Publications by authors named "Christopher I Richards"

31 Publications

Early satellite cell communication creates a permissive environment for long-term muscle growth.

iScience 2021 Apr 29;24(4):102372. Epub 2021 Mar 29.

The Center for Muscle Biology, University of Kentucky, Lexington, KY 40536, USA.

Using muscle stem cell (satellite cell)-specific extracellular vesicle (EV) tracking, satellite cell depletion, cell culture, and single-cell RNA sequencing, we show satellite cells communicate with other cells in skeletal muscle during mechanical overload. Early satellite cell EV communication primes the muscle milieu for proper long-term extracellular matrix (ECM) deposition and is sufficient to support sustained hypertrophy in adult mice, even in the absence of fusion to muscle fibers. Satellite cells modulate chemokine gene expression across cell types within the first few days of loading, and EV delivery of miR-206 to fibrogenic cells represses expression required for appropriate ECM remodeling. Late-stage communication from myogenic cells during loading is widespread but may be targeted toward endothelial cells. Satellite cells coordinate adaptation by influencing the phenotype of recipient cells, which extends our understanding of their role in muscle adaptation beyond regeneration and myonuclear donation.
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http://dx.doi.org/10.1016/j.isci.2021.102372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080523PMC
April 2021

Nicotine induces morphological and functional changes in astrocytes via nicotinic receptor activity.

Glia 2021 Apr 14. Epub 2021 Apr 14.

Department of Chemistry, University of Kentucky, Lexington, Kentucky, USA.

Nicotine is a highly addictive compound present in tobacco, which causes the release of dopamine in different regions of the brain. Recent studies have shown that astrocytes express nicotinic acetylcholine receptors (nAChRs) and mediate calcium signaling. In this study, we examine the morphological and functional adaptations of astrocytes due to nicotine exposure. Utilizing a combination of fluorescence and atomic force microscopy, we show that nicotine-treated astrocytes exhibit time-dependent remodeling in the number and length of both proximal and fine processes. Blocking nAChR activity with an antagonist completely abolishes nicotine's influence on astrocyte morphology indicating that nicotine's action is mediated by these receptors. Functional studies show that 24-hr nicotine treatment induces higher levels of calcium activity in both the cell soma and the processes with a more substantial change observed in the processes. Nicotine does not induce reactive astrocytosis even at high concentrations (10 μM) as determined by cytokine release and glial fibrillary acidic protein expression. We designed tissue clearing experiments to test whether morphological changes occur in vivo using astrocyte specific Aldh1l1-tdTomato knock in mice. We find that nicotine induces a change in the volume of astrocytes in the prefrontal cortex, CA1 of the hippocampus, and the substantia nigra. These results indicate that nicotine directly alters the functional and morphological properties of astrocytes potentially contributing to the underlying mechanism of nicotine abuse.
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http://dx.doi.org/10.1002/glia.24011DOI Listing
April 2021

Photoluminescence Enhancement, Blinking Suppression, and Improved Biexciton Quantum Yield of Single Quantum Dots in Zero Mode Waveguides.

J Phys Chem Lett 2021 Apr 25;12(13):3303-3311. Epub 2021 Mar 25.

Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506, United States.

The capability of quantum dots to generate both single and multiexcitons can be harnessed for a wide variety of applications, including those that require high optical gain. Here, we use time-correlated photoluminescence (PL) spectroscopy to demonstrate that the isolation of single CdSeTe/ZnS core-shell, nanocrystal quantum dots (QDs) in Zero Mode Waveguides (ZMWs) leads to a significant modification in PL intensity, blinking dynamics, and biexciton behavior. QDs in aluminum ZMWs (AlZMWs) exhibited a 15-fold increase in biexciton emission, indicating a preferential enhancement of the biexciton radiative decay rate as compared to the single exciton rate. The increase in biexciton behavior was accompanied by a decrease in blinking events due to a shortening in the dark state residence time. These results indicate that plasmon mediated enhanced decay rates of QDs in AlZMWs lead to substantial changes in the photophysical properties of single quantum dots, including an increase in biexciton behavior.
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http://dx.doi.org/10.1021/acs.jpclett.1c00450DOI Listing
April 2021

Macrophage-Engineered Vesicles for Therapeutic Delivery and Bidirectional Reprogramming of Immune Cell Polarization.

ACS Omega 2021 Feb 26;6(5):3847-3857. Epub 2021 Jan 26.

Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506, United States.

