Abstract
Mitochondria, the double membrane-bound organelles found in most eukaryotic cells, are highly dynamic structures that continuously change their morphologies through fusion and fission processes, collectively known as mitochondrial remodelling. While the involvement of proteins in this process have been extensively studied, the roles of different lipids in mitochondrial remodelling remain less understood due to challenges such as a lack of techniques to visualize and manipulate lipids within cells. Currently, one of the most comprehensive ways to perturb lipid levels is to inactivate the enzymes involved in lipid biosynthesis.Our research project was driven by the central hypothesis that various lipid species play distinct roles in mitochondrial remodelling, and alterations in lipid composition can lead to changes in mitochondrial morphology and function. To test this hypothesis, we conducted an siRNA screen targeting 258 different lipid biosynthetic enzymes, aiming to uncover potential lipid species or lipids that are involved in mitochondrial remodelling. We employed microscopy-based phenotypic screening to identify and document any abnormal mitochondrial morphologies resulting from these lipid perturbations. We then validated and selected the most interesting hits based on their phenotypes and penetrance.
Upon confirming these hits, we employed mass spectrometry to analyse lipidomic changes both in whole HeLa cells and isolated mitochondria. In parallel, we conducted a series of mitochondrial functional assays, including assessments of key protein expression, cell viability, the JC-1 assay to measure mitochondrial membrane potential, the Seahorse cellular mitochondrial stress test to evaluate respiration, and ATP production assays. These experiments aimed to investigate how the identified hits influenced mitochondrial dynamics and functions.
Our findings demonstrated that the cellular lipid composition plays a crucial role in maintaining both mitochondrial morphology and function. Specifically, we identified that alterations in the lipids produced by enzymes such as HMGCS1, GBA3, ORMDL3, ACOT8, and PLA2G4F not only led to changes in mitochondrial morphology but also had a significant impact on mitochondrial functions. These effects encompassed alterations in cell viability, ATP production, and maximal respiration.
In summary, our study offers valuable insights into the impact of lipid composition on both the structural and functional aspects of mitochondria. Significantly, our research marks the first systematic analysis of the relationship between lipid composition and mitochondrial morphology and function.
| Date of Award | 1 Jun 2024 |
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| Original language | English |
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| Supervisor | Ulrike Eggert (Supervisor) & Jeremy Carlton (Supervisor) |