NADH, best known for electron transfer during energy metabolism, also functions in development, aging, and carcinogenesis. Intracellular NADH levels can be monitored by the molecule's autofluorescence, but the signal is weak and specificity is a concern. This state of affairs inspired two groups to develop fluorescent protein–based NADH sensors. The reports, from Hung et al. and Zhao et al., appear back to back in the journal Cell Metabolism. Both groups combined a circularly permuted fluorescent protein with a bacterial NADH-binding protein. Circular permutation relies upon the fact that many proteins have their termini in close proximity in space, meaning that in some cases the termini can be linked and new ends opened up elsewhere without fully disrupting the overall structure and function. Circularly permuted proteins do tend to be more sensitive to their environment, including to conformational changes. Thus, both groups utilized subunits of the homodimeric NADH-binding protein Rex, which shifts from an open to closed form when