Dissecting subcellular and cellular organization by spatial molecular neighborhood networks

Subcellular organization of intracellular molecules regulates basic cell functions such as growth, division, proliferation, and differentiation. By regulating subcellular neighborhoods of RNAs and proteins, cells survive but also specialize in a multicellular system. While subcellular localization mechanisms are widely studied, the role of subcellular neighborhoods in determining the cellular response to stimuli and multiple drugs has been limited to a few low-throughput and low-sensitivity molecular studies. Little is known about how these highly coordinated subcellular neighborhoods give rise to tissue-specificity, tissue-sate, and how they regulate cell function in distinct subcellular volumes of tissue states and phenotypes. Image-based visualization of molecular neighborhoods has explored only a few target molecules, yielding evidence of the importance of subcellular localization of transcripts and protein factors. One bottleneck in subcellular regulation is the lack of models explaining neighborhood interactions of molecules that are driving cellular response in tissues from distinct abnormalities. Previous work established multiplexed gene expression measurements to model subcellular RNA neighborhoods and multiparameter protein analysis to quantify spatial signaling protein neighborhoods that are altered in response to perturbations. However, dominant signaling protein and RNA neighborhoods have not been established at a physically meaningful resolution in widely employed mesenchymal and epithelial cells as they respond to perturbations, experience differentiation events, or adapt to tissue environments of individuals treated with cell-based or small molecule interventions for correcting abnormalities due to genetic or molecular dysfunction. Thus, the long-term goal of this project is to leverage advanced high-throughput screening and spatially resolved genomic and protein measurements in single cells for modeling molecular neighborhoods in populations and tissue contexts. To achieve these tasks, using recent advances in sequential fluorescence in situ hybridization and multiplexed protein imaging, this proposal plans to 1) mechanistically dissect the molecular RNA and protein neighborhoods using proximity ligation assays, 2) enhance the spatial information capacity of molecular neighborhoods using 3D super-resolution microscopy and temporal evolution of molecular neighborhoods by pseudo-temporal differentiation models, 3) evaluate the outcome of subcellular neighborhood organization and tissue context of mesenchymal cells near vessels and epithelial cells near linings from human specimens isolated from health restoring interventions. Molecular benchmarking and normal tissue annotations will be performed in collaboration with signaling biology, bioinformatics, biomanufacturing, and pathology experts. Cross-scale molecular control from subcellular neighborhoods to tissue organization will reveal how a systemic response to treatments can be re-stratified by network variance of molecular neighborhoods. This MIRA proposal sheds light on the spatially resolved subcellular organization in health and disease, providing a predictive metric for deciphering tissue-state control and alterations in many disorders.