Macrophages, one of the most important phagocytic cells of the immune system, are highly plastic and are known to exhibit diverse roles under different pathological conditions. The ability to repolarize macrophages from pro-inflammatory (M1) to anti-inflammatory (M2) or offers a promising therapeutic approach for treating various diseases such as traumatic injury and cancer. Herein, it is demonstrated that macrophage-engineered vesicles (MEVs) generated by disruption of macrophage cellular membranes can be used as nanocarriers capable of reprogramming macrophages and microglia toward either pro- or anti-inflammatory phenotypes. MEVs can be produced at high yields and easily loaded with diagnostic molecules or chemotherapeutics and delivered to both macrophages and cancer cells and . Overall, MEVs show promise as potential delivery vehicles for both therapeutics and their ability to controllably modulate macrophage/microglia inflammatory phenotypes.
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http://dx.doi.org/10.1021/acsomega.0c05632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876833PMC
February 2021

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells.

J Vis Exp 2020 08 11(162). Epub 2020 Aug 11.

Department of Mechanical Engineering, University of Kentucky;

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe "pokes" the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.
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http://dx.doi.org/10.3791/61433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7680637PMC
August 2020

Fusion-Independent Satellite Cell Communication to Muscle Fibers During Load-Induced Hypertrophy.

Function (Oxf) 2020 6;1(1):zqaa009. Epub 2020 Jul 6.

The Center for Muscle Biology, University of Kentucky, Lexington, KY 40536, USA.

The "canonical" function of Pax7+ muscle stem cells (satellite cells) during hypertrophic growth of adult muscle fibers is myonuclear donation via fusion to support increased transcriptional output. In recent years, however, emerging evidence suggests that satellite cells play an important secretory role in promoting load-mediated growth. Utilizing genetically modified mouse models of delayed satellite cell fusion and extracellular vesicle (EV) tracking, we provide evidence for satellite cell communication to muscle fibers during hypertrophy. Myogenic progenitor cell-EV-mediated communication to myotubes influences extracellular matrix (ECM)-related gene expression, which is congruent with overload experiments involving satellite cell depletion, as well as analyses. Satellite cell-derived EVs can transfer a Cre-induced, cytoplasmic-localized fluorescent reporter to muscle cells as well as microRNAs that regulate ECM genes such as matrix metalloproteinase 9 (), which may facilitate growth. Delayed satellite cell fusion did not limit long-term load-induced muscle hypertrophy indicating that early fusion-independent communication from satellite cells to muscle fibers is an underappreciated aspect of satellite cell biology. We cannot exclude the possibility that satellite cell-mediated myonuclear accretion is necessary to maintain prolonged growth, specifically in the later phases of adaptation, but these data collectively highlight how EV delivery from satellite cells can directly contribute to mechanical load-induced muscle fiber hypertrophy, independent of cell fusion to the fiber.
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http://dx.doi.org/10.1093/function/zqaa009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448100PMC
July 2020

Single-Molecule Detection of Nanoparticles for Multiphoton Fluorescence Correlation Spectroscopy to Quantify Cerebral Blood Flow.

Nano Lett 2020 08 8;20(8):6135-6141. Epub 2020 Jul 8.

Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506, United States.

We present the application of multiphoton fluorescence correlation spectroscopy (FCS) of fluorescent nanoparticles for the measurement of cerebral blood flow with excellent spatial and temporal resolution. Through the detection of single nanoparticles within the complex vessel architecture of a live mouse, this new approach enables the quantification of nanoparticle dynamics occurring within the vasculature along with simultaneous measurements of blood flow properties in the brain. In addition to providing high resolution blood flow measurements, this approach enables real-time quantification of nanoparticle concentration, degradation, and transport. This method is capable of quantifying flow rates at each pixel with submicron resolution to enable monitoring of dynamic changes in flow rates in response to changes in the animal's physiological condition. Scanning the excitation beam using FCS provides pixel by pixel mapping of flow rates with subvessel resolution across capillaries 300 μm deep in the brains of mice.
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http://dx.doi.org/10.1021/acs.nanolett.0c02280DOI Listing
August 2020

High-throughput fluorescence correlation spectroscopy enables analysis of surface components of cell-derived vesicles.

Anal Bioanal Chem 2020 Apr 7;412(11):2589-2597. Epub 2020 Mar 7.

Department of Chemistry, University of Kentucky, 161 Jacobs Science Building, Lexington, KY, 40506, USA.

Extracellular vesicles (EVs) and cell-derived vesicles (CDVs), generated by fragmenting cellular membranes, have both been explored as therapeutic delivery vehicles. Surface proteins on these vesicles are of great importance as they are characteristic to the cell of origin and modulate vesicle interactions with target cells. Here, we introduced a high-throughput fluorescence correlation spectroscopy (ht-FCS) approach capable of characterizing vesicle surface proteins across a large number of samples. We used automated screening and acquisition of FCS data to profile surface proteins of cell-derived vesicles with high fidelity based on changes in diffusion time upon antibody-vesicle interactions. We characterized vesicles generated from 4 cell types using antibodies for known exosome biomarkers. The ht-FCS technique presented here offers the capability to screen EVs or cell-derived vesicles against a library of surface markers or to screen a library of cell-derived vesicles for a specific identifying marker at a high speed.
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http://dx.doi.org/10.1007/s00216-020-02485-zDOI Listing
April 2020

Inhibition of Bacteroidetes and Firmicutes by select phytochemicals.

