Quantitative Single-Molecule Analysis of Ryanodine Receptor 2 Subunit Assembly in Cardiac and Neuronal Tissues.

We developed a method for ex vivo receptor encapsulation and single-molecule imaging techniques from neuronal and cardiac tissues, illustrating the method's broad applicability for measuring membrane receptor assembly. Ryanodine receptor 2 (RyR2) is a tetrameric Ca2+ channel governing intracellular Ca2+ dynamics, which is critical for muscle contraction. Employing GFP-RyR2 knock-in mice, we isolated individual receptor proteins in tissue-specific nanovesicles and performed subunit counting analyses to yield quantitative assessment of stoichiometric distributions across different organs. With this method, we explored the potential heterogeneity of brain-derived RyR2, which has been reported to form heteromeric assemblies with other ryanodine receptor isoforms.

Impact of nicotine and cotinine on macrophage inflammatory plasticity via vesicular modifications in gastrointestinal bacteria.

OBJECTIVES: This study aimed to elucidate mechanistic explanation(s) for compositional changes to enteric microbiota by determining the impacts of continuous nicotine/cotinine exposure on representative gastrointestinal bacteria and how these alterations impact innate immune cell plasticity. METHODS: In vitro cultures of the gastrointestinal bacteria (Bacteroides fragilis 25285, Prevotella bryantii B14, and Acetoanaerobium sticklandii SR) were continuously exposed to nicotine or cotinine. Supernatant samples were collected for fermentation acid analysis. Vesicles were collected and analyzed for physiological changes in number, size, and total protein cargo. Cultured macrophages were stimulated to a tolerogenic phenotype, exposed to control or altered (nicotine or cotinine - exposed) vesicles, and inflammatory plasticity assessed via inflammatory cytokine production. RESULTS: Nicotine/cotinine exposure differentially affected metabolism of all bacteria tested in a Gram (nicotine) and concentration-dependent (cotinine) manner. Physiological studies demonstrated changes in vesiculation number and protein cargo following nicotine/cotinine exposures. Continuous exposure to 1 µM nicotine and 10 µM cotinine concentrations reduced total protein cargo of Gram (-) - 25285 and B14 vesicles, while cotinine generally increased total protein in Gram (+) - SR vesicles. We found that theses physiological changes to the vesicles of 25285 and SR formed under nicotine and cotinine, respectively, challenged the plasticity of tolerogenic macrophages. Tolerogenic macrophages exposed to vesicles from 1 µM nicotine, and 5 or 10 µΜ cotinine cultures produced significantly less IL-12p70, TNFα, or KC/GRO, regardless of macrophage exposure to nicotine/cotinine. CONCLUSIONS: Nicotine/cotinine exposure differentially alters bacterial metabolism and vesicle physiology, ultimately impacting the inflammatory response of tolerogenic macrophages.

ER-GCaMP6f: An Endoplasmic Reticulum-Targeted Genetic Probe to Measure Calcium Activity in Astrocytic Processes.

Ca2+ is a major second messenger involved in cellular and subcellular signaling in a wide range of cells, including astrocytes, which use calcium ions to communicate with other cells in the brain. Even though a variety of genetically encoded Ca2+ indicators have been developed to study astrocyte calcium signaling, understanding the dynamics of endoplasmic reticulum calcium signaling is greatly limited by the currently available tools. To address this, we developed an endoplasmic reticulum-targeted calcium indicator, ER-GCaMP6f, which is anchored to the cytosolic side of the organelle and measures signaling that occurs in close proximity to the endoplasmic reticulum of astrocytes. Using a combination of confocal and super-resolution microscopy techniques, we demonstrate the localization of the indicator in the endoplasmic reticulum in both cell soma and processes of astrocytes. Combining ER-GCaMP6f with total internal reflection fluorescence microscopy, we show that Ca2+ fluctuations in small astrocytic processes can be detected, which are otherwise not observable with existing indicators and standard wide-field and confocal techniques. We also compared the ER-GCaMP6f indicator against currently used plasma membrane-tethered and cytosolic GCaMP6f indicators. ER-GCaMP6f identifies dynamics in calcium signaling of endoplasmic reticulum resident receptors that are missed by plasma membrane-anchored indicators. We also generated an adeno-associated virus (AAV5) and demonstrate that ER-GCaMP6f can be expressed in vivo and by measured calcium activity in brain slices. ER-GCaMP6f provides a powerful tool to study calcium signaling in close proximity to the endoplasmic reticulum in astrocyte cell soma and processes both in culture and in brain slices.

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

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.

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

We present the application of multiphoton in vivo 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.

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

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.

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

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.

Inhibition of Bacteroidetes and Firmicutes by select phytochemicals.

