Search

Significance Here, we present unequivocal evidence of physical association of AMPK holoenzymes with mitochondrial reticulum (mitoAMPK) across multiple mouse tissues with evidence of conservation in human skeletal muscle and heart. We demonstrate that mitoAMPK is activated heterogeneously across the mitochondrial reticulum by mitochondrial energetic stress. Finally, we present evidence that suggests activation of mitoAMPK in skeletal muscle is required for mitophagy. We propose that mitoAMPK responds to mitochondrial microenvironment cues to maintain energetic homeostasis through mitochondrial quality control., Mitochondria form a complex, interconnected reticulum that is maintained through coordination among biogenesis, dynamic fission, and fusion and mitophagy, which are initiated in response to various cues to maintain energetic homeostasis. These cellular events, which make up mitochondrial quality control, act with remarkable spatial precision, but what governs such spatial specificity is poorly understood. Herein, we demonstrate that specific isoforms of the cellular bioenergetic sensor, 5′ AMP-activated protein kinase (AMPKα1/α2/β2/γ1), are localized on the outer mitochondrial membrane, referred to as mitoAMPK, in various tissues in mice and humans. Activation of mitoAMPK varies across the reticulum in response to energetic stress, and inhibition of mitoAMPK activity attenuates exercise-induced mitophagy in skeletal muscle in vivo. Discovery of a mitochondrial pool of AMPK and its local importance for mitochondrial quality control underscores the complexity of sensing cellular energetics in vivo that has implications for targeting mitochondrial energetics for disease treatment.

Altered mitochondrial DNA (mtDNA) occurs in neurodegenerative disorders like Alzheimer’s disease (AD); how mtDNA synthesis is linked to neurodegeneration is poorly understood. We discovered Nutrient-induced Mitochondrial Activity (NiMA), an inter-organelle signaling pathway where nutrient-stimulated lysosomal mTORC1 activity regulates mtDNA replication in neurons by a mechanism sensitive to amyloid-β oligomers (AβOs), a primary factor in AD pathogenesis. Using 5-ethynyl-2’-deoxyuridine (EdU) incorporation into mtDNA of cultured neurons, along with photoacoustic and mitochondrial metabolic imaging of cultured neurons and mouse brains, we show these effects being mediated by mTORC1-catalyzed T40 phosphorylation of superoxide dismutase 1 (SOD1). Mechanistically, tau, another key factor in AD pathogenesis and other tauopathies, reduced the lysosomal content of the tuberous sclerosis complex (TSC), thereby increasing NiMA and suppressing SOD1 activity and mtDNA synthesis. AβOs inhibited these actions. Dysregulation of mtDNA synthesis was observed in fibroblasts derived from TS patients, who lack functional TSC and elevated SOD1 activity was also observed in human AD brain. Together, these findings imply that tau and SOD1 couple nutrient availability to mtDNA replication, linking mitochondrial dysfunction to AD.

With different states of two intrinsic fluorophores, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), we developed a single-layer autoencoder (AE) for feature extraction, which outputs condensed features representing the full metabolic FLIM information with lower dimensionality. We also described distributions of AE features and fluorescence lifetime redox ratio (FLIRR) from single cells by Gaussian mixture models (GMM), and predicted the values of FLIRR based on feature data from each time point for the HeLa cell lines and Caucasian-American (LNCaP) prostate cancer cell lines by the polynomial regression model and the random forest regression model.

This chapter discusses the best to apply fluorescence lifetime microscopy (FLIM) to those autofluorescent cellular components and analyzes the data by individual, segmented cells using HeLa cervical cancer cells as a model. nicotinamide adenine dinucleotide and flavin adenine dinucleotide channels also contain 690 nm short pass filter in the beam path. Evolution has provided life on earth with several methods of generating cellular energy. Applications of FLIM have grown exponentially in a broad range of life sciences and industrial fields, a reflection of specific advantages over intensity-based microscopy. The effects on lifetime of fixing specimens with formaldehyde or methanol have been published with mixed results for fluorescently labeled proteins, depending on types of buffers and/or mounting media. The fluorescence lifetime parameters are derived from a model containing lifetime components and fractions used to fit the measured data. The measured decay data are fitted with the convolution of the model function and the instrument response function.

