Curated Optogenetic Publication Database

Search precisely and efficiently by using the advantage of the hand-assigned publication tags that allow you to search for papers involving a specific trait, e.g. a particular optogenetic switch or a host organism.

Showing 1 - 7 of 7 results

An optogenetic switch for the Set2 methyltransferase provides evidence for transcription-dependent and -independent dynamics of H3K36 methylation.

blue AsLOV2 S. cerevisiae Epigenetic modification
Genome Res, 5 Oct 2020 DOI: 10.1101/gr.264283.120 Link to full text
Abstract: Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification associated with transcription and DNA repair. Although the effects of H3K36 methylation have been studied, the genome-wide dynamics of H3K36me deposition and removal are not known. We established rapid and reversible optogenetic control for Set2, the sole H3K36 methyltransferase in yeast, by fusing the enzyme with the light-activated nuclear shuttle (LANS) domain. Light activation resulted in efficient Set2-LANS nuclear localization followed by H3K36me3 deposition in vivo, with total H3K36me3 levels correlating with RNA abundance. Although genes showed disparate levels of H3K36 methylation, relative rates of H3K36me3 accumulation were largely linear and consistent across genes, suggesting that H3K36me3 deposition occurs in a directed fashion on all transcribed genes regardless of their overall transcription frequency. Removal of H3K36me3 was highly dependent on the demethylase Rph1. However, the per-gene rate of H3K36me3 loss weakly correlated with RNA abundance and followed exponential decay, suggesting H3K36 demethylases act in a global, stochastic manner. Altogether, these data provide a detailed temporal view of H3K36 methylation and demethylation that suggests transcription-dependent and -independent mechanisms for H3K36me deposition and removal, respectively.

Cells lay their own tracks: optogenetic Cdc42 activation stimulates fibronectin deposition supporting directed migration.

blue iLID isolated MEFs mouse IA32 fibroblasts Control of cytoskeleton / cell motility / cell shape
J Cell Sci, 28 Jul 2017 DOI: 10.1242/jcs.205948 Link to full text
Abstract: Rho GTPase family members are known regulators of directed migration and therefore play key roles in processes including development, immune response and cancer metastasis. However, their individual contributions to these processes are complex. Here, we regulate the activity of two family members, Rac and Cdc42, by optogenetically recruiting specific GEF DH/PH domains to defined regions on the cell membrane. We find that the localized activation of both GTPases produce lamellipodia in cells plated on a fibronectin substrate. Using a novel optotaxis assay, we show that biased activation can drive directional migration. Interestingly, in the absence of exogenous fibronectin, Rac activation is insufficient to produce stable lamellipodia or directional migration while Cdc42 activation is sufficient. We find that a remarkably small amount of fibronectin (<10 puncta per protrusion) is necessary to support stable GTPase-driven lamellipodia. Cdc42 bypasses the need for exogenous fibronectin by stimulating cellular fibronectin deposition under the newly formed lamellipodia.

Tuning the Binding Affinities and Reversion Kinetics of a Light Inducible Dimer Allows Control of Transmembrane Protein Localization.

blue iLID in vitro mouse IA32 fibroblasts rat hippocampal neurons
Biochemistry, 8 Sep 2016 DOI: 10.1021/acs.biochem.6b00529 Link to full text
Abstract: Inducible dimers are powerful tools for controlling biological processes through colocalizing signaling molecules. To be effective, an inducible system should have a dissociation constant in the "off" state that is greater (i.e., weaker affinity) than the concentrations of the molecules that are being controlled, and in the "on" state a dissociation constant that is less (i.e., stronger affinity) than the relevant protein concentrations. Here, we reengineer the interaction between the light inducible dimer, iLID, and its binding partner SspB, to better control proteins present at high effective concentrations (5-100 μM). iLID contains a light-oxygen-voltage (LOV) domain that undergoes a conformational change upon activation with blue light and exposes a peptide motif, ssrA, that binds to SspB. The new variant of the dimer system contains a single SspB point mutation (A58V), and displays a 42-fold change in binding affinity when activated with blue light (from 3 ± 2 μM to 125 ± 40 μM) and allows for light-activated colocalization of transmembrane proteins in neurons, where a higher affinity switch (0.8-47 μM) was less effective because more colocalization was seen in the dark. Additionally, with a point mutation in the LOV domain (N414L), we lengthened the reversion half-life of iLID. This expanded suite of light induced dimers increases the variety of cellular pathways that can be targeted with light.

