Purinoreceptors and ectonucleotidases control ATP-induced calcium waveforms and calcium-dependent responses in microglia: Roles of P2 receptors and CD39 in ATP-stimulated microglia.

Adenosine triphosphate (ATP) and its metabolites drive microglia migration and cytokine production by activating P2X- and P2Y- class purinergic receptors. Purinergic receptor activation gives rise to diverse intracellular calcium (Ca2+ signals, or waveforms, that differ in amplitude, duration, and frequency. Whether and how these characteristics of diverse waveforms influence microglia function is not well-established. We developed a computational model trained with data from published primary murine microglia studies. We simulate how purinoreceptors influence Ca2+ signaling and migration, as well as, how purinoreceptor expression modifies these processes. Our simulation confirmed that P2 receptors encode the amplitude and duration of the ATP-induced Ca2+ waveforms. Our simulations also implicate CD39, an ectonucleotidase that rapidly degrades ATP, as a regulator of purinergic receptor-induced Ca2+ responses. Namely, it was necessary to account for CD39 metabolism of ATP to align the model's predicted purinoreceptor responses with published experimental data. In addition, our modeling results indicate that small Ca2+ transients accompany migration, while large and sustained transients are needed for cytokine responses. Lastly, as a proof-of-principal, we predict Ca2+ transients and cell membrane displacements in a BV2 microglia cell line using published P2 receptor mRNA data to illustrate how our computer model may be extrapolated to other microglia subtypes. These findings provide important insights into how differences in purinergic receptor expression influence microglial responses to ATP.

Mammalian Cell-derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies.

Cell-derived vesicles facilitate the isolation of transmembrane proteins in their physiological membrane maintaining their structural and functional integrity. These vesicles can be generated from different cellular organelles producing, housing, or transporting the proteins. Combined with single-molecule imaging, isolated organelle specific vesicles can be employed to study the trafficking and assembly of the embedded proteins. Here we present a method for organelle specific single molecule imaging via isolation of ER and plasma membrane vesicles from HEK293T cells by employing OptiPrep gradients and nitrogen cavitation. The isolation was validated through Western blotting, and the isolated vesicles were used to perform single molecule studies of oligomeric receptor assembly.

Advances in micro- and nanotechnologies for the GLP-1-based therapy and imaging of pancreatic beta-cells.

Therapies to prevent diabetes in particular the progressive loss of ß-cell mass and function and/or to improve the dysregulated metabolism associated with diabetes are highly sought. The incretin-based therapy comprising GLP-1R agonists and DPP-4 inhibitors have represented a major focus of pharmaceutical R&D over the last decade. The incretin hormone GLP-1 has powerful antihyperglycemic effect through direct stimulation of insulin biosynthesis and secretion within the ß-cells; it normalizes ß-cell sensitivity to glucose, has an antiapoptotic role, stimulates ß-cell proliferation and differentiation, and inhibits glucagon secretion. However, native GLP-1 therapy is inappropriate due to the rapid post-secretory inactivation by DPP-4. Therefore, incretin mimetics developed on the backbone of the GLP-1 or exendin-4 molecule have been developed to behave as GLP-1R agonists but to display improved stability and clinical efficacy. New formulations of incretins and their analogs based on micro- and nanomaterials (i.e., PEG, PLGA, chitosan, liposomes and silica) and innovative encapsulation strategies have emerged to achieve a better stability of the incretin, to improve its pharmacokinetic profile, to lower the administration frequency or to allow another administration route and to display fewer adverse effects. An important advantage of these formulations is that they can also be used at the targeted non-invasive imaging of the beta-cell mass. This review therefore focuses on the current state of these efforts as the next step in the therapeutic evolution of this class of antidiabetic drugs.

A High-Throughput Screening Assay Using a Photoconvertable Protein for Identifying Inhibitors of Transcription, Translation, or Proteasomal Degradation.