Redox changes in live HeLa cervical cancer cells after doxorubicin treatment can either be analyzed by a novel fluorescence lifetime microscopy (FLIM)-based redox ratio NAD(P)H-a2%/FAD-a1%, called fluorescence lifetime redox ratio or one of its components (NAD(P)H-a2%), which is actually driving that ratio and offering a simpler and alternative metric and are both compared. Auto-fluorescent NAD(P)H, FAD lifetime is acquired by 2- photon excitation and Tryptophan by 3-photon, at 4 time points after treatment up to 60 min demonstrating early drug response to doxorubicin. Identical Fields-of-view (FoV) at each interval allows single-cell analysis, showing heterogeneous responses to treatment, largely based on their initial control redox state. Based on a discrete ROI selection method, mitochondrial OXPHOS and cytosolic glycolysis are discriminated. Furthermore, putative FRET interaction and energy transfer between tryptophan residue carrying enzymes and NAD(P)H correlate with NAD(P)H-a2%, as does the NADPH/NADH ratio, highlighting a multi-parametric assay to track metabolic changes in live specimens. © 2019 International Society for Advancement of Cytometry.

Increasingly, the auto-fluorescent coenzymes NAD(P)H and FAD are being tracked by multi-photon fluorescence lifetime microscopy (FLIM) and used as versatile markers for changes in mammalian metabolism. The cellular redox state of different cell model systems, organoids and tissue sections is investigated in a range of pathologies where the metabolism is disrupted or reprogrammed; the latter is particularly relevant in cancer biology. Yet, the actual optimized process of acquiring images by FLIM, execute a correct lifetime fitting procedure and subsequent processing and analysis can be challenging for new users. Questions remain of how to optimize FLIM experiments, whether any potential photo-bleaching affects FLIM results and whether fixed specimens can be used in experiments. We have broken down the multi-step sequence into best-practice application of FLIM for NAD(P)H and FAD imaging, with images generated by a time-correlated-single-photon-counting (TCSPC) system, fitted with Becker & Hickl software and further processed with open-source ImageJ/Fiji and Python software.

Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture autofluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. Among these intrinsic fluorophores, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD)—essential coenzymes in cellular respiration—have been used as intrinsic fluorescent biomarkers for metabolic states in cancer and other pathologies. Traditional FLIM imaging for NAD(P)H, FAD, and in particular fluorescence lifetime redox ratio (FLIRR) requires a sequential multiwavelength excitation to avoid spectral bleed-through (SBT). This sequential imaging complicates image acquisition, may introduce motion artifacts, and reduce temporal resolution. Testing several two-photon excitation wavelengths in combination with optimized emission filters, we have proved a FLIM imaging protocol, allowing simultaneous image acquisition with a single 800-nm wavelength excitation for NADH and FAD with negligible SBT. As a first step, standard NADH and FAD single and mixed solutions were tested that mimic biological sample conditions. After these optimization steps, the assay was applied to two prostate cancer live cell lines: African-American (AA) and Caucasian-American (LNCaP), used in our previous publications. FLIRR result shows that, in cells, the 800-nm two-photon excitation wavelength is suitable for NADH and FAD FLIM imaging with negligible SBT. While NAD(P)H signals are decreased, sufficient photons are present for accurate lifetime fitting and FAD signals are measurably increased at lower laser power, compared with the common 890-nm excitation conditions. This single wavelength excitation allows a simplification of NADH and FAD FLIM imaging data analysis, decreasing the total imaging time. It also avoids motion artifacts and increases temporal resolution. This simplified assay will also make it more suitable to be applied in a clinical setting.

A major obstacle to pre-symptomatic diagnosis and disease-modifying therapy for Alzheimer's disease (AD) is inadequate understanding of molecular mechanisms of AD pathogenesis. For example, impaired brain insulin signaling is an AD hallmark, but whether and how it might contribute to the synaptic dysfunction and neuron death that underlie memory and cognitive impairment has been mysterious. Neuron death in AD is often caused by cell cycle re-entry (CCR) mediated by amyloid-β oligomers (AβOs) and tau, the precursors of plaques and tangles. We now report that CCR results from AβO-induced activation of the protein kinase complex, mTORC1, at the plasma membrane and mTORC1-dependent tau phosphorylation, and that CCR can be prevented by insulin-stimulated activation of lysosomal mTORC1. AβOs were also shown previously to reduce neuronal insulin signaling. Our data therefore indicate that the decreased insulin signaling provoked by AβOs unleashes their toxic potential to cause neuronal CCR, and by extension, neuron death.

Fluorescence lifetime imaging microscopy (FLIM) techniques are widely used in auto-fluorescence imaging to investigate dynamic metabolic states of the cells. Traditional FLIM imaging requires the cells to adhere to the coverslip to collect enough photons for FLIM data processing. Such conditions pose challenges to non-adherent cells, such as acute myeloid leukemia (AML) cells, because of cell motility. We developed a proto-type micro cell trapping array (MCTA) to immobilize cells in picoliter-size wells with location references. The array keeps non-adherent cells in referenced well locations, allows treatment on stage and re-imaging after time for ultimate cell segmentation analysis. Individual wells are analyzed by a pixel-based region-of-interest (ROI) to analyze cellular redox states. This single well trapping and analysis method allows to isolate treatment responses of a small number of cells, compare their range and predict early effect, which may have clinical applications in the context of cancer aggressiveness and treatment outcomes. We expanded the common intensity-based assay for cellular redox state by a Fluorescence Lifetime measurement, NAD(P)H-a2%, serving as an alternative metric. The new assay is flexible and can be applied to other non-adherent cell lines, expanding FLIM applications in both research and the clinic.

