Transbilayer transport of phospholipids by scramblases and flippases: a key role in neurotransmission – LipidTrans4NeuroTraffic

Neurons and neuroendocrine cells release neurotransmitters, neuropeptides and hormones through calcium-regulated exocytosis. The ultimate step of exocytosis involves fusion of secretory vesicles with the plasma membrane, leading to the merging of these two compartments. Following exocytosis, the excess of membrane supply is compensated through membrane fission process by endocytosis. Although much has been learned concerning the proteins regulating vesicle fission and fusion at donor and acceptor compartments, relatively little attention has been paid to the necessary role of lipids. In particular, how membrane phospholipids asymmetry is regulated during exo-endocytosis remain a fundamental yet unresolved question. This aspect is particularly important in neurosecretory cells that display intense membrane trafficking and mixing between organelle membranes to support crucial neuro-physiological functions such as neurotransmission.

One of the key features of the plasma and the exo-endo membranes is the asymmetric distribution of lipids between the leaflets with for example, the aminophospholipids (APLs) phosphatidylserine and phosphatidylethanolamine primarily restricted to the cytosolic leaflet. This selective asymmetry is involved in many cellular processes and is controlled by proteins like the P4-type ATPases, flippases that catalyze the transport of APLs from the exoplasmic to the cytoplasmic leaflet of membranes. On the contrary, proteins named “scramblases” catalyze the bidirectional transbilayer movement of a broad range of phospholipids resulting in the disruption of lipid asymmetry.

Preliminary data from our consortium indicate that i) APLs scrambling occurs at the close vicinity of the secretory vesicle fusion sites in neuroendocrine cells, ii) scramblase protein is somehow important to control exo-endocytic processes in neurons and neuroendocrine cells and iii) that P4-ATPases control various endocytic processes. However, the nature of the flippases and scramblases involved in neuro-secretory processes, their functional role and the importance of transbilayer lipid movement remains unknown. Therefore, our project’s central aim is to delineate which, why and how P4-ATPases and scramblases controls hormone secretion in neuroendocrine cells and neurotransmission in neurons.

Investigating lipids dynamic is clearly a challenge and to succeed in this ambitious task, we have built a consortium able to lift the main technical limitations away. We indeed propose here a unique collaborative strategy that combines the generation of CRISPR/Cas9-engineered neuroendocrine cell lines knock-in or knock out for various scramblase and P4-ATPases, ultrastructural electron microscopy imaging on freeze-fracture replica of membrane layer fragments and the super-resolution imaging STED and SMLM methods with the use of specific lipid probes, membrane deformation assay and in cellulo exploration of secretory exo-endocytic events. Such synergistic combination will allow us to unravel for the first time the importance of lipid transbilayer transport regulation during vesicular membrane trafficking events in neuro-secretory cells. Moreover, we believe that this project will not only provide new insight into exo- and endocytic processes, but will create general and novel tools and methods for understanding complex mechanisms of lipid biology and biomembrane dynamics.