The conserved tetrameric subunit stoichiometry of Slc26 proteins.

The Slc26 family proteins, with one possible exception, transport anions across membranes in a wide variety of tissues in vertebrates, invertebrates, and plants. Mutations in human members of the family are a significant cause of disease. Slc26 family proteins are thought to be oligomers, but their stoichiometry of association is in dispute. A recent study, using sequential bleaching of single fluorophore-coupled molecules in membrane fragments, demonstrated that mammalian Slc26a5 (prestin) is a tetramer. In this article, the stoichiometry of two nonmammalian prestins and three human SLC26 proteins has been analyzed by the same method, including the evolutionarily-distant SLC26A11. The analysis showed that tetramerization is common and likely to be ubiquitous among Slc26 proteins, at least in vertebrates. The implication of the findings is that tetramerization is present for functional reasons.

Prestin in HEK cells is an obligate tetramer.

The unusual membrane motor protein prestin is essential for mammalian hearing and for the survival of cochlear outer hair cells. While prestin has been demonstrated to be a homooligomer, by Western blot and FRET analyses, the stoichiometry of self association is unclear. Prestin, coupled to the enhanced green fluorescent protein, was synthesized and membrane targeted in human embryonic kidney cells by plasmid transfection. Fragments of membrane containing immobilized fluorescent molecules were isolated by osmotic lysis. Diffraction-limited fluorescent spots consistent in size with single molecules were observed. Under continuous excitation, the spots bleached to background in sequential and approximately equal-amplitude steps. The average step count to background levels was 2.7. A binomial model of prestin oligomerization indicated that prestin was most likely a tetramer, and that a fraction of the green fluorescent protein molecules was dark. As a positive control, the same procedure was applied to cells transfected with plasmids coding for the human cyclic nucleotide-gated ion channel A3 subunit (again coupled to the enhanced green fluorescent protein), which is an obligate tetramer. The average step count for this molecule was also 2.7. This result implies that in cell membranes prestin oligomerizes to a tetramer.

Single molecule imaging approach to membrane protein stoichiometry.

Recent technical advances have enabled the imaging of single fluorescent molecules. The application of single molecule visualization techniques has opened up new avenues of experimentation in biology at the molecular level. In this article, we review the application of single fluorescent molecule visualization and analysis to an important problem, that of subunit stoichiometry in membrane proteins, with particular emphasis on our approach. Single fluorescent molecules, coupled to fluorescent proteins, are localized in the membranes of cells. The molecules are then exposed to continuous low-level excitation until their fluorescent emissions reach background levels. The high sensitivity of modern instrumentation has enabled direct observations of discrete step decreases in the fluorescence of single molecules, which represent the bleaching of single fluorophores. By counting the number of steps over a large number of single molecules, an average step count is determined from which the stoichiometry is deduced using a binomial model. We examined the stoichiometry of a protein, prestin, that is central to mammalian hearing. We discuss how we prepared, identified, and imaged single molecules of prestin. The methodological considerations behind our approach are described and compared to similar procedures in other laboratories.

Metabolic imaging using two-photon excited NADH intensity and fluorescence lifetime imaging.

Metabolism and mitochondrial dysfunction are known to be involved in many different disease states. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, fluorescence intensity has commonly been used as an indicator of the NADH concentration in cells and tissues. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. Since NADH binding changes with metabolic state, this approach presents a new opportunity to track the cellular metabolic state.

Development and characterization of phasor-based analysis for FLIM to evaluate the metabolic and epigenetic impact of HER2 inhibition on squamous cell carcinoma cultures.

