Identifying heterogeneity in organelle populations using multiplexed DNA-PAINT super-resolution microscopy.

Abstract

Cells are highly compartmentalized with distinct membrane-bound organelles controlling unique aspects of cellular homeostasis. Despite an abundance of literature highlighting diverse functions of these organelles, they are typically studied as a homogenous group. Fluorescence microscopy often relies on labeling organelle populations via a canonical surface protein marker, but studies in recent years have shown that two canonical markers known to be enriched on a given organelle do not always mark the same organelle populations at the single cell level. Hence, the heterogeneity of cellular organelles in terms of their membrane protein makeup and how that heterogeneity relates to diverse organelle functions is an open question. DNA-PAINT super-resolution microscopy combines high sensitivity and target specificity with high-order multiplexing capabilities, making it perfectly suited for systematic, quantitative studies of organelle heterogeneity. In addition, lysosomes are a great model system as they are typically at or below the diffraction limit (200-300 nm in size) and are important cellular recycling compartments, mediators of metabolic control, and centers for protein sorting and targeting. Using DNA-PAINT imaging of several canonical lysosomal membrane protein markers (LAMP1, LAMP2, CD63, Rab7 and TMEM 192), we investigated the intracellular organization of lysosomal compartments in HeLa cells. These experiments identified distinct sub-populations of lysosomes enriched with subsets of these proteins. Most strikingly, only 8% of LAMP1 positive organelles are also positive for LAMP2. This heterogeneity will inform the future study of distinct subpopulations of lysosomes, focused on linking them to discrete cellular functions. Furthermore, this study can serve as a model for identification of functionally distinct subpopulations of other organelles.