Optogenetics in cell biology

Optogenetics in cell biology

Optogenetics is the use of genetically encoded light-inducible switches that are engineered to exert a certain biological function only after absorption of light of a specific wavelength. The greatest advantage of using light as input to control biological processes is the possibility of local activation with down to subcellular resolution. A very promising natural photoreceptor that is being often used in optogenetic tools is the second Light Oxygen Voltage domain of Avena sativa phototropin 1 (AsLOV2). This is a small (12 kDa) soluble domain that undergoes a conformational change when absorbing blue light (450-480 nm), so that its N- and C-terminal helices unfold and undock from theOptogenetics is the use of genetically encoded light-inducible switches that are engineered to exert a certain biological function only after absorption of light of a specific wavelength. The greatest advantage of using light as input to control biological processes is the possibility of local activation with down to subcellular resolution. A very promising natural photoreceptor that is being often used in optogenetic tools is the second Light Oxygen Voltage domain of Avena sativa phototropin 1 (AsLOV2). This is a small (12 kDa) soluble domain that undergoes a conformational change when absorbing blue light (450-480 nm), so that its N- and C-terminal helices unfold and undock from the core LOV domain. The AsLOV2 domain – as well as other LOV domains from different organisms – has been successfully fused to other proteins to control their activity via light. Another approach consists in exploiting the light-induced unfolding and undocking of the C-terminal Jα helix of the AsLOV2 domain to control via light the exposure of small peptides that are either appended to or embedded into the Jα helix itself.

Our contribution to this field

Tool development

We have engineered over the years several blue light-responsive optogenetic tools, such as the transcription factor BLADE for use in E. coli, and the nuclear import and export tools LINuS and LEXY. Our most recent effort is the engineering of blue light-responsive split inteins, namely gp41-1 and NrdJ-1 (see also “Intein-mediated protein splicing”).

Figure 1: Examples of optogenetic tools developed in our lab. A. Domain composition of wild-type (left) and light-inducible (right) AraC. B. Representative differential interference contrast (DIC) images of E. coli KC717 cells transformed with pBLADEONLY_C (pBAD33 expressing BLADE but deprived of the PBAD promoter) at the indicated time points. Induction was performed with 460-nm light (5 W m−2) for the indicated time. Recovery was performed by putting cells in the darkness for the indicated time. Scale bar, 5 μm. . C. Bacteriograph. A lawn of E. coli MG1655 cells transformed with pBLADE(FP6*) driving the expression of superfolder GFP was grown overnight at 37 °C while being exposed to blue light through a photomask reproducing the famous painting by Michelangelo. A total of 160 individual images were taken with a fluorescent microscope and stitched together with Zen Blue 2.3 software. Scale bar, 1 cm. D. Schematic of LINuS function. In the dark state, the hybrid Ja helix is folded and interacts with the AsLOV2 core domain. Upon blue light exposure, it unfolds rendering the NLS accessible to endogenous importins. E.Representative fluorescence microscopy images of HEK 293T cells transiently transfected with mCherry-biLINuS2 variant bearing the indicated NES before and after 20 min of illumination. PKIt, truncated cAMP-dependent protein kinase inhibitor alpha NES. Scale bar, 15 mm. F. Fluorescence images of HEK 293T cells
transiently transfected with NLS-mCherry-LEXY and H2B-GFP before and after 15 min of pulsatile blue light irradiation. Scale bar, 20 mm. Modified from Romano, Baumschlager et al., Nat Chem Biol, 2021; Niopek et al., Nat Commun, 2014; and Niopek et al., Nat Commun, 2016. Created with BioRender.

Applying optogenetics to study the role of transcription factor dynamics in the selective activation of target genes in mammalian cells

Beyond continuing to develop tools, we currently focus on employing optogenetics to study the role of transcription factor dynamics in gene expression in mammalian cells.

Indeed, over the last few decades, it has become evident that transcription factors (TFs) display different dynamics depending on the signal sensed by the cells. With dynamics here we mean the shape of the curve describing the temporal change of TF activity, which in many cases corresponds to its nuclear localization. An important question was whether mammalian promoters sense directly TF dynamics, thus being activated by certain dynamics rather than others, or whether additional information such as post-translational modifications of the TF are needed.

