Endocannabinoid Signaling Dynamics Probed with Optical Tools

Intravital microscopic imaging (IVM) allows for the study of interactions between immune cells and tumor cells in a dynamic, physiologically relevant system in vivo. Current IVM strategies primarily use fluorescence imaging; however, with the advances in bioluminescence imaging and the development of new bioluminescent reporters with expanded emission spectra, the applications for bioluminescence are extending to single cell imaging. Herein, we describe a molecular imaging window chamber platform that uniquely combines both bioluminescent and fluorescent genetically encoded reporters, as well as exogenous reporters, providing a powerful multi-plex strategy to study molecular and cellular processes in real-time in intact living systems at single cell resolution all in one system. We demonstrate that our molecular imaging window chamber platform is capable of imaging signaling dynamics in real-time at cellular resolution during tumor progression. Importantly, we expand the utility of IVM by modifying an off-the-shelf commercial system with the addition of bioluminescence imaging achieved by the addition of a CCD camera and demonstrate high quality imaging within the reaches of any biology laboratory.

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A rationally engineered decoder of transient intracellular signals

Nature Communications ◽

10.1038/s41467-021-22190-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Claude Lormeau ◽

Fabian Rudolf ◽

Jörg Stelling

Keyword(s):

Rapid Prototyping ◽

Positive Feedback ◽

Budding Yeast ◽

Short Pulse ◽

Design Principles ◽

Computational Method ◽

Transient Signals ◽

Temporal Filtering ◽

Signaling Dynamics ◽

Intracellular Signals

AbstractCells can encode information about their environment by modulating signaling dynamics and responding accordingly. Yet, the mechanisms cells use to decode these dynamics remain unknown when cells respond exclusively to transient signals. Here, we approach design principles underlying such decoding by rationally engineering a synthetic short-pulse decoder in budding yeast. A computational method for rapid prototyping, TopoDesign, allowed us to explore 4122 possible circuit architectures, design targeted experiments, and then rationally select a single circuit for implementation. This circuit demonstrates short-pulse decoding through incoherent feedforward and positive feedback. We predict incoherent feedforward to be essential for decoding transient signals, thereby complementing proposed design principles of temporal filtering, the ability to respond to sustained signals, but not to transient signals. More generally, we anticipate TopoDesign to help designing other synthetic circuits with non-intuitive dynamics, simply by assembling available biological components.

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