[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.

Stepwise synaptic plasticity events drive the early phase of memory consolidation.

Memories are initially encoded in the hippocampus but subsequently consolidated to the cortex. Although synaptic plasticity is key to these processes, its precise spatiotemporal profile remains poorly understood. Using optogenetics to selectively erase long-term potentiation (LTP) within a defined temporal window, we found that distinct phases of synaptic plasticity play differential roles. The first wave acts locally in the hippocampus to confer context specificity. The second wave, during sleep on the same day, organizes these neurons into synchronously firing assemblies. Finally, LTP in the anterior cingulate cortex during sleep on the second day is required for further stabilization of the memory. This demonstrates the precise localization, timing, and characteristic contributions of the plasticity events that underlie the early phase of memory consolidation.

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.

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.

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.

Photoactivated chromophore for infectious keratitis - Corneal cross-linking (PACK-CXL): A systematic review and meta-analysis.

PURPOSE: To examine the efficacy of adjuvant photoactivated chromophore for infectious keratitis-corneal cross-linking (PACK-CXL) for the treatment of infectious keratitis (IK). METHODS: Electronic databases, including MEDLINE, EMBASE and Cochrane Central, were searched for articles related to PACK-CXL. All clinical studies, including randomized controlled trials (RCTs), non-randomized controlled studies, case series and case reports, were included. A meta-analysis was further performed when there were sufficient similarities in the included RCTs. Primary outcome measure was time to complete corneal healing and secondary outcome measures included size of epithelial defect and infiltrate, corrected-distance-visual-acuity (CDVA), and adverse events. RESULTS: Forty-six eligible studies (including four RCTs) with 435 patients were included. When compared to standard antimicrobial treatment (SAT) alone, adjuvant PACK-CXL resulted in shorter mean time to complete corneal healing (-7.44 days; 95% CI, -10.71 to -4.16) and quicker resolution of the infiltrate at 7 days (-5.49 mm2; 95% CI, -7.44 to -3.54) and at 14-30 days (-5.27 mm2; 95% CI, -9.12 to -1.41). There was no significant difference in the size of epithelial defect, CDVA and risk of adverse events. Evidence on the use of PACK-CXL in acanthamoeba and mixed IK was insufficient. CONCLUSIONS: Our study demonstrates that adjuvant PACK-CXL expedites the healing of IK when compared to SAT alone (low-quality evidence). Further adequately powered, high-quality RCTs are required to fully ascertain the therapeutic effect of PACK-CXL.

[Novel Visible Light Photoactivatable Caged Neurotransmitters Based on a N-Methyl Quinolinium Chromophore].

The development of novel photolabile protecting groups with practical levels of photolytic efficiency and hydrophilicity can provide smart photochemical tools, such as caged compounds. One of the long-standing problems of most reported photolabile protecting groups is the requirement for one-photon activation, of ultraviolet light (250-400 nm), that is harmful to living cells and has low tissue penetration power. An attractive approach to overcome this would be the use of longer-wavelength light for one-photon activation; advantages would include both lower phototoxicity and higher tissue penetration power than UV irradiation. As part of our research aimed at developing new photochemical tools, we have developed the N-methyl-7-hydroxyquinolinium (N-Me-7-HQm) caging chromophore as a novel photocage, sensitive to visible light. A key to the success of the development of the N-Me-7-HQm photocage was simple N-methylation of the 7-hydroxyquinoline chromophore. This modification allows access to visible light absorbance, facile photoactivation by blue-LED light (458 nm) with high photolytic efficiency, excellent water solubility, and high resistance to spontaneous hydrolysis. The success of the late stage upgrading of a chromophore in the synthetic sequence suggests that further functionalization of the caging chromophore will be possible, and should aid in the rapid generation of structurally diverse libraries of visible light-sensitive photocages.

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.

Using fluorescence detected two-dimensional spectroscopy to investigate initial exciton delocalization between coupled chromophores.