Anaerobe 2020 Feb 24;61:102145. Epub 2019 Dec 24.

USDA Agricultural Research Service Forage-Animal Production Research Unit, Lexington, KY, USA; Department of Animal and Food Sciences, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA. Electronic address:

Current research indicates that changes in gut microbiota can impact the host, but it is not always clear how dietary and environmental factors alter gut microbiota. One potential factor is antimicrobial activity of compounds ingested by the host. The goal of this study was to determine the antimicrobial activity of common plant secondary metabolites against pure cultures of paired, structurally and phylogenetically distinct gastrointestinal bacteria of human or bovine origin: Prevotella bryantii B4, Bacteroides fragilis 25285, Acetoanaerobium (Clostridium) sticklandii SR and Clostridioides difficile 9689. When growth media were amended with individual phytochemicals (the alkaloids: berberine, capsaicin, nicotine, piperine and quinine and the phenolic: curcumin), growth of each species was inhibited to varying degrees at the three greatest concentrations tested (0.10-10.00 mg mL). The viable cell numbers of all the cultures were reduced, ≥4-logs, by berberine at concentrations ≥1.00 mg mL. Quinine performed similarly to berberine for B4, 25285, and SR at the same concentrations. The other phytochemicals were inhibitory, but not as much as quinine or berberine. Nicotine had activity against all four species (≥2-log reduction in viable cell number at 10.00 mg mL), but had stronger activity against the Gram-positive bacteria, SR and 9689, (≥4-log reductions at 10.00 mg mL). In conclusion, the phytochemicals had varying spectra of antimicrobial activity. These results are consistent with the hypothesis that ingested phytochemicals have the ability to differentially impact gut microbiota through antimicrobial activity.
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http://dx.doi.org/10.1016/j.anaerobe.2019.102145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441489PMC
February 2020

Heat shock protein 47 (HSP47) binds to discoidin domain-containing receptor 2 (DDR2) and regulates its protein stability.

J Biol Chem 2019 11 30;294(45):16846-16854. Epub 2019 Sep 30.

Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536

Cell-collagen interactions are crucial for cell migration and invasion during cancer development and progression. Heat shock protein 47 (HSP47) is an endoplasmic reticulum-resident molecular chaperone that facilitates collagen maturation and deposition. It has been previously shown that HSP47 expression in cancer cells is crucial for cancer invasiveness. However, exogenous collagen cannot rescue cell invasion in HSP47-silenced cancer cells, suggesting that other HSP47 targets contribute to cancer cell invasion. Here, we show that HSP47 expression is required for the stability and cell-surface expression of discoidin domain-containing receptor 2 (DDR2) in breast cancer tissues. HSP47 silencing reduced DDR2 protein stability, accompanied by suppressed cell migration and invasion. Co-immunoprecipitation results revealed that HSP47 binds to the DDR2 ectodomain. Using a photoconvertible technique and total internal reflection fluorescence microscopy, we further demonstrate that HSP47 expression significantly sustains the membrane localization of the DDR2 protein. These results suggest that binding of HSP47 to DDR2 increases DDR2 stability and regulates its membrane dynamics and thereby enhances cancer cell migration and invasion. Given that DDR2 has a crucial role in the epithelial-to-mesenchymal transition and cancer progression, targeting the HSP47-DDR2 interaction might be a potential strategy for inhibiting DDR2-dependent cancer progression.
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http://dx.doi.org/10.1074/jbc.RA119.009312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851340PMC
November 2019

Cell-Derived Vesicles for in Vitro and in Vivo Targeted Therapeutic Delivery.

ACS Omega 2019 Jul 24;4(7):12657-12664. Epub 2019 Jul 24.

Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506, United States.

Efficient delivery of therapeutics across the cell membrane to the interior of the cell remains a challenge both in vitro and in vivo. Here, we demonstrate that vesicles derived from cellular membranes can be efficiently loaded with cargo that can then be delivered to the interior of the cell. These vesicles demonstrated cell-targeting specificity as well as the ability to deliver a wide range of different cargos. We utilized this approach to deliver both lipophilic and hydrophilic cargos including therapeutics and DNA in vitro. We further demonstrated in vivo targeting and delivery using fluorescently labeled vesicles to target tumor xenografts in an animal. Cell-derived vesicles can be generated in high yields and are easily loaded with a variety of cargos. The ability of these vesicles to specifically target the same cell type from which they originated provides an efficient means of delivering cargo, such as therapeutics, both in vitro and in vivo.
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http://dx.doi.org/10.1021/acsomega.9b01353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681979PMC
July 2019

High resolution measurement of membrane receptor endocytosis.