Light-induced nuclear export reveals rapid dynamics of epigenetic modifications.

blue AsLOV2 iLID C. elegans in vivo Cos-7 HeLa mouse IA32 fibroblasts S. cerevisiae Epigenetic modification
Nat Chem Biol, 18 Apr 2016 DOI: 10.1038/nchembio.2068 Link to full text
Abstract: We engineered a photoactivatable system for rapidly and reversibly exporting proteins from the nucleus by embedding a nuclear export signal in the LOV2 domain from phototropin 1. Fusing the chromatin modifier Bre1 to the photoswitch, we achieved light-dependent control of histone H2B monoubiquitylation in yeast, revealing fast turnover of the ubiquitin mark. Moreover, this inducible system allowed us to dynamically monitor the status of epigenetic modifications dependent on H2B ubiquitylation.

Correlating in Vitro and in Vivo Activities of Light-Inducible Dimers: A Cellular Optogenetics Guide.

blue CRY2/CIB1 iLID TULIP in vitro mouse IA32 fibroblasts S. cerevisiae Control of cytoskeleton / cell motility / cell shape Benchmarking
ACS Synth Biol, 30 Oct 2015 DOI: 10.1021/acssynbio.5b00119 Link to full text
Abstract: Light-inducible dimers are powerful tools for cellular optogenetics, as they can be used to control the localization and activity of proteins with high spatial and temporal resolution. Despite the generality of the approach, application of light-inducible dimers is not always straightforward, as it is frequently necessary to test alternative dimer systems and fusion strategies before the desired biological activity is achieved. This process is further hindered by an incomplete understanding of the biophysical/biochemical mechanisms by which available dimers behave and how this correlates to in vivo function. To better inform the engineering process, we examined the biophysical and biochemical properties of three blue-light-inducible dimer variants (cryptochrome2 (CRY2)/CIB1, iLID/SspB, and LOVpep/ePDZb) and correlated these characteristics to in vivo colocalization and functional assays. We find that the switches vary dramatically in their dark and lit state binding affinities and that these affinities correlate with activity changes in a variety of in vivo assays, including transcription control, intracellular localization studies, and control of GTPase signaling. Additionally, for CRY2, we observe that light-induced changes in homo-oligomerization can have significant effects on activity that are sensitive to alternative fusion strategies.

Control of Protein Activity and Cell Fate Specification via Light-Mediated Nuclear Translocation.

blue AsLOV2 C. elegans in vivo Cos-7 HEK293 HeLa S. cerevisiae Developmental processes
PLoS ONE, 17 Jun 2015 DOI: 10.1371/journal.pone.0128443 Link to full text
Abstract: Light-activatable proteins allow precise spatial and temporal control of biological processes in living cells and animals. Several approaches have been developed for controlling protein localization with light, including the conditional inhibition of a nuclear localization signal (NLS) with the Light Oxygen Voltage (AsLOV2) domain of phototropin 1 from Avena sativa. In the dark, the switch adopts a closed conformation that sterically blocks the NLS motif. Upon activation with blue light the C-terminus of the protein unfolds, freeing the NLS to direct the protein to the nucleus. A previous study showed that this approach can be used to control the localization and activity of proteins in mammalian tissue culture cells. Here, we extend this result by characterizing the binding properties of a LOV/NLS switch and demonstrating that it can be used to control gene transcription in yeast. Additionally, we show that the switch, referred to as LANS (light-activated nuclear shuttle), functions in the C. elegans embryo and allows for control of nuclear localization in individual cells. By inserting LANS into the C. elegans lin-1 locus using Cas9-triggered homologous recombination, we demonstrated control of cell fate via light-dependent manipulation of a native transcription factor. We conclude that LANS can be a valuable experimental method for spatial and temporal control of nuclear localization in vivo.

Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins.

blue AsLOV2 iLID in vitro mouse IA32 fibroblasts Control of cytoskeleton / cell motility / cell shape
Proc Natl Acad Sci USA, 22 Dec 2014 DOI: 10.1073/pnas.1417910112 Link to full text
Abstract: The discovery of light-inducible protein-protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
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