Chromophore-assisted Light Inactivation Research Articles

Optical inactivation of intracellular molecules by fast-maturating photosensitizing fluorescence protein, HyperNova

Photosensitizing fluorescence protein is a promising tool for chromophore-assisted light inactivation (CALI) that enables specific oxidation and inactivation of intracellular molecules. However, a commonly used monomeric photosensitizing fluorescent protein, SuperNova, shows a low CALI efficiency due to its insufficient maturation at 37 °C, thereby limiting the application of CALI to various molecules, especially in mammalian cells. Here, we present a photosensitizing fluorescence protein, HyperNova, with markedly improved maturation at 37 °C, leading to greatly enhanced CALI efficiency. Exploiting this quality, HyperNova enables the application of CALI to variety of molecules such as a mitotic kinase and transcriptional factors that were highly challenging with conventional SuperNova. To further demonstrate the utility of HyperNova, we have also succeeded in developing novel CALI techniques for MAP kinases by HyperNova. Our findings suggest that HyperNova has the potential to expand the molecular toolbox for manipulating biological events in living cells, providing new avenues for investigating cellular signaling pathways.

Chromophore-Assisted Light Inactivation for Protein Degradation and Its Application in Biomedicine.

The functional investigation of proteins holds immense significance in unraveling physiological and pathological mechanisms of organisms as well as advancing the development of novel pharmaceuticals in biomedicine. However, the study of cellular protein function using conventional genetic manipulation methods may yield unpredictable outcomes and erroneous conclusions. Therefore, precise modulation of protein activity within cells holds immense significance in the realm of biomedical research. Chromophore-assisted light inactivation (CALI) is a technique that labels photosensitizers onto target proteins and induces the production of reactive oxygen species through light control to achieve precise inactivation of target proteins. Based on the type and characteristics of photosensitizers, different excitation light sources and labeling methods are selected. For instance, KillerRed forms a fusion protein with the target protein through genetic engineering for labeling and inactivates the target protein via light activation. CALI is presently predominantly employed in diverse biomedical domains encompassing investigations into protein functionality and interaction, intercellular signal transduction research, as well as cancer exploration and therapy. With the continuous advancement of CALI technology, it is anticipated to emerge as a formidable instrument in the realm of life sciences, yielding more captivating outcomes for fundamental life sciences and precise disease diagnosis and treatment.

Chromophore-assisted light inactivation of target proteins for singularity biology.

Singularity phenomena are rare events that occur only with a probability of one in tens of thousands and yet play an important role in the fate of the entire system. Recently, an ultra-wide-field microscopy imaging systems, AMATERAS, have been developed to reliably capture singularity phenomena. However, to determine whether a rare phenomenon captured by microscopy is a true singularity phenomenon-one with a significant impact on the entire system-, causal analysis is required. In this section, we introduce the CALI method, which uses light to inactivate molecules as one of the techniques enabling causal analysis. In addition, we discuss the technical innovations of the CALI method that are required to contribute to the future development of singularity biology.

Enabling Photo-Crosslinking and Photo-Sensitizing Properties for Synthetic Fluorescent Protein Chromophores.

Synthetic fluorescent protein chromophores have been reported for their singlet state fluorescence properties and applications in bioimaging, but rarely for the triplet state chemistries. Herein, we enabled their photo-sensitizing and photo-crosslinking properties through rational modulations. Extension of molecular conjugation and introduction of heavy atoms promoted the generation of reactive oxygen species. Unlike other photosensitizers, these chromophores selectively photo-crosslinked aggregated proteins and uncovered the interactome profiles. We also exemplified their general applications in chromophore-assisted light inactivation, photodynamic therapy and photo induced polymerization. Theoretical calculation, pathway analysis and transient absorption spectroscopy provided mechanistic insights for this triplet state chemistry. Overall, this work expands the function and application of synthetic fluorescent protein chromophores by enabling their triplet excited state properties.

Control of actin polymerization via reactive oxygen species generation using light or radiation.

Actin is one of the most prevalent proteins in cells, and its amino acid sequence is remarkably conserved from protozoa to humans. The polymerization-depolymerization cycle of actin immediately below the plasma membrane regulates cell function, motility, and morphology. It is known that actin and other actin-binding proteins are targets for reactive oxygen species (ROS), indicating that ROS affects cells through actin reorganization. Several researchers have attempted to control actin polymerization from outside the cell to mimic or inhibit actin reorganization. To modify the polymerization state of actin, ultraviolet, visible, and near-infrared light, ionizing radiation, and chromophore-assisted light inactivation have all been reported to induce ROS. Additionally, a combination of the fluorescent protein KillerRed and the luminescent protein luciferase can generate ROS on actin fibers and promote actin polymerization. These techniques are very useful tools for analyzing the relationship between ROS and cell function, movement, and morphology, and are also expected to be used in therapeutics. In this mini review, we offer an overview of the advancements in this field, with a particular focus on how to control intracellular actin polymerization using such optical approaches, and discuss future challenges.