Förster theory describes electronic exciton energy migration in molecular assemblies as an incoherent hopping process between donor and acceptor molecules. The rate is expressed in terms of the overlap integral between donor fluorescence and acceptor absorption spectra. Typical time scales for systems like photosynthetic antennae are on the order of a few picoseconds. Prior to transfer, it is assumed that the initially excited donor molecule has equilibrated with respect to the local environment. However, upon excitation and during the equilibration phase, the state of the system needs to be described by the full density matrix, including coherences between donor and acceptor states. While being intuitively clear, addressing this regime experimentally has been a challenge until the recently reported advances in fluorescence detected two-dimensional spectroscopy. Here, we demonstrate using fourth order perturbation theory the conditions for the presence of donor-acceptor coherence induced cross-peaks at zero waiting time between the first and the second pair of pulses. The approach is illustrated for a heterodimer model which facilitates an analytical solution.

An experimental methodology to measure the reaction rate constants of processes sensitised by the triplet state of 4-carboxybenzophenone as a proxy of the triplet states of chromophoric dissolved organic matter, under steady-state irradiation conditions.

By a combination of transient absorption spectroscopy and steady-state irradiation experiments, we investigated the transformation of phenol and furfuryl alcohol (FFA) sensitised by irradiated 4-carboxybenzophenone (CBBP). The latter is a reasonable proxy molecule to assess the reactivity of the excited triplet states of the chromophoric dissolved organic matter that occurs in natural waters. The main reactive species for the transformation of both phenol and FFA was the CBBP triplet state, despite the fact that FFA is a commonly used probe for 1O2. In the case of FFA it was possible to develop a simple kinetic model that fitted well the experimental data obtained by steady-state irradiation, in a wide range of FFA concentration values. In the case of phenol the model was made much more complex by the likely occurrence of back reactions between radical species (e.g., phenoxyl and superoxide). This problem can be tackled by considering only the experimental data at low phenol concentration, where the degradation rate increases linearly with concentration. We do not recommend the use of 1O2 scavengers/quenchers such as sodium azide to elucidate CBBP photoreaction pathways, because the azide provides misleading results by also acting as a triplet-state quencher. Based on the experimental data, we propose a methodology for the measurement of the CBBP triplet-sensitisation rate constants from steady-state irradiation experiments, allowing for a better assessment of the triplet-sensitised degradation of emerging contaminants.

Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex.

Quantitative measurement of transcription rates in live cells is important for revealing mechanisms of transcriptional regulation. This is particularly challenging when measuring the activity of RNA polymerase III (Pol III), which transcribes growth-promoting small RNAs. To address this issue, we developed Corn, a genetically encoded fluorescent RNA reporter suitable for quantifying RNA transcription in cells. Corn binds and induces fluorescence of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime, which resembles the fluorophore found in red fluorescent protein (RFP). Notably, Corn shows high photostability, enabling quantitative fluorescence imaging of mTOR-dependent Pol III transcription. We found that, unlike actinomycin D, mTOR inhibitors resulted in heterogeneous transcription suppression in individual cells. Quantitative imaging of Corn-tagged Pol III transcript levels revealed distinct Pol III transcription 'trajectories' elicited by mTOR inhibition. Together, these studies provide an approach for quantitative measurement of Pol III transcription by direct imaging of Pol III transcripts containing a photostable RNA-fluorophore complex.

The N6-Position of Adenine Is a Blind Spot for TAL-Effectors That Enables Effective Binding of Methylated and Fluorophore-Labeled DNA.

