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

Abstract

The organization of the eukaryotic cell into discrete membrane-bound organelles allows for the separation of incompatible biochemical processes, yet the activities of these organelles must be coordinated. For example, lipid metabolism is distributed between the endoplasmic reticulum (ER) for lipid synthesis, lipid droplets (LDs) for storage and transport, mitochondria and peroxisomes for β-oxidation, and lysosomes for lipid hydrolysis and recycling1–5. Organelle contacts are increasingly understood to be vital for diverse cellular functions5–8. However, the spatial and temporal organization of organelles within the cell remains poorly characterized due to the inability of fluorescence imaging-based approaches to distinguish more than a few fluorescent labels in a single image9. 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 employed confocal and lattice light sheet (LLS)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 fibroblast cells. We describe the frequency and locality of two-, three-, four-, and five-way interactions among six different membrane-bound organelles (ER, Golgi, lysosome, peroxisome, mitochondria and LD) and show how these relationships change over time. We demonstrate that each organelle has a characteristic distribution and dispersion pattern in three-dimensional space and that there is a reproducible pattern of contacts among the six organelles, impacted by microtubule and cell nutrient status. These live-cell confocal and LLS spectral-imaging approaches are applicable to any cell system expressing multiple fluorescent probes, whether in normal conditions or when cells are exposed to disturbances such as drugs, pathogens or stress. This methodology thus offers a powerful new descriptive tool and source for hypotheses about cellular organization and dynamics.

A role for Mitochondrial Rho GTPase 1 (MIRO1) in motility and membrane dynamics of peroxisomes

“…Similar to mitochondria, peroxisomes are oxidative organelles that fulfil important functions in lipid metabolism and ROS homeostasis rendering them essential for human health and development 2, 3. Peroxisomes metabolically cooperate and physically interact with a variety of subcellular organelles including the ER, mitochondria, lipid droplets and other peroxisomes 4, 5, 6. These functions require peroxisome positioning and movement within eukaryotic cells.…”

Whole-Cell Scale Dynamic Organization of Lysosomes Revealed by Spatial Statistical Analysis

“…The size of a lysosome typically is limited to several hundred nanometers (Bandyopadhyay et al, 2014; Yu et al, 2010). The number of lysosomes in a mammalian cell typically is limited to several hundred (Valm et al, 2017). Furthermore, lysosomes are specialized compartments that depend on interactions with partner organelles to fulfill their functions (Bonifacino and Neefjes, 2017; Luzio et al, 2007).…”

“…Positioning of individual lysosomes is mediated by mechanisms that include their active transport along microtubules and their interactions with the actin cytoskeleton and partner organelles, especially the ER (Bonifacino and Neefjes, 2017; Pu et al, 2016; Valm et al, 2017). Under normal conditions, lysosomes in non-polarized mammalian cells often cluster in a perinuclear region surrounding the microtubule-organizing center (MTOC), forming what is called the perinuclear cloud (Jongsma et al, 2016; Korolchuk et al, 2011; Pu et al, 2016), but they also spread into peripheral regions of cells, with some approaching the plasma membrane.…”

Mitochondrial signalling, physiology and pathophysiology

“…Mitochondria are involved in the formation of junctions with a number of cellular organelles (including peroxisomes, lipid droplets, lysosomes/endosomes and the plasma membrane) but the ER is certainly one of their preferred junctional partners (e.g. [4] reviewed in [3]). The junctions between the mitochondria and the ER and signalling mechanisms operating in these junctions (particularly Ca 2+ and ROS signalling) are the subject of the last review in this group of papers focused on mitochondrial signalling.…”