SIGNIFICANCE: Deranged metabolism and dysregulated growth factor signaling are closely associated with abnormal levels of proliferation, a recognized hallmark in tumorigenesis. Fluorescence lifetime imaging microscopy (FLIM) of endogenous nicotinamide adenine dinucleotide (NADH), a key metabolic coenzyme, offers a non-invasive, diagnostic indicator of disease progression, and treatment response. The model-independent phasor analysis approach leverages FLIM to rapidly evaluate cancer metabolism in response to targeted therapy. AIM: We combined lifetime and phasor FLIM analysis to evaluate the influence of human epidermal growth factor receptor 2 (HER2) inhibition, a prevalent cancer biomarker, on both nuclear and cytoplasmic NAD(P)H of two squamous cell carcinoma (SCC) cultures. While better established, the standard lifetime analysis approach is relatively slow and potentially subject to intrinsic fitting errors and model assumptions. Phasor FLIM analysis offers a rapid, model-independent alternative, but the sensitivity of the bound NAD(P)H fraction to growth factor signaling must also be firmly established. APPROACH: Two SCC cultures with low- and high-HER2 expression, were imaged using multiphoton-excited NAD(P)H FLIM, with and without treatment of the HER2 inhibitor AG825. Cells were challenged with mitochondrial inhibition and uncoupling to investigate AG825's impact on the overall metabolic capacity. Phasor FLIM and lifetime fitting analyses were compared within nuclear and cytoplasmic compartments to investigate epigenetic and metabolic impacts of HER2 inhibition. RESULTS: NAD(P)H fluorescence lifetime and bound fraction consistently decreased following HER2 inhibition in both cell lines. High-HER2 SCC74B cells displayed a more significant response than low-HER2 SCC74A in both techniques. HER2 inhibition induced greater changes in nuclear than cytoplasmic compartments, leading to an increase in NAD(P)H intensity and concentration. CONCLUSIONS: The use of both, complementary FLIM analysis techniques together with quantitative fluorescence intensity revealed consistent, quantitative changes in NAD(P)H metabolism associated with inhibition of growth factor signaling in SCC cell lines. HER2 inhibition promoted increased reliance on oxidative phosphorylation in both cell lines.

Low-intensity light-induced drug release from a dual delivery system comprising of a drug loaded liposome and a photosensitive conjugate.

This study reports the development of a binary drug delivery system consisting of charged liposomes and an oppositely charged peptide-photosensitiser conjugate. Liposomes were prepared with phosphatidyl-l-serine as a negatively charged lipid. Calcein, a fluorophore marker, and doxorubicin, an anticancer drug, were used as model hydrophilic loads. The conjugate consisted of a positively charged arginine-rich peptide synthesised by solid-phase peptide synthesis, and a phthalocyanine derivative with characteristic absorption around 685 nm. Illumination of the binary system with far-red light of 12-15 mW/cm2 intensity resulted in 5- to 15-fold increase in release of payloads from the liposomes. The mechanism of drug release was based on photosensitised oxidation of lipids destabilising the liposomal membrane. The cytotoxicity of the liposomes loaded with doxorubicin was tested on B16-F10 melanoma and Y79 retinoblastoma cells. The cytotoxicity of the illuminated binary system in melanoma cell line was significantly higher as compared to the system without illumination. The components of the binary system can be individually prepared and stored with greater storage stability. However, their combination will allow for substantial release of hydrophilic payload from the liposomes under externally applied light.

Ultrasmall Mixed Eu-Gd Oxide Nanoparticles for Multimodal Fluorescence and Magnetic Resonance Imaging of Passive Accumulation and Retention in TBI.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. TBI can have a long-term impact on the quality of life for survivors of all ages. However, there remains no approved treatment that improves outcomes following TBI, which is partially due to poor delivery of therapies into the brain. Therefore, there is a significant unmet need to develop more effective delivery strategies that increase the accumulation and retention of potentially efficacious treatments in the injured brain. Recent work has revealed that nanoparticles (NPs) may offer a promising approach for site-specific delivery; however, a detailed understanding of the specific NP properties that promote brain accumulation and retention are still being developed. Multimodal imaging plays a vital role in the understanding of physicochemical properties that initiate the uptake and accumulation of NPs in the brain at both high spatial (e.g., fluorescence imaging) and temporal (e.g., magnetic resonance imaging, MRI) frequency. However, many NP systems that are currently used in TBI only provide contrast in a single imaging modality limiting the imaging data that can be obtained, and those that offer multimodal imaging capabilities have complicated multistep synthesis methods. Therefore, the goal of this work was to develop an ultrasmall NP with simple fabrication capable of multimodal imaging. Here, we describe the development, characterization, accumulation, and retention of poly(ethylene glycol) (PEG)-coated europium-gadolinium (Eu-Gd) mixed magnetic NPs (MNPs) in a controlled cortical impact mouse model of TBI. We find that these NPs having an ultrasmall core size of 2 nm and a small hydrodynamic size of 13.5 nm can be detected in both fluorescence and MR imaging modalities and rapidly accumulate and are retained in injured brain parenchyma. These NPs should allow for further testing of NP physicochemical properties that promote accumulation and retention in TBI and other disease models.

