The dynamic remodeling of the endoplasmic reticulum (ER) is key to maintaining cellular homeostasis upon changing physiological conditions. For instance, cells can increase ER size in response to the accumulation of misfolded proteins in the ER, a condition known as ER stress. Previous studies in Saccharomyces cerevisiae have provided evidence that ER expansion serves to improve cellular fitness upon ER stress. In mammalian cells, the range of ER stress-induced ER phenotypes and the mechanisms promoting such changes are unclear. Proteins involved in lipid metabolism have the potential to regulate ER size by controlling ER membrane biogenesis. Lipins, a conserved family of proteins acting as phosphatidic acid phosphatases and transcriptional co- regulators, have been shown to play an important role in regulating ER membrane biogenesis in Saccharomyces cerevisiae. However, their possible functions in ER membrane biogenesis in mammalian cells have not been directly explored. In this study, I investigated ER membrane remodeling in human U2OS and SUM159 cells by confocal fluorescence microscopy and showed that three commonly used chemical ER stressors (Tunicamycin, Thapsigargin and Cyclopiazonic acid) can increase apparent ER size while inducing different ER morphology phenotypes, ranging from the formation of a denser peripheral tubular network to peripheral ER sheets. Moreover, I contributed to the development of a semi- automated image analysis tool to quantify ER size and morphology in an unbiased manner in large cell populations. In the second part of this study, I investigated the roles of Lipin1, Lipin2 and Lipin3 in ER size and shape control in U2OS cells, using the above-mentioned image analysis tool. I showed that simultaneous knockdown of Lipin1, Lipin2 and Lipin3, or treatment with a candidate Lipin inhibitor, induce ER membrane expansion and an increase in peripheral ER sheets. My findings also suggest that while Lipin1 and Lipin2 may have reciprocal compensatory functions, Lipin3 may play distinct roles in regulating ER size. Furthermore, I showed that Lipin1 and Lipin2 levels increase upon ER stress. This revealed that both Lipin upregulation and downregulation can correlate with ER membrane expansion. However, I showed that the ER expansion phenotype induced by Tunicamycin and the one caused by Lipin perturbation likely arise from distinct molecular mechanisms. This raises the possibility that, depending on the physiological context, cells may regulate the individual Lipins and their activities differently to achieve ER expansion. Furthermore, in this study I showed that triple knockdown of Lipin1+2+3 also impairs the formation of lipid droplets. All in all, my study identifies ER stress conditions inducing robust ER membrane remodeling in human cells and lays the foundation for investigating the mechanisms driving ER membrane remodeling and their physiological significance. Moreover, my study provides a system to investigate the contributions of the individual Lipins and their different activities in ER size and shape control as well as in lipid droplet biogenesis, at steady state and upon ER stress.
Giulia Ruffini (Thu,) studied this question.