Segmented cell analyses to measure redox states of autofluorescent NAD(P)H, FAD &amp; Trp in cancer cells by FLIM

Wallrabe, Horst; Švindrych, Zdeněk; Alam, Shagufta Rehman; Siller, Karsten H.; Wang, Tianxiong; Kashatus, David F.; Hu, Song; Periasamy, Ammasi

doi:10.1038/s41598-017-18634-x

Cited by 87 publications

(107 citation statements)

References 35 publications

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“…It is becoming increasingly apparent that organelles “talk” to each other to regulate key cellular functions (Murley & Nunnari, ; Quirós et al , ; Valm et al , ), but the mechanisms involved and potential roles in disease pathogenesis remain largely unexplored. Using a 2P‐FLIM assay for label‐free imaging of mitochondrial activity in live cells (Lakowicz, ; Alam et al , ; Wallrabe et al , ) (Fig A and B), we describe here a previously unknown form of inter‐organelle communication, from lysosomes to mitochondria, and reveal details of its function and mechanism of action, and how its dysregulation may represent a seminal process in AD pathogenesis and a defining molecular defect in tuberous sclerosis.…”

Section: Discussionmentioning

confidence: 94%

A novel lysosome‐to‐mitochondria signaling pathway disrupted by amyloid‐β oligomers

et al. 2018

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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.

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Section: Discussionmentioning

confidence: 94%

A novel lysosome‐to‐mitochondria signaling pathway disrupted by amyloid‐β oligomers

et al. 2018

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“…Furthermore, the fluorescence lifetimes and redox ratio of these natural biomarkers have been used to study various live tissues and organisms such as rat cardiac trabeculae (32), cochlear coils (33), and zebrafish embryos (34). Most importantly, multiphoton imaging of NADH and FAD was able to provide insightful analysis regarding morphological and metabolic changes in cancer cells at different metastatic stages, as well as interactions between them and neighboring fibroblasts (35)(36)(37)(38)(39). Recently, Lukina et al (40) employed NAD(P)H and FAD autofluorescence microscopy for metabolic studies of multicellular spheroids, grown in liquid suspension culture, as a function of Paclitaxel (a widely used antimicrotubular agent) treatment.…”

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Two‐photon fluorescence lifetime imaging of intrinsic NADH in three‐dimensional tumor models

et al. 2018

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Most studies using intrinsic NAD(P)H as biomarkers for energy metabolism and mitochondrial anomalies have been conducted in routine two-dimensional (2D) cell culture formats. Cellular metabolism and cell behavior, however, can be significantly different in 2D cultures from that in vivo. As a result, there are emerging interests in integrating noninvasive, quantitative imaging techniques of NAD(P)H with in vivo-like threedimensional (3D) models. The overall features and metabolic responses of the murine breast cancer cells line 4T1 in 2D cultures were compared with those in 3D collagen matrix using integrated optical micro-spectroscopy. The metabolic responses to two novel compounds, MD1 and TPPBr, that target metabolism by disrupting monocarboxylate transporters or oxidative phosphorylation (OXPHOS), respectively, were investigated using two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intracellular NAD(P)H in 2D and 3D cultures. 4T1 cells exhibit distinct behaviors in a collagenous 3D matrix from those in 2D culture, forming anastomosing multicellular networks and spherical acini in 3D culture, as opposed to simple flattened epithelial plaques in 2D culture. The cellular NAD(P)H in 3D collagen matrix exhibits a longer fluorescence lifetime as compared with 2D culture, which is attributed to an enhanced population of enzyme-bound NAD(P)H in the 3D culture. TPPBr induces mitochondrial hyperpolarization in 2D culture of 4T1 cells along with an enhanced free NAD(P)H population, which suggest an interference with OXPHOS. In contrast, 2P-FLIM of cellular NAD(P)H revealed an enhanced autofluorescence lifetime in 3D 4T1 cultures after MD1 treatment as compared with MD1-treated 2D culture and the control 3D culture. Physical and chemical microenvironmental signaling are critical factors in understanding how therapeutic compounds target cancer cells by disrupting their metabolic pathways. Integrating 2P-FLIM of intrinsic NAD(P)H with refined 3D tumor-matrix in vitro models promises to advance our understanding of the roles of metabolism and metabolic plasticity in tumor growth and metastatic behavior. © 2018International Society for Advancement of Cytometry Key terms NAD(P)H; 4T1; 3D collagen matrix; MD1; TPPBr derivative; two-photon microscopy; FLIM CHANGES in cellular metabolism have long been recognized as a fundamental feature, and are now considered a hallmark, of cancer. A resurgent interest in this area has recently been driven in large measure by significant breakthroughs elucidating relevant mechanisms of cross-talk between not only tumor and stromal cells but also between distinct subpopulations of tumor cells themselves within heterogeneous tumors. In particular, advanced metabolic imaging methods promise to significantly enhance our understanding of how specific microenvironmental factors influence the regulation of cancer cell metabolism on a cell-by-cell basis. Overall, it is becoming clear that a better understanding of the metabolic features of cancer cells-and in particular those that may ...

