Tetramethylalloxazines as efficient singlet oxygen photosensitizers and potential redox-sensitive agents.

Tetramethylalloxazines (TMeAll) have been found to have a high quantum yield of singlet oxygen generation when used as photosensitizers. Their electronic structure and transition energies (S0 → Si, S0 → Ti, T1 → Ti) were calculated using DFT and TD-DFT methods and compared to experimental absorption spectra. Generally, TMeAll display an energy diagram similar to other derivatives belonging to the alloxazine class of compounds, namely π,π* transitions are accompanied by closely located n,π* transitions. Photophysical data such as quantum yields of fluorescence, fluorescence lifetimes, and nonradiative rate constants were also studied in methanol (MeOH), acetonitrile (ACN), and 1,2-dichloroethane (DCE). The transient absorption spectra were also analyzed. To assess cytotoxicity of new compounds, a hemolytic assay was performed using human red blood cells (RBC) in vitro. Subsequently, fluorescence lifetime imaging experiments (FLIM) were performed on RBC under physiological and oxidative stress conditions alone or in the presence of TMeAll allowing for pinpointing changes caused by those compounds on the intracellular environment of these cells.

5-Deazaalloxazine as photosensitizer of singlet oxygen and potential redox-sensitive agent.

Flavins are a unique class of compounds that combine the features of singlet oxygen generators and redox-dependent fluorophores. From a broad family of flavin derivatives, deazaalloxazines are significantly underdeveloped from the point of view of photophysical properties. Herein, we report photophysics of 5-deazaalloxazine (1a) in water, acetonitrile, and some other solvents. In particular, triplet excited states of 1a in water and in acetonitrile were investigated using ultraviolet-visible (UV-Vis) transient absorption spectroscopy. The measured triplet lifetimes for 1a were all on the microsecond time scale (≈ 60 µs) in deoxygenated solutions. The quantum yield of S1 → T1 intersystem crossing for 1a in water was 0.43 based on T1 energy transfer from 1a to indicaxanthin (5) acting as acceptor and on comparative actinometric measurements using benzophenone (6). 1a was an efficient photosensitizer for singlet oxygen in aerated solutions, with quantum yields of singlet oxygen in methanol of about 0.76, compared to acetonitrile ~ 0.74, dichloromethane ~ 0.64 and 1,2-dichloroethane ~ 0.54. Significantly lower singlet oxygen quantum yields were obtained in water and deuterated water (Ð¤Δ ~ 0.42 and 0.44, respectively). Human red blood cells (RBC) were used as a cell model to study the antioxidant capacity in vitro and cytotoxic activity of 1a. Fluorescence-lifetime imaging microscopy (FLIM) data were analyzed by fluorescence lifetime parameters and distribution for different parts of the emission spectrum. Comparison of multidimensional fluorescent properties of RBC under physiological-like and oxidative-stress conditions in the presence and absence of 1a suggests its dual activity as probe and singlet-oxygen generator and opens up a pathway for using FLIM to analyze complex intracellular behavior of flavin-like compounds. These new data on structure-property relationship contribute to the body of information required for a rational design of flavin-based tools for future biological and biochemical applications.

Elucidating regulators of astrocytic Ca2+ signaling via multi-threshold event detection (MTED).

Recent achievements in indicator optimization and imaging techniques promote the advancement of functional imaging to decipher complex signaling processes in living cells, such as Ca2+ activity patterns. Astrocytes are important regulators of the brain network and well known for their highly complex morphology and spontaneous Ca2+ activity. However, the astrocyte community is lacking standardized methods to analyze and interpret Ca2+ activity recordings, hindering global comparisons. Here, we present a biophysically-based analytical concept for deciphering the complex spatio-temporal changes of Ca2+ biosensor fluorescence for understanding the underlying signaling mechanisms. We developed a pixel-based multi-threshold event detection (MTED) analysis of multidimensional data, which accounts for signal strength as an additional signaling dimension and provides the experimenter with a comprehensive toolbox for a differentiated and in-depth characterization of fluorescence signals. MTED was validated by analyzing astrocytic Ca2+ activity across Ca2+ indicators, imaging setups, and model systems from primary cell culture to awake, head-fixed mice. We identified extended Ca2+ activity at 25°C compared to 37°C physiological body temperature and dissected how neuronal activity shapes long-lasting astrocytic Ca2+ activity. Our MTED strategy, as a parameter-free approach, is easily transferrable to other fluorescent indicators and biosensors and embraces the additional dimensionality of signaling activity strength. It will also advance the definition of standardized procedures and parameters to improve comparability of research data and reports.

Serotonin receptor 4 regulates hippocampal astrocyte morphology and function.

Astrocytes are an important component of the multipartite synapse and crucial for proper neuronal network function. Although small GTPases of the Rho family are powerful regulators of cellular morphology, the signaling modules of Rho-mediated pathways in astrocytes remain enigmatic. Here we demonstrated that the serotonin receptor 4 (5-HT4 R) is expressed in hippocampal astrocytes, both in vitro and in vivo. Through fluorescence microscopy, we established that 5-HT4 R activation triggered RhoA activity via Gα13 -mediated signaling, which boosted filamentous actin assembly, leading to morphological changes in hippocampal astrocytes. We investigated the effects of these 5-HT4 R-mediated changes in mixed cultures and in acute slices, in which 5-HT4 R was expressed exclusively in astrocytes. In both systems, 5-HT4 R-RhoA signaling changed glutamatergic synaptic transmission: It increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in mixed cultures and reduced the paired-pulse-ratio (PPR) of field excitatory postsynaptic potentials (fEPSPs) in acute slices. Overall, our present findings demonstrate that astrocytic 5-HT4 R-Gα13 -RhoA signaling is a previously unrecognized molecular pathway involved in the functional regulation of excitatory synaptic circuits.

