clearance pathways. Functional perturbation demonstrated that inhibition of individual pathways was insufficient to compromise survival, whereas simultaneous disruption of all ceramide clearance routes resulted in cell death, revealing a degenerate metabolic architecture. This network-level flexibility enables cancer cells to maintain fitness by switching between alternative metabolic states under acidic stress. Together, our findings identify sphingolipid metabolic plasticity as an adaptive strategy that supports tumor persistence in acidic ecosystems and suggest that targeting metabolic flexibility, rather than individual pathways, may provide a more effective therapeutic approach.

cancer biology↗

bioRxiv2025

DDHD2 possesses both lipase and transacylase capacities that remodel triglyceride acyl chains

Hereditary spastic paraplegia subtype SPG54 is a genetic neurological disorder caused by mutations in the DDHD2 gene. Excessive lipid droplet accumulation is observed in the brains of SPG54 patients and DDHD2 knockout mice, consistent with DDHD2s reported neutral lipase activity. Here, we find recombinant human DDHD2 preferentially hydrolyzes diacylglycerol (DAG) over phospholipids, with a slight preference for DAG over triacylglycerol (TAG). DDHD2 also exhibits transacylase activity, which enables transfer of acyl chains from triacylglycerols to diacylglycerols and monoacylglycerols to remodel the acyl chains of triglycerides. A predicted hydrophobic amphipathic helix on DDHD2 is essential for lipid droplet binding in vitro and in cells, and its lack reduces the enzymatic activity and triglyceride acyl chain remodeling. Adipose triglyceride lipase (ATGL), but not hormone sensitive lipase (HSL), also has transacylation activity and can remodel triglyceride acyl chains, but to a lesser extent than DDHD2. Taken together, this provides evidence that DDHD2 is a neutral lipid lipase and transacylase whose broad specificity enables triglyceride acyl-chain remodeling. SIGNIFICANCE STATEMENTTriglycerides (TAGs), the primary form of long-term energy storage, have acyl chain compositions crucial for diverse cellular processes. Lipases typically hydrolyze TAGs into free fatty acids. Here, we reveal a novel function for the neutral lipid lipase DDHD2: a transacylase activity. Instead of releasing fatty acids, DDHD2 transfers them between neutral lipids, altering TAG acyl chain composition. This transacylation requires the unique oil environment of lipid droplets (LDs), which excludes water from DDHD2s lipolytic active site, favoring transacylation over hydrolysis. DDHD2s lipase and transacylase activities enable TAG acyl-chain remodeling, demonstrating the possibility that a single enzyme can catalyze TAG cycling. This finding has implications for understanding lipid metabolism, LD dynamics, and specific motor neuron diseases implicating DDHD2.

biochemistry↗

bioRxiv2025

Diet-induced phospholipid remodeling dictates ferroptosis sensitivity and tumorigenesis in the pancreas

High-fat diet (HFD) intake has been linked to an increased risk of pancreatic ductal adenocarcinoma (PDAC), a lethal and therapy-resistant cancer. However, whether and how specific dietary fats drive cancer development remains unresolved. Leveraging an oncogenic Kras-driven mouse model that closely mimics human PDAC progression, we screened a dozen isocaloric HFDs differing solely in fat source and representing the diversity of human fat consumption. Unexpectedly, diets rich in oleic acid - a monounsaturated fatty acid (MUFA) typically associated with good health - markedly enhanced tumorigenesis. Conversely, diets high in polyunsaturated fatty acids (PUFAs) suppressed tumor progression. Relative dietary fatty acid saturation levels (PUFA/MUFA) governed pancreatic membrane phospholipid composition, lipid peroxidation, and ferroptosis sensitivity in mice, concordant with circulating PUFA/MUFA levels being linked to altered PDAC risk in humans. These findings directly implicate dietary unsaturated fatty acids in controlling ferroptosis susceptibility and tumorigenesis, supporting potential "precision nutrition" strategies for PDAC prevention.

cancer biology↗

bioRxiv2025

A Critical Role for Neutral Sphingomyelinase-2 in Doxorubicin-induced Cardiotoxicity

Cardiotoxicity is a major side effect of Doxorubicin (Dox) that has hampered its clinical utility, and strategies to mitigate this cardiotoxicity are limited. Sphingolipids (SL) are central to the chemotherapy response in cancer but their role in normal tissue is less clear. Here, we identified the SL enzyme neutral sphingomyelinase-2 (nSMase2) as a critical mediator of chronic Dox-induced cardiotoxicity, establishing nSMase2 as a key downstream effector of Dox in cardiomyocytes (CM) and showing that in vivo loss of nSMase2 activity is protecting against chronic Dox-induced cardiac damage and dysfunction. Biologically, these studies link nSMase2 with Dox-induced CM senescence both in vitro and in vivo and identify the dual specificity phosphatase DUSP4 as a novel effector of nSMase2 in the Dox response. In addition to cementing a role for SL metabolism in Dox effects in normal tissue, this study advances nSMase2 as a target of interest for cardioprotection.

cell biology↗

bioRxiv2025

Critical Evaluation of Sphingolipids Detection by MALDI-MSI

The increasing interest in the role of sphingolipids in (patho)physiology has led to the demand for visualization of these lipids within tissue samples (both from animal models and patient specimens) using techniques such as matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). While increasingly adopted, detection of sphingolipids with MALDI-MSI is challenging due to: i) the significant structural variations of sphingolipid molecules, ii) the potential breakdown of the more complex molecules into structurally simpler species which may confound the analysis, and iii) the great difference in levels among sphingolipid classes and subspecies, with the low-abundant ones often being close to the detection limit. In this study, we adopted a multi-pronged approach to establish a robust pipeline for the detection of sphingolipids by MALDI-MSI and to establish best practices and limitations of this technology. First, we evaluated the more commonly adopted methods [2,5-Dihydroxyacetophenon (DHA) or 2,5-Dihydroxybenzoic acid (DHB) matrix in positive ion mode and 1,5-Diaminonaphthalene (DAN) matrix in negative ion mode] using MALDI-MS on reference standards. These standards were used at ratios similar to their relative levels in biological samples to evaluate signal artifacts originating from fragmentation of more complex sphingolipids and impacting low level species. Next, by applying the most appropriate protocol for each sphingolipid class, MALDI-MSI signals were validated in cell culture by modulating specific sphingolipid species using sphingolipid enzymes and inhibitors. Finally, the optimized parameters were utilized on breast cancer tissue from the PyMT mouse model. We report the optimal signal for sphingomyelin (SM) and, for the first time, Sph in DHB positive ion mode (in cells and PyMT tissue), and the validated detection of ceramides and glycosphingolipids in DAN negative ion mode. We document the extensive fragmentation of SM into sphingosine-1-phosphate (S1P) and even more so into ceramide-1-phosphate (C1P) using DAN in negative ion mode and its effect in generating an artifactual C1P tissue signal; we also report the lack of detectable signal for S1P and C1P in biological samples (cells and tissue) using the more suitable DHB positive ion mode protocol.

cell biology↗