Dysregulated transcription, translation, and protein degradation are common features of cancer cells, regardless of specific genetic profiles. Several clinical anticancer agents take advantage of this characteristic vulnerability and interfere with the processes of transcription and translation or inhibit protein degradation. However, traditional assays that follow the process of protein production and removal require multistep processing and are not easily amenable to high-throughput screening. The use of recombinant fluorescent proteins provides a convenient solution to this problem, and moreover, photoconvertable fluorescent proteins allow for ratiometric detection of both new protein production and removal of existing proteins. Here, the photoconvertable protein Dendra2 is used in the development of in-cell assays of protein production and degradation that are optimized and validated for high-throughput screening. Conversion from the green to red emissive form can be achieved using a high-intensity light-emitting diode array, producing a stable pool of the red fluorescent form of Dendra2. This allows for rates of protein production or removal to be quantified in a plate reader or by fluorescence microscopy, providing a means to measure the potencies of inhibitors that affect these key processes.

Signal Discrimination Between Fluorescent Proteins in Live Cells by Long-wavelength Optical Modulation.

Fluorescent proteins (FPs) have revolutionized molecular and cellular biology; yet, discrimination over cellular autofluorescence, spectral deconvolution, or detection at low concentrations remain challenging problems in many biological applications. By optically depopulating a photoinduced dark state with orange secondary laser co-excitation, the higher-energy green AcGFP fluorescence is dynamically increased. Modulating this secondary laser then modulates the higher-energy, collected fluorescence; enabling its selective detection by removing heterogeneous background from other FPs. Order-of-magnitude reduction in obscuring fluorophore background emission has been achieved in both fixed and live cells. This longwavelength modulation expands the dimensionality to discriminate FP emitters based on dark state lifetimes and enables signal of interest to be recovered by removing heterogeneous background emitter signals. Thus, AcGFP is not only useful for extracting weak signals from systems plagued by high background, but it is a springboard for further FP optimization and utilization for improving sensitivity and selectivity in biological fluorescence imaging.

FRET-enabled optical modulation for high sensitivity fluorescence imaging.

Fluorescence resonance energy transfer is utilized to engineer donor photophysics for facile signal amplification and selective fluorescence recovery from high background. This is generalized such that many different fluorophores can be used in optical modulation schemes to drastically improve fluorescence imaging sensitivity. Dynamic, simultaneous, and direct excitation of the acceptor brightens and optically modulates higher energy donor emission. The externally imposed modulation waveform enables selective donor fluorescence extraction through demodulation. By incorporating an acceptor with significant, spectrally shifted, dark-state population, necessary excitation intensities are quite low and agree well with simulated enhancements. Enhancement versus modulation frequency directly yields dark-state lifetimes in a simple ensemble measurement. Using the long-lived Cy5 dark state in conjunction with Cy3 donors, we demonstrate image extraction from a large background to yield >>10-fold sensitivity improvements through synchronously amplified fluorescence image recovery (SAFIRe).

Synchronously amplified fluorescence image recovery (SAFIRe).

Fluorescence intermittency severely limits brightness in both single molecule and bulk fluorescence. Herein, we demonstrate that optical depopulation of organic fluorophore triplet states opens a path to significantly increased sensitivity by simultaneously increasing brightness and greatly reducing background through synchronously detected fluorescence modulation. Image recovery is achieved through selective fluorescence enhancement via modulating a secondary laser excitation at much lower energy than the observed emission in order to depopulate the long-lived triplet states. A series of xanthene dyes that exhibit efficient triplet-state formation demonstrate that this method of selective signal extraction can be achieved at moderate primary and secondary excitation intensities through tailoring dye photophysics and imaging conditions. Up to 5-fold increases in solution-based fluorescence over primary laser excitation alone was achieved upon secondary laser excitation, and dynamic control of signal modulation was demonstrated over a wide time range simply by varying the modulation frequency of the laser used for depopulation of the triplet state. We identify the photophysical characteristics that enable existing or to-be-designed fluorophores to be used in synchronously amplified fluorescence image recovery (SAFIRe) microscopy.