Serotonin 5-HT4 receptor boosts functional maturation of dendritic spines via RhoA-dependent control of F-actin.

Activity-dependent remodeling of excitatory connections underpins memory formation in the brain. Serotonin receptors are known to contribute to such remodeling, yet the underlying molecular machinery remains poorly understood. Here, we employ high-resolution time-lapse FRET imaging in neuroblastoma cells and neuronal dendrites to establish that activation of serotonin receptor 5-HT4 (5-HT4R) rapidly triggers spatially-restricted RhoA activity and G13-mediated phosphorylation of cofilin, thus locally boosting the filamentous actin fraction. In neuroblastoma cells, this leads to cell rounding and neurite retraction. In hippocampal neurons in situ, 5-HT4R-mediated RhoA activation triggers maturation of dendritic spines. This is paralleled by RhoA-dependent, transient alterations in cell excitability, as reflected by increased spontaneous synaptic activity, apparent shunting of evoked synaptic responses, and enhanced long-term potentiation of excitatory transmission. The 5-HT4R/G13/RhoA signaling thus emerges as a previously unrecognized molecular pathway underpinning use-dependent functional remodeling of excitatory synaptic connections.

Graphene biointerfaces for optical stimulation of cells.

Noninvasive stimulation of cells is crucial for the accurate examination and control of their function both at the cellular and the system levels. To address this need, we present a pioneering optical stimulation platform that does not require genetic modification of cells but instead capitalizes on unique optoelectronic properties of graphene, including its ability to efficiently convert light into electricity. We report the first studies of optical stimulation of cardiomyocytes via graphene-based biointerfaces (G-biointerfaces) in substrate-based and dispersible configurations. The efficiency of stimulation via G-biointerfaces is independent of light wavelength but can be tuned by changing the light intensity. We demonstrate that an all-optical evaluation of use-dependent drug effects in vitro can be enabled using substrate-based G-biointerfaces. Furthermore, using dispersible G-biointerfaces in vivo, we perform optical modulation of the heart activity in zebrafish embryos. Our discovery is expected to empower numerous fundamental and translational biomedical studies.

Measurement of intracellular concentration of fluorescently-labeled targets in living cells.

Estimations of intracellular concentrations of fluorescently-labeled molecules within living cells are very important for guidance of biological experiments and interpretation of their results. Here we propose a simple and universal approach for such estimations. The approach is based upon common knowledge that the dye fluorescence is directly proportional to its quantum yield and the number of its molecules and that a coefficient of proportionality is determined by spectral properties of the dye and optical equipment used to record fluorescent signals. If two fluorescent dyes are present in the same volume, then a ratio of their concentrations is equal to a ratio of their fluorescence multiplied by some dye- and equipment-dependent coefficient. Thus, if the coefficient and concentration of one dye is known then the concentration of another dye can be determined. Here we have demonstrated how to calculate this coefficient (called a ratio factor) and how to use it for concentration measurements of fluorescently tagged molecules. As an example of how this approach can be used, we estimated a concentration of exogenously expressed neuronal Ca2+ sensor protein, hippocalcin, tagged by a fluorescent protein in a dendritic tree of rat hippocampal neurons loaded via a patch pipette with Alexa Fluor dye of known concentration. The new approach should allow performing a fast, inexpensive and reliable quantitative analysis of fluorescently-labeled targets in different parts of living cells.

Endocytic adaptor protein intersectin 1 forms a complex with microtubule stabilizer STOP in neurons.

Intersectin 1 (ITSN1) is a multidomain adaptor protein that functions in clathrin-mediated endocytosis and signal transduction. This protein is highly abundant in neurons and is implicated in Down syndrome, Alzheimer's disease and, possibly, other neurodegenerative disorders. Here we used an in vitro binding assay combined with MALDI-TOF mass spectrometry to identify novel binding partners of ITSN1. We found that the neuron-specific isoform of the stable tubule-only polypeptide (STOP) interacts with SH3A domain of ITSN1. STOP and ITSN1 were shown to form a complex in vivo and to partially co-localize in rat primary hippocampal neurons. As STOP is a microtubule-stabilizing protein that is required for several forms of synaptic plasticity in the hippocampus, identification of this interaction raises the possibility of ITSN1 participation in this process.

Hippocalcin signaling via site-specific translocation in hippocampal neurons.

Hippocalcin is a Ca(2+)-binding protein, which belongs to the family of neuronal Ca(2+) sensors. It is highly expressed in the hippocampus but molecular mechanisms underlying its action in this part of the brain have not been investigated in detail. To study whether intrinsic neuronal activity could result in hippocalcin-mediated signal transduction we examined spontaneous and action potential (AP)-dependent changes in fluorescence of yellow fluorescent protein-tagged hippocalcin (HPCA-YFP) in transiently transfected hippocampal cultured neurons. In 6-12 DIV neurons HPCA-YFP spontaneously translocated longitudinally to specific sites within diffusionally confined domains of neuronal processes. The translocations to these sites were expressed as fast, reversible increases in HPCA-YFP fluorescence coincided with a decrease in adjacent sites indicating genuine protein translocation. Physiologically relevant neuronal stimulation with short trains of action potentials also resulted in fast, simultaneous, reversible, and [Ca(2+)](i)-dependent translocations of HPCA-YFP to several sites synchronizing hippocalcin signaling in different parts of neuronal processes. The amount of translocated protein increased with the number of action potentials in a train decoding the number of APs into the amount of translocated protein. We conclude that hippocalcin may signal within diffusionally restricted domains of neuronal processes in which it might play a physiological role in Ca(2+)-dependent local activation of specific molecular targets.