To investigate this question, we employed our previously developed light-inducible nuclear import tool LINuS to engineer a synthetic TF whose nuclear accumulation is externally controlled by us with blue light. Using this synthetic TF in combination with various synthetic promoters, we unravelled that TF dynamics can be read by mammalian promoters provided the coupling between TF binding and transcription pre-initiation complex formation is inefficient. We moreover found that inefficient translation amplifies the ability of a promoter to distinguish TF dynamics.

Figure 2: Using optogenetics to study promoter selectivity to TF dynamics. A. Schematic representation of the expected behavior of the synthetic transcription factor (synTF) in darkness (left) and during blue
light illumination (right). Blue light leads to the exposure of the nuclear localization signal (NLS), which, in turn, leads to the nuclear import of synTF. LexA, full-length bacterial repressor used as DNA binding domain (DBD) within synTF. VP48, 3x residues 436–47 of the VP16 transactivation domain (TAD). NES, nuclear export signal.  B. Schematic representation of the reporter libraries. C. Quantification of synTF nuclear translocation over time for the indicated TF dynamics. Light blue shadowing, blue light illumination phase. Together with the experimental data (black dots), fitted (violet line; for sustained dynamics) and simulated (pink line; for both pulsatile dynamics) values are shown. The mathematical model used can be found in Antwi et al., NAR, 2021. D. Left, Quantification of mean nascent RNA per cell per minute for the indicated synTF dynamics. Right, Quantification of mean reporter protein expression levels at the end of the experiment for the indicated synTF dynamics. P-values were calculated with the Welch’s t-test. ns, non-significant (P> 0.05). *, P-value (from left to right) = 0.0217, 0.0212. Data represent mean± s.e.m. of at least n= 20 individual cells, imaged on at least n= 3 biologically independent experiments. The promoter used in these experiments was p4. Modified from Antwi et al., NAR, 2021. Created with BioRender.

Currently, we are carrying out a purely theoretical analysis to find out which promoter configurations (meant as the interplay between topology and regulation of the promoter transitions) are able to distinguish TF dynamics.

In parallel to our study of synthetic TFs, we aimed to control with light the dynamics of a natural TF, namely the p65 subunit of the NF-kB transcription factor. In this case, we managed to engineer opto-p65 constructs whose nuclear accumulation is dictated by light; however, we needed to shine at least 24 hours of continuous light to get a robust expression from a p65-responsive promoter, suggesting that the protein is only mildly active in the absence of post-translational modifications which occur when the pathway is activated at the receptor level.

We did not find a statistically significant difference in the expression of endogenous NF-kB target genes between samples exposed to light or kept in the dark. The difference is, instead, seen when we use a plasmid-borne p65 reporter

Figure 3: Optogenetic control of p65. ASchematic representation of the system, illustrating expected construct localization in cells under dark and illuminated conditions. Nb@GFP, nanobody against GFP. B. Diagram of the single plasmid used, encoding both constructs. This design ensures a higher expression of the first coding sequence compared to the second coding sequence following the IRES. C.Representative mCherry and GFP channel microscopy images are shown acquired at the following time points: before illumination (t=0′), after 30 minutes of blue light illumination (t=30′), and 30 minutes post-illumination (t=60′). Scale bar= 10µm. D. Quantification of nuclear-to- cytoplasmic fluorescence ratio for each construct: Nb-mCherry-biLINuS (red line), GFP-p65 (green line). Data represent mean ± SEM of n = 20 cells, from two independent experiments. Scale bar = 10 µm. E.Schematic of the light regimes used. F. Luciferase activity in HeLa RELA-/- NFKBIA-/- cells co-transfected with the indicated constructs, the reporter construct, and a construct for normalization, either left in the dark or illuminated with constant light for 24 hours. Data represent mean ± SD from three independent experiments N+F: Nanoluc (NF-κB reporter) + Firefly (transfection control); biLINuS10: CMV-NES-mCherry-biLINuS10; Nb + EGFP-p65: CMV_Nb(@GFP)-mCherry-biLINuS10 + EGFP-p65. G. qPCR results for the indicated genes in HeLa RELA-/- NFKBIA-/- cells transfected with the CMV_Nb(@GFP)-mCherry-biLINuS10 + EGFP-p65 system. Cells were kept in the dark or illuminated for 24 hours using constant or pulsed light. Data represent mean ± SEM from three independent experiments. Statistical significance (One-way ANOVA) is denoted above relevant conditions: ns, p-value ≥ 0.5, *, p-value < 0.5, ** p-value < 0.01, ***, p-value < 0.001, ****, p-value < 0.0001. Modified from Forlani, G. PhD thesis. Created with BioRender.

