Biology subjects

Elowitz, M. B.

Publications and source records attributed to Elowitz, M. B..

27 records · Page 2Linked to original sources

bioRxiv2021

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 signalling pathways. We created 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. The design allows functional regulation of multiple synthetic and natural proteins by synthetic protease circuits to realize diverse signal processing capabilities, including logic operation and threshold tuning. By linking RELEASE to additional novel sensing and processing circuits, we were able to achieve elevated protein secretion in response to "undruggable" oncogene KRAS mutants. RELEASE should enable the local, programmable delivery of intercellular cues for a broad variety of fields such as neurobiology, cancer immunotherapy and cell transplantation.

synthetic biology↗

bioRxiv2021

Ratiometric RNA labeling allows dynamic multiplexed analysis of gene circuits in single cells

Biological processes are highly dynamic and are regulated by genes that connect with one and another, forming regulatory circuits and networks. Understanding how gene regulatory circuits operate dynamically requires monitoring the expression of multiple genes in the same cell. However, it is limited by the relatively few distinguishable fluorescent proteins. Here, we developed a multiplexed real-time transcriptional imaging method based on two RNA stem-loop binding proteins, and employed it to analyze the temporal dynamics of synthetic gene circuits. By incorporating different ratios of MS2 and PP7 stem-loops, we were able to monitor the real-time nascent transcriptional activities of up to five genes in the same cell using only two fluorescent proteins. Applying this multiplexing capability to synthetic linear or branched gene regulatory cascades revealed that propagation of transcriptional dynamics is enhanced by non-stationary dynamics and is dictated by the slowest regulatory branch in the presence of combinatorial regulation. Mathematical modeling provided further insight into temporal multi-gene interactions and helped to understand potential challenges in regulatory inference using snapshot single-cell data. Ratiometric multiplexing should scale exponentially with additional labelling channels, providing a way to track the dynamics of larger circuits.

systems biology↗

bioRxiv2020

Ligand-receptor promiscuity enables cellular addressing

In multicellular organisms, secreted ligands selectively activate, or "address," specific target cell populations to control cell fate decision-making and other processes. Key cell-cell communication pathways use multiple promiscuously interacting ligands and receptors, provoking the question of how addressing specificity can emerge from molecular promiscuity. To investigate this issue, we developed a general mathematical modeling framework based on the bone morphogenetic protein (BMP) pathway architecture. We find that promiscuously interacting ligand-receptor systems allow a small number of ligands, acting in combinations, to address a larger number of individual cell types, each defined by its receptor expression profile. Promiscuous systems outperform seemingly more specific one-to-one signaling architectures in addressing capacity. Combinatorial addressing extends to groups of cell types, is robust to receptor expression noise, grows more powerful with increasing receptor multiplicity, and is maximized by specific biochemical parameter relationships. Together, these results identify fundamental design principles governing cell addressing by ligand combinations.

systems biology↗

bioRxiv2020

The context-dependent, combinatorial logic of BMP signaling

Cell-cell communication systems typically comprise families of ligand and receptor variants that function together in combinations. Pathway activation depends in a complex way on which ligands are present and what receptors are expressed by the signal-receiving cell. To understand the combinatorial logic of such a system, we systematically measured pairwise Bone Morphogenetic Protein (BMP) ligand interactions in cells with varying receptor expression. Ligands could be classified into equivalence groups based on their profile of positive and negative synergies with other ligands. These groups varied with receptor expression, explaining how ligands can functionally replace each other in one context but not another. Context-dependent combinatorial interactions could be explained by a biochemical model based on competitive formation of alternative signaling complexes with distinct activities. Together, these results provide insights into the roles of BMP combinations in developmental and therapeutic contexts and establish a framework for analyzing other combinatorial, context-dependent signaling systems.

systems biology↗

bioRxiv2020

Synthetic mammalian signaling circuits for robust cell population control

In multicellular organisms, cells actively sense and control their own population density. Synthetic mammalian quorum sensing circuits could provide insight into principles of population control and extend cell therapies. However, a key challenge is reducing their inherent sensitivity to "cheater" mutations that evade control. Here, we repurposed the plant hormone auxin to enable orthogonal mammalian cell-cell communication and quorum sensing. We designed a paradoxical population control circuit, termed Paradaux, in which auxin stimulates and inhibits net cell growth at different concentrations. This circuit limited population size over extended timescales, of up to 42 days of continuous culture. By contrast, when operating in a non-paradoxical regime, the same cells limited population growth, but were more susceptible to mutational escape. These results establish auxin as a versatile "private" communication system, and demonstrate that paradoxical circuit architectures can provide robust population control.