J Biol Methods 2018 12;5(4):e105. Epub 2018 Dec 12.

Department of Chemistry, University of Kentucky, 505 Rose Street, Lexington, KY 40506, USA.

We present a new approach to quantify the half-life of membrane proteins on the cell surface, through tagging the protein with the photoconvertible fluorescent protein, Dendra2. Upon exposure to 405 nm light, Dendra2 is photoconverted from green to red emission. Total internal reflection fluorescence microscopy (TIRF) is applied to limit visualization of fluorescence to proteins located on the plasma membrane. Conversion of Dendra2 works as a pulse chase experiment through monitoring only the population of protein that has been photoconverted. As the protein is endocytosed the red emission decreases due to the protein leaving the TIRF field of view. This method is not impacted by the insertion of new protein into the plasma membrane as newly synthesized protein only exhibits green emission. We used this approach to determine the half-life of ENaC on the plasma membrane illustrating the high temporal resolution capability of this technique compared to current methods.
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http://dx.doi.org/10.14440/jbm.2018.266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706155PMC
December 2018

Brain Region Specific Single-Molecule Fluorescence Imaging.

Anal Chem 2019 08 24;91(15):10125-10131. Epub 2019 Jul 24.

Department of Chemistry , University of Kentucky , Lexington , Kentucky 40506 , United States.

We developed an approach utilizing nanoscale vesicles extracted from brain regions combined with single molecule imaging to monitor how an animal's physiological condition regulates the dynamics of protein distributions in different brain regions. This method was used to determine the effect of nicotine on the distribution of receptor stoichiometry in different mouse brain regions. Nicotine-induced upregulation of α4β2 nicotinic acetylcholine receptors (nAChRs) is associated with changes in their expression, trafficking, and stoichiometry. The structural assembly of nAChRs has been quantified in cell culture based systems using single molecule techniques. However, these methods are not capable of quantifying biomolecule assembly that takes place in a live animal. Both nicotine-induced upregulation and changes in nAChR stoichiometry differ across brain regions. Our single molecule approach revealed that nicotine acts differentially across brain regions to alter assembly in response to exposure and withdrawal.
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http://dx.doi.org/10.1021/acs.analchem.9b02133DOI Listing
August 2019

Bacterial Cytological Profiling Reveals the Mechanism of Action of Anticancer Metal Complexes.

Mol Pharm 2018 08 30;15(8):3404-3416. Epub 2018 Jun 30.

Department of Chemistry , University of Kentucky , Lexington , Kentucky 40506 , United States.

Target identification and mechanistic studies of cytotoxic agents are challenging processes that are both time-consuming and costly. Here we describe an approach to mechanism of action studies for potential anticancer compounds by utilizing the simple prokaryotic system, E. coli, and we demonstrate its utility with the characterization of a ruthenium polypyridyl complex [Ru(bpy)dmbpy]. Expression of the photoconvertible fluorescent protein Dendra2 facilitated both high throughput studies and single-cell imaging. This allowed for simultaneous ratiometric analysis of inhibition of protein production and phenotypic investigations. The profile of protein production, filament size and population, and nucleoid morphology revealed important differences between inorganic agents that damage DNA vs more selective inhibitors of transcription and translation. Trace metal analysis demonstrated that DNA is the preferred nucleic acid target of the ruthenium complex, but further studies in human cancer cells revealed altered cell signaling pathways compared to the commonly administrated anticancer agent cisplatin. This study demonstrates E. coli can be used to rapidly distinguish between compounds with disparate mechanisms of action and also for more subtle distinctions within in studies in mammalian cells.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b00407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6083414PMC
August 2018

Organelle-specific single-molecule imaging of α4β2 nicotinic receptors reveals the effect of nicotine on receptor assembly and cell-surface trafficking.

J Biol Chem 2017 12 26;292(51):21159-21169. Epub 2017 Oct 26.

