Inhibition of Osteoclastogenesis by Mechanically Loaded Osteocytes: Involvement of MEPE

In regions of high bone loading, the mechanoresponsive osteocytes inhibit osteoclastic bone resorption by producing signaling molecules. One possible candidate is matrix extracellular phosphoglycoprotein (MEPE) because acidic serine- and aspartate-rich MEPE-associated motif peptides upregulate osteoprotegerin (OPG) gene expression, a negative regulator of osteoclastogenesis. These peptides are cleaved from MEPE when relatively more MEPE than PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome) is present. We investigated whether mechanical loading of osteocytes affects osteocyte-stimulated osteoclastogenesis by involvement of MEPE. MLO-Y4 osteocytes were mechanically loaded by 1-h pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) or kept under static control conditions. Recombinant MEPE (0.05, 0.5, or 5 μg/ml) was added to some static cultures. Mouse bone marrow cells were seeded on top of the osteocytes to determine osteoclastogenesis. Gene expression of MEPE, PHEX, receptor activator of nuclear factor kappa-B ligand (RANKL), and OPG by osteocytes was determined after PFF. Osteocytes supported osteoclast formation under static control conditions. Both PFF and recombinant MEPE inhibited osteocyte-stimulated osteoclastogenesis. PFF upregulated MEPE gene expression by 2.5-fold, but not PHEX expression. PFF decreased the RANKL/OPG ratio at 1-h PFF treatment. Our data suggest that mechanical loading induces changes in gene expression by osteocytes, which likely contributes to the inhibition of osteoclastogenesis after mechanical loading of bone. Because mechanical loading upregulated gene expression of MEPE but not PHEX, possibly resulting in the upregulation of OPG gene expression, we speculate that MEPE is a soluble factor involved in the inhibition of osteoclastogenesis by osteocytes

Design and Application of Photoinduced-Electron Transfer-Based Voltage-Sensitive Dyes to Biological Imaging

Voltage-sensitive dyes have provided recent promise for affording new methods to interrogate the electrical activity of biological circuits. In particular, small-molecule based approaches have proven useful for designing sensitive molecules that can observe neuronal activity in a noninvasive, highly-parallel manner. However, currently-available voltage-sensitive dyes are marred by low sensitivity, brightness, and/or poor solubility. In order to overcome these challenges, we designed and synthesized voltage-sensitive dyes based on a new family of photoinduced-electron transfer-based voltage sensors, or VoltageFluors. Using the VoltageFluor scaffold, we designed two new families of rhodol- and rhodamine-based voltage-sensitive dyes optimized for two-photon voltage imaging in thick biological tissues, such as brain slices and intact brains. Using these dyes, we characterized the neuronal phenotype associated with the epileptic disorder tuberous sclerosis and monitored spiking activity in awake, behaving mice. We then developed a synergistic computational and experimental approach to guide the rational design of new VoltageFluor dyes. Through this approach, we design and test the most voltage-sensitive VoltageFluor dye to date and also the brightest, highest signal-to-noise fluorescein-based VoltageFluor dye. This work lays the foundation for the diverse array of biological applications of VoltageFluor dyes and develops the guiding principles for future dye design

Voltage-sensitive rhodol with enhanced two-photon brightness.

We have designed, synthesized, and applied a rhodol-based chromophore to a molecular wire-based platform for voltage sensing to achieve fast, sensitive, and bright voltage sensing using two-photon (2P) illumination. Rhodol VoltageFluor-5 (RVF5) is a voltage-sensitive dye with improved 2P cross-section for use in thick tissue or brain samples. RVF5 features a dichlororhodol core with pyrrolidyl substitution at the nitrogen center. In mammalian cells under one-photon (1P) illumination, RVF5 demonstrates high voltage sensitivity (28% ΔF/F per 100 mV) and improved photostability relative to first-generation voltage sensors. This photostability enables multisite optical recordings from neurons lacking tuberous sclerosis complex 1, Tsc1, in a mouse model of genetic epilepsy. Using RVF5, we show that Tsc1 KO neurons exhibit increased activity relative to wild-type neurons and additionally show that the proportion of active neurons in the network increases with the loss of Tsc1. The high photostability and voltage sensitivity of RVF5 is recapitulated under 2P illumination. Finally, the ability to chemically tune the 2P absorption profile through the use of rhodol scaffolds affords the unique opportunity to image neuronal voltage changes in acutely prepared mouse brain slices using 2P illumination. Stimulation of the mouse hippocampus evoked spiking activity that was readily discerned with bath-applied RVF5, demonstrating the utility of RVF5 and molecular wire-based voltage sensors with 2P-optimized fluorophores for imaging voltage in intact brain tissue

A modular platform to develop peptoid-based selective fluorescent metal sensors.

Despite the reduction in industrial use of toxic heavy metals, there remain contaminated natural water sources across the world. Herein we present a modular platform for developing selective sensors for toxic metal ions using N-substituted glycine, or peptoid, oligomers coupled to a fluorophore. As a preliminary evaluation of this strategy, structures based on previously identified metal-binding peptoids were synthesized with terminal pyrene moieties. Both derivatives of this initial design demonstrated a turn-off response in the presence of various metal ions. A colorimetric screen was designed to identify a peptoid ligand that chelates Hg(ii). Multiple ligands were identified that were able to deplete Hg(ii) from a solution selectively in the presence of an excess of competing ions. The C-terminal fluoropeptoid derivatives demonstrated similar selectivity to their label-free counterparts. This strategy could be applied to develop sensors for many different metal ions of interest using a variety of fluorophores, leading to a panel of sensors for identifying various water source contaminants

Isomerically Pure Tetramethylrhodamine Voltage Reporters

We present the design, synthesis, and application of a new family of fluorescent voltage indicators based on isomerically pure tetramethylrhodamines. These new <b>Rho</b>damine <b>V</b>oltage <b>R</b>eporters, or RhoVRs, use photoinduced electron transfer (PeT) as a trigger for voltage sensing, display excitation and emission profiles in the green to orange region of the visible spectrum, demonstrate high sensitivity to membrane potential changes (up to 47% Δ<i>F</i>/<i>F</i> per 100 mV), and employ a tertiary amide derived from sarcosine, which aids in membrane localization and simultaneously simplifies the synthetic route to the voltage sensors. The most sensitive of the RhoVR dyes, RhoVR 1, features a methoxy-substituted diethylaniline donor and phenylenevinylene molecular wire at the 5′-position of the rhodamine aryl ring, exhibits the highest voltage sensitivity to date for red-shifted PeT-based voltage sensors, and is compatible with simultaneous imaging alongside green fluorescent protein-based indicators. The discoveries that sarcosine-based tertiary amides in the context of molecular-wire voltage indicators prevent dye internalization and 5′-substituted voltage indicators exhibit improved voltage sensitivity should be broadly applicable to other types of PeT-based voltage-sensitive fluorophores

Isomerically Pure Tetramethylrhodamine Voltage Reporters

We present the design, synthesis, and application of a new family of fluorescent voltage indicators based on isomerically pure tetramethylrhodamines. These new <b>Rho</b>damine <b>V</b>oltage <b>R</b>eporters, or RhoVRs, use photoinduced electron transfer (PeT) as a trigger for voltage sensing, display excitation and emission profiles in the green to orange region of the visible spectrum, demonstrate high sensitivity to membrane potential changes (up to 47% Δ<i>F</i>/<i>F</i> per 100 mV), and employ a tertiary amide derived from sarcosine, which aids in membrane localization and simultaneously simplifies the synthetic route to the voltage sensors. The most sensitive of the RhoVR dyes, RhoVR 1, features a methoxy-substituted diethylaniline donor and phenylenevinylene molecular wire at the 5′-position of the rhodamine aryl ring, exhibits the highest voltage sensitivity to date for red-shifted PeT-based voltage sensors, and is compatible with simultaneous imaging alongside green fluorescent protein-based indicators. The discoveries that sarcosine-based tertiary amides in the context of molecular-wire voltage indicators prevent dye internalization and 5′-substituted voltage indicators exhibit improved voltage sensitivity should be broadly applicable to other types of PeT-based voltage-sensitive fluorophores

In Vivo Two-Photon Voltage Imaging with Sulfonated Rhodamine Dyes

Optical methods that rely on fluorescence for mapping changes in neuronal membrane potential in the brains of awake animals provide a powerful way to interrogate the activity of neurons that underlie neural computations ranging from sensation and perception to learning and memory. To achieve this goal, fluorescent indicators should be bright, highly sensitive to small changes in membrane potential, nontoxic, and excitable with infrared light. We report a new class of fluorescent, voltage-sensitive dyes: sulfonated rhodamine voltage reporters (sRhoVR), synthetic fluorophores with high voltage sensitivity, excellent two-photon performance, and compatibility in intact mouse brains. sRhoVR dyes are based on a tetramethyl rhodamine fluorophore coupled to a phenylenevinylene molecular wire/diethyl aniline voltage-sensitive domain. When applied to cells, sRhoVR dyes localize to the plasma membrane and respond to membrane depolarization with a fluorescence increase. The best of the new dyes, sRhoVR 1, displays a 44% ΔF/F increase in fluorescence per 100 mV change, emits at 570 nm, and possesses excellent two-photon absorption of approximately 200 GM at 840 nm. sRhoVR 1 can detect action potentials in cultured rat hippocampal neurons under both single- and two-photon illumination with sufficient speed and sensitivity to report on action potentials in single trials, without perturbing underlying physiology or membrane properties. The combination of speed, sensitivity, and brightness under two-photon illumination makes sRhoVR 1 a promising candidate for in vivo imaging in intact brains. We show sRhoVR powerfully complements electrode-based modes of neuronal activity recording in the mouse brain by recording neuronal transmembrane potentials from the neuropil of layer 2/3 of the mouse barrel cortex in concert with extracellularly recorded local field potentials (LFPs). sRhoVR imaging reveals robust depolarization in response to whisker stimulation; concurrent electrode recordings reveal negative deflections in the LFP recording, consistent with the canonical thalamocortical response. Importantly, sRhoVR 1 can be applied in mice with chronic optical windows, presaging its utility in dissecting and resolving voltage dynamics using two-photon functional imaging in awake, behaving animals