Orthogonalized human protease control of secreted signals.

Synthetic circuits that regulate protein secretion in human cells could support cell-based therapies by enabling control over local environments. While protein-level circuits enable such potential clinical applications, featuring orthogonality and compactness, their non-human origin poses a potential immunogenic risk. Here, we developed Humanized Drug Induced Regulation of Engineered CyTokines (hDIRECT) as a platform to control cytokine activity exclusively using human-derived proteins.

Tunable, self-contained gene dosage control via proteolytic cleavage of CRISPR-Cas systems.

Gene therapy holds great therapeutic potential. Yet, controlling cargo expression in single cells is limited due to the variability of delivery methods. We implement an incoherent feedforward loop based on proteolytic cleavage of CRISPR-Cas activation or inhibition systems to reduce gene expression variability against the variability of vector delivery.

Perspectives on Synthetic Protein Circuits in Mammalian Cells.

Mammalian synthetic biology aims to engineer cellular behaviors for therapeutic applications, such as enhancing immune cell efficacy against cancers or improving cell transplantation outcomes. Programming complex biological functions necessitates an understanding of molecular mechanisms governing cellular responses to stimuli. Traditionally, synthetic biology has focused on transcriptional circuits, but recent advances have led to the development of synthetic protein circuits, leveraging programmable binding, proteolysis, or phosphorylation to modulate protein interactions and cellular functions.

Protease-Driven Phase Separation of Elastin-Like Polypeptides.

Elastin-like polypeptides (ELPs) are a promising material platform for engineering stimuli-responsive biomaterials, as ELPs undergo phase separation above a tunable transition temperature. ELPs with phase behavior that is isothermally regulated by biological stimuli remain attractive for applications in biological systems. Herein, we report protease-driven phase separation of ELPs.

Post-Transcriptional Modular Synthetic Receptors.

Inspired by the power of transcriptional synthetic receptors and hoping to complement them to expand the toolbox for cell engineering, we establish LIDAR (Ligand-Induced Dimerization Activating RNA editing), a modular post-transcriptional synthetic receptor platform that harnesses RNA editing by ADAR. LIDAR is compatible with various receptor architectures in different cellular contexts, and enables the sensing of diverse ligands and the production of functional outputs. Furthermore, LIDAR can sense orthogonal signals in the same cell and produce synthetic spatial patterns, potentially enabling the programming of complex multicellular behaviors.

Compact Programmable Control of Protein Secretion in Mammalian Cells.

Synthetic biology currently holds immense potential to engineer the spatiotemporal control of intercellular signals for biomedicine. Programming behaviors using protein-based circuits has advantages over traditional gene circuits such as compact delivery and direct interactions with signaling proteins. Previously, we described a generalizable platform called RELEASE to enable the control of intercellular signaling through the proteolytic removal of ER-retention motifs compatible with pre-existing protease-based circuits.

Modular, programmable RNA sensing using ADAR editing in living cells.

With the increasing availability of single-cell transcriptomes, RNA signatures offer a promising basis for targeting living cells. Molecular RNA sensors would enable the study of and therapeutic interventions for specific cell types/states in diverse contexts, particularly in human patients and non-model organisms. Here we describe a modular, programmable system for live RNA sensing using adenosine deaminases acting on RNA (RADAR).

Protease-controlled secretion and display of intercellular signals.

To program intercellular communication for biomedicine, it is crucial to regulate the secretion and surface display of signaling proteins. If such regulations are at the protein level, there are additional advantages, including compact delivery and direct interactions with endogenous signaling pathways. Here we create a modular, generalizable design called Retained Endoplasmic Cleavable Secretion (RELEASE), with engineered proteins retained in the endoplasmic reticulum and displayed/secreted in response to specific proteases.

Topological Organization of Ventral Tegmental Area Connectivity Revealed by Viral-Genetic Dissection of Input-Output Relations.