Imaging natural fluorescence of the endogenous molecules is of clinical importance. Amino acids like Phenylalanine, Tyrosine and Tryptophan, structural proteins like collagen and elastin, and coenzymes directly involved in cellular metabolism like Nicotinamide Adenine Dinucleotide Phosphate (NAD(P)H) and Flavin Adenine Dinucleotide (FAD). Each endogenous molecule absorbs and emits at different wavelengths and has been used to detect various cancer including prostate cancer (PCa), based on fluorescence intensity. NAD(P)H and FAD provide detectable natural autofluorescence signals to monitor metabolic flux. Among listed amino acids, tryptophan (Trp) provides the strongest fluorescence signals. It has been shown by biochemical methods that degradation or reduction in the abundance of the Trp indicates the presence of cancer in biological systems. The one-photon absorption of Trp is 260 nm which is phototoxic to the cells. The emission of Trp is 300-400 nm. Most of the optical microscopy system transmits maximally starting from 400 nm. To meet the Trp requirement, we configured the Zeiss 780 to provide maximum emission signal about 80% at 340 nm at the ND output port for three-photon excitation. The spectral sensitivity of the detector used for Trp imaging is 300-650 nm which is integrated with the Zeiss780 system. A ROI-based segmented cell FLIRR assays was developed to investigate the interaction between NAD(P)H and Trp.

Abnormal folding and aggregation of the microtubule-associated protein, tau, is a hallmark of several neurodegenerative disorders, including Alzheimer's disease (AD). Although normal tau is an intrinsically disordered protein, it does exhibit tertiary structure whereby the N- and C-termini are often in close proximity to each other and to the contiguous microtubule-binding repeat domains that extend C-terminally from the middle of the protein. Unfolding of this paperclip-like conformation might precede formation of toxic tau oligomers and filaments, like those found in AD brain. While there are many ways to monitor tau aggregation, methods to monitor changes in tau folding are not well established. Using full length human 2N4R tau doubly labeled with the Forster resonance energy transfer (FRET) compatible fluorescent proteins, Venus and Teal, on the N- and C-termini, respectively (Venus-Tau-Teal), intensity and lifetime FRET measurements were able to distinguish folded from unfolded tau in living cells independently of tau-tau intermolecular interactions. When expression was restricted to low levels in which tau-tau aggregation was minimized, Venus-Tau-Teal was sensitive to microtubule binding, phosphorylation, and pathogenic oligomers. Of particular interest is our finding that amyloid-β oligomers (AβOs) trigger Venus-Tau-Teal unfolding in cultured mouse neurons. We thus provide direct experimental evidence that AβOs convert normally folded tau into a conformation thought to predominate in toxic tau aggregates. This finding provides further evidence for a mechanistic connection between Aβ and tau at seminal stages of AD pathogenesis.

Redox changes in live HeLa cervical cancer cells after doxorubicin treatment can either be analyzed by a novel fluorescence lifetime microscopy (FLIM)-based redox ratio NAD(P)H-a2%/FAD-a1%, called fluorescence lifetime redox ratio or one of its components (NAD(P)H-a2%), which is actually driving that ratio and offering a simpler and alternative metric and are both compared. Auto-fluorescent NAD(P)H, FAD lifetime is acquired by 2- photon excitation and Tryptophan by 3-photon, at 4 time points after treatment up to 60 min demonstrating early drug response to doxorubicin. Identical Fields-of-view (FoV) at each interval allows single-cell analysis, showing heterogeneous responses to treatment, largely based on their initial control redox state. Based on a discrete ROI selection method, mitochondrial OXPHOS and cytosolic glycolysis are discriminated. Furthermore, putative FRET interaction and energy transfer between tryptophan residue carrying enzymes and NAD(P)H correlate with NAD(P)H-a2%, as does the NADPH/NADH ratio, highlighting a multi-parametric assay to track metabolic changes in live specimens. © 2019 International Society for Advancement of Cytometry.

