Abstract
Advances in single cell RNA sequencing have allowed for the identification and characterization of cellular subtypes based on quantification of the number of transcripts in each cell. However, cells may differ not only in the number of mRNA transcripts that they exhibit, but also in their spatial and temporal distribution, intrinsic to the definition of their cellular state. Here we describe DypFISH, an approach to quantitatively investigate the spatial and temporal subcellular localization of RNA and protein, by combining micropatterning of cells with fluorescence microscopy at high resolution. We introduce a range of analytical techniques for quantitatively interrogating single molecule RNA FISH data in combination with protein immunolabeling over time. Strikingly, our results show that constraining cellular architecture reduces variation in subcellular mRNA and protein distributions, allowing the characterization of their localization and dynamics with high reproducibility. Many tissues contain cells that exist in similar constrained architectures. Thus DypFISH reveals reproducible patterns of clustering, strong correlative influences of mRNA-protein localization on MTOC orientation when they are present and interdependent dynamics globally and at specific subcellular locations which can be extended to physiological systems.