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 B14, 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-1). The viable cell numbers of all the cultures were reduced, ≥4-logs, by berberine at concentrations ≥1.00 mg mL-1. Quinine performed similarly to berberine for B14, 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-1), but had stronger activity against the Gram-positive bacteria, SR and 9689, (≥4-log reductions at 10.00 mg mL-1). 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.

Brain Region Specific Single-Molecule Fluorescence Imaging.

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 α4ß2 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.

Organelle-specific single-molecule imaging of α4ß2 nicotinic receptors reveals the effect of nicotine on receptor assembly and cell-surface trafficking.

Nicotinic acetylcholine receptors (nAChRs) assemble in the endoplasmic reticulum (ER) and traffic to the cell surface as pentamers composed of α and ß 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 versus 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 α4ß2 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.

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

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)2dmbpy2+]. 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.

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

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.

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

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 α4ß2 receptors on the plasma membrane and causes a redistribution of intracellular receptors. In contrast to this, cotinine exposure down-regulates α6ß2ß3 receptors. We also used single molecule fluorescence studies to show that cotinine and nicotine both alter the assembly of α4ß2 receptors to favor the high sensitivity (α4)2(ß2)3 stoichiometry.

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

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.

Lynx1 shifts α4ß2 nicotinic receptor subunit stoichiometry by affecting assembly in the endoplasmic reticulum.

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 α4ß2 nAChRs within the endoplasmic reticulum. Lynx1 affects assembly of nascent α4 and ß2 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(ß2)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.

Single-particle fluorescence intensity fluctuations of carbon nanodots.

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.

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

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 α3ß4 nicotinic receptors. The method provides the capability to extend single-molecule studies to previously inaccessible classes of receptors.

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

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.

The Role of Microglia in Sex- and Region-Specific Blood-Brain Barrier Integrity During Nicotine Withdrawal.

Background: Smoking is the largest preventable cause of death and disease in the United States, with <5% of quit attempts being successful. Microglia activation and proinflammatory neuroimmune signaling in reward neurocircuitry are implicated in nicotine withdrawal symptomology. Microglia are integral regulators of blood-brain barrier (BBB) functionality as well; however, whether the effects of nicotine withdrawal on microglia function impact BBB integrity is unknown. Methods: Mice were treated with chronic nicotine (12 mg/kg/day) and subjected to 48 hours nicotine withdrawal. Regional BBB permeability, together with messenger RNA and protein expression of tight junction proteins, were assessed. PLX5622 chow was used to deplete microglia to evaluate the role of microglia in regulating BBB integrity and nicotine withdrawal symptomology. Results: Female mice had higher baseline BBB permeability in the prefrontal cortex and hippocampus than males. Nicotine withdrawal further exacerbated the BBB permeability selectively in the prefrontal cortex of females. These effects were concurrent with prefrontal cortex alterations in a subset of tight junction proteins with increased proinflammatory responses following nicotine withdrawal in females. Depletion of microglia via PLX5622 treatment prevented all these molecular effects and attenuated withdrawal-induced anxiety-like behavior in female mice. Conclusions: These results are the first to show sex differences in regional BBB permeability during nicotine withdrawal. This represents a possible link to both the reduced smoking cessation success seen in women and women's increased risk for smoking-related neurovascular disorders. Furthermore, these findings open an avenue for sex-specific therapeutics that target microglia and BBB dysfunction during nicotine withdrawal in women.

Organelle Specific Macrophage Engineered Vesicles Differentially Reprogram Macrophage Polarization.

Tumor-associated macrophages (TAMs) represent the majority of the immune cells present in the tumor microenvironment. These macrophages exhibit an anti-inflammatory (M2)-like physiological state and execute immune-suppressive and tumor-supporting properties. With TAMs being plastic, there is a growing interest in reprogramming them toward a pro-inflammatory (M1)-like phenotype that exhibits anti-tumoral properties. Recent studies have demonstrated that both engineered vesicles derived from macrophages and endogenous extracellular vesicles produced by macrophages can be programmed to alter macrophage phenotype. Here it is demonstrated that pro-inflammatory macrophage-engineered subcellular vesicles (MEVs) have differential properties based on their organelle of origin. Endoplasmic reticulum specific MEVs (erMEVs) treated M2 macrophages exhibit enhanced pro-inflammatory cytokine production compared to plasma membrane specific MEVs (pmMEVs) treated M2 macrophages. In addition, under in vitro co-culture conditions, erMEVs elicit superior efficacy in suppressing the viability of cancer cells compared to the same concentration of pmMEVs. Furthermore, erMEVs and pmMEVs maintain differences in their membrane proteins, that regulate the repolarization efficacy of M2 macrophages toward an M1-like phenotype. In addition, The M2 to M1 repolarizing efficacy of MEVs can be altered by changing the activity of the membrane proteins present on erMEVs or pmMEVs.