Applying systems-level spectral imaging and analysis to reveal the organelle interactome

Using confocal and lattice light sheet microscopy, the authors perform systems-level analysis of the organelle interactome in live cells, allowing them to visualize the frequency and locality of up to five-way interactions between different organelles. Mapping organelle interactions Various cell components, or organelles, make contacts that are not mediated by trafficking vesicles, and which result in changes to their physical behaviour, biochemical composition and functionality. Imaging is a powerful tool for studying inter-organelle contact sites, but work by Jennifer Lippincott-Schwartz and colleagues take such analysis to a new level. Using confocal and lattice light sheet microscopy, as well as a multispectral image acquisition and analysis method, they perform systems-level analysis of the organelle interactome in live cells. The approach allows them to visualize the frequency and locality of up to five-way interactions among six different organelles (endoplasmic reticulum, Golgi, lysosome, peroxisome, mitochondria and lipid droplet), providing unexpected insights into the dynamics of these interactions. The method could prove a useful tool for further analysis of non-vesicular communication within the cell. The organization of the eukaryotic cell into discrete membrane-bound organelles allows for the separation of incompatible biochemical processes, but the activities of these organelles must be coordinated. For example, lipid metabolism is distributed between the endoplasmic reticulum for lipid synthesis, lipid droplets for storage and transport, mitochondria and peroxisomes for β-oxidation, and lysosomes for lipid hydrolysis and recycling 1 , 2 , 3 , 4 , 5 . It is increasingly recognized that organelle contacts have a vital role in diverse cellular functions 5 , 6 , 7 , 8 . However, the spatial and temporal organization of organelles within the cell remains poorly characterized, as fluorescence imaging approaches are limited in the number of different labels that can be distinguished in a single image 9 . Here we present a systems-level analysis of the organelle interactome using a multispectral image acquisition method that overcomes the challenge of spectral overlap in the fluorescent protein palette. We used confocal and lattice light sheet 10 instrumentation and an imaging informatics pipeline of five steps to achieve mapping of organelle numbers, volumes, speeds, positions and dynamic inter-organelle contacts in live cells from a monkey fibro