Correlating <i>in Vitro</i> and <i>in Vivo</i> Activities of Light-Inducible Dimers: A Cellular Optogenetics Guide

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 <i>in vivo</i> 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 <i>in vivo</i> 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 <i>in vivo</i> 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 vulval development via photoactivatable LIN-1.

<p>(A) Simplified schematic of the role of LIN-1 in vulval fate specification. (B) Top: Schematic of the wild type LIN-1 protein. Bottom: Schematic of the LIN-1::LANS protein produced after modification of the native <i>lin-1</i> locus using Cas9-triggered homologous recombination. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#pone.0128443.s004" target="_blank">S4 Fig</a>. (C) DIC Images of the developing vulvae in mid-L4 larvae from the indicted strains and conditions. Top panel: Black arrow indicates the normal, symmetric vulval invagination. Middle panel: Black arrow indicates the main vulval invagination, and green arrowhead indicates an extra vulval invagination. Bottom panel: Orange arrowheads indicate small invaginations produced by the secondary vulval precursors, and black arrow indicates the plug of tissue derived from the failed primary cell. Scale bars represent 20 μm. (D) Quantification of phenotypes in the indicated strains and conditions. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#sec011" target="_blank">Methods</a> for detailed definitions of each phenotype. Numbers at the top of each bar indicate the total number of animals scored in this experiment. These data are from a single experiment; the experiment was repeated three times, using two independently isolated <i>lin-1</i>::<i>lans</i> alleles, with similar results.</p

Light activated nuclear translocation in <i>C</i>. <i>elegans</i> embryo.

<p>(A) Schematic of the mKate2::LANS construct that was expressed in <i>C</i>. <i>elegans</i> embryos (B) Confocal images of an embryo expressing mKate2::LANS ubiquitously and subjected to photoactivation with blue light. Scale bars represent 10 μm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#pone.0128443.s008" target="_blank">S4 Movie</a>. (C) Left: Confocal images of four mKate2::LANS expressing MS lineage cells on the ventral surface of a late gastrulation-stage embryo. The blue box in the center image indicates the region that was photoactivated with blue light. Brightness and contrast were adjusted to compensate for photobleaching. Scale bar represents 5 μm. Right: Sketches summarizing the observed localization. Numbers correspond to the cell numbers in (D). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#pone.0128443.s009" target="_blank">S5 Movie</a>. (D) Quantification of nuclear and cytoplasmic fluorescence intensities as a function of time for the two cells labelled in (C). Cell 1 was illuminated with blue light, and Cell 2 is a neighboring cell. These measurements were corrected for photobleaching (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#sec011" target="_blank">materials and methods</a>).</p

We FRET so You Don’t Have To: New Models of the Lipoprotein Lipase Dimer

Lipoprotein lipase (LPL) is a dimeric enzyme that is responsible for clearing triglyceride-rich lipoproteins from the blood. Although LPL plays a key role in cardiovascular health, an experimentally derived three-dimensional structure has not been determined. Such a structure would aid in understanding mutations in LPL that cause familial LPL deficiency in patients and help in the development of therapeutic strategies to target LPL. A major obstacle to structural studies of LPL is that LPL is an unstable protein that is difficult to produce in the quantities needed for nuclear magnetic resonance or crystallography. We present updated LPL structural models generated by combining disulfide mapping, computational modeling, and data derived from single-molecule Förster resonance energy transfer (smFRET). We pioneer the technique of smFRET for use with LPL by developing conditions for imaging active LPL and identifying positions in LPL for the attachment of fluorophores. Using this approach, we measure LPL–LPL intermolecular interactions to generate experimental constraints that inform new computational models of the LPL dimer structure. These models suggest that LPL may dimerize using an interface that is different from the dimerization interface suggested by crystal packing contacts seen in structures of pancreatic lipase

Light induced transcription via light mediated nuclear translocation in yeast.