Photodynamic Therapy by Glucose Transporter 1-Selective Light Inactivation

Chromophore-assisted light inactivation (CALI) was applied to molecule-targeted photodynamic therapy (PDT). In order to identify organic photosensitizers suitable for CALI, the carbonic anhydrase II (CAII) ligand, 4-sulfamoylbenzoic acid 1, was conjugated with several photosensitizers to produce compounds 2–7, whose CALI ability was evaluated by measuring their effect on CAII enzymatic activity. Di-iodinated BODIPY (I2BODIPY) exhibited excellent CAII inactivation ability, similar to that of Ru(bpy)3. The glucose–I2BODIPY conjugate (8) was synthesized as an inactivation of glucose transporter 1 (GLUT1), a protein overexpressed in many cancer cells. Under light irradiation, 8 exhibited concentration-dependent cytotoxicity with half maximal inhibitory concentration (IC50) values of 5.49, 11.14, and 8.73 μM, against human cervical carcinoma (HeLa), human lung carcinoma (A549), and human hepatocellular carcinoma (HepG2) cell lines, respectively. The GLUT1 inhibitor phloretin suppressed the cytotoxicity induced by 8 under light irradiation in a concentration-dependent manner. Western blot analysis indicated that GLUT1 was not detected in cell lines treated with 10 μM 8 under light irradiation. Furthermore, 8 reduced the levels of epidermal growth factor receptor tyrosine kinase (EGFR), phospho-ERK (Y204), and GLUT1 without affecting ERK, α-tubulin, and PCNA protein levels, whereas talaporfin sodium, a clinically approved photosensitizer for PDT, nonspecifically reduced intracellular protein levels in HeLa cells, indicating that 8 has a GLUT1-specific inactivation ability and causes light-induced cytotoxicity by modulating the EGFR/MAPK signaling pathway.

Intermitochondrial signaling regulates the uniform distribution of stationary mitochondria in axons

In the central nervous system (CNS), many neurons develop axonal arbors that are crucial for information processing. Previous studies have demonstrated that premature axons contain motile and stationary mitochondria, and their balance is important for axonal arborization. However, the mechanisms by which neurons determine the positions of stationary mitochondria as well as their turnover remain to be elucidated. We observed that the distribution of stationary mitochondrial spots along the unmyelinated and nonsynaptic axons is not random but rather relatively uniform both in primary cultured neurons and in tissues. Intriguingly, whereas the positions of each mitochondrial spot changed over time, the overall distribution remained uniform. In addition, local inactivation of mitochondria by KillerRed mediated chromophore-assisted light inactivation (CALI) inhibited the translocation of mitochondrial spots in adjacent axonal regions, suggesting that functional mitochondria enhance the motility of other mitochondria in the vicinity. Signals of ATP:ADP sensor, PercevalHR indicated that the ATP:ADP ratio was relatively high around mitochondria, and treating axons with phosphocreatine (PCr), which supplies ATP, reduced the immobile mitochondria induced by the local mitochondrial inactivation. In a mathematical model, we found that the ATP gradient generated by mitochondria, and ATP dependent regulation of mitochondrial motility could establish uniform mitochondrial distribution. These observations suggest that axons in the CNS possess the system that distributes mitochondria uniformly, and intermitochondrial signaling contribute to the regulation. In addition, our results suggest the possibility that ATP might be one of the molecules mediating the signaling.

Optical inactivation of molecular functions in vivo by chromophore-assisted light inactivation

Many biological phenomena have spatio-temporal characteristics, such as the expression of molecular activity locally or at a limited time. Such phenomena have been observed in various organisms from slime mold to mammals, and are considered to be one of the basic patterns in biological reactions. Live imaging studies using the fluorescent protein GFP and fluorescence microscopy have become a standard technique in the life sciences to reveal the dynamics of these characteristic biological phenomena. On the other hand, the characteristic behaviors of molecules and cells captured by microscopy only correlate with life phenomena, and the causal relationship of whether they really matter is unknown. It is unclear whether they are really important or not. Therefore, to elucidate their physiological significance, it is important to introduce spatiotemporal manipulation techniques to manipulate molecules and cells locally and at arbitrary timing, and to perform causal analysis in vivo. The chromophore-assisted light inactivation (CALI) method, which uses light to inactivate molecular functions, is an optical technology that enables such spatiotemporal manipulation, and has recently been used in vivo in various model organisms, attracting widespread attention. In this section, we will review the principle of the CALI method, actual research examples, in particular, its in vivo application, and future prospects.

