Optogenetic Approaches for the Spatiotemporal Control of Signal Transduction Pathways

Kramer, Markus M.; Lataster, Levin; Weber, Wilfried; Radziwill, Gerald

doi:10.3390/ijms22105300

Cited by 20 publications

(14 citation statements)

References 110 publications

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“…Optogenetic tools have been widely applied in eukaryotic cells in conjunction with diffraction-limited fluorescence microscopy to obtain population-averaged, phenotypic readouts 52,53 . Therefore, we sought to quantify the optogenetic response of the iLID system in K12 E.coli using diffraction-limited imaging as a first step.…”

Section: Resultsmentioning

confidence: 99%

Dimerization of iLID Optogenetic Proteins Observed Using 3D Single-Molecule Tracking in Live Bacterial Cells

Achimovich

Yan

Gahlmann

2022

Preprint

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3D single molecule tracking microscopy has enabled measurements of protein diffusion in living cells, offering information about protein dynamics and the cellular environment. For example, different diffusive states can be resolved and assigned to protein complexes of different size and composition. However, substantial statistical power and biological validation, often through genetic deletion of binding partners, are required to support diffusive state assignments. When investigating some cellular processes, transient perturbation to protein spatial distributions is preferable to permanent genetic deletion of an essential protein. Optogenetic dimerization systems can be used to manipulate protein spatial distributions which could offer a means to deplete specific diffusive states observed in single molecule tracking experiments. Here, we evaluate the performance of the iLID optogenetic system in living E. coli cells using diffraction-limited microscopy and 3D single molecule tracking. We observed a robust optogenetic response in protein spatial distribution after 488 nm laser activation. Surprisingly, 3D single molecule tracking results indicate activation of the optogenetic response at high intensity wavelengths for which there is evidence of minimal photon absorbance by the LOV2 domain. However, the preactivation response was minimized through the use of iLID system mutants, and titration of protein expression levels.

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Section: Resultsmentioning

confidence: 99%

Dimerization of iLID Optogenetic Proteins Observed Using 3D Single-Molecule Tracking in Live Bacterial Cells

Achimovich

Yan

Gahlmann

2022

Preprint

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“…Optogenetic activation of signaling cascades typically relies on a genetically encoded light responsive dimerizing/oligomerizing protein fused to a signaling effector to mimic the effect of upstream ligand binding (Kramer et al, 2021 ), and it can do so with spatiotemporal control. In particular, several optogenetic systems controlling morphogen signals relevant to development have recently been reported, leveraging Cry2 (Kennedy et al, 2010 ; Bugaj et al, 2013 ).…”

Section: Optogenetics In Organismal Developmentmentioning

confidence: 99%

Optogenetic Application to Investigating Cell Behavior and Neurological Disease

Zhu

Johnson

Schaffer

2022

Front. Cell. Neurosci.

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Cells reside in a dynamic microenvironment that presents them with regulatory signals that vary in time, space, and amplitude. The cell, in turn, interprets these signals and accordingly initiates downstream processes including cell proliferation, differentiation, migration, and self-organization. Conventional approaches to perturb and investigate signaling pathways (e.g., agonist/antagonist addition, overexpression, silencing, knockouts) are often binary perturbations that do not offer precise control over signaling levels, and/or provide limited spatial or temporal control. In contrast, optogenetics leverages light-sensitive proteins to control cellular signaling dynamics and target gene expression and, by virtue of precise hardware control over illumination, offers the capacity to interrogate how spatiotemporally varying signals modulate gene regulatory networks and cellular behaviors. Recent studies have employed various optogenetic systems in stem cell, embryonic, and somatic cell patterning studies, which have addressed fundamental questions of how cell-cell communication, subcellular protein localization, and signal integration affect cell fate. Other efforts have explored how alteration of signaling dynamics may contribute to neurological diseases and have in the process created physiologically relevant models that could inform new therapeutic strategies. In this review, we focus on emerging applications within the expanding field of optogenetics to study gene regulation, cell signaling, neurodevelopment, and neurological disorders, and we comment on current limitations and future directions for the growth of the field.

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“…The inserts and the backbone vector digested with restriction enzymes (NEB) according to the cloning scheme. The non-coding backbone sequences including CMV promoter was ampli ed from the Addgene #92390 plasmid vector described in a previous study 16,17,22 . The digested backbone vector and inserts were ligated with T4 ligase (NEB) and the ligation mixture was transformed into DH5α competent cells (Enzynomics).…”

Section: Plasmid Vector Design and Cloningmentioning

confidence: 99%

“…The complementation of split proteases occurs when chemically interacting proteins bind to each other via small-molecule drugs, such as rapamycin 15 . Like CID, non-invasive optogenetic heterodimer complementation allows unprecedented approaches to mediate fast signal transduction with high spatiotemporal resolution including blue-lightinducible TEV protease (BLITz) 16,17,22 . However, complementation strategies consisting of separate two…”

mentioning

confidence: 99%

LAUNCHER: A single-component, light-assisted uncaging switch for endoproteolytic release

Cui¹,

Lee²,

Ban³

et al. 2022

Preprint

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Proteases can serve as molecular switches to initiate a wide spectrum of biological events. High efficiency and specificity enhance the potential of proteases as versatile elements in synthetic biological circuits. To address the unmet need for a novel protease-based toolkit with modular features, we developed a single-component photocleavable switch, termed LAUNCHER (Light-Assisted UNcaging switCH for Endoproteolytic Release), combining a circularly permuted potyviral protease and a blue-light-gated substrate with variable intermodular linkers. LAUNCHER enables user-controllable release of payloads that can serve in various cellular applications, such as transgene expression, multilayer-based G protein-coupled receptor (GPCR) activity sensing, and optochemogenetic manipulation. Moreover, LAUNCHER orthologs from the orthogonal potyviral protease family can be utilized in a multiple protease-based circuit without mutual interference. LAUNCHER provides a generalized method to implant optogenetic manipulations into existing synthetic biological circuits in a plug-and-play manner and adds to optogenetic toolboxes for the expansion of biomedical fields.

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Optogenetic Approaches for the Spatiotemporal Control of Signal Transduction Pathways

Cited by 20 publications

References 110 publications

Dimerization of iLID Optogenetic Proteins Observed Using 3D Single-Molecule Tracking in Live Bacterial Cells

Dimerization of iLID Optogenetic Proteins Observed Using 3D Single-Molecule Tracking in Live Bacterial Cells

Optogenetic Application to Investigating Cell Behavior and Neurological Disease

LAUNCHER: A single-component, light-assisted uncaging switch for endoproteolytic release

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