synthetic biology↗

bioRxiv2020

Engineering multiple levels of specificity in an RNA viral vector

Synthetic molecular circuits could provide powerful therapeutic capabilities, but delivering them to specific cell types and controlling them remains challenging. An ideal “smart” viral delivery system would enable controlled release of viral vectors from “sender” cells, conditional entry into target cells based on cell-surface proteins, conditional replication specifically in target cells based on their intracellular protein content, and an evolutionarily robust system that allows viral elimination with drugs. Here, combining diverse technologies and components, including pseudotyping, engineered bridge proteins, degrons, and proteases, we demonstrate each of these control modes in a model system based on the rabies virus. This work shows how viral and protein engineering can enable delivery systems with multiple levels of control to maximize therapeutic specificity.Competing Interest StatementX.J.G, L.S.C., M.S.K., and M.B.E. are inventors on a U.S. patent provisional application related to this work.View Full Text

synthetic biology↗

bioRxiv2020

Imaging cell lineage with a synthetic digital recording system

Multicellular development depends on the differentiation of cells into specific fates with precise spatial organization. Lineage history plays a pivotal role in cell fate decisions, but is inaccessible in most contexts. Engineering cells to actively record lineage information in a format readable in situ would provide a spatially resolved view of lineage in diverse developmental processes. Here, we introduce a serine integrase-based recording system that allows in situ readout, and demonstrate its ability to reconstruct lineage relationships in cultured stem cells and flies. The system, termed intMEMOIR, employs an array of independent three-state genetic memory elements that can recombine stochastically and irreversibly, allowing up to 59,049 distinct digital states. intMEMOIR accurately reconstructed lineage trees in stem cells and enabled simultaneous analysis of single cell clonal history, spatial position, and gene expression in Drosophila brain sections. These results establish a foundation for microscopy-readable clonal analysis and recording in diverse systems. One sentence summaryA new genetic editing system termed intMEMOIR reveals the lineage histories of individual cells directly within their native tissue context.

synthetic biology↗

bioRxiv2018

A stochastic epigenetic switch controls the dynamics of T-cell lineage commitment

Cell fate decisions occur through the switch-like, irreversible activation of fate-specifying genes. These activation events are often assumed to be tightly-coupled to changes in upstream transcription factors, but could also be constrained by cis-epigenetic mechanisms at individual gene loci. Here, we studied the activation of Bcl11b, which controls T-cell fate commitment. To disentangle cis and trans effects, we generated mice where two Bcl11b copies are tagged with distinguishable fluorescent proteins. Quantitative live microscopy of progenitors from these mice revealed that Bcl11b turned on after a stochastic delay averaging multiple days, which varied not only between cells but also between Bcl11b alleles within the same cell. Genetic perturbations, together with mathematical modeling, showed that a distal enhancer controls the rate of epigenetic activation, while a parallel Notch-dependent trans-acting step stimulates expression from activated loci. These results show that developmental fate transitions can be controlled by stochastic cis-acting events on individual loci.

bioinformatics↗

bioRxiv2018

Cis-activation in the Notch signaling pathway

The Notch signaling pathway consists of transmembrane ligands and receptors that can interact both within the same cell (cis) and across cell boundaries (trans). Previous work has shown that cis-interactions act to inhibit productive signaling. Here, by analyzing Notch activation in single cells while controlling cell density and ligand expression level, we show that cis-ligands can in fact activate Notch receptors. This cis-activation process resembles trans-activation in its ligand level dependence, susceptibility to cis-inhibition, and sensitivity to Fringe modification. Cis-activation occurred for multiple ligand-receptor pairs, in diverse cell types, and affected survival and differentiation in neural stem cells. Finally, mathematical modeling shows how cis-activation could potentially expand the capabilities of Notch signaling, for example enabling \"negative\" signaling. These results establish cis-activation as a prevalent mode of signaling in the Notch pathway, and should contribute to a more complete understanding of how Notch signaling functions in developmental, physiological, and biomedical contexts.

bioinformatics↗