Light-induced, spatiotemporal control of protein in the developing embryo of the sea urchin

Differential protein regulation is a critical biological process that regulates cellular activity and controls cell fate determination. It is especially important during early embryogenesis when post-transcriptional events predominate differential fate specification in many organisms. Light-induced approaches have been a powerful technology to interrogate protein functions with temporal and spatial precision, even at subcellular levels within a cell by controlling laser irradiation on the confocal microscope. However, application and efficacy of these tools need to be tested for each model system or for the cell type of interest because of the complex nature of each system. Here, we introduce two types of light-induced approaches to track and control proteins at a subcellular level in the developing embryo of the sea urchin. We found that the photoconvertible fluorescent protein Kaede is highly efficient to distinguish pre-existing and newly synthesized proteins with no apparent phototoxicity, even when interrogating proteins associated with the mitotic spindle. Further, chromophore-assisted light inactivation (CALI) using miniSOG successfully inactivated target proteins of interest in the vegetal cortex and selectively delayed or inhibited asymmetric cell division. Overall, these light-induced manipulations serve as important molecular tools to identify protein function for for subcellular interrogations in developing embryos.

The Axonal Glycolytic Pathway Contributes to Sensory Axon Extension and Growth Cone Dynamics

Understanding the bioenergetics of axon extension and maintenance has wide ranging implications for neurodevelopment and disease states. Glycolysis is a pathway consisting of 10 enzymes and separated into preparatory and payoff phases, the latter producing ATP. Using embryonic chicken sensory neurons, we report that glycolytic enzymes are found through the axon and the growth cone. Pharmacological inhibition of glycolysis in the presence of NGF impairs axon extension and growth cone dynamics within minutes without affecting axon maintenance. Experiments using microfluidic chambers show that the effect of inhibiting glycolysis on axon extension is local along distal axons and can be reversed by promoting mitochondrial respiration. Knockdown of GAPDH simplifies growth cone morphology and is rescued by shRNA-resistant GAPDH expression. Rescue of GAPDH using KillerRed fused to GAPDH followed by localized chromophore-assisted light inactivation of KillerRed-GAPDH in distal axons halts growth cone dynamics. Considering filament polymerization requires ATP, inhibition of glycolysis results in a paradoxical increase in axonal actin filament levels. The effect on actin filaments is because of enzymes before GAPDH, the first enzyme in the payoff phase. In the absence of NGF, inhibition of glycolysis along distal axons results in axon degeneration independent of cell death. These data indicate that the glycolytic pathway is operative in distal axons and contributes to the rate of axon extension and growth cone dynamics in the presence of NGF and that, in the absence of NGF, the axonal glycolytic pathway is required for axon maintenance.SIGNIFICANCE STATEMENT Elucidation of the sources of ATP required for axon extension and maintenance has implications for understanding the mechanism of neuronal development and diseases of the nervous system. While recent work has emphasized the importance of mitochondrial oxidative phosphorylation, the role of the glycolytic pathway in axon morphogenesis and maintenance remains minimally understood. The data reveal that the glycolytic pathway is required for normal sensory axon extension in the presence of NGF, while in the absence of NGF the glycolytic pathway is required for axon maintenance. The results have implications for the understanding of the bioenergetics of axon morphogenesis and plasticity and indicate that NGF has protective effects on sensory axon maintenance in hypoglycemic states.

Optical manipulation of molecular function by chromophore-assisted light inactivation.

In addition to simple on/off switches for molecular activity, spatiotemporal dynamics are also thought to be important for the regulation of cellular function. However, their physiological significance and in vivo importance remain largely unknown. Fluorescence imaging technology is a powerful technique that can reveal the spatiotemporal dynamics of molecular activity. In addition, because imaging detects the correlations between molecular activity and biological phenomena, the technique of molecular manipulation is also important to analyze causal relationships. Recent advances in optical manipulation techniques that artificially perturb molecules and cells via light can address this issue to elucidate the causality between manipulated target and its physiological function. The use of light enables the manipulation of molecular activity in microspaces, such as organelles and nerve spines. In this review, we describe the chromophore-assisted light inactivation method, which is an optical manipulation technique that has been attracting attention in recent years.

