Project description:mRNAs associated with microtubules during interphase, metaphase and the midbody stage of cytokinesis were sequenced. Selective midbody-localized RNAs were identified and their translational characteristics were studied.
Project description:The compartmentalisation of distinct organelles within eukaryotic cells is essential for their diverse functions, however, how their structures and functions depend on each other has not been systematically explored. We combined a fluorescent reporter of mitochondrial stress with genome-wide CRISPR knockout screening and identified networks of genes involved in the biogenesis and metabolism of diverse organelles. Targeted organelle gene knockouts identified that defects in peroxisomes, Golgi, and ER cause mitochondrial fragmentation and dysfunction. Correlative light and electron microscopy analysed using artificial intelligence-directed voxel extraction revealed in unprecedented detail how impaired mitochondrial interactions with diverse organelles caused cell-wide defects in their morphology and biogenesis. Multi-omics analyses identified a unified proteome stress response and global shifts in lipid and glycoprotein homeostasis that are elicited when organelle biogenesis is compromised. Our comprehensive resource has defined metabolic and morphological interactions between organelles that can be mined to understand how changes in organelle components drive diverse cellular pathologies.
Project description:Eukaryotic cells contain several membrane-separated organelles to compartmentalize distinct metabolic reactions. However, it has remained unclear how these organelle systems are coordinated, when cells adapt metabolic pathways to support their development, survival or effector functions. Here we present OrgaPlexing, a multispectral organelle imaging approach for the comprehensive mapping of six key metabolic organelles and their interactions. We use this analysis on macrophages, immune cells that undergo rapid metabolic switches upon sensing bacterial and inflammatory stimuli. Our results identify lipid droplets (LDs) as primary inflammatory responder organelle, which forms three- and four-way interactions with other organelles. While clusters with endoplasmic reticulum (ER) and mitochondria (M-ER-LD unit) help supply fatty acids for LD growth, the additional recruitment of peroxisomes (M-ER-P-LD unit) supports fatty acid efflux from LDs. Interference with individual components of these units has direct functional consequences for inflammatory lipid synthesis. Together, we show that macrophages form functional multi-organellar units (MOUs) to support metabolic adaptation, and provide an experimental strategy to identify organelle-metabolic signaling hubs.