The mechanisms of mitochondrial dysfunction in Alzheimer's disease are incompletely understood. Using two-photon fluorescence lifetime microscopy of the coenzymes, NADH and NADPH, and tracking brain oxygen metabolism with multi-parametric photoacoustic microscopy, we show that activation of lysosomal mechanistic target of rapamycin complex 1 (mTORC1) by insulin or amino acids stimulates mitochondrial activity and regulates mitochondrial DNA synthesis in neurons. Amyloid-β oligomers, which are precursors of amyloid plaques in Alzheimer's disease brain and stimulate mTORC1 protein kinase activity at the plasma membrane but not at lysosomes, block this Nutrient-induced Mitochondrial Activity (NiMA) by a mechanism dependent on tau, which forms neurofibrillary tangles in Alzheimer's disease brain. NiMA was also disrupted in fibroblasts derived from two patients with tuberous sclerosis complex, a genetic disorder that causes dysregulation of lysosomal mTORC1. Thus, lysosomal mTORC1 couples nutrient availability to mitochondrial activity and links mitochondrial dysfunction to Alzheimer's disease by a mechanism dependent on the soluble building blocks of the poorly soluble plaques and tangles.

Multiphoton FLIM microscopy offers many opportunities to investigate processes in live cells, tissue and animal model systems. For redox measurements, FLIM data is mostly published by cell mean values and intensity-based redox ratios. Our method is based entirely on FLIM parameters generated by 3-detector time domain microscopy capturing autofluorescent signals of NAD(P)H, FAD and novel FLIM-FRET application of Tryptophan and NAD(P)H-a2%/FAD-a1% redox ratio. Furthermore, image data is analyzed in segmented cells thresholded by 2 × 2 pixel Regions of Interest (ROIs) to separate mitochondrial oxidative phosphorylation from cytosolic glycolysis in a prostate cancer cell line. Hundreds of data points allow demonstration of heterogeneity in response to intervention, identity of cell responders to treatment, creating thereby different sub-populations. Histograms and bar charts visualize differences between cells, analyzing whole cell versus mitochondrial morphology data, all based on discrete ROIs. This assay method allows to detect subtle differences in cellular and tissue responses, suggesting an advancement over means-based analyses.

The mechanisms of mitochondrial dysfunction in Alzheimer’s Disease (AD) are incompletely understood. Using two-photon fluorescence lifetime microscopy of NAD(P)H and tracking brain oxygen metabolism with multi-parametric photoacoustic microscopy, we show that activation of lysosomal mechanistic target of rapamycin complex 1 (mTORC1) by insulin or amino acids stimulates mitochondrial activity and regulates mitochondrial DNA synthesis in neurons. Amyloid-β oligomers, which are precursors of amyloid plaques in AD brain and stimulate mTORC1 protein kinase activity at the plasma membrane, but not at lysosomes, block this nutrient-induced mitochondrial activity (NiMA) by a mechanism dependent on tau, which forms neurofibrillary tangles in AD brain. NiMA was also disrupted in fibroblasts derived from a patient with tuberous sclerosis complex, a genetic disorder that causes dysregulation of lysosomal mTORC1. Thus, lysosomal mTORC1 couples nutrient availability to mitochondrial activity, and links mitochondrial dysfunction to AD by a mechanism dependent on soluble building blocks of plaques and tangles.

Experiments using live cell 3-color Forster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. In addition, this study emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells. © 2015 International Society for Advancement of Cytometry

Prostate cancer (PCa) is one of the leading cancers in men in the USA. Lack of experimental tools that predict therapy response is one of the limitations of current therapeutic regimens. Mitochondrial dysfunctions including defective oxidative phosphorylation (OXPHOS) in cancer inhibit apoptosis by modulating ROS production and cellular signaling. Thus, correction of mitochondrial dysfunction and induction of apoptosis are promising strategies in cancer treatment. We have used Fluorescence Lifetime Imaging Microscopy (FLIM) to quantify mitochondrial metabolic response in PCa cells by tracking auto-fluorescent NAD(P)H, FAD and tryptophan (Trp) lifetimes and their enzyme-bound fractions as markers, before and after treatment with anti-cancer drug doxorubicin. A 3-channel FLIM assay and quantitative analysis of these markers for cellular metabolism show in response to doxorubicin, NAD(P)H mean fluorescence lifetime (τm) and enzyme-bound (a2%) fraction increased, FAD enzyme-bound (a1%) fraction was decreased, NAD(P)H-a2%/FAD-a1% FLIM-based redox ratio and ROS increased, followed by induction of apoptosis. For the first time, a FRET assay in PCa cells shows Trp-quenching due to Trp-NAD(P)H interactions, correlating energy transfer efficiencies (E%) vs NAD(P)H-a2%/FAD-a1% as sensitive parameters in predicting drug response. Applying this FLIM assay as early predictor of drug response would meet one of the important goals in cancer treatment.

A region of interest (ROI) based quantitative FLIM-FRET image analysis is developed to quantitate the autofluorescence signals of the essential amino acid tryptophan as a biomarker to investigate the metabolism in prostate cancer cells.