Optical inactivation of synaptic molecules by chromophore-assisted light inactivation

It is suggested that spatiotemporal dynamics of molecular activities such as locality and timing are important as well as simple ON / OFF system in the living cells. On the other hand, the physiological significance of these activities and the extent of their contribution to the in vivo remain largely unknown. Recently, live imaging has been used as a method to approach these issues. Although imaging technique has revealed the correlation between molecular and cellular dynamics and biological phenomena, new methods are needed to elucidate the causal relationship between them. The CALI (chromophore-assisted light inactivation) is one of the optical technologies for molecular inactivation by using photosensitizers that release reactive oxygen species upon irradiation. Such locally and temporally inactivation of target molecules by light is effective in elucidating the spatiotemporal properties of molecules, which are difficult to approach by genome manipulation. Our new CALI method is attracting a lot of attention as a next-generation technology for neuroscience (Humeau Y et al. Nat. Neurosci. 2019, Frank JA et al. Nat. Biotechnol. 2019, Paoletti P et al. Nat. Rev. Neurosci. 2019 etc.). In this presentation, we will first introduce the elemental technology of our highly efficient and versatile CALI methods. In addition, the application of the CALI method to novel molecules will be introduced in detail, including our recently reported work on elucidating the memory mechanism using the CALI method (Takemoto K et al. Nat. Biotechnol. 2017).

Genetically Encoded Photosensitizer for Destruction of Protein or Cell Function.

There are several paths when excited molecules return to the ground state. In the case of fluorescent molecules, the dominant path is fluorescence emission that is greatly contributing to bioimaging. Meanwhile, photosensitizers transfer electron or energy from chromophore to the surrounding molecules, including molecular oxygen. Generated reactive oxygen species has potency to attack other molecules by oxidation. In this chapter, we introduce the chromophore-assisted light inactivation (CALI) method using a photosensitizer to inactivate proteins in a spatiotemporal manner and development of CALI tools, which is useful for investigation of protein functions and dynamics, by inactivation of the target molecules. Moreover, photosensitizers with high efficiency make it possible optogenetic control of cell ablation in living organisms and photodynamic therapy. Further development of photosensitizers with different excitation wavelengths will contribute to the investigation of multiple proteins or cell functions through inactivation in the different positions and timings.

Double-Stranded DNA Matrix for Photosensitization Switching

Photosensitization, originated from the activation of triplet states, is the basis of many photodynamic applications, but often competes with a series of nonradiative processes. Herein, we communic...

Rational Design of Bioavailable Photosensitizers for Manipulation and Imaging of Biological Systems

Light-mediated chemical reactions are powerful methods for manipulating and interrogating biological systems. Photosensitizers, compounds that generate reactive oxygen species upon excitation with light, can be utilized for numerous biological experiments, but the repertoire of bioavailable photosensitizers is limited. Here, we describe the synthesis, characterization, and utility of two photosensitizers based upon the widely used rhodamine scaffold and demonstrate their efficacy for chromophore-assisted light inactivation, cell ablation in culture and invivo, and photopolymerization of diaminobenzidine for electron microscopy. These chemical tools will facilitate a broad range of applications spanning from targeted destruction of proteins to high-resolution imaging.

Growing tool-kit of photosensitizers for clinical and non-clinical applications.

Photosensitizers are photosensitive molecules utilized in clinical and non-clinical applications by taking advantage of light-mediated reactive oxygen generation, which triggers local and systemic cellular toxicity. Photosensitizers are used for diverse biological applications such as spatio-temporal inactivation of a protein in a living system by chromophore-assisted light inactivation, localized cell photoablation, photodynamic and immuno-photodynamic therapy, and correlative light-electron microscopy imaging. Substantial efforts have been made to develop several genetically encoded, chemically synthesized, and nanotechnologically driven photosensitizers for successful implementation in redox biology applications. Genetically encoded photosensitizers (GEPS) or reactive oxygen species (ROS) generating proteins have the advantage of using them in the living system since they can be manipulated by genetic engineering with a variety of target-specific genes for the precise spatio-temporal control of ROS generation. The GEPS variety is limited but is expanding with a variety of newly emerging GEPS proteins. Apart from GEPS, a large variety of chemically- and nanotechnologically-empowered photosensitizers have been developed with a major focus on photodynamic therapy-based cancer treatment alone or in combination with pre-existing treatment methods. Recently, immuno-photodynamic therapy has emerged as an effective cancer treatment method using smartly designed photosensitizers to initiate and engage the patient's immune system so as to empower the photosensitizing effect. In this review, we have discussed various types of photosensitizers, their clinical and non-clinical applications, and implementation toward intelligent efficacy, ROS efficiency, and target specificity in biological systems.