Determination of cell elasticity through hybrid ray optics and continuum mechanics modeling of cell deformation in the optical stretcher.

The optical stretcher is a dual-beam trap capable of stretching individual cells. Previous studies have used either ray- or wave-optical models to compute the optical pressure on the surface of a spherical cell. We have extended the ray-optics model to account for focusing by the spherical interface and the effects of multiple internal reflections. Simulation results for red-blood cells (RBCs) show that internal reflections can lead to significant perturbation of the deformation, leading to a systematic error in the determination of cellular elasticity. Calibration studies show excellent agreement between the predicted and measured escape force, and RBC stiffness measurements are consistent with literature values. Measurements of the elasticity of murine osteogenic cells reveal that these cells are approximately 5.4 times stiffer than RBCs.

Low-intensity light-induced paclitaxel release from lipid-based nano-delivery systems.

Light-induced drug release has been explored as a strategy for externally modulating the release of drug from delivery systems. This study reports the development of a solid lipid nanoparticulate system (SLN) for paclitaxel (PTX), where photosensitizer-mediated oxidation of lipids was used as a mechanism for controlling the drug release. Low-intensity (23 mW/cm2) near-infrared (around 730 nm) illumination was externally applied as the light source. PTX release was less than 10% within 4 h from these SLN and was 8-fold higher after application of light at time zero. The other advantages of this approach include the use of ascorbic acid (ASC) as an antioxidant for enhancing the release and storage stability of the delivery system. Antioxidant like ASC in the SLN decrease the degradation of lipid by 8-fold within 4 months of storage. Presence of ASC and light illumination of SLN containing PTX further decreased the IC50 by 2 times in A549 cells. The uniqueness of this approach allows the possibility of external modulation to achieve pulsatile release from the delivery system. The light used in the NIR spectral range of 700-850 nm, which has the greatest tissue penetration ability, with a low intensity will be safe for normal tissues.

Aminoglycosides rapidly inhibit NAD(P)H metabolism increasing reactive oxygen species and cochlear cell demise.

Despite causing permanent hearing loss by damaging inner ear sensory cells, aminoglycosides (AGs) remain one of the most widely used classes of antibiotics in the world. Although the mechanisms of cochlear sensory cell damage are not fully known, reactive oxygen species (ROS) are clearly implicated. Mitochondrial-specific ROS formation was evaluated in acutely cultured murine cochlear explants exposed to gentamicin (GM), a representative ototoxic AG antibiotic. Superoxide (O2·-) and hydrogen peroxide (H2O2) were measured using MitoSOX Red and Dihydrorhodamine 123, respectively, in sensory and supporting cells. A 1-h GM exposure significantly increased O2·- formation in IHCs and increased H2O2 formation in all cell types. At the same time point, GM significantly increased manganese superoxide dismutase (MnSOD) levels while significantly decreasing copper/zinc superoxide dismutase (CuZnSOD) in cochlear sensory cells. This suggests (1) a rapid conversion of highly reactive O2·- to H2O2 during the acute stage of ototoxic antibiotic exposure and (2) that the endogenous antioxidant system is significantly altered by AGs. Fluorescence intensity-based measurements of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and mitochondrial membrane potential were measured to determine if increases in GM-induced ROS production were correlated with changes in mitochondrial metabolism. This project provides a basis for understanding the mechanisms of mitochondrial ROS production in cochlear cells exposed to ototoxic antibiotics. Understanding the nature of ototoxic antibiotic-induced changes in mitochondrial metabolism is critical for developing hearing loss treatment and prevention strategies.

Determination of hair cell metabolic state in isolated cochlear preparations by two-photon microscopy.

