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The Rise of Molecular Optogenetics

2021; Wiley; Volume: 5; Issue: 5 Linguagem: Inglês

10.1002/adbi.202100776

2701-0198

Barbara Di Ventura, Wilfried Weber,

Photosynthetic Processes and Mechanisms

The ability to control biological systems via light as the external stimulus is revolutionizing fundamental and applied life sciences. Light represents the ideal trigger to steer biological processes with unmatched spatial and temporal precision in a dose-dependent manner, offering the possibility of multiplexing by using light of different wavelengths. The development and use of genetically encoded, light-responsive switches define the rapidly growing field of optogenetics. First optogenetic approaches were pioneered by the Quail group using the Arabidopsis phytochrome system to control gene expression in yeast. Nonetheless, the field of optogenetics truly took off with the discovery that light-sensitive ion channels could be used to trigger action potentials in neuronal and muscle cells. For about a decade, indeed, optogenetics was deeply intertwined and immediately associated with neurobiology. Such optogenetic technologies allow dissecting and understanding the function of neuronal networks but also controlling neuronal processes in optically triggered brain-machine interfaces. After this initial phase in which it was mostly applied in neurobiology, optogenetics became widespread among cell biologists to control intracellular processes. Nowadays, we see interesting applications spanning many different cell types and even organisms; for instance, muscle cells have been engineered for light-inducible contraction and used as optically stimulated actuators in soft robotics. The second wave of innovation in optogenetics, capitalizing on the initial phytochrome-based optogenetic work, focuses on photoreceptors that translate light stimuli into a molecular output. We call it, therefore, molecular optogenetics. The output can be a change in the interaction state between two biomolecules, an altered enzyme activity, or the regulated availability of a protein motif or peptide. Molecular optogenetics is exponentially growing with an annual publication rate that doubles every 2 years (Figure 1). This growth in publications reflects the exponentially growing community of scientists harnessing and advancing the intriguing opportunities of molecular optogenetics. Innovations in this field have yielded technologies allowing controlling every step of the cellular signaling cascade. This molecular optogenetic toolbox includes light-responsive membrane-bound receptors, intracellular kinase cascades, second messenger-producing enzymes, light-inducible transcriptional regulators, or tools to steer the translocation of proteins between different compartments as well as their degradation, just to name the most predominant ones. More recently, tools from molecular optogenetics have been applied in the extracellular space for the development of light-responsive synthetic extracellular matrices or nanobodies/monobodies with an optically tunable affinity for their antigens. In order to keep track of the vertiginous selection of molecular optogenetic tools, the publicly available database www.optobase.org was established. In this issue dedicated to molecular optogenetics, the contributions of experts in the field in the form of reviews of the latest advances or original research are collected with the aim to help interested readers enter the exciting world of light-triggered biological devices learning how to apply them for their own purposes. One of the most often targeted cellular processes for light control since the early days of optogenetics is gene expression. By now, we have a multitude of tools for use in different organisms, ranging from bacteria to living mice. Baumschlager and Khammash discuss in article 2000256 past and recently developed strategies to control gene expression in bacteria, which will help readers find their way in the intricate jungle of possibilities to choose from. In article 2000234, Tucker and Pearce review recent approaches in endowing light-regulated dimerization systems with a second control layer. These approaches are used to decrease background leakiness and increase the dynamic range of naturally occurring photoreceptors for advanced optogenetic applications. One way to control protein activity with light is by allosteric coupling of the protein of interest to a photosensor. The successful engineering of fusions whereby the light-triggered conformational change of the photoreceptor propagates down to the protein of interest is not straightforward and often requires trial and error. In article 2000181, Mathony and Niopek give an exciting guided tour of the most recent advances in methodologies that support the design of optogenetic allosteric switches. Among the discussed strategies, the one based on the insertion of LOV domains into surface-exposed loops is particularly promising, as demonstrated by its successful application to the light-mediated control of kinases, Cas9, and nanobodies. In article 2000180, Zhang and colleagues comprehensively review recent new optogenetic switches as well as improved, engineered variants of previous switches. They further compile experimental conditions, biophysical properties and application fields of different switches, representing a highly valuable resource when planning future