Viral-genetic tracing techniques have enabled mesoscale mapping of neuronal connectivity by teasing apart inputs to defined neuronal populations in regions with heterogeneous cell types. We previously observed input biases to output-defined ventral tegmental area dopamine (VTA-DA) neurons. Here, we further dissect connectivity in the VTA by defining input-output relations of neurochemically and output-defined neuronal populations.

Programmable protein circuits in living cells.

Synthetic protein-level circuits could enable engineering of powerful new cellular behaviors. Rational protein circuit design would be facilitated by a composable protein-protein regulation system in which individual protein components can regulate one another to create a variety of different circuit architectures. In this study, we show that engineered viral proteases can function as composable protein components, which can together implement a broad variety of circuit-level functions in mammalian cells.

Circuit Architecture of VTA Dopamine Neurons Revealed by Systematic Input-Output Mapping.

Dopamine (DA) neurons in the midbrain ventral tegmental area (VTA) integrate complex inputs to encode multiple signals that influence motivated behaviors via diverse projections. Here, we combine axon-initiated viral transduction with rabies-mediated trans-synaptic tracing and Cre-based cell-type-specific targeting to systematically map input-output relationships of VTA-DA neurons. We found that VTA-DA (and VTA-GABA) neurons receive excitatory, inhibitory, and modulatory input from diverse sources.

Viral-genetic tracing of the input-output organization of a central noradrenaline circuit.

Deciphering how neural circuits are anatomically organized with regard to input and output is instrumental in understanding how the brain processes information. For example, locus coeruleus noradrenaline (also known as norepinephrine) (LC-NE) neurons receive input from and send output to broad regions of the brain and spinal cord, and regulate diverse functions including arousal, attention, mood and sensory gating. However, it is unclear how LC-NE neurons divide up their brain-wide projection patterns and whether different LC-NE neurons receive differential input.

A transcriptional reporter of intracellular Ca(2+) in Drosophila.

Intracellular Ca(2+) is a widely used neuronal activity indicator. Here we describe a transcriptional reporter of intracellular Ca(2+) (TRIC) in Drosophila that uses a binary expression system to report Ca(2+)-dependent interactions between calmodulin and its target peptide. We found that in vitro assays predicted in vivo properties of TRIC and that TRIC signals in sensory systems depend on neuronal activity.

Extremely sparse olfactory inputs are sufficient to mediate innate aversion in Drosophila.

Innate attraction and aversion to odorants are observed throughout the animal kingdom, but how olfactory circuits encode such valences is not well understood, despite extensive anatomical and functional knowledge. In Drosophila melanogaster, ~50 types of olfactory receptor neurons (ORNs) each express a unique receptor gene, and relay information to a cognate type of projection neurons (PNs). To examine the extent to which the population activity of ORNs is required for olfactory behavior, we developed a genetic strategy to block all ORN outputs, and then to restore output in specific types.

Drosophila chemotaxis: a first look with neurogenetics.

Chemotaxis, the ability to direct movements according to chemical cues in the environment, is important for the survival of most organisms. In our original article, we combined a quantitative behavioral assay with genetic manipulations to dissect the neural substrate for chemotaxis. In this Extra View article, we offer a more chronological narration of the findings leading to our key conclusion that aversion engages specific motor-related circuits and kinematics.

Specific kinematics and motor-related neurons for aversive chemotaxis in Drosophila.

Background: Chemotaxis, the ability to direct movements according to chemical cues in the environment, is important for the survival of most organisms. The vinegar fly, Drosophila melanogaster, displays robust olfactory aversion and attraction, but how these behaviors are executed via changes in locomotion remains poorly understood. In particular, it is not clear whether aversion and attraction bidirectionally modulate a shared circuit or recruit distinct circuits for execution.

A versatile in vivo system for directed dissection of gene expression patterns.

Tissue-specific gene expression using the upstream activating sequence (UAS)–GAL4 binary system has facilitated genetic dissection of many biological processes in Drosophila melanogaster. Refining GAL4 expression patterns or independently manipulating multiple cell populations using additional binary systems are common experimental goals. To simplify these processes, we developed a convertible genetic platform, the integrase swappable in vivo targeting element (InSITE) system.