From the Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506

Nicotinic acetylcholine receptors (nAChRs) assemble in the endoplasmic reticulum (ER) and traffic to the cell surface as pentamers composed of α and β subunits. Many nAChR subtypes can assemble with varying subunit ratios, giving rise to multiple stoichiometries exhibiting different subcellular localization and functional properties. In addition to the endogenous neurotransmitter acetylcholine, nicotine also binds and activates nAChRs and influences their trafficking and expression on the cell surface. Currently, no available technique can specifically elucidate the stoichiometry of nAChRs in the ER those in the plasma membrane. Here, we report a method involving single-molecule fluorescence measurements to determine the structural properties of these membrane proteins after isolation in nanoscale vesicles derived from specific organelles. These cell-derived nanovesicles allowed us to separate single membrane receptors while maintaining them in their physiological environment. Sorting the vesicles according to the organelle of origin enabled us to determine localized differences in receptor structural properties, structural influence on transport between organelles, and changes in receptor assembly within intracellular organelles. These organelle-specific nanovesicles revealed that one structural isoform of the α4β2 nAChR was preferentially trafficked to the cell surface. Moreover, nicotine altered nAChR assembly in the ER, resulting in increased production of the receptor isoform that traffics more efficiently to the cell surface. We conclude that the combined effects of the increased assembly of one nAChR stoichiometry and its preferential trafficking likely drive the up-regulation of nAChRs on the cell surface upon nicotine exposure.
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http://dx.doi.org/10.1074/jbc.M117.801431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743088PMC
December 2017

Real-Time Sensing of Single-Ligand Delivery with Nanoaperture-Integrated Microfluidic Devices.

ACS Omega 2017 Jul 25;2(7):3858-3867. Epub 2017 Jul 25.

Department of Chemistry, University of Kentucky, 505 Rose Street, Lexington, Kentucky 40506, United States.

The measurement of biological events on the surface of live cells at the single-molecule level is complicated by several factors including high protein densities that are incompatible with single-molecule imaging, cellular autofluorescence, and protein mobility on the cell surface. Here, we fabricated a device composed of an array of nanoscale apertures coupled with a microfluidic delivery system to quantify single-ligand interactions with proteins on the cell surface. We cultured live cells directly on the device and isolated individual epidermal growth factor receptors (EGFRs) in the apertures while delivering fluorescently labeled epidermal growth factor. We observed single ligands binding to EGFRs, allowing us to quantify the ligand turnover in real time. These results demonstrate that this nanoaperture-coupled microfluidic device allows for the spatial isolation of individual membrane proteins while maintaining them in their cellular environment, providing the capability to monitor single-ligand binding events while maintaining receptors in their physiological environment. These methods should be applicable to a wide range of membrane proteins.
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http://dx.doi.org/10.1021/acsomega.7b00934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5537690PMC
July 2017

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking.

J Vis Exp 2017 03 16(121). Epub 2017 Mar 16.

Department of Chemistry, University of Kentucky;

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.
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http://dx.doi.org/10.3791/55466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409030PMC
March 2017

Nicotinic Acetylcholine Receptors as Targets for Tobacco Cessation Therapeutics: Cutting-Edge Methodologies to Understand Receptor Assembly and Trafficking.

Neuromethods 2016 30;117:119-132. Epub 2016 Sep 30.

Tobacco dependence is a chronic relapsing disorder and nicotine, the primary alkaloid in tobacco, acts at nicotinic receptors to stimulate dopamine release in brain, which is responsible for the reinforcing properties of nicotine, leading to addiction. Although the majority of tobacco users express the desire to quit, only a small percentage of those attempting to quit are successful using the currently available pharmacotherapies. Nicotine upregulates the number of specific nicotinic receptors on the neuronal cell surface. An increase in receptor trafficking or preferential stoichiometric assembly of receptor subunits involves changes in assembly, endoplasmic reticulum export, vesicle transport, decreased degradation, desensitization, enhanced maturation of functional pentamers, and pharmacological chaperoning. Understanding these changes on a mechanistic level is important to the development of nicotinic receptors as drug targets. For this reason, cutting-edge methodologies are being developed and employed to pinpoint distinct changes in localization, assembly, export, vesicle trafficking, and stoichiometry in order to further understand the physiology of these receptors and to evaluate the action of novel therapeutics for smoking cessation.
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http://dx.doi.org/10.1007/978-1-4939-3768-4_7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5180357PMC
September 2016

Direct Measurement of Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator to the Cell Surface and Binding to a Chemical Chaperone.

Biochemistry 2017 Jan 21;56(1):240-249. Epub 2016 Dec 21.