A genetically encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling

BackgroundIntrabodies enable targeting of proteins in live cells, but generating specific intrabodies against the thousands of proteins in a proteome poses a challenge. We leverage the widespread availability of fluorescently labelled proteins to visualize and manipulate intracellular signalling pathways in live cells by using nanobodies targeting fluorescent protein tags.ResultsWe generated a toolkit of plasmids encoding nanobodies against red and green fluorescent proteins (RFP and GFP variants), fused to functional modules. These include fluorescent sensors for visualization of Ca2+, H+ and ATP/ADP dynamics; oligomerising or heterodimerising modules that allow recruitment or sequestration of proteins and identification of membrane contact sites between organelles; SNAP tags that allow labelling with fluorescent dyes and targeted chromophore-assisted light inactivation; and nanobodies targeted to lumenal sub-compartments of the secretory pathway. We also developed two methods for crosslinking tagged proteins: a dimeric nanobody, and RFP-targeting and GFP-targeting nanobodies fused to complementary hetero-dimerizing domains. We show various applications of the toolkit and demonstrate, for example, that IP3 receptors deliver Ca2+ to the outer membrane of only a subset of mitochondria and that only one or two sites on a mitochondrion form membrane contacts with the plasma membrane.ConclusionsThis toolkit greatly expands the utility of intrabodies and will enable a range of approaches for studying and manipulating cell signalling in live cells.

Chromophore-Assisted Light Inactivation of the V-ATPase V0c Subunit Inhibits Neurotransmitter Release Downstream of Synaptic Vesicle Acidification

Synaptic vesicle proton V-ATPase is an essential component in synaptic vesicle function. Active acidification of synaptic vesicles, triggered by the V-ATPase, is necessary for neurotransmitter storage. Independently from its proton transport activity, an additional important function of the membrane-embedded sector of the V-ATPase has been uncovered over recent years. Subunits a and c of the membrane sector of this multi-molecular complex have been shown to interact with SNARE proteins and to be involved in modulating neurotransmitter release. The c-subunit interacts with the v-SNARE VAMP2 and facilitates neurotransmission. In this study, we used chromophore-assisted light inactivation and monitored the consequences on neurotransmission on line in CA3 pyramidal neurons. We show that V-ATPase c-subunit V0c is a key element in modulating neurotransmission and that its specific inactivation rapidly inhibited neurotransmission.

Quantification of light-induced miniSOG superoxide production using the selective marker, 2-hydroxyethidium.

Genetically-encoded photosensitizers produce reactive oxygen species (ROS) in response to light. Transgenic expression of fusion proteins can target the photosensitizers to specific cell regions and permit the spatial and temporal control of ROS production. These ROS-generating proteins (RGPs) are widely used for cell ablation, mutagenesis and chromophore-assisted light inactivation of target proteins. However, the species produced by RGPs are unclear due to indirect measures with confounding interpretations. Recently, the RGP mini "Singlet Oxygen Generator" (miniSOG) was engineered from Arabidopsis thaliana phototropin 2. While miniSOG produces singlet oxygen (1O2), the contribution of superoxide (O2•-) to miniSOG-generated ROS remains unclear. We measured the light-dependent O2•- production of purified miniSOG using HPLC separation of dihydroethidium (DHE) oxidation products. We demonstrate that DHE is insensitive to 1O2 and establish that DHE is a suitable indicator to measure O2•- production in a system that produces both 1O2 and O2•-. We report that miniSOG produces both 1O2 and O2•-, as can its free chromophore, flavin mononucleotide. miniSOG produced O2•- at a rate of ~4.0µmol O2•-/min/µmol photosensitizer for an excitation fluence rate of 5.9mW/mm2 at 470 ± 20nm, and the rate remained consistent across fluences (light doses). Overall, the contribution of O2•- to miniSOG phenotypes should be considered.

Conjugated polyelectrolytes with a label-free aptamer for specific protein photoinactivation

A novel and convenient chromophore-assisted light inactivation strategy is developed for specific and efficient protein inactivation based on conjugated polyelectrolytes and a label-free aptamer.