Currently there is no accepted method to measure the metabolic status of the organ of Corti. Since metabolism and mitochondrial dysfunction are expected to play a role in many different hearing disorders, here for the first time we employ two-photon metabolic imaging to assess the metabolic status of the cochlea. When excited with ultrashort pulses of 740-nm light, both inner and outer hair cells in isolated murine cochlear preparations exhibited intrinsic fluorescence. This fluorescence is characterized and shown to be consistent with a mixture of oxidized flavoproteins (Fp) and reduced nicotinamide adenine dinucleotide (NADH). The location of the fluorescence within hair cells is also consistent with the different mitochondrial distributions in these cell types. Treatments with cyanide and mitochondrial uncouplers show that hair cells are metabolically active. Both NADH and Fp in inner hair cells gradually become completely oxidized within 50 min from the time of death of the animal. Outer hair cells show similar trends but are found to have greater variability. We show that it is possible to use two-photon metabolic imaging to assess metabolism in the mouse organ of Corti.

Studying inner ear protein-protein interactions using FRET and FLIM.

Molecular genetic studies of the inner ear have recently revealed a large number of previously undescribed proteins, but their functions remain unclear. Optical methods such as FRET and FLIM are just beginning to be applied to the study of functional interactions between novel inner ear proteins. This review discusses the various methods for employing FRET and FLIM in protein-protein interaction studies, their advantages and pitfalls, with examples drawn from inner ear studies.

Photobleaching of reduced nicotinamide adenine dinucleotide and the development of highly fluorescent lesions in rat basophilic leukemia cells during multiphoton microscopy.

Endogenous reduced nicotinamide adenine dinucleotide (NADH) fluorescence provides an intrinsic indicator of the cellular metabolic state, but prolonged monitoring is limited by photobleaching and/or phototoxicity. Multiphoton excitation of NADH by ultrashort, 740-nm laser pulses provides a significant improvement over UV excitation by eliminating peripheral photobleaching; however, molecules within the subfemtoliter excitation volume remain susceptible. We have investigated the photophysical mechanisms responsible for multiphoton photobleaching of NADH in living cells to permit the imaging technique to be optimized. The loss of fluorescence because of multiphoton photobleaching was measured by repetitively imaging individual planes within rat basophilic leukemia cells. The photobleaching rate was proportional to the fourth power of the laser intensity. Based on these measurements, we propose a double-biphotonic, four-photon photobleaching mechanism and estimate the quantum yield of photobleaching of intracellular NADH to be 0.0073 +/- 0.0002 by this mechanism. In addition to photobleaching, the development of bright, punctate fluorescent lesions can also be observed. The frequency of lesion formation also increased approximately as the fourth power of the laser intensity after an intensity-dependent threshold number of images had been exceeded. The consequences for two-photon metabolic imaging are discussed.

The Single-Molecule Approach to Membrane Protein Stoichiometry.

The advent of techniques for imaging solitary fluorescent molecules has made possible many new kinds of biological experiments. Here, we describe the application of single-molecule imaging to the problem of subunit stoichiometry in membrane proteins. A membrane protein of unknown stoichiometry, prestin, is coupled to the fluorescent enhanced green fluorescent protein (eGFP) and synthesized in the human embryonic kidney (HEK) cell line. We prepare adherent membrane fragments containing prestin-eGFP by osmotic lysis. The molecules are then exposed to continuous low-level excitation until their fluorescence reaches background levels. Their fluorescence decreases in discrete equal-amplitude steps, consistent with the photobleaching of single fluorophores. We count the number of steps required to photobleach each molecule. The molecular stoichiometry is then deduced using a binomial model.

A comparison of the sensitivity of photodamage assays in rat basophilic leukemia cells.

