کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
31116 44547 2009 12 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level
موضوعات مرتبط
مهندسی و علوم پایه مهندسی شیمی بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level
چکیده انگلیسی

Reduced nicotinamide adenine dinucleotide, NADH, is a major electron donor in the oxidative phosphorylation and glycolytic pathways in cells. As a result, there has been recent resurgence in employing intrinsic NADH fluorescence as a natural probe for a range of cellular processes that include apoptosis, cancer pathology, and enzyme kinetics. Here, we report on two-photon fluorescence lifetime and polarization imaging of intrinsic NADH in breast cancer (Hs578T) and normal (Hs578Bst) cells for quantitative analysis of the concentration and conformation (i.e., free-to-enzyme-bound ratios) of this coenzyme. Two-photon fluorescence lifetime imaging of intracellular NADH indicates sensitivity to both cell pathology and inhibition of the respiratory chain activities using potassium cyanide (KCN). Using a newly developed non-invasive assay, we estimate the average NADH concentration in cancer cells (168 ± 49 μM) to be ∼1.8-fold higher than in breast normal cells (99 ± 37 μM). Such analyses indicate changes in energy metabolism and redox reactions in normal breast cells upon inhibition of the respiratory chain activity using KCN. In addition, time-resolved associated anisotropy of cellular autofluorescence indicates population fractions of free (0.18 ± 0.08) and enzyme-bound (0.82 ± 0.08) conformations of intracellular NADH in normal breast cells. These fractions are statistically different from those in breast cancer cells (free: 0.25 ± 0.08; bound: 0.75 ± 0.08). Comparative studies on the binding kinetics of NADH with mitochondrial malate dehydrogenase and lactate dehydrogenase in solution mimic our findings in living cells. These quantitative studies demonstrate the potential of intracellular NADH dynamics (rather than intensity) imaging for probing mitochondrial anomalies associated with neurodegenerative diseases, cancer, diabetes, and aging. Our approach is also applicable to other metabolic and signaling pathways in living cells, without the need for cell destruction as in conventional biochemical assays.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Photochemistry and Photobiology B: Biology - Volume 95, Issue 1, 2 April 2009, Pages 46–57
نویسندگان
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