1.
An improved FLARE system for recording and manipulating neuronal activity.
Abstract:
To address the need for methods for tagging and manipulating neuronal ensembles underlying specific behaviors, we present an improved version of FLARE, termed cytoFLARE (cytosol-expressed FLARE). cytoFLARE incorporates cytosolic tethering of a transcription factor and expression of a more sensitive pair of calcium-sensing domains. We show that cytoFLARE captures more calcium- and light-dependent signals in HEK293T cells and higher signal-to-background ratios in neuronal cultures. We further establish cytoFLARE transgenic Drosophila models and apply cytoFLARE to label activated neurons upon sensory or optogenetic stimulation within a defined time window. Notably, through the cytoFLARE-driven expression of optogenetic actuators, we successfully reactivated and inhibited neurons involved in the larval nociceptive system. Our findings demonstrate the characterization and application of time-gated calcium integrators for both recording and manipulating neuronal activity in Drosophila larvae.
2.
Computational framework for single-cell spatiotemporal dynamics of optogenetic membrane recruitment.
Abstract:
We describe a modular computational framework for analyzing cell-wide spatiotemporal signaling dynamics in single-cell microscopy experiments that accounts for the experiment-specific geometric and diffractive complexities that arise from heterogeneous cell morphologies and optical instrumentation. Inputs are unique cell geometries and protein concentrations derived from confocal stacks and spatiotemporally varying environmental stimuli. After simulating the system with a model of choice, the output is convolved with the microscope point-spread function for direct comparison with the observable image. We experimentally validate this approach in single cells with BcLOV4, an optogenetic membrane recruitment system for versatile control over cell signaling, using a three-dimensional non-linear finite element model with all parameters experimentally derived. The simulations recapitulate observed subcellular and cell-to-cell variability in BcLOV4 signaling, allowing for inter-experimental differences of cellular and instrumentation origins to be elucidated and resolved for improved interpretive robustness. This single-cell approach will enhance optogenetics and spatiotemporally resolved signaling studies.
