A quantitative 3D Subcellular Map of Human Retinal Pigment Epithelium Discovers Non-stochastic Cell State Transition in Establishment of Apical/Basal Polarity

Abstract The retinal pigment epithelium (RPE) is a specialized cell monolayer that forms the barrier between the subretinal and the choroidal spaces. During development RPE cells polarize perpendicular to the monolayer plane such that organelles attain specific intracellular locations. This allows the RPE to differentially interact with overlying photoreceptors and underlying choriocapillaris. When RPE polarity is disrupted, tissue homeostasis is disturbed leading to retinal degeneration. The subcellular organizational principles of RPE polarity are unknown. We used high-content imaging, machine-learning-based image-segmentation, phenotypic quantification, and mathematical modeling to develop quantitative 3-dimensional maps of subcellular structures during the establishment of RPE apical/basal polarity. Leveraging the statistical power of large imaging datasets, we discovered holistic RPE cell phenotypic states at four polarization timepoints. We discovered, during apical/basal polarization, cells constrict along the lateral axis and elongate apically, nuclear volume decreases while nuclear envelope develops invaginations, junctional complexes consolidate to the lateral membrane, the endoplasmic reticulum and mitochondria increase in volume and translocate towards the nucleus, and lysosomes move towards the central-apical side. Mathematical analysis using generalized linear models suggests non-stochastic cell state transitions and spatial organelle interactions during RPE polarization. These integrated quantitative morphometric data provide reference maps to discover intracellular defects in diseased RPE.