Curated Optogenetic Publication Database

Abstract: Inducible protein switches are used throughout the biosciences to allow on-demand control of proteins in response to chemical or optical inputs. However, these inducers either cannot be controlled with precision in space and time or cannot be applied in optically dense settings, limiting their application in tissues and organisms. Here we introduce a protein module whose active state can be reversibly toggled with a small change in temperature, a stimulus that is both penetrant and dynamic. This protein, called Melt (Membrane localization through temperature), exists as a monomer in the cytoplasm at elevated temperatures but both oligomerizes and translocates to the plasma membrane when temperature is lowered. Using custom devices for rapid and high-throughput temperature control during live-cell microscopy, we find that the original Melt variant fully switches states between 28-32°C, and state changes can be observed within minutes of temperature changes. Melt was highly modular, permitting thermal control over diverse intracellular processes including signaling, proteolysis, and nuclear shuttling through straightforward end-to-end fusions with no further engineering. Melt was also highly tunable, giving rise to a library of Melt variants with switch point temperatures ranging from 30-40°C. The variants with higher switch points allowed control of molecular circuits between 37°C-41°C, a well-tolerated range for mammalian cells. Finally, Melt could thermally regulate important cell decisions over this range, including cytoskeletal rearrangement and apoptosis. Thus Melt represents a versatile thermogenetic module that provides straightforward, temperature-based, real-time control of mammalian cells with broad potential for biotechnology and biomedicine.

Abstract: Optogenetic tools respond to light through one of a small number of behaviors including allosteric changes, dimerization, clustering, or membrane translocation. Here, we describe a new class of optogenetic actuator that simultaneously clusters and translocates to the plasma membrane in response to blue light. We demonstrate that dual translocation and clustering of the BcLOV4 photoreceptor can be harnessed for novel single-component optogenetic tools, including for control of the entire family of epidermal growth factor receptor (ErbB1-4) tyrosine kinases. We further find that clustering and membrane translocation are mechanistically linked. Stronger clustering increased the magnitude of translocation and downstream signaling, increased sensitivity to light by ~threefold-to-fourfold, and decreased the expression levels needed for strong signal activation. Thus light-induced clustering of BcLOV4 provides a strategy to generate a new class of optogenetic tools and to enhance existing ones.

Abstract: Protein clustering plays numerous roles in cell physiology and disease. However, protein oligomers can be difficult to detect because they are often small and fall below the detection limits of conventional fluorescence microscopy. Existing techniques to visualize such aggregates require specialized microscopy and may require overexpression of the protein of interest, which can introduce clustering artifacts that are not representative of the endogenous protein. Here we describe a fluorescent reporter strategy that detects endogenous protein clustering with high sensitivity, called CluMPS (Clusters Magnified by Phase Separation). A CluMPS reporter detects and visually amplifies even small clusters of a binding partner, generating large, easily quantifiable phase-separated condensates as a readout. We use optogenetic clustering to show that the CluMPS approach can reliably report on target clusters as small as tetramers. Experiments and simulations showed that CluMPS activation depends on the affinity for the target, the target cluster size, and the cluster size distribution. CluMPS detected small aggregates of pathological proteins where the corresponding GFP fusions appeared diffuse. Uniquely, CluMPS permitted visualization of clusters of endogenous proteins, allowing the measurement of drug response kinetics of oncogenic condensates in patient-derived cancer cells. Finally, CluMPS could be multiplexed to report on distinct clustered species in the same cell. CluMPS thus provides a powerful yet straightforward approach to observe higher-order protein assembly in its native cellular context.

Abstract: Understanding how cells self-organize into functional higher-order structures is of great interest, both towards deciphering animal development, as well as for our ability to predictably build custom tissues to meet research and therapeutic needs. The proper organization of cells across length-scales results from interconnected and dynamic networks of molecules and cells. Optogenetic probes provide dynamic and tunable control over molecular events within cells, and thus represent a powerful approach to both dissect and control collective cell behaviors. Here we emphasize the breadth of the optogenetic toolkit and discuss how these methods have already been used to reverse-engineer the design rules of developing organisms. We also offer our perspective on the rich potential for optogenetics to power forward-engineering of tissue assembly towards the generation of bespoke tissues with user-defined properties.