optogenetic experiments. In addition to these reviews, this special issue features new optogenetic technologies applied at different levels of intracellular signal processing but also in the extracellular space to control the assembly of nanomaterials. In article 2000307, the Zurbriggen group describes a blue light-responsive system for mRNA knockdown. The system is based on the blue light-inducible expression of an engineered Cas13b to degrade target mRNAs specifically. By combining three blue light-inducible switches for mRNA degradation, suppressing transcription, and target protein destabilization, the protein levels can rapidly be reduced by >99%. In article 2000147, Zhou and colleagues bring us into the world of the innate immune system. They adapt two previously established optogenetic tools, namely CRY2-mediated protein oligomerization and LOVTRAP-mediated protein heterodimerization, to control with blue light the assembly of MyDDsomes and MAVsomes, two types of supramolecular organizing centers (SMOC). Consequently, the authors can trigger the activation of nuclear factor-kB (NF-κB) and type-I interferons (IFNs) with blue light, opening up new avenues to study the innate immune system and potentially counteract immune diseases and cancer. In article 2000196, Radziwill and colleagues develop and characterize a multichromatic optogenetic system, where blue, red, and far-red light is used to activate either RAF/ERK or AKT signaling orthogonally. This system is useful in dissecting and steering the fine-grained and intricate processes involved in cell fate decisions. In article 2000134, Yi, Zhang and colleagues apply optogenetics in hair-follicle-derived stem cells (HSCs) to study how activation of the tropomyosin receptor kinase A (TrkA) receptor impacts several processes such as migration and differentiation. Making use of the blue light-inducible TrkA (OptoTrkA) previously engineered by the Zhang group, they show that HSCs with OptoTrkA activity proliferate and migrate more, as well as differentiate more quickly into neuronal and glial cells. The Grosse group was the first to engineer optogenetic control of the endogenous formin mDia2 back in 2013 to show the involvement of this formin in the formation of dynamic actin filaments in the nucleus. Continuing with their tradition to develop optogenetic molecular tools to study the actin cytoskeleton and related cellular processes, Zhao and Grosse present in article 2000208 a novel light-controllable MRTFA, a transcription factor involved in the regulation of the actin cytoskeleton. By fusing the blue light-inducible nuclear export signal LEXY to MRTFA, they are able to steer with blue light the ability of different mammalian cells to invade and bleb. In article 2000541, the Zhou group develops so-called sunbodies consisting of nanobodies with engineered blue light-responsive light-oxygen-voltage (LOV) domains. They show that the affinity of sunbodies towards intracellular antigens can be switched by blue light, which they use for light-inducible targeting of intracellular proteins. The Wegner group moves optogenetics out of the cell in article 2000199. They target the green light photoreceptor CarH to the surface of breast cancer cell lines, which induces cell-cell interactions in the dark. By the subsequent illumination of the sample with green light, CarH tetramers dissociate leading to the dissolution of the cell aggregates. Similarly, in article 2000179, the Möglich group harvests the ability of some LOV photoreceptors to homodimerize with blue light to control the aggregation state of gold nanoparticles. They conjugate two well-characterized LOV domains – Phaeodactylum tricornutum aureochrome 1a (Ptaur) and Neurospora crassa Vivid (NcVVD) – to the surface of the gold nanoparticles. By shining blue light, they trigger the assembly of large aggregates, which are instead monodispersed in darkness. This article nicely contributes to the growing field of optogenetics for material science. We hope the readers will be as fascinated as we are by this excellent collection and eagerly await the next developments in the field of molecular optogenetics the tools described here will surely spark. Barbara Di Ventura Barbara Di Ventura is the leader of the “Molecular and Cellular Engineering group” and is BIOSS professor of Biological Signaling at the University of Freiburg. Her team is interested in understanding the mechanisms used by cells to control processes in space and time, especially gene expression in mammalian cells. The group uses an interdisciplinary approach that combines molecular and cellular biology with synthetic biology and mathematical modeling. A special focus of the lab is optogenetics, that is, the use of light to externally control protein function and localization in individual living cells. Wilfried Weber Wilfried Weber is a BIOSS professor of Synthetic Biology at the University of Freiburg, Germany. His research is positioned at the intersection of synthetic biology and materials sciences. He is a pioneer in molecular optogenetics and develops light-triggered switches to control cell signaling but also to steer the mechanical properties of synthetic extracellular matrices. He is applying such technologies towards the development of applications in drug delivery, tissue engineering, or point-of-care analytics. He is a founding spokesperson of the Centre for Integrative Biological Signalling Studies, CIBSS.