Spatiotemporal Subcellular Characterization of Absolute NADH Concentration Over the Dynamic Course of Metaphase, Anaphase, Telophase and Cytokinesis using the Phasor Approach to FLIM

Abstract

Absolute concentration measurements of free and bound nicotinamide adenine dinucleotide hydride (NADH) using the phasor approach to fluorescence lifetime imaging microscopy (phasor FLIM) is a powerful method for discerning metabolic robustness in living cells with subcellular resolution. Cell cycle and metabolism are at the forefront of ongoing cancer research to explore new targets for cancer therapeutics. A key feature of cancer cells is continual mitotic division. Completion of mitosis ends with the progression of cells through metaphase, anaphase, telophase and cytokinesis. Each of these phases last approximately 3 minutes, presenting a large challenge for researchers to assess metabolism at these stages using available biochemical approaches. Using the phasor FLIM paired with Digital Frequency Domain (DFD) FLIM hardware, we spatiotemporally measure the absolute concentration of NADH (a measure of metabolic robustness) and free/bound NADH ratio (a measure of metabolic mode) in individual mitochondria of cells progressing from metaphase through cytokinesis. Our timelapse FLIM measurements of HeLa cells characterize the metabolic heterogeneity of mitochondria metabolism based on spatial location at the cell equator or spindle poles during the temporal progression of cells from metaphase through cytokinesis. We observe mitochondria NADH concentration and free/bound NADH at the spindle equator and pole correlate in early metaphase, but diverge starting late metaphase to early anaphase coinciding with onset of anaphase cell elongation. Our subcellular NADH FLIM analysis results support the existence of a spatiotemporal specific mechanism for mitochondria function during the course of cell division. Supported by NIGMS P41GM103540 to EG and MD.