Intra- and inter-cellular communication driven by membrane contact sites within a virus microenvironment  

Our goal is to develop tunable tools to define principles underlying MCS-driven intra- and inter-cellular communication. We will leverage our biological focus on infection-driven organelle remodeling with our technical expertise in proteomics, optogenetics, genome editing, and super-resolution microscopy. In a virus microenvironment system we established, we will define intracellular and cell-cell communication events in 2D and 3D (organoid) cell systems at the nexus of MCSs, metabolic regulation, and immunity. Using single-cell proteomic technology, we will design assays for concomitant quantification of all known MCS proteins, as well as for monitoring site-specific phosphorylations and protein interactions underlying MCS formation and functions. Photoactivatable optogenetic tags will be adapted for the reversible, light-induced tethering of MCSs with spatial and temporal precision. Molecular assays will probe the MCS-induced signaling and functional outcomes. To understand the coordinated roles of MCSs, we will develop assays for monitoring and manipulating three-way contacts in real-time. An endogenous tagging system will be optimized via CRISPR knock-in and fluorophores with diverse properties. Dual split tagging will be adapted for visualizing and identifying three-way ER MCSs (e.g., ER-mitochondria-peroxisome). Contacts will be validated by CLEM and Cryo-ET, and genetic manipulations will clarify downstream consequence. Finally, as an MCS model of an antiviral-to-proviral switch, we will identify and characterize peroxisomal contacts. 

• Establish tools for intra- & inter-cellular MCS profiling and dynamic manipulation. 

• Real-time analysis of three-way MCSs via optimized endogenous tagging system. 

• Identify and characterize peroxisomal contacts. 

This project is part of the 2024 Organelle Communication cohort

These researchers will explore a thrilling frontier in cell biology emerging from the discovery that organelles (cellular compartments) can directly connect to each other to exchange materials and information, forming complex and dynamic networks. Much of how these interactions occur remains unknown due to the profound challenges of observing rapid events on a nanometer scale. This cohort will pioneer new tools to directly observe and model the organelle ‘interactome’ across time, space, and cell type. Their findings will expand our understanding of core biological principles, with powerful implications for fields ranging from regenerative medicine to virology.