Evidence from multiple sources supports the hypothesis that Schwann cells in the peripheral nervous system transfer messenger RNA and ribosomes to the axons they ensheath. Several technical and methodological difficulties exist for investigators to unravel this process in myelinated axons - a complex two-cell unit. We present an experimental design to demonstrate that newly synthesized RNA is transferred from Schwann cells to axons in association with Myosin Va. The use of quantitative confocal FRET microscopy to track newly-synthesized RNA and determine the molecular association with Myosin Va, is described in detail.

IQGAP1 stimulates branched actin filament nucleation by activating N-WASP, which then activates the Arp2/3 complex. N-WASP can be activated by other factors, including GTP-bound Cdc42 or Rac1, which also bind IQGAP1. Here we report the use of purified proteins for in vitro binding and actin polymerization assays, and Forster (or fluorescence) resonance energy transfer (FRET) microscopy of cultured cells to illuminate functional interactions among IQGAP1, N-WASP, actin, and either Cdc42 or Rac1. In pyrene-actin assembly assays containing N-WASP and Arp2/3 complex, IQGAP1 plus either small G protein cooperatively stimulated actin filament nucleation by reducing the lag time before 50% maximum actin polymerization was reached. Similarly, Cdc42 and Rac1 modulated the binding of IQGAP1 to N-WASP in a dose-dependent manner, with Cdc42 enhancing the interaction and Rac1 reducing the interaction. These in vitro reconstitution results suggested that IQGAP1 interacts by similar, yet distinct mechanisms with Cdc42 versus Rac1 to regulate actin filament assembly through N-WASP in vivo. The physiological relevance of these multi-protein interactions was substantiated by 3-color FRET microscopy of live MDCK cells expressing various combinations of fluorescent N-WASP, IQGAP1, Cdc42, Rac1, and actin. This study also establishes 3-color FRET microscopy as a powerful tool for studying dynamic intermolecular interactions in live cells.

NAD(P)H is a known biomarker for cellular metabolism; a higher ratio of enzyme-bound NAD(P)H to free/unbound NAD(P)H indicates an increase in metabolic activity. Free NADH has a shorter fluorescence lifetime (τ1), the bound version (τ2) a longer lifetime. FLIM’s unique capability to establish inter alia the relative fractions of τ1 (a1%) and τ2 (a2%) in each pixel, determines the level of metabolic activity. The relative abundances of bound NAD(P)H were analyzed for single cells, confluent and partially confluent cells within 3 Fields-of-View (FoVs). A gradient of increasing a 2% levels of bound NAD(P)H from single, partially confluent to confluent cells was observed.

Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to identify changes in metabolic activity during cancer progression and upon anti-cancer drug treatment. Prostate cancer (PCa) is one of the leading cancers in men in the USA. This research focusses on understanding the lifetime changes of endogenous biomarkers: NAD(P)H, FAD and Trp in LNCaP cells upon treatment with doxorubicin using our 3-channel FLIM approach. The LNCaP cells were treated with doxorubicin for 24hr. Images using FLIM of LNCaP control and treated cells were acquired on Zeiss 780 multiphoton confocal microscope coupled with B and H TCSPC FLIM board. After FLIM data fitting and processing we observed increase in the mean fluorescence lifetime of Trp, NAD(P)H and FAD with doxorubicin treatment. Additionally, we saw reduction in the NAD(P)H/FAD redox ratio with doxorubicin treatment. Our results identify the changes in the lifetime of these endogenous biomarkers and in the cellular redox state as a metabolic response with doxorubicin treatment in prostate cancer cells.

S-Nitrosoglutathione (GSNO) reductase regulates cell signaling pathways relevant to asthma and protects cells from nitrosative stress. Recent evidence suggests that this enzyme may prevent human hepatocellular carcinoma arising in the setting of chronic hepatitis. We hypothesized that GSNO reductase may also protect the lung against potentially carcinogenic reactions associated with nitrosative stress. We report that wild-type Ras is S-nitrosylated and activated by nitrosative stress and that it is denitrosylated by GSNO reductase. In human lung cancer, the activity and expression of GSNO reductase are decreased. Further, the distribution of the enzyme (including its colocalization with wild-type Ras) is abnormal. We conclude that decreased activity of GSNO reductase could leave the human lung vulnerable to the oncogenic effects of nitrosative stress, as is the case in the liver. This potential should be considered when developing therapies that inhibit pulmonary GSNO reductase to treat asthma and other conditions.

