Showing 1 - 12 of 12 results
Signaling, Deconstructed: Using Optogenetics to Dissect and Direct Information Flow in Biological Systems.
Cells receive enormous amounts of information from their environment. How they act on this information-by migrating, expressing genes, or relaying signals to other cells-comprises much of the regulatory and self-organizational complexity found across biology. The "parts list" involved in cell signaling is generally well established, but how do these parts work together to decode signals and produce appropriate responses? This fundamental question is increasingly being addressed with optogenetic tools: light-sensitive proteins that enable biologists to manipulate the interaction, localization, and activity state of proteins with high spatial and temporal precision. In this review, we summarize how optogenetics is being used in the pursuit of an answer to this question, outlining the current suite of optogenetic tools available to the researcher and calling attention to studies that increase our understanding of and improve our ability to engineer biology. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 23 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Dual Systems for Enhancing Control of Protein Activity through Induced Dimerization Approaches.
To reveal the underpinnings of complex biological systems, a variety of approaches have been developed that allow switchable control of protein function. One powerful approach for switchable control is the use of inducible dimerization systems, which can be configured to control activity of a target protein upon induced dimerization triggered by chemicals or light. Individually, many inducible dimerization systems suffer from pre‐defined dynamic ranges and overwhelming sensitivity to expression level and cellular context. Such systems often require extensive engineering efforts to overcome issues of background leakiness and restricted dynamic range. To address these limitations, recent tool development efforts have explored overlaying dimerizer systems with a second layer of regulation. Albeit more complex, the resulting layered systems have enhanced functionality, such as tighter control that can improve portability of these tools across platforms.
Steering Molecular Activity with Optogenetics: Recent Advances and Perspectives.
Optogenetics utilizes photosensitive proteins to manipulate the localization and interaction of molecules in living cells. Because light can be rapidly switched and conveniently confined to the sub‐micrometer scale, optogenetics allows for controlling cellular events with an unprecedented resolution in time and space. The past decade has witnessed an enormous progress in the field of optogenetics within the biological sciences. The ever‐increasing amount of optogenetic tools, however, can overwhelm the selection of appropriate optogenetic strategies. Considering that each optogenetic tool may have a distinct mode of action, a comparative analysis of the current optogenetic toolbox can promote the further use of optogenetics, especially by researchers new to this field. This review provides such a compilation that highlights the spatiotemporal accuracy of current optogenetic systems. Recent advances of optogenetics in live cells and animal models are summarized, the emerging work that interlinks optogenetics with other research fields is presented, and exciting clinical and industrial efforts to employ optogenetic strategy toward disease intervention are reported.
Improved Photocleavable Proteins with Faster and More Efficient Dissociation.
The photocleavable protein (PhoCl) is a green-to-red photoconvertible fluorescent protein that, when illuminated with violet light, undergoes main chain cleavage followed by spontaneous dissociation of the resulting fragments. The first generation PhoCl (PhoCl1) exhibited a relative slow rate of dissociation, potentially limiting its utilities for optogenetic control of cell physiology. In this work, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast ratio and PhoCl2f with faster dissociation. We characterized the performance of these new variants as purified proteins and expressed in cultured cells. Our results demonstrate that PhoCl2 variants exhibit faster and more efficient dissociation, which should enable improved optogenetic manipulations of protein localization and protein-protein interactions in living cells.
A light way for nuclear cell biologists.
The nucleus is a very complex organelle present in eukaryotic cells. Having the crucial task to safeguard, organize and manage the genetic information, it must tightly control its molecular constituents, its shape and its internal architecture at any given time. Despite our vast knowledge of nuclear cell biology, much is yet to be unraveled. For instance, only recently we came to appreciate the existence of a dynamic nuclear cytoskeleton made of actin filaments that regulates processes such as gene expression, DNA repair and nuclear expansion. This suggests further exciting discoveries ahead of us. Modern cell biologists embrace a new methodology relying on precise perturbations of cellular processes that require a reversible, highly spatially-confinable, rapid, inexpensive and tunable external stimulus: light. In this review, we discuss how optogenetics, the state-of-the-art technology that uses genetically-encoded light-sensitive proteins to steer biological processes, can be adopted to specifically investigate nuclear cell biology.
SPLIT: Stable Protein Coacervation using a Light Induced Transition.
Protein coacervates serve as hubs to concentrate and sequester proteins and nucleotides and thus function as membrane-less organelles to manipulate cell physiology. We have engineered a coacervating protein to create tunable, synthetic membrane-less organelles that assemble in response to a single pulse of light. Coacervation is driven by the intrinsically disordered RGG domain from the protein LAF-1, and opto-responsiveness is coded by the protein PhoCl which cleaves in response to 405 nm light. We developed a fusion protein containing a solubilizing maltose binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellular-sized water-in-oil emulsions. An optimized version of this system displayed light-induced coacervation in Saccharomyces cerevisiae. The methods described here provide novel strategies for inducing protein phase separation using light.
Recent advances in the use of genetically encodable optical tools to elicit and monitor signaling events.
Cells rely on a complex network of spatiotemporally regulated signaling activities to effectively transduce information from extracellular cues to intracellular machinery. To probe this activity architecture, researchers have developed an extensive molecular tool kit of fluorescent biosensors and optogenetic actuators capable of monitoring and manipulating various signaling activities with high spatiotemporal precision. The goal of this review is to provide readers with an overview of basic concepts and recent advances in the development and application of genetically encodable biosensors and optogenetic tools for understanding signaling activity.
