Uncovering Theorganelle Interactome: Dynamic Imaging of Multiple Organelles

Abstract

Numerous aspects of cell biology and biophysics are becoming clearer due to emerging visualization technologies, which can capture processes at the level of whole organisms down to single molecules. Here, I will discuss developments in probes and microscopes that are dramatically expanding productive imaging. To surmount fluorophore bleed-through, a combined excitation-based spectral unmixing and lattice light sheet microscopy was used to visualize up to six organelles (i.e., ER, Golgi, mitochondria, lysosomes, peroxisomes and lipid droplets) simultaneously within cells. This allowed us to track these organelles through time and analyze their inter-organelle contacts, providing a systems-level map of the organelle interactome and how it is perturbed under different physiological conditions. To increase temporal resolution during imaging, we employed total internal reflection fluorescence combined with structured illumination microscopy to visualize organelle dynamics at very high temporal-spatial resolution. Examining the ER, we observed that many peripheral ER sheets seen using diffraction-limited imaging are actually highly perforated structures comprised of tightly latticed groups of dynamic tubules. Within the latticed ER tubule meshwork, subdiffraction-limited holes were observed (∼150-250 nm diameter) having transient lifespans (∼250 msec). Viewed at higher resolution using lattice light sheet microscopy combined with point accumulation for nanoscale topology (PAINT), the peripheral ER sheets represented a complex meshwork of tightly cross-linked ER tubules, with potentially important roles in ER function.