Transcription-activator-like effectors (TALEs) are programmable DNA binding proteins widely used for genome targeting. TALEs consist of multiple concatenated repeats, each selectively recognizing one nucleobase via a defined repeat variable diresidue (RVD). Effective use of TALEs requires knowledge about their binding ability to epigenetic and other modified nucleobases occurring in target DNA. However, aside from epigenetic cytosine-5 modifications, the binding ability of TALEs to modified DNA is unknown. We here study the binding of TALEs to the epigenetic nucleobase N6-methyladenine (6mA) found in prokaryotic and recently also eukaryotic genomes. We find that the natural, adenine (A)-binding RVD NI is insensitive to 6mA. Model-assisted structure-function studies reveal accommodation of 6mA by RVDs with altered hydrophobic surfaces and abilities of hydrogen bonding to the N6-amino group or N7 atom of A. Surprisingly, this tolerance of N6 substitution was transferrable to bulky N6-alkynyl substituents usable for click chemistry and even to a large rhodamine dye, establishing the N6 position of A as the first site of DNA that offers label introduction within TALE target sites without interference. These findings will guide future in vivo studies with TALEs and expand their applicability as DNA capture probes for analytical applications in vitro.

Blue fluorescent amino acid for biological spectroscopy and microscopy.

Many fluorescent proteins are currently available for biological spectroscopy and imaging measurements, allowing a wide range of biochemical and biophysical processes and interactions to be studied at various length scales. However, in applications where a small fluorescence reporter is required or desirable, the choice of fluorophores is rather limited. As such, continued effort has been devoted to the development of amino acid-based fluorophores that do not require a specific environment and additional time to mature and have a large fluorescence quantum yield, long fluorescence lifetime, good photostability, and an emission spectrum in the visible region. Herein, we show that a tryptophan analog, 4-cyanotryptophan, which differs from tryptophan by only two atoms, is the smallest fluorescent amino acid that meets these requirements and has great potential to enable in vitro and in vivo spectroscopic and microscopic measurements of proteins.

Performing Chromophore-Assisted Laser Inactivation in Drosophila Embryos Using GFP.

Chromophore-assisted laser inactivation (CALI) is an optogenetic technique in which light-induced release of reactive oxygen species triggers acute inactivation of a protein of interest, with high spatial and temporal resolution. At its simplest, selective protein inactivation can be achieved via the genetic fusion of the protein to a photosensitizer such as EGFP, and using standard optical setups such as laser scanning confocal microscopes. Although use of CALI in Drosophila is relatively recent, this technique can be a powerful complement to developmental genetics, especially in vivo as it allows visualization of the immediate consequences of local protein inactivation when coupled to time-lapse microscopy analysis. In addition to providing examples of protocols, this chapter is intended as a conceptual framework to support the rational design of CALI experiments.

Chromophore-Assisted Light Inactivation of Mitochondrial Electron Transport Chain Complex II in Caenorhabditis elegans.

Mitochondria play critical roles in meeting cellular energy demand, in cell death, and in reactive oxygen species (ROS) and stress signaling. Most Caenorhabditis elegans loss-of-function (lf) mutants in nuclear-encoded components of the respiratory chain are non-viable, emphasizing the importance of respiratory function. Chromophore-Assisted Light Inactivation (CALI) using genetically-encoded photosensitizers provides an opportunity to determine how individual respiratory chain components contribute to physiology following acute lf. As proof-of-concept, we expressed the 'singlet oxygen generator' miniSOG as a fusion with the SDHC subunit of respiratory complex II, encoded by mev-1 in C. elegans, using Mos1-mediated Single Copy Insertion. The resulting mev-1::miniSOG transgene complemented mev-1 mutant phenotypes in kn1 missense and tm1081(lf) deletion mutants. Complex II activity was inactivated by blue light in mitochondria from strains expressing active miniSOG fusions, but not those from inactive fusions. Moreover, light-inducible phenotypes in vivo demonstrated that complex II activity is important under conditions of high energy demand, and that specific cell types are uniquely susceptible to loss of complex II. In conclusion, miniSOG-mediated CALI is a novel genetic platform for acute inactivation of respiratory chain components. Spatio-temporally controlled ROS generation will expand our understanding of how the respiratory chain and mitochondrial ROS influence whole organism physiology.

