A fast-acting lipid checkpoint in G1 prevents mitotic defects

Lipid synthesis must increase during the cell cycle to double membrane mass, but how insufficient synthesis restricts cell-cycle entry is not understood. Here, we identify a lipid checkpoint in G1 phase of the mammalian cell cycle by using live single-cell imaging, lipidome, and transcriptome analysis in non-transformed cells. We show that synthesis of fatty acids in G1 not only increases lipid mass but extensively shifts the lipid composition to unsaturated phospholipids and neutral lipids. Strikingly, acute lowering of lipid synthesis rapidly activates the PERK/ATF4 endoplasmic reticulum (ER) stress pathway that blocks cell-cycle entry by increasing p21 levels, decreasing Cyclin D levels, and suppressing Retinoblastoma protein phosphorylation. Together, our study identifies a rapid anticipatory ER lipid checkpoint in G1 that prevents cells from starting the cell cycle as long as lipid synthesis is low, thereby preventing mitotic failure and cell death, which are triggered by low lipid synthesis much later in mitosis. To investigate the effect of fatty acid metabolism inhibition on cell cycle progression, we treated MCF-10A cells that were previously synchronized through starvation and released into the cell cycle with epidermal growth factor (EGF) with compounds inhibiting fatty acid metabolism. We performed gene expression profiling on MCF-10A cells released with EGF for 9, 12, and 15 hours. Cells were treated with fatty acid synthase inhibitors C75 (30µM), or GSK2194069 (50µM), or stearoyl-CoA desaturase (SCD) inhibitor (32µM) or DMSO (control) for 3 or 6 hours after 9 hours of EGF release. Comparative gene expression profiling analysis of RNA-Seq data was performed between inhibitor treatment and DMSO control.