Fluorescence Lifetime Imaging (FLIM) can be used to understand the metabolic activity in cancer. Prostate cancer is one of the leading cancers in men in the USA. This research focuses on FLIM measurements of NAD(P)H and Tryptophan, used as biomarkers to understand the metabolic activity in prostate cancer cells. Two prostate cancers and one normal cell line were used for live-cell FLIM measurements on Zeiss780 2P confocal microscope with SPCM FLIM board. Glucose uptake and glycolysis proceeds about ten times faster in cancer than in non-cancerous tissues. Therefore, we assessed the glycolytic activity in the prostate cancer in comparison to the normal cells upon glucose stimulation by analyzing the NAD(P)H and Trp lifetime distribution and efficiency of energy transfer (E%). Furthermore, we treated the prostate cancer cells with 1μM Doxorubicin, a commonly used anti-cancer chemotherapeutic. Increase in NADH a2%, an indicator of increased glycolysis and increased E% between Trp and NAD(P)H were seen upon glucose stimulation for 30min. The magnitude of shift to the right for NAD(P)H a2% and E% distribution was higher in prostate cancer versus the normal cells. Upon treatment with Doxorubicin decrease in cellular metabolism was seen at 15 and 30 minutes. The histogram for NAD(P)H a2% post-treatment for prostate cancer cells showed a left shift compared to the untreated control suggesting decrease in glycolysis and metabolic activity opposite to what was observed after glucose stimulation. Hence, NAD(P)H and Trp lifetimes can be used biomarkers to understand metabolic activity in prostate cancer and upon chemotherapeutic interventions.

Fluorescence lifetime imaging microscopy (FLIM) is one of the most sensitive techniques to measure metabolic activity in living cells, tissues and whole animals. We used two- and three-photon fluorescence excitation together with time-correlated single photon counting (TCSPC) to acquire FLIM signals from normal and prostate cancer cell lines. FLIM requires complex data fitting and analysis; we explored different ways to analyze the data to match diverse cellular morphologies. After non-linear least square fitting of the multi-photon TCSPC images by the SPCImage software (Becker & Hickl), all image data are exported and further processed in ImageJ. Photon images provide morphological, NAD(P)H signal-based autofluorescent features, for which regions of interest (ROIs) are created. Applying these ROIs to all image data parameters with a custom ImageJ macro, generates a discrete, ROI specific database. A custom Excel (Microsoft) macro further analyzes the data with charts and statistics. Applying this highly automated assay we compared normal and cancer prostate cell lines with respect to their glycolytic activity by analyzing the NAD(P)H-bound fraction (a2%), NADPH/NADH ratio and efficiency of energy transfer (E%) for Tryptophan (Trp). Our results show that this assay is able to differentiate the effects of glucose stimulation and Doxorubicin in these prostate cell lines by tracking the changes in a2% of NAD(P)H, NADPH/NADH ratio and the changes in Trp E%. The ability to isolate a large, ROI-based data set, reflecting the heterogeneous cellular environment and highlighting even subtle changes — rather than whole cell averages - makes this assay particularly valuable.

Autofluorescence of endogeneous fluorophores, such as nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and tryptophan can provide valuable information about metabolic processes and states of cells and tissues. When combined with time-correlated single photon counting (TCSPC), the amount of bound and free NADH and relative amount of NADPH can be readily extracted from the fluorescence decay data. These three FLIM detectors allow us to monitor the three endogenous molecules simultaneously in living cells.One of the challenges in analyzing endogenous fluorescence is the spectral overlap of generally broad emission spectra of the fluorophores. To separate individual components of multi-photon excited autofluorescence (three-photon excited tryptophan and two-photon excited NADH and FAD) we have employed three independent FLIM channels with spectral ranges centered around 360 nm, 480 nm and 540 nm.The calculation of lifetimes of individual components is complicated due to the inevitable emission spectral overlap between NADH and FAD and the inherent instability of multiple-exponential fitting problem. Because TCSPC decay curves can be easily transformed into phasor plots at various harmonic frequencies, we attempt to solve this problem using global analysis methods based both on exponential fitting with global parameters and phasor plot analyses. This work summarizes the main properties and the performance of both methods, and suggests optimal parameters (such as excitation wavelengths and collection times) during image acquisition.

Previously, FRET confocal microscopy has shown that polymeric IgA-receptor (pIgA-R) is distributed in a clustered manner in apical endosomes. To test whether different membrane-bound components form clusters during membrane trafficking, live-cell quantitative FRET was used to characterize the organization of pIgA-R and transferrin receptor (TFR) in endocytic membranes of polarized MDCK cells upon internalization of donor- and acceptor-labeled ligands. We show that pIgA-R and TFR complexes form increasingly organized clusters during cotransport from basolateral to perinuclear endosomes. The organization of these receptor clusters in basolateral versus perinuclear/apical endosomes is significantly different; the former showing a mixed random/clustered distribution while the latter highly organized clusters. Our results indicate that although both perinuclear and apical endosomes comprise pIgA-R and TFR clusters, their E% levels are significantly different suggesting that these receptors are packed into clusters in a distinct manner. The quantitative FRET-based assay presented here suggests that different receptor complexes form clusters, with diverse levels of organization, while being cotransported via the polarized endocytic pathways.