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“…These endogenous fluorophores are metabolic coenzymes/ cofactors important to all cells for the production of ATP, and have been optically characterized for decades (1,20,21). It is this optical fluctuation during metabolic changes that makes them favorable for research in applications related to cancer (26,27), cell proliferation (28)(29)(30), apoptosis (31), and cell differentiation (32,33). Therefore, when FAD and NAD(P)H are optically tracked (where NAD(P)H refers to both NAD cofactors), their fluorescence is correlated to changes in cellular respiration.…”

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“…FAD and NAD(P)H cycle from reduced to oxidized forms, thus facilitating redox reactions during energy production (e.g., oxidative phosphorylation and glycolysis). Examples of this include diagnostic imaging to reveal boundaries of demarcation between normal and cancerous tissues or measurement of optical redox ratios (29,32,(34)(35)(36)(37)(38)(39)(40)(41). The signals that result vary depending on the NAD(P)H reduced or oxidized form (20,22), location inside of the cell (e.g., mitochondria, cytosol) (23), and binding status with enzymes (e.g., malate, lactate, isocitrate, and succinate dehydrogenases) (24,25).…”

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confidence: 99%

“…The signals that result vary depending on the NAD(P)H reduced or oxidized form (20,22), location inside of the cell (e.g., mitochondria, cytosol) (23), and binding status with enzymes (e.g., malate, lactate, isocitrate, and succinate dehydrogenases) (24,25). It is this optical fluctuation during metabolic changes that makes them favorable for research in applications related to cancer (26,27), cell proliferation (28)(29)(30), apoptosis (31), and cell differentiation (32,33). In some cases, the bound enzyme is known or has been validated by additional biochemical analysis.…”

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confidence: 99%

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Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time‐resolved flow cytometry

et al. 2018

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Autofluorescence from the intracellular metabolite, NAD(P)H, is a biomarker that is widely used and known to reliably screen and report metabolic activity as well as metabolic fluctuations within cells. As a ubiquitous endogenous fluorophore, NAD(P)H has a unique rate of fluorescence decay that is altered when bound to coenzymes. In this work we measure the shift in the fluorescence decay, or average fluorescence lifetime (1–3 ns), of NAD(P)H and correlate this shift to changes in metabolism that cells undergo during apoptosis. Our measurements are made with a flow cytometer designed specifically for fluorescence lifetime acquisition within the ultraviolet to violet spectrum. Our methods involved culture, treatment, and preparation of cells for cytometry and microscopy measurements. The evaluation we performed included observations and quantification of the changes in endogenous emission owing to the induction of apoptosis as well as changes in the decay kinetics of the emission measured by flow cytometry. Shifts in NAD(P)H fluorescence lifetime were observed as early as 15 min post‐treatment with an apoptosis inducing agent. Results also include a phasor analysis to evaluate free to bound ratios of NAD(P)H at different time points. We defined the free to bound ratios as the ratio of ‘short‐to‐long’ (S/L) fluorescence lifetime, where S/L was found to consistently decrease with an increase in apoptosis. With a quantitative framework such as phasor analysis, the short and long lifetime components of NAD(P)H can be used to map the cycling of free and bound NAD(P)H during the early‐to‐late stages of apoptosis. The combination of lifetime screening and phasor analyses provides the first step in high throughput metabolic profiling of single cells and can be leveraged for screening and sorting for a range of applications in biomedicine. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

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Segmented cell analyses to measure redox states of autofluorescent NAD(P)H, FAD & Trp in cancer cells by FLIM

Cited by 87 publications

References 35 publications

A novel lysosome‐to‐mitochondria signaling pathway disrupted by amyloid‐β oligomers

A novel lysosome‐to‐mitochondria signaling pathway disrupted by amyloid‐β oligomers

Two‐photon fluorescence lifetime imaging of intrinsic NADH in three‐dimensional tumor models

Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time‐resolved flow cytometry

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