We conclude that our opto-p65, while being transcriptionally active, lacks the necessary modifications to cooperate with co-regulators, such as epigenetic modifiers, which are needed to open up the chromatin and allow robust activation of endogenous promoters.

Thus, our optogenetic approach has given us some important insights into the biology of p65, despite not having given us the results we were hoping for.

Publications related to this topic

Requirements for mammalian promoters to decode transcription factor dynamics
E. B. Antwi, Y. Marrakchi, Ö. Cicek, T. Brox, B. Di Ventura
Nucleic Acids Research, 2023
https://doi.org/10.1093/nar/gkad273

Analysis of slow-cycling variants of the light-inducible nuclear protein export system LEXY in mammalian cells
G. Forlani, E. B. Antwi, D. Weis, M. A. Öztürk, B. A. W. Queck, D. Brecht, B. Di Ventura
ACS Synthetic Biology, 2022
https://doi.org/10.1021/acssynbio.2c00232

Experimental characterization of in silico red-shift predicted iLOVL470T/Q489K and iLOVV392K/F410V/A426S mutants
P. Wehler, D. Armbruster, A. Günter, E. Schleicher B. Di Ventura, M. A. Öztürk
ACS Omega, 2022
https://doi.org/10.1021/acsomega.2c01283

Light express
G. Forlani and B. Di Ventura
Current Opinion in Systems Biology, 2021
https://doi.org/10.1016/j.coisb.2021.100356

Engineering AraC to make it responsive to light instead of arabinose
G. E. Romano, A. Baumschlager, E. B. Akmeriç, N. Palanisamy, M. Houmani, G. Schmidt, M. A. Öztürk, L. Ernst, M. Khammash and B. Di Ventura
Nature Chemical Biology, 2021
https://doi.org/10.1038/s41589-021-00787-6

A light way for nuclear cell biologists
G. Forlani and B. Di Ventura
The Journal of Biochemistry, 2020
doi:10.1093/jb/mvaa1396

Optogenetic control of nucleocytoplasmic protein transport
D. Weis and B. Di Ventura
Photoswitching Proteins: Methods and Protocols, Methods in Molecular Biology, 2020
https://doi.org/10.1007/978-1-0716-0755-8_8

Optogenetic repressors of gene expression in yeasts using light-controlled nuclear localization
S. H. Geller, E. B. Antwi, B. Di Ventura and M. N. McClean
Cellular and Molecular Bioengineering, 2019
https://doi.org/10.1007/s12195-019-00598-9_8

Optogenetic control of nuclear protein import in living cells using Light-Inducible Nuclear Localization Signals (LINuS)
P. Wehler, D. Niopek, R. Eils and B. Di Ventura
Current Protocols in Chemical Biology, 2016
https://doi.org/10.1002/cpch.4

Optogenetic control of nuclear protein export
D. Niopek, P. Wehler, J. Roensch, R. Eils and B. Di Ventura
Nature Communications, 2016
https://doi.org/10.1038/ncomms10624

Go in! Go out! Inducible control of nuclear localization
B. Di Ventura and B. Kuhlman
Current Opinion in Chemical Biology, 2016
(https://doi.org/10.1016/j.cbpa.2016.06.009

Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells
D. Niopek, D. Benzinger, J. Roensch, T. Draebing, P. Wehler, R. Eils and B. Di Ventura
Nature Communications, 2014
https://doi.org/10.1038/ncomms5404