Many aspects of cellular function or physiology can be used to indicate the level of damage resulting from the application of potentially deleterious agents such as drugs, solvents or even light. The dose required to reach a specific biological endpoint will necessarily depend on the characteristics of the damage induced by the agent. By using multiple biological probes, it is possible to get a more complete description of the type of damage induced. Photodamage was induced in rat basophilic leukemia cells by either 254-nm UVC light exposure or rose bengal photosensitization. Damage was measured by three quantitative assays employing fluorescent probes: calcein, to measure nonspecific esterase activity, propidium iodide (PI), to measure loss of plasma membrane integrity, rhodamine 123 (R123) to measure mitochondrial depolarization, and the incorporation of 5'-bromodeoxyuridine (BrdU), to measure the progress of cell replication. BrdU incorporation was found to be the most sensitive indicator for both forms of photodamage. For UVC photodamage, the BrdU assay was 330 times more sensitive than the other two assays. For rose bengal photosensitization, the BrdU assay was 48 or 62 times more sensitive than either the R123 or calcein/PI assays, respectively.

Reduction in DNA synthesis during two-photon microscopy of intrinsic reduced nicotinamide adenine dinucleotide fluorescence.

Two-photon laser scanning microscopy (TPLSM) of endogenous reduced nicotinamide adenine dinucleotide (NAD(P)H) provides important information regarding the cellular metabolic state. When imaging the punctate mitochondrial fluorescence originating from NAD(P)H in a rat basophilic leukemia (RBL) cell at low laser powers, no morphological changes are evident, and photobleaching is not observed when many images are taken. At higher powers, mitochondrial NAD(P)H fluorescence bleaches rapidly. To assess the limitations of this technique and to quantify the extent of photodamage, we have measured the effect of TPLSM on DNA synthesis. Although previous reports have indicated a threshold power for "safe" two-photon imaging, we find the laser power to be an insufficient indicator of photodamage. A more meaningful metric is a two-photon-absorbed dose that is proportional to the number of absorbed photon pairs. A temporary reduction of DNA synthesis in RBL cells occurs whenever a threshold dose of approximately 2 x 10(53) photon2 cm-4 s-1 is exceeded. This threshold is independent of laser intensity when imaging with average powers ranging from 5 to 17 mW at 740 nm. Beyond this threshold, the extent of the reduction is intensity dependent. DNA synthesis returns to control levels after a recovery period of several hours.

Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging.

Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs.

Gentamicin rapidly inhibits mitochondrial metabolism in high-frequency cochlear outer hair cells.

Aminoglycosides (AG), including gentamicin (GM), are the most frequently used antibiotics in the world and are proposed to cause irreversible cochlear damage and hearing loss (HL) in 1/4 of the patients receiving these life-saving drugs. Akin to the results of AG ototoxicity studies, high-frequency, basal turn outer hair cells (OHCs) preferentially succumb to multiple HL pathologies while inner hair cells (IHCs) are much more resilient. To determine if endogenous differences in IHC and OHC mitochondrial metabolism dictate differential sensitivities to AG-induced HL, IHC- and OHC-specific changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH) fluorescence during acute (1 h) GM treatment were compared. GM-mediated decreases in NADH fluorescence and succinate dehydrogenase activity were observed shortly after GM application. High-frequency basal turn OHCs were found to be metabolically biased to rapidly respond to alterations in their microenvironment including GM and elevated glucose exposures. These metabolic biases may predispose high-frequency OHCs to preferentially produce cell-damaging reactive oxygen species during traumatic challenge. Noise-induced and age-related HL pathologies share key characteristics with AG ototoxicity, including preferential OHC loss and reactive oxygen species production. Data from this report highlight the need to address the role of mitochondrial metabolism in regulating AG ototoxicity and the need to illuminate how fundamental differences in IHC and OHC metabolism may dictate differences in HC fate during multiple HL pathologies.

Metabolic imaging of the organ of corti--a window on cochlea bioenergetics.

Hair cell loss is a major cause of sensorineural hearing loss. We have developed a method to examine metabolic events in hair cells in response to stimuli known to cause hair cell loss, such as acoustic trauma and aminoglycoside administration. The method employs two-photon excitation of the metabolic intermediate, reduced nicotinamide adenine dinucleotide (NADH), in hair cell mitochondria in an explanted mouse cochlea. Using this method, we show evidence that the aminoglycoside gentamicin selectively affects the level of mitochondrial NADH in outer hair cells, but not inner hair cells, within minutes of administration.