Optically modulated fluorophores for selective fluorescence signal recovery.

Fluorescence imaging in biological sciences is hindered by significant depth-dependent signal attenuation and highly fluorescent backgrounds. We have developed optically modulated near-IR-emitting few-atom Ag nanodots that are selectively and dynamically photobrightened upon simultaneous excitation with a secondary laser, enabling high-sensitivity image extraction to reveal only the demodulated fluorophores. Image demodulation is demonstrated in high-background environments to extract weak signals from completely obscuring background emission.

Electron transfer-induced blinking in Ag nanodot fluorescence.

Various single-standed DNA-encapsulated Ag nanoclusters (nanodots) exhibit strong, discrete fluorescence with solvent polarity-dependent absorption and emission throughout the visible and near-IR. All species examined, regardless of their excitation and emission energies, show similar µs single-molecule blinking dynamics and near IR transient absorptions. The polarity dependence, µsec blinking, and indistinguishable µsec-decaying transient absorption spectra for multiple nanodots suggest a common charge transfer-based mechanism that gives rise to nanodot fluorescence intermittency. Photoinduced charge transfer that is common to all nanodot emitters is proposed to occur from the Ag cluster into the nearby DNA bases to yield a long-lived charge-separated trap state that results in blinking on the single molecule level.

Live cell surface labeling with fluorescent Ag nanocluster conjugates.

DNA-encapsulated silver clusters are readily conjugated to proteins and serve as alternatives to organic dyes and semiconductor quantum dots. Stable and bright on the bulk and single molecule levels, Ag nanocluster fluorescence is readily observed when staining live cell surfaces. Being significantly brighter and more photostable than organics and much smaller than quantum dots with a single point of attachment, these nanomaterials offer promising new approaches for bulk and single molecule biolabeling.

Water-soluble Ag nanoclusters exhibit strong two-photon-induced fluorescence.

Water-soluble ssDNA-encapsulated Ag clusters exhibit large two-photon cross sections reaching 50 000 GM, with high quantum yields in the red and near-IR. Three distinct, spectrally pure, several atom clusters emitting at 660, 680, or 710 nm have been created with two-photon cross sections rivaling those of much larger water-soluble semiconductor quantum dots. Their stability, biocompatibility, and small size offer the promise of nontoxic, sensitive high-resolution biological imaging.

Oligonucleotide-stabilized Ag nanocluster fluorophores.

Single-stranded oligonucleotides stabilize highly fluorescent Ag nanoclusters, with emission colors tunable via DNA sequence. We utilized DNA microarrays to optimize these scaffold sequences for creating nearly spectrally pure Ag nanocluster fluorophores that are highly photostable and exhibit great buffer stability. Five different nanocluster emitters have been created with tunable emission from the blue to the near-IR and excellent photophysical properties. Ensemble and single molecule fluorescence studies show that oligonucleotide encapsulated Ag nanoclusters exhibit significantly greater photostability and higher emission rates than commonly used cyanine dyes.

Strongly emissive individual DNA-encapsulated Ag nanoclusters as single-molecule fluorophores.

The water-soluble, near-IR-emitting DNA-encapsulated silver nanocluster presented herein exhibits extremely bright and photostable emission on the single-molecule and bulk levels. The photophysics have been elucidated by intensity-dependent correlation analysis and suggest a heavy atom effect of silver that rapidly depopulates an excited dark level before quenching by oxygen, thereby conferring great photostability, very high single-molecule emission rates, and essentially no blinking on experimentally relevant time scales (0.1 to >1,000 ms). Strong antibunching is observed from these biocompatible species, which emit >10(9) photons before photobleaching. The significant dark-state quantum yield even enables bunching from the emissive state to be observed as a dip in the autocorrelation curve with only a single detector as the dark state precludes emission from the emissive level. These species represent significant improvements over existing dyes, and the nonpower law blinking kinetics suggest that these very small species may be alternatives to much larger and strongly intermittent semiconductor quantum dots.