Hydrogels With Tunable Mechanical Properties Based on Photocleavable Proteins.
Hydrogels with photo-responsive mechanical properties have found broad biomedical applications, including delivering bioactive molecules, cell culture, biosensing, and tissue engineering. Here, using a photocleavable protein, PhoCl, as the crosslinker we engineer two types of poly(ethylene glycol) hydrogels whose mechanical stability can be weakened or strengthened, respectively, upon visible light illumination. In the photo weakening hydrogels, photocleavage leads to rupture of the protein crosslinkers, and decrease of the mechanical properties of the hydrogels. In contrast, in the photo strengthening hydrogels, by properly choosing the crosslinking positions, photocleavage does not rupture the crosslinking sites but exposes additional cryptical reactive cysteine residues. When reacting with extra maleimide groups in the hydrogel network, the mechanical properties of the hydrogels can be enhanced upon light illumination. Our study indicates that photocleavable proteins could provide more designing possibilities than the small-molecule counterparts. A proof-of-principle demonstration of spatially controlling the mechanical properties of hydrogels was also provided.
Functional Modulation of Receptor Proteins on Cellular Interface with Optogenetic System.
In multicellular organisms, living cells cooperate with each other to exert coordinated complex functions by responding to extracellular chemical or physical stimuli via proteins on the plasma membrane. Conventionally, chemical signal transduction or mechano-transduction has been investigated by chemical, genetic, or physical perturbation; however, these methods cannot manipulate biomolecular reactions at high spatiotemporal resolution. In contrast, recent advances in optogenetic perturbation approaches have succeeded in controlling signal transduction with external light. The methods have enabled spatiotemporal perturbation of the signaling, providing functional roles of the specific proteins. In this chapter, we summarize recent advances in the optogenetic tools that modulate the function of a receptor protein. While most optogenetic systems have been devised for controlling ion channel conductivities, the present review focuses on the other membrane proteins involved in chemical transduction or mechano-transduction. We describe the properties of natural or artificial photoreceptor proteins used in optogenetic systems. Then, we discuss the strategies for controlling the receptor protein functions by external light. Future prospects of optogenetic tool development are discussed.
Photocleavable Cadherin Inhibits Cell-to-Cell Mechanotransduction by Light.
Precise integration of individual cell behaviors is indispensable for collective tissue morphogenesis and maintenance of tissue integrity. Organized multicellular behavior is achieved via mechanical coupling of individual cellular contractility, mediated by cell adhesion molecules at the cell-cell interface. Conventionally, gene depletion or laser microsurgery has been used for functional analysis of intercellular mechanotransduction. Nevertheless, these methods are insufficient to investigate either the spatiotemporal dynamics or the biomolecular contribution in cell-cell mechanical coupling within collective multicellular behaviors. Herein, we present our effort in adaption of PhoCl for attenuation of cell-to-cell tension transmission mediated by E-cadherin. To release intercellular contractile tension applied on E-cadherin molecules with external light, a genetically encoded photocleavable module called PhoCl was inserted into the intracellular domain of E-cadherin, thereby creating photocleavable cadherin (PC-cadherin). In response to light illumination, the PC-cadherin cleaved into two fragments inside cells, resulting in attenuating mechanotransduction at intercellular junctions in living epithelial cells. Light-induced perturbation of the intercellular tension balance with surrounding cells changed the cell shape in an epithelial cell sheet. The method is expected to enable optical manipulation of force-mediated cell-to-cell communications in various multicellular behaviors, which contributes to a deeper understanding of embryogenesis and oncogenesis.
Genetically Encoded Photocleavable Linkers for Patterned Protein Release from Biomaterials.
Given the critical role that proteins play in almost all biological processes, there is great interest in controlling their presentation within and release from biomaterials. Despite such outstanding enthusiasm, previously developed strategies in this regard result in ill-defined and heterogeneous populations with substantially decreased activity, precluding their successful application to fragile species including growth factors. Here, we introduce a modular and scalable method for creating monodisperse, genetically encoded chimeras that enable bioactive proteins to be immobilized within and subsequently photoreleased from polymeric hydrogels. Building upon recent developments in chemoenzymatic reactions, bioorthogonal chemistry, and optogenetics, we tether fluorescent proteins, model enzymes, and growth factors site-specifically to gel biomaterials through a photocleavable protein (PhoCl) that undergoes irreversible backbone photoscission upon exposure to cytocompatible visible light (λ ≈ 400 nm) in a dose-dependent manner. Mask-based and laser-scanning lithographic strategies using commonly available light sources are employed to spatiotemporally pattern protein release from hydrogels while retaining their full activity. The photopatterned epidermal growth factor presentation is exploited to promote anisotropic cellular proliferation in 3D. We expect these methods to be broadly useful for applications in diagnostics, drug delivery, and regenerative medicine.
Optogenetic control with a photocleavable protein, PhoCl.
To expand the range of experiments that are accessible with optogenetics, we developed a photocleavable protein (PhoCl) that spontaneously dissociates into two fragments after violet-light-induced cleavage of a specific bond in the protein backbone. We demonstrated that PhoCl can be used to engineer light-activatable Cre recombinase, Gal4 transcription factor, and a viral protease that in turn was used to activate opening of the large-pore ion channel Pannexin-1.