<p>(A) NMY51 contains <i>his3</i>, <i>ade2</i> and <i>lacZ</i> genomic reporter genes under the control of LexAop. (B) Schematic of the LANS controlled artificial transcription factor in yeast (C) Growth assay of LANS controlled transcription factor in NMY51. The left panel shows growth on media lacking leucine, which confers plasmid resistance and demonstrates that the light used does not affect regular yeast growth. The right panel demonstrates light dependent growth on media lacking leucine, histidine and adenine. (D) β-galactosidase activity measurements upon blue light induced transcription activation with LANS4 n = 3 each, mean reported ± SEM and statistical significance is calculated with unpaired two-tailed t-student’s test (p = 0.0019).</p

Real time light induced nuclear translocation of LANS4 in mammalian tissue culture cells.

<p>(A) Representative images for light activation and reversion in HeLa cells and Cos7 (B) (scale bar = 25 μm); (c) Plotting the fold change of nuclear accumulations in HeLa, Cos7 and HEK293 (n = 4 each, mean reported ± SEM with dashed line). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#pone.0128443.s005" target="_blank">S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#pone.0128443.s006" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128443#pone.0128443.s007" target="_blank">S3</a> Movies. The blue shaded region indicates pulsed blue light activation (see Supplemental experimental procedures). (C) Multple activation reversion cycles in Cos7 (n = 2, mean reported ± SEM with shaded grey area). The blue shaded regions indicate pulsed blue light activation.</p

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

<div><p>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 <i>Avena sativa</i>. 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 <i>C</i>. <i>elegans</i> embryo and allows for control of nuclear localization in individual cells. By inserting LANS into the <i>C</i>. <i>elegans lin-1</i> 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 <i>in vivo</i>.</p></div

Design and biophysical characterization of light conditioned nuclear localization signal.

<p>(A) Schematic of the Light Activated Nuclear Shuttle (LANS) design for light activated nuclear import (B) Sequence alignment of the wild type AsLOV2 and the designed AsLOV2cNLS (sequence identity and homology is marked according to CLUSTALW scheme). (C) Fluorescence polarization competitive binding assay of AsLOV2cNLS against human importin α5 and importin α7.</p

Confocal microscopy of LANS in HeLa cells.

<p>(A) Schematic of the LANS constructs (B) List of the nuclear export signals tested. (C) Representative nuclear optical slices of cells used for the quantification of the nuclear/cytoplasmic distribution of the switch (scale bar = 15 μm). (D) Quantification of the effect of the nuclear export signal on the nuclear/cytoplasmic distribution of LANS (D—wild type construct imaged in the dark, L—lit mimetic I539E). Mean is reported ±SEM and statistical significance calculated with unpaired two-tailed t-students test; NS—Not Significant.</p

We FRET so You Don’t Have To: New Models of the Lipoprotein Lipase Dimer

Lipoprotein lipase (LPL) is a dimeric enzyme that is responsible for clearing triglyceride-rich lipoproteins from the blood. Although LPL plays a key role in cardiovascular health, an experimentally derived three-dimensional structure has not been determined. Such a structure would aid in understanding mutations in LPL that cause familial LPL deficiency in patients and help in the development of therapeutic strategies to target LPL. A major obstacle to structural studies of LPL is that LPL is an unstable protein that is difficult to produce in the quantities needed for nuclear magnetic resonance or crystallography. We present updated LPL structural models generated by combining disulfide mapping, computational modeling, and data derived from single-molecule Förster resonance energy transfer (smFRET). We pioneer the technique of smFRET for use with LPL by developing conditions for imaging active LPL and identifying positions in LPL for the attachment of fluorophores. Using this approach, we measure LPL–LPL intermolecular interactions to generate experimental constraints that inform new computational models of the LPL dimer structure. These models suggest that LPL may dimerize using an interface that is different from the dimerization interface suggested by crystal packing contacts seen in structures of pancreatic lipase