Department of Chemistry, University of Kentucky , Lexington, Kentucky 40506, United States.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) result in the disease cystic fibrosis. Deletion of Phe508, the most prevalent mutation associated with this disease, disrupts trafficking of the protein. Small molecule correctors yield moderate improvements in the trafficking of ΔF508-CFTR to the plasma membrane. It is currently not known if correctors increase the level of trafficking through improved cargo loading of transport vesicles or through direct binding to CFTR. Real-time measurements of trafficking were utilized to identify the mechanistic details of chemical, biochemical, and thermal factors that impact CFTR correction, using the corrector molecule VX-809, a secondary mutation (I539T), and low-temperature conditions. Each individually improved trafficking of ΔF508-CFTR to approximately 10% of wild-type levels. The combination of VX-809 with either low temperature or the I539T mutation increased the amount of CFTR on the plasma membrane to nearly 40%, indicating synergistic activity. The number of vesicles reaching the surface was significantly altered; however, the amount of channel in each vesicle remained the same. Direct binding measurements of VX-809 in native membranes using backscattering interferometry indicate tight binding to CFTR, which occurred in a manner independent of mutation. The similar values obtained for all forms of the channel indicate that the binding site is not compromised or enhanced by these mutations.
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http://dx.doi.org/10.1021/acs.biochem.6b00853DOI Listing
January 2017

A Comparison of Single-Molecule Emission in Aluminum and Gold Zero-Mode Waveguides.

J Phys Chem A 2016 Sep 17;120(34):6719-27. Epub 2016 Aug 17.

Department of Chemistry, University of Kentucky , 505 Rose Street, Lexington, Kentucky 40506, United States.

The effect of gold and aluminum zero-mode waveguides (ZMWs) on the brightness of immobilized single emitters was characterized by probing fluorophores that absorb in the green and red regions of the visible spectrum. Aluminum ZMWs enhance the emission of Atto565 fluorophores upon green excitation, but they do not enhance the emission of Atto647N fluorophores upon red excitation. Gold ZMWs increase emission of both fluorophores with Atto647N showing enhancement that is threefold higher than that observed for Atto565. This work indicates that 200 nm gold ZMWs are better suited for single-molecule fluorescence studies in the red region of the visible spectrum, while aluminum appears more suited for the green region of the visible spectrum.
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http://dx.doi.org/10.1021/acs.jpca.6b03309DOI Listing
September 2016

The nicotine metabolite, cotinine, alters the assembly and trafficking of a subset of nicotinic acetylcholine receptors.

J Biol Chem 2015 Oct 12;290(40):24403-12. Epub 2015 Aug 12.

From the Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506

Exposure to nicotine alters the trafficking and assembly of nicotinic receptors (nAChRs), leading to their up-regulation on the plasma membrane. Although the mechanism is not fully understood, nicotine-induced up-regulation is believed to contribute to nicotine addiction. The effect of cotinine, the primary metabolite of nicotine, on nAChR trafficking and assembly has not been extensively investigated. We utilize a pH-sensitive variant of GFP, super ecliptic pHluorin, to differentiate between intracellular nAChRs and those expressed on the plasma membrane to quantify changes resulting from cotinine and nicotine exposure. Similar to nicotine, exposure to cotinine increases the number of α4β2 receptors on the plasma membrane and causes a redistribution of intracellular receptors. In contrast to this, cotinine exposure down-regulates α6β2β3 receptors. We also used single molecule fluorescence studies to show that cotinine and nicotine both alter the assembly of α4β2 receptors to favor the high sensitivity (α4)2(β2)3 stoichiometry.
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http://dx.doi.org/10.1074/jbc.M115.661827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4591823PMC
October 2015

The nicotinic α6 subunit gene determines variability in chronic pain sensitivity via cross-inhibition of P2X2/3 receptors.

Sci Transl Med 2015 May;7(287):287ra72

Neurobiologie Intégrative des Systèmes Cholinergiques, CNRS UMR 3571, Département de Neuroscience, Institute Pasteur, 75724 Paris, France.

Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6's role in analgesia is at least partially due to direct interaction and cross-inhibition of α6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish the relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical and temporomandibular pain.
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http://dx.doi.org/10.1126/scitranslmed.3009986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5018401PMC
May 2015

Cell-derived vesicles for single-molecule imaging of membrane proteins.

Angew Chem Int Ed Engl 2015 Jan 31;54(2):481-4. Epub 2014 Oct 31.

Department of Chemistry, University of Kentucky, 505 Rose Street, Lexington, KY 40506 (USA).

A new approach is presented for the application of single-molecule imaging to membrane receptors through the use of vesicles derived from cells expressing fluorescently labeled receptors. During the isolation of vesicles, receptors remain embedded in the membrane of the resultant vesicles, thus allowing these vesicles to serve as nanocontainers for single-molecule measurements. Cell-derived vesicles maintain the structural integrity of transmembrane receptors by keeping them in their physiological membrane. It was demonstrated that receptors isolated in these vesicles can be studied with solution-based fluorescence correlation spectroscopy (FCS) and can be isolated on a solid substrate for single-molecule studies. This technique was applied to determine the stoichiometry of α3β4 nicotinic receptors. The method provides the capability to extend single-molecule studies to previously inaccessible classes of receptors.
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http://dx.doi.org/10.1002/anie.201408707DOI Listing
January 2015

Lynx1 shifts α4β2 nicotinic receptor subunit stoichiometry by affecting assembly in the endoplasmic reticulum.

