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Lights up on organelles: Optogenetic tools to control subcellular structure and organization

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Abstract Since the neurobiological inception of optogenetics, light‐controlled molecular perturbations have been applied in many scientific disciplines to both manipulate and observe cellular function. Proteins exhibiting light‐sensitive conformational changes provide researchers with avenues for spatiotemporal control over the cellular environment and serve as valuable alternatives to chemically inducible systems. Optogenetic approaches have been developed to target proteins to specific subcellular compartments, allowing for the manipulation of nuclear translocation and plasma membrane morphology. Additionally, these tools have been harnessed for molecular interrogation of organelle function, location, and dynamics. Optogenetic approaches offer novel ways to answer fundamental biological questions and to improve the efficiency of bioengineered cell factories by controlling the assembly of synthetic organelles. This review first provides a summary of available optogenetic systems with an emphasis on their organelle‐specific utility. It then explores the strategies employed for organelle targeting and concludes by discussing our perspective on the future of optogenetics to control subcellular structure and organization. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Physiology > Physiology of Model Organisms Biological Mechanisms > Regulatory Biology Models of Systems Properties and Processes > Cellular Models
Strategies for optogenetic protein degradation through proteasomal targeting. (a) Photosensitive degrons (PSDs) are composed of degradation sequences (deg) fused to the Jα helix of a LOV2 domain. These deg sequences are protected in the dark and exposed upon blue light activation of LOV2, which targets the tagged protein of interest (POI) to the proteasome for degradation. (b) A generalizable light‐modulated protein stabilization system (GLIMPSe) uses an evolved AsLOV2 (eLOV) domain to photocage Tobacco Etch Virus (TEV) protease cleavage sites. In the same system, a TEV protease fused to both LEXY and an NLS is sequestered in the nucleus until blue light exposure releases it to cleave the degron tags from POIs. Created with BioRender
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Optogenetic tools to control nuclear translocation. (a) The light‐inducible nuclear localization signal (LINuS) and the light activated nuclear shuttle (LANS) mask a nuclear localization signal (NLS) fused to the Jα helix of AsLOV2 domain, which induces translocation of the fused protein of interest (POI) into the nucleus in response to blue light. (b) The light‐inducible nuclear export system (LEXY) and the light‐inducible nuclear exporter (LINX) utilize AsLOV2 fused to a nuclear export signal (NES) to translocate a POI to the cytosol in blue light. (c) Upon red light exposure, PhyB fused to a POI and an NES binds to PIF3 fused to an NLS. The resulting complex is imported into the nucleus with red light whereas far‐red light induces dissociation from PIF3 and subsequent nuclear export of the POI fusion. (d) Homodimers of UVR8‐POI fusions are dissociated in UVB light, allowing them to heterodimerize with NLS‐tagged COP1 and be imported into the nucleus. Created with BioRender
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Light‐controlled protein phase transitions. (a) CRY2 or CRY2olig fused to intrinsically disordered regions (IDRs) result in light‐induced liquid condensates called optoDroplets. (b) Corelets are light‐inducible liquid condensates composed of iLID modules fused to a ferritin heavy chain monomer (FTH1) and SspB‐IDR fusions that bind to the ferritin cores in the light to nucleate condensates and dissociate in the dark to dissolve them. (c) PixELLs are light‐dissociable liquid condensates composed of IDRs fused to PixE and PixD, which can nucleate liquid condensates in the dark when fused to IDRs. Created with BioRender
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Monomeric, dimeric, and multimeric optogenetic systems with demonstrated subcellular applications in eukaryotes. (a) Blue light‐induced opening of channelrhodopsins (ChRs) allows for cation translocation. (b) Loss of Jα helix secondary structure in light, oxygen, voltage (LOV) domains upon blue light exposure leads to the uncaging of a fused protein or an amino acid sequence. (c) Blue light stimulation exposes positive charges within the Jα helix of BcLOV4 to electrostatic interaction with the negatively charged plasma membrane. (d) Photoactivation exposes SsrA to SspB binding in the AsLOV2‐based iLID system. (e) Blue light allows for heterodimerization of complementary Magnet monomers—variants of LOV‐based Vivid monomers with either a positively charged Ncap (pMag) or a negatively charged Ncap (nMag). (f) Blue light induces cryptochrome 2 (CRY2)—cryptochrome‐interacting basic helix–loop–helix (CIB) heterodimerization. (g) OptoBNDRs, including AsLOV2‐nanobody (OptoNB) and AsLOV2‐monobody (OptoMB) chimeras, enable light‐inhibited binding of a protein of interest (POI). (h) OptoNBs can also be designed to have light‐inducible binding to POIs. (i) Phytochrome B (PhyB)—phytochrome‐interacting factor (PIF) heterodimers form in response to red light and dissociate when exposed to far‐red light. (j) Bacterial phytochrome photoreceptor 1 (BphP1) homodimerizes in the dark and does so more quickly when red light is introduced. Far‐red light dissociates these dimers and frees BphP1 to bind with its engineered interaction partner Q‐PAS1. (k) Ultraviolet response locus 8 (UVR8) homodimers dissociate in the presence of UVB light and UVR8 monomers are free to bind constitutive photomorphogenesis 1 (COP1). In the absence of UVB light, repressors of UVB photomorphogenesis (RUPs) interact with UVR8 monomers and induce their homodimerization. (l) Both wildtype CRY2 and mutant CRY2olig cluster following blue light induced conformational shifts. (m) Blue light dissociates multimers composed of PixD dimers and PixE monomers, which re‐associate in the dark. (n) Light‐activated reversible inhibition by assembled trap (LARIAT) utilizes CIB functionalization of multivalent assembly domain subunits to sequester CRY2‐bound proteins into multimeric clusters. Created with BioRender
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Optogenetic control of subcellular structure and organization. Light‐induced modules allow for photosensitive control over protein secretion, accumulation, and degradation in addition to nuclear import and export. Optogenetic systems also allow for manipulation of plasma membrane morphology, organelle transport, cell death signaling, and mitochondrial membrane potential. Created with BioRender
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Select applications of synthetic organelles. (a) Enzymatically dead Cas9 (dCas9) fused to SunTag (ST) targets gRNA‐specified genomic loci and recruits ST‐specific antibody fragments (scFvs) fused to iLID. SspB‐IDR fusions form nuclear condensates on those specific genomic loci following blue light exposure (adapted from Shin et al., 2018). (b) Optogenetic condensates colocalize metabolic enzymes to increase the conversion of chemical intermediates to the desired product (adapted from Bracha et al., 2019). Created with BioRender
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Select strategies for optogenetic mitochondrial manipulation. (a) Activation of channelrhodopsins (ChRs) targeted to the inner mitochondrial membrane leads to light‐induced disruption of the electrochemical gradient. (b) Light‐dependent recruitment of CRY2‐BAX fusions to CIB monomers tagged with TOM20 mitochondrial localization sequences enables light‐induced apoptosis. (c) Light‐triggered dimerization of iLID fused to an ActA outer mitochondrial membrane (OMM) linker sequence and SspB tagged with an ER anchor sequence from cytochrome b5 has been employed to induce mitochondrial‐ER tethering. Created with BioRender
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Models of Systems Properties and Processes > Cellular Models
Biological Mechanisms > Regulatory Biology
Physiology > Physiology of Model Organisms

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