Showing 1 - 8 of 8 results
Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs.
Fluorescent RNAs (FRs), aptamers that bind and activate fluorescent dyes, have been used to image abundant cellular RNA species. However, limitations such as low brightness and limited availability of dye/aptamer combinations with different spectral characteristics have limited use of these tools in live mammalian cells and in vivo. Here, we develop Peppers, a series of monomeric, bright and stable FRs with a broad range of emission maxima spanning from cyan to red. Peppers allow simple and robust imaging of diverse RNA species in live cells with minimal perturbation of the target RNA's transcription, localization and translation. Quantification of the levels of proteins and their messenger RNAs in single cells suggests that translation is governed by normal enzyme kinetics but with marked heterogeneity. We further show that Peppers can be used for imaging genomic loci with CRISPR display, for real-time tracking of protein-RNA tethering, and for super-resolution imaging. We believe these FRs will be useful tools for live imaging of cellular RNAs.
A calcium- and light-gated switch to induce gene expression in activated neurons.
Despite recent advances in optogenetics, it remains challenging to manipulate gene expression in specific populations of neurons. We present a dual-protein switch system, Cal-Light, that translates neuronal-activity-mediated calcium signaling into gene expression in a light-dependent manner. In cultured neurons and brain slices, we show that Cal-Light drives expression of the reporter EGFP with high spatiotemporal resolution only in the presence of both blue light and calcium. Delivery of the Cal-Light components to the motor cortex of mice by viral vectors labels a subset of excitatory and inhibitory neurons related to learned lever-pressing behavior. By using Cal-Light to drive expression of the inhibitory receptor halorhodopsin (eNpHR), which responds to yellow light, we temporarily inhibit the lever-pressing behavior, confirming that the labeled neurons mediate the behavior. Thus, Cal-Light enables dissection of neural circuits underlying complex mammalian behaviors with high spatiotemporal precision.
A light- and calcium-gated transcription factor for imaging and manipulating activated neurons.
Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high- to low-calcium signal ratio of 10 after 10 min of stimulation. Opsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor-activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.
Optogenetic control of endogenous Ca(2+) channels in vivo.
Calcium (Ca(2+)) signals that are precisely modulated in space and time mediate a myriad of cellular processes, including contraction, excitation, growth, differentiation and apoptosis. However, study of Ca(2+) responses has been hampered by technological limitations of existing Ca(2+)-modulating tools. Here we present OptoSTIM1, an optogenetic tool for manipulating intracellular Ca(2+) levels through activation of Ca(2+)-selective endogenous Ca(2+) release-activated Ca(2+) (CRAC) channels. Using OptoSTIM1, which combines a plant photoreceptor and the CRAC channel regulator STIM1 (ref. 4), we quantitatively and qualitatively controlled intracellular Ca(2+) levels in various biological systems, including zebrafish embryos and human embryonic stem cells. We demonstrate that activating OptoSTIM1 in the CA1 hippocampal region of mice selectively reinforced contextual memory formation. The broad utility of OptoSTIM1 will expand our mechanistic understanding of numerous Ca(2+)-associated processes and facilitate screening for drug candidates that antagonize Ca(2+) signals.
Photoactivatable CRISPR-Cas9 for optogenetic genome editing.
We describe an engineered photoactivatable Cas9 (paCas9) that enables optogenetic control of CRISPR-Cas9 genome editing in human cells. paCas9 consists of split Cas9 fragments and photoinducible dimerization domains named Magnets. In response to blue light irradiation, paCas9 expressed in human embryonic kidney 293T cells induces targeted genome sequence modifications through both nonhomologous end joining and homology-directed repair pathways. Genome editing activity can be switched off simply by extinguishing the light. We also demonstrate activation of paCas9 in spatial patterns determined by the sites of irradiation. Optogenetic control of targeted genome editing should facilitate improved understanding of complex gene networks and could prove useful in biomedical applications.
In silico feedback for in vivo regulation of a gene expression circuit.
We show that difficulties in regulating cellular behavior with synthetic biological circuits may be circumvented using in silico feedback control. By tracking a circuit's output in Saccharomyces cerevisiae in real time, we precisely control its behavior using an in silico feedback algorithm to compute regulatory inputs implemented through a genetically encoded light-responsive module. Moving control functions outside the cell should enable more sophisticated manipulation of cellular processes whenever real-time measurements of cellular variables are possible.
Induction of protein-protein interactions in live cells using light.
Protein-protein interactions are essential for many cellular processes. We have developed a technology called light-activated dimerization (LAD) to artificially induce protein hetero- and homodimerization in live cells using light. Using the FKF1 and GIGANTEA (GI) proteins of Arabidopsis thaliana, we have generated protein tags whose interaction is controlled by blue light. We demonstrated the utility of this system with LAD constructs that can recruit the small G-protein Rac1 to the plasma membrane and induce the local formation of lamellipodia in response to focal illumination. We also generated a light-activated transcription factor by fusing domains of GI and FKF1 to the DNA binding domain of Gal4 and the transactivation domain of VP16, respectively, showing that this technology is easily adapted to other systems. These studies set the stage for the development of light-regulated signaling molecules for controlling receptor activation, synapse formation and other signaling events in organisms.
A light-switchable gene promoter system.
Regulatable transgene systems providing easily controlled, conditional induction or repression of expression are indispensable tools in biomedical and agricultural research and biotechnology. Several such systems have been developed for eukaryotes. Most of these rely on the administration of either exogenous chemicals or heat shock. Despite the general success of many of these systems, the potential for problems, such as toxic, unintended, or pleiotropic effects of the inducing chemical or treatment, can impose limitations on their use. We have developed a promoter system that can be induced, rapidly and reversibly, by short pulses of light. This system is based on the known red light-induced binding of the plant photoreceptor phytochrome to the protein PIF3 and the reversal of this binding by far-red light. We show here that yeast cells expressing two chimeric proteins, a phytochrome-GAL4-DNA-binding-domain fusion and a PIF3-GAL4-activation-domain fusion, are induced by red light to express selectable or "scorable" marker genes containing promoters with a GAL4 DNA-binding site, and that this induction is rapidly abrogated by subsequent far-red light. We further show that the extent of induction can be controlled precisely by titration of the number of photons delivered to the cells by the light pulse. Thus, this system has the potential to provide rapid, noninvasive, switchable control of the expression of a desired gene to a preselected level in any suitable cell by simple exposure to a light signal.