Photoactivatable aggregation-induced emission fluorophores with multiple-color fluorescence and wavelength-selective activation.

Photoactivatable (caged) fluorophores are widely used in chemistry, materials, and biology. However, the development of such molecules exhibiting photoactivable solid-state fluorescence is still challenging due to the aggregation-caused quenching (ACQ) effect of most fluorophores in their aggregate or solid states. In this work, we developed caged salicylaldehyde hydrazone derivatives, which are of aggregation-induced emission (AIE) characteristics upon light irradiation, as efficient photoactivatable solid-state fluorophores. These compounds displayed multiple-color emissions and ratiometric (photochromic) fluorescence switches upon wavelength-selective photoactivation, and were successfully applied for photopatterning and photoactivatable cell imaging in a multiple-color and stepwise manner.

Chromophore-assisted laser inactivation--towards a spatiotemporal-functional analysis of proteins, and the ablation of chromatin, organelle and cell function.

Chromophore-assisted laser or light inactivation (CALI) has been employed as a promising technique to achieve spatiotemporal knockdown or loss-of-function of target molecules in situ. CALI is performed using photosensitizers as generators of reactive oxygen species (ROS). There are two CALI approaches that use either transgenic tags with chemical photosensitizers, or genetically encoded fluorescent protein fusions. Using spatially restricted microscopy illumination, CALI can address questions regarding, for example, protein isoforms, subcellular localization or phase-specific analyses of multifunctional proteins that other knockdown approaches, such as RNA interference or treatment with chemicals, cannot. Furthermore, rescue experiments can clarify the phenotypic capabilities of CALI after the depletion of endogenous targets. CALI can also provide information about individual events that are involved in the function of a target protein and highlight them in multifactorial events. Beyond functional analysis of proteins, CALI of nuclear proteins can be performed to induce cell cycle arrest, chromatin- or locus-specific DNA damage. Even at organelle level - such as in mitochondria, the plasma membrane or lysosomes - CALI can trigger cell death. Moreover, CALI has emerged as an optogenetic tool to switch off signaling pathways, including the optical depletion of individual neurons. In this Commentary, we review recent applications of CALI and discuss the utility and effective use of CALI to address open questions in cell biology.

Permeation of fluorophore-conjugated phalloidin into live hair cells of the inner ear is modulated by P2Y receptors.

Phalloidin, a toxin isolated from the death cap mushroom, Amanita phalloides, binds to filamentous actin with high affinity, and this has made fluorophore-conjugated phalloidin a useful tool in cellular imaging. Hepatocytes take up phalloidin via the liver-specific organic anion transporting polypeptide 1b2, but phalloidin does not permeate most living cells. Rapid entry of styryl dyes into live hair cells has been used to evaluate function, but the usefulness of those fluorescence dyes is limited by broad and fixed absorption spectra. Since phalloidin can be conjugated to fluorophores with various spectra, we investigated whether it would permeate living hair cells. When we incubated mouse utricles in 66 nM phalloidin-CF488A and followed that by washes in phalloidin-free medium, we observed that it entered a subset of hair cells and labeled entire hair bundles fluorescently after 20 min. Incubations of 90 min labeled nearly all the hair bundles. When phalloidin-treated utricles were cultured for 24 h after washout, the label disappeared from the hair cells and progressively but heterogeneously labeled filamentous actin in the supporting cells. We investigated how phalloidin may enter hair cells and found that P2 receptor antagonists, pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid and suramin, blocked phalloidin entry, while the P2Y receptor ligands, uridine-5'-diphosphate and uridine-5'-triphosphaste, stimulated uptake. Consistent with that, the P2Y6 receptor antagonist, MRS 2578, decreased phalloidin uptake. The results show that phalloidin permeates live hair cells through a pathway that requires metabotropic P2Y receptor signaling and suggest that phalloidin can be transferred from hair cells to supporting cells in culture.