IQGAP1 has been implicated as a regulator of cell motility because its overexpression or underexpression stimulates or inhibits cell migration, respectively, but the underlying mechanisms are not well understood. Here, we present evidence that IQGAP1 stimulates branched actin filament assembly, which provides the force for lamellipodial protrusion, and that this function of IQGAP1 is regulated by binding of type 2 fibroblast growth factor (FGF2) to a cognate receptor, FGFR1. Stimulation of serum-starved MDBK cells with FGF2 promoted IQGAP1-dependent lamellipodial protrusion and cell migration, and intracellular associations of IQGAP1 with FGFR1 – and two other factors – the Arp2/3 complex and its activator N-WASP, that coordinately promote nucleation of branched actin filament networks. FGF2 also induced recruitment of IQGAP1, FGFR1, N-WASP and Arp2/3 complex to lamellipodia. N-WASP was also required for FGF2-stimulated migration of MDBK cells. In vitro, IQGAP1 bound directly to the cytoplasmic tail of FGFR1 and to N-WASP, and stimulated branched actin filament nucleation in the presence of N-WASP and the Arp2/3 complex. Based on these observations, we conclude that IQGAP1 links FGF2 signaling to Arp2/3 complex-dependent actin assembly by serving as a binding partner for FGFR1 and as an activator of N-WASP.

Average lifetime between the usually bi-exponential double -label specimen and a mono -exponential single donor sample serves as a basis for the calculation of the average energy transfer efficiency (E ). T his semi-quantitative approach however does not fully explore cellular functions, such as endosomal pH differences, specific morphological features, ex amin ing sub-po pulations and the like. W e applied a different, quantitative Regi on -of-Interest (ROI)-based method in 2 live -cell assays by TCSPC FLIM-FRET microscopy: a 5 amino-acid linked FRET standard and mouse pituitary cells expressing a GLPHUL]HG& (%3. -bZip transcription factor in the nucleus , both tagged with Cerulean (C) and Venus (V). ROIs with different selection thresholds were generated and compared. Average lifetimes are similar , but ratios between them and other subtle differences are revealed by compreh ensive distribution information. Following published references, we also explored 3 different methods to calculate FLIM-FRET energy transfer efficiencies for the Cerulean-Venus constructs, producing differences and supporting the long-held notion that is called 'apparent' E . efficiencyFRET's greatest contribution es to be exploringcontinu changes taking place at the cellular level and quantifying differences in relative terms between control and variables .

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

With traditional 2-color Forster Resonance Energy Transfer (FRET) microscopy, valuable quantitative analyses can be conducted. Correlations of donor (D), acceptor (A) and their ratios (D:A) with energy transfer efficiency (E%) or distance (r) allows measurement of changes between control and experimental samples; also, clustered vs. random assembly of cellular components can be differentiated. Essentially, only the above three parameters D, A and D:A vs. E% are the basis for these deductions. 3-color FRET uses the same basic parameters, but exponentially expands the opportunities to quantify interrelationships among 3 cellular components. We investigated a number of questions based on the results of a triple combination (F1-F2-F3) of TFP-NWASP/Venus-IQGAP1/mCherry-Actin - all involved in the nucleation of actin - to apply the extensive analysis assay possible with 3-color FRET. How do changing N-WASP or IQGAP1 fluorescence levels affect actin fluorescence? What is the effect on E% of NWASP-actin by IQGAP1 or E% of IQGAP1-actin by N-WASP? These and other questions are explored in the context of all proteins of interest being in FRET distance vs. any two in the absence of the third. 4 cases are compared based on bleed-through corrected FRET: (1) all 3 interact, (2) only F1-F3 and F2-F3 [not F1-F2], (3) only F1-F2 and F2-F3 interact [not F1-F3], (4) only F1-F2 and F1-F3 interact [not F2-F3]. Other than describing the methodology in detail, several biologically relevant results are presented showing how E% (i.e. distance), fluorescence levels and ratios are affected in each of the cases. These correlations can only be observed in a 3-fluorophore combination. 3-color FRET will greatly expand the investigative range of quantitative analysis for the life-science researcher.

