TACLE

Beyond their structural role, lipids (and particularly phospholipids (PLs)) are key regulators of membrane trafficking processes such as vesicular exocytosis and endocytosis, which are essential for hormone secretion and neurotransmission. A key feature of the plasma membrane (PM) is the asymmetric distribution of PLs between its two leaflets. While flippases and floppases maintain this asymmetry, scramblases disrupt it by catalyzing bidirectional translocation of lipids. We were the first to demonstrate that the phospholipid scramblase-1 (PLSCR1) is required for local disruption of lipid asymmetry near vesicle fusion sites, thereby regulating neurotransmitter and hormone release most likely through vesicle recycling by compensatory endocytosis. However, the mechanisms linking PM lipid asymmetry and membrane trafficking remain poorly understood, largely due to key technical and conceptual limitations. First, commercially available synthetic phospholipids are typically modified at the fatty acyl chains or head group, which compromises their ability to mimic natural PL functions and distorts experimental outcomes. Second, current scrambling assays rely on such unappropriated analogues and neglect the acyl chain composition. Third, the role of PLSCR1 remains controversial: its classification as a bona fide scramblase is still debated and its function may instead be indirect, mediated through interaction with other proteins. Finally, the causal link between lipid scrambling and vesicle recycling via endocytosis has not been firmly established. Bringing together chemists, biologists and biophysicists, the TACLE project (Trafficking and Asymmetry of Cellular Lipid Environment) aims to address these limitations and to elucidate how the regulation of PM phospholipid asymmetry governs key membrane trafficking events such as exocytosis and endocytosis, using adrenal chromaffin cells and cerebellum granule neurons as neuroendocrine and neuronal experimental models, respectively. To do so, the TACLE project will pursue the following specific objectives: 1) Design novel synthetic clickable azide-based PL analogues, preserving both native head group and acyl chain composition. 2) Develop a next-generation scrambling assay using these analogues to monitor PL scrambling dynamics and localization during exocytosis and endocytosis in neurosecretory cells, and assess the influence of acyl chain composition on scrambling behavior. 3) Investigate how lipid scrambling impacts plasma membrane properties, using established biophysical measurements to provide complementary insights into membrane behavior during vesicle trafficking. 4) Clarify the molecular role of PLSCR1, by identifying its potential protein partners and determining whether its effect in lipid remodeling is direct or mediated through protein interactions. We will also examine how modulating PLSCR1 activity, or scrambling more broadly, affects compensatory endocytosis and PL–protein interactions. The TACLE project will significantly advance our understanding of how dynamic modulation of membrane asymmetry regulates cellular secretion and may help resolve the longstanding debate over the scramblase activity of PLSCR1. In doing so, it will also deliver new broadly applicable tools and methods for investigating complex mechanisms of lipid biology and biomembrane dynamics in living cells.