J Biol Chem 2014 Nov 5;289(45):31423-32. Epub 2014 Sep 5.

From the Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, the Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015

Glycosylphosphatidylinositol-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on α4β2 nAChRs within the endoplasmic reticulum. Lynx1 affects assembly of nascent α4 and β2 subunits and alters the stoichiometry of the receptor population that reaches the plasma membrane. Additionally, these data suggest that lynx1 shifts nAChR stoichiometry to low sensitivity (α4)3(β2)2 pentamers primarily through this interaction in the endoplasmic reticulum, rather than solely via direct modulation of activity on the plasma membrane. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any glycosylphosphatidylinositol-anchored protein, could act within the cell to alter assembly of a multisubunit protein.
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http://dx.doi.org/10.1074/jbc.M114.573667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4223341PMC
November 2014

Pharmacological chaperoning of nAChRs: a therapeutic target for Parkinson's disease.

Pharmacol Res 2014 May 1;83:20-9. Epub 2014 Mar 1.

Department of Chemistry, University of Kentucky, Lexington, KY, United States.

Chronic exposure to nicotine results in an upregulation of neuronal nicotinic acetylcholine receptors (nAChRs) at the cellular plasma membrane. nAChR upregulation occurs via nicotine-mediated pharmacological receptor chaperoning and is thought to contribute to the addictive properties of tobacco as well as relapse following smoking cessation. At the subcellular level, pharmacological chaperoning by nicotine and nicotinic ligands causes profound changes in the structure and function of the endoplasmic reticulum (ER), ER exit sites, the Golgi apparatus and secretory vesicles of cells. Chaperoning-induced changes in cell physiology exert an overall inhibitory effect on the ER stress/unfolded protein response. Cell autonomous factors such as the repertoire of nAChR subtypes expressed by neurons and the pharmacological properties of nicotinic ligands (full or partial agonist versus competitive antagonist) govern the efficiency of receptor chaperoning and upregulation. Together, these findings are beginning to pave the way for developing pharmacological chaperones to treat Parkinson's disease and nicotine addiction.
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http://dx.doi.org/10.1016/j.phrs.2014.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075820PMC
May 2014

Single-particle fluorescence intensity fluctuations of carbon nanodots.

Nano Lett 2014 Feb 10;14(2):620-5. Epub 2014 Jan 10.

Department of Chemistry, University of Kentucky , 505 Rose Street, Lexington, Kentucky, United States.

Fluorescent carbon nanodots (CNDs) were synthesized in oxidized and reduced forms and were analyzed at the single-particle level. Images of single CNDs at different excitation energies revealed significant heterogeneity in the lower energy trap sites between particles. We observed that a high percentage of reduced CND particles transitioned between multiple fluorescence intensity levels indicative of multichromophoric systems. Despite this behavior, individual CNDs exhibit single-step photobleaching and transient blinking to the background level suggesting single-molecule behavior.
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http://dx.doi.org/10.1021/nl403820mDOI Listing
February 2014

Nicotine exploits a COPI-mediated process for chaperone-mediated up-regulation of its receptors.

J Gen Physiol 2014 Jan;143(1):51-66

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.

Chronic exposure to nicotine up-regulates high sensitivity nicotinic acetylcholine receptors (nAChRs) in the brain. This up-regulation partially underlies addiction and may also contribute to protection against Parkinson's disease. nAChRs containing the α6 subunit (α6* nAChRs) are expressed in neurons in several brain regions, but comparatively little is known about the effect of chronic nicotine on these nAChRs. We report here that nicotine up-regulates α6* nAChRs in several mouse brain regions (substantia nigra pars compacta, ventral tegmental area, medial habenula, and superior colliculus) and in neuroblastoma 2a cells. We present evidence that a coat protein complex I (COPI)-mediated process mediates this up-regulation of α6* or α4* nAChRs but does not participate in basal trafficking. We show that α6β2β3 nAChR up-regulation is prevented by mutating a putative COPI-binding motif in the β3 subunit or by inhibiting COPI. Similarly, a COPI-dependent process is required for up-regulation of α4β2 nAChRs by chronic nicotine but not for basal trafficking. Mutation of the putative COPI-binding motif or inhibition of COPI also results in reduced normalized Förster resonance energy transfer between α6β2β3 nAChRs and εCOP subunits. The discovery that nicotine exploits a COPI-dependent process to chaperone high sensitivity nAChRs is novel and suggests that this may be a common mechanism in the up-regulation of nAChRs in response to chronic nicotine.
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http://dx.doi.org/10.1085/jgp.201311102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874574PMC
January 2014

Förster resonance energy transfer (FRET) correlates of altered subunit stoichiometry in cys-loop receptors, exemplified by nicotinic α4β2.

