PROJECT SUMMARY It is known that at the level of a single cell, intracellular pressure governs motility, shape, volume, and proliferation. Mounting evidence suggests that pressure can vary within a single cell and the compartmentalization of pressure may be essential for dynamic cell function. Thus, it is essential to develop approaches to map the heterogeneous intracellular pressure within a single cell at submicron resolution given the technical limitations of the current technology. Specifically, it is challenging to study how intracellular pressure regulates cellular processes, such as protrusion of the cell cortex, heterogeneously and dynamically, to result in certain phenotypes, such as directional migration. This challenge stems from the lack of nano-sized sensors that are compatible for high- throughput multiplexing imaging so that local intracellular pressure and other dynamic processes can be simultaneously measured across the cell. Herein we propose to develop a high-throughput, multiplexing-ready intracellular probe in the form of nano- sized liposomes enclosed by DNA-based scaffold with aquaporin molecules distributed in the lipid bilayer. Joining the DNA scaffold and the aquaporin-embedded liposome are elastic DNA tethers conjugated with Foster Resonance Energy Transfer (FRET) donor and acceptor fluorophores at prescribed spacing, which extend or contract as the result of pressure-dependent changes to liposome volume. The nano-sized pressure sensor, coined “aquaporin-laced liposome pressure sensor (ALPS)”, will be delivered to the cytoplasm in quantity. Upon pressure changes in the cytoplasm, the internalized ALPS will change its volume by water efflux or influx through the aquaporin, while the DNA scaffold stabilizes the liposome to prevent collapse or rupture. As a proof of concept, we will then use ALPS to map the dynamic pressure field induced within single cells using compartmentalized pressure to migrate within 3D matrix; the results will be compared to the direct measurements obtained by 0.5-μm micro-electrodes with limited spatial resolution, the current state-of-art. If successful, we will generate a novel tool for measuring intracellular pressure with unprecedented spatiotemporal resolution, which promises to provide insights on how local intracellular pressure changes dynamically as cells navigate the 3D terrain.

PROJECT NARRATIVE Mounting evidences suggest that the intracellular pressure is dynamic and heterogeneous across the cell, and cell motility, cell shape, cell size and cell division are regulated the dynamic and heterogeneous intracellular pressure, which cannot be effective mapped with desirable spatiotemporal resolution, due to the lack of adequate sensors. Leverage our expertise in cell biology, DNA nanotechnology and imaging, we propose to develop a nano-sized intracellular pressure sensor, which will be delivered to the cytoplasm and measure local pressure while other cellular processes, such as cytoskeleton dynamics, can be simultaneously measured. If successful, we will generate a novel tool for measuring intracellular pressure with unprecedented spatiotemporal resolution, paving the way to gain insights on how local intracellular pressure changes dynamically as cells navigate the 3D terrain in our proof-of-concept study.