The What in Within Prevents the Breach from Without: The Make-up of the Plasma Membrane is Integral in the Pathways of Lipotoxic Cell Death

The plasma membrane (PM) experiences a dynamic interplay of lipids and proteins to fulfil its functions of barrier and regulated transport. The dual leaflets of this bilayer face vastly different environments that require the balancing of forces alongside their own interleaflet pressures. Thus, the composition of the membrane not only defines the lateral lipid environment but also influences the opposing layer. We examine how the lipotoxic stressors of exogenous ceramides and ferroptosis affect this microcosm to ultimately decide cell survival or death./nCeramides are a class of bioactive sphingolipids involved in crucial cellular processes that range from proliferation to death. This crux point makes ceramide regulation pivotal in determining cellular fate and endogenous ceramide concentrations are kept compartmentalised to avoid triggering an apoptotic cascade. However, not all ceramide species are alike and we explore the nuances of chain-length and the see-saw balance of life or death. Exogenous short-chain ceramides (C6-Cer) can trigger cell death and we utilised a CRISPR-Cas9 knockout (KO) screen to identify novel targets of C6-Cer toxicity. Our findings implicated transmembrane protein 30A (TMEM30A), a crucial component in functional P4-ATPases, and ADP ribosylation factor like GTPase 5A (ARL5A), a possible recruiter for tethering complexes in endosome to Golgi retrograde transport./nThrough the combination of genetic, biochemical and mass spectrometry approaches, we formulate possible mechanisms for these genes in relation to exogenous ceramide toxicity. Our results indicate that KO of TMEM30A creates an exoplasmic surface high in phosphatidylserine (PS). Literature shows that high concentrations of PS incorporates and stabilises cholesterol within its layer. This suggests that the PS phenotype of KOTMEM30A may draw cholesterol to the PM outer leaflet to create conditions that favour C24-Ceramide production. The high amounts of C24-Ceramide could offer protection against the death cascades of C6-Cer. KO of ARL5A possibly interferes with sterol regulation to cause the accumulation of cholesterol in the PM’s cytoplasmic leaflet. Cholesterol alters membrane fluidity through increased membrane packing; this rigidity may increase levels of C16-Ceramide, the endogenous species responsible for cell death. Addition of exogenous C6-Cer jumpstarts these respective pathways, allowing KOTMEM30A to resist C6-Cer death whilst death in KOARL5A cells is accelerated./nTurning to another form of lipid-mediated death, ferroptosis is a form of regulated cell death that is dependent on iron and the accumulation of lipid peroxides. These lipid radicals are readily formed upon abstraction of a hydrogen from the easily accessible tails of poly-unsaturated fatty acids (PUFAs). Accumulation of peroxidated PUFA-containing phospholipids on the PM will cause membrane rupture and lead to cell death./nAcyl-CoA synthetase long-chain 4 (ACSL4) converts free long-chain fatty acids into the substrates required from PUFA synthesis. The ACSL4 gene can produce two isoenzymes that differ in length and final protein localisation. The long-form associates with lipid droplets (LDs) whilst the short-form concentrates on the PM. We propose that this differential localisation will lead to altered subcellular localisation of PUFA-containing phospholipids and protection of PUFA translocation to the PM with alter the cell’s response to ferroptotic insults./nThis body of work explores the intricate dance of specific gene expression and its effect upon PM composition. It is the make-up of the membrane that determines if a cell is able to survive acute lipotoxic stressors. In essence, we explore the philosophical ponderings that cell death by C6-Cer or ferroptosis is a merit of the underlying structure of the PM.

Advisor: Olzmann, James