Theodor Forster would have been 100 years old this year, and he would have been astounded to see the impact of his scientific achievement, which is still evolving. Combining his quantitative approach of (Forster) resonance energy transfer (FRET) with state-of-the-art digital imaging techniques allows scientists to breach the resolution limits of light (ca. 200 nm) in light microscopy. The ability to deduce molecular or particle distances within a range of 1-10 nm in real time and to prove or disprove interactions between two or more components is of vital interest to researchers in many branches of science. While Forster's groundbreaking theory was published in the 1940s, the availability of suitable fluorophores, instruments, and analytical tools spawned numerous experiments in the last 20 years, as demonstrated by the exponential increase in publications. These cover basic investigation of cellular processes and the ability to investigate them when they go awry in pathological states, the dynamics involved in genetics, and following events in environmental sciences and methods in drug screening. This review covers the essentials of Theodor Forster's theory, describes the elements for successful implementation of FRET microscopy, the challenges and how to overcome them, and a leading-edge example of how Forster's scientific impact is still evolving in many directions. While this review cannot possibly do justice to the burgeoning field of FRET microscopy, a few interesting applications such as threecolor FRET, which greatly expands the opportunities for investigating interactions of cellular components compared with the traditional two-color method, are described, and an extensive list of references is provided for the interested reader to access.

We developed a FRET-based assay to analyze multiple focal planes from z-staks collected using confocal imaging of polarized epithelial MDCK cells. To establish the imaging assay for multiple focal planes, we have used fixed cells, where changes in the organization and conformation of transferrin-receptor complexes between endosomes are expected to be minimized. Therefore, we indeed only see minor changes in E% throughout the endocytic pathway of polarized cells. In the future, we will apply this FRET-based assay to live polarized epithelial cells to investigate the factors involved in the regulation of the organization and conformation of membrane-bound receptors during endocytic trafficking.

Forster resonance energy transfer (FRET) is an effective and high resolution method to monitor protein-protein interactions in live or fixed specimens. FRET can be used to estimate the distance between interacting protein molecules in vivo or in vitro using laser-scanning confocal FRET microscopy. The spectral overlap of donor and acceptor-essential for FRET-also generates a contamination of the FRET signal, which should be removed in order to carry out quantitative data analysis with confidence. Quantitative FRET data analysis addresses the wealth of information contained in the data set, once optimized FRET imaging has been completed. In this chapter, we describe step-by-step what the issues are in quantitative FRET data analysis, using membrane receptor trafficking and organization as an example. The assays described are applicable to many other biological applications.

Forster (or fluorescence) resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM) have moved center stage and are increasingly forming part of multifaceted imaging approaches. They are complementary methodologies that can be applied to advanced quantitative analyses. The widening application of FRET and FLIM has been driven by the availability of suitable fluorophores, increasingly sophisticated microscopy systems, methodologies to correct spectral bleed-through, and the ease with which FRET can be combined with other techniques. FRET and FLIM have recently found use in several applications: in the analysis of protein-protein interactions with high spatial and temporal specificity (e.g. clustering), in the study of conformational changes, in the analysis of binding sequences, and in applications such as high-throughput screening.

This chapter discusses the use of confocal Forster resonance (radiationless) energy transfer (C-FRET) microscopic techniques to study the clustered distribution of receptor–ligand complexes in endocytic membranes. Confocal microscopy is capable of producing images free of out-of-focus information. It provides a significant improvement in lateral resolution and in the capacity for direct, noninvasive serial optical sectioning of intact, thick living specimens. FRET microscopy helps to follow the endocytic and transcytotic transport of membrane-bound receptors internalized from opposite membranes in the epithelial polarized MDCK cell. In FRET-based assay of receptor distribution and sorting, pIgA-R ligands, labeled with different fluorophores, are internalized from opposite PMs in the MDCK cells, which are stably transfected with rabbit pIgA-R. Upon binding, these receptor–ligand complexes are transcytosed from the basolateral PM and endocytosed from the apical PM to eventually co-localize in the apical recycling endosomal compartment. The receptor distribution and sorting the FRET-based assay provides a snapshot of receptor distribution and sorting throughout the endocytic pathway of polarized cells.

Here, we have investigated the molecular mechanisms underlying the dynamics of protein distribution within membranes using Fluorescence Resonance Energy Transfer Microscopy (FRET). We have developed a one-photon (1-P) and two-photon (2-P) FRET assay to differentiate between the clustered and random distribution of membrane-bound fluorophore-labeled receptor-ligand complexes. Our results demonstrate that polymeric IgA-receptor-ligand complexes are organized in clusters within apical endocytic membranes of polarized MDCK cells, since energy transfer efficiency (E%) levels are independent from acceptor fluorescence, a standard parameter to confirm clustered distribution. We also describe a second parameter: E% decreases with increasing unquenched donor fluorescence and unquenched donor:acceptor ratios, a phenomenon which we ascribe to some donors preventing others from interacting with an acceptor. We call this effect 'donor geometric exclusion.' Going beyond the determination of clustered vs. random distribution of protein complexes, mathematical models have been developed, tailored to large, tightly packed molecular clusters, estimating their local densities with an adjustable parameter 's.'