Int J Mol Sci 2012 10;13(8):10022-40. Epub 2012 Aug 10.

Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA; E-Mails: (R.S.); (C.I.R.); (C.D.); (F.J.M.).

We provide a theory for employing Förster resonance energy transfer (FRET) measurements to determine altered heteropentameric ion channel stoichiometries in intracellular compartments of living cells. We simulate FRET within nicotinic receptors (nAChRs) whose α4 and β2 subunits contain acceptor and donor fluorescent protein moieties, respectively, within the cytoplasmic loops. We predict FRET and normalized FRET (NFRET) for the two predominant stoichiometries, (α4)(3)(β2)(2)vs. (α4)(2)(β2)(3). Studying the ratio between FRET or NFRET for the two stoichiometries, minimizes distortions due to various photophysical uncertainties. Within a range of assumptions concerning the distance between fluorophores, deviations from plane pentameric geometry, and other asymmetries, the predicted FRET and NFRET for (α4)(3)(β2)(2) exceeds that of (α4)(2)(β2)(3). The simulations account for published data on transfected Neuro2a cells in which α4β2 stoichiometries were manipulated by varying fluorescent subunit cDNA ratios: NFRET decreased monotonically from (α4)(3)(β2)(2) stoichiometry to mostly (α4)(2)(β2)(3). The simulations also account for previous macroscopic and single-channel observations that pharmacological chaperoning by nicotine and cytisine increase the (α4)(2)(β2)(3) and (α4)(3)(β2)(2) populations, respectively. We also analyze sources of variability. NFRET-based monitoring of changes in subunit stoichiometry can contribute usefully to studies on Cys-loop receptors.
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http://dx.doi.org/10.3390/ijms130810022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431844PMC
September 2015

Live-cell imaging of single receptor composition using zero-mode waveguide nanostructures.

Nano Lett 2012 Jul 8;12(7):3690-4. Epub 2012 Jun 8.

Division of Biology 156-29, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, United States.

We exploit the optical and spatial features of subwavelength nanostructures to examine individual receptors on the plasma membrane of living cells. Receptors were sequestered in portions of the membrane projected into zero-mode waveguides. Using single-step photobleaching of green fluorescent protein incorporated into individual subunits, the resulting spatial isolation was used to measure subunit stoichiometry in α4β4 and α4β2 nicotinic acetylcholine and P2X2 ATP receptors. We also show that nicotine and cytisine have differential effects on α4β2 stoichiometry.
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http://dx.doi.org/10.1021/nl301480hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3397148PMC
July 2012

Pharmacological chaperoning of nicotinic acetylcholine receptors reduces the endoplasmic reticulum stress response.

Mol Pharmacol 2012 Jun 29;81(6):759-69. Epub 2012 Feb 29.

Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.

We report the first observation that endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) can decrease when a central nervous system drug acts as an intracellular pharmacological chaperone for its classic receptor. Transient expression of α4β2 nicotinic receptors (nAChRs) in Neuro-2a cells induced the nuclear translocation of activating transcription factor 6 (ATF6), which is part of the UPR. Cells were exposed for 48 h to the full agonist nicotine, the partial agonist cytisine, or the competitive antagonist dihydro-β-erythroidine; we also tested mutant nAChRs that readily exit the ER. Each of these four manipulations increased Sec24D-enhanced green fluorescent protein fluorescence of condensed ER exit sites and attenuated translocation of ATF6-enhanced green fluorescent protein to the nucleus. However, we found no correlation among the manipulations regarding other tested parameters [i.e., changes in nAChR stoichiometry (α4(2)β2(3) versus α4(3)β2(2)), changes in ER and trans-Golgi structures, or the degree of nAChR up-regulation at the plasma membrane]. The four manipulations activated 0 to 0.4% of nAChRs, which shows that activation of the nAChR channel did not underlie the reduced ER stress. Nicotine also attenuated endogenously expressed ATF6 translocation and phosphorylation of eukaryotic initiation factor 2α in mouse cortical neurons transfected with α4β2 nAChRs. We conclude that, when nicotine accelerates ER export of α4β2 nAChRs, this suppresses ER stress and the UPR. Suppression of a sustained UPR may explain the apparent neuroprotective effect that causes the inverse correlation between a person's history of tobacco use and susceptibility to developing Parkinson's disease. This suggests a novel mechanism for neuroprotection by nicotine.
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http://dx.doi.org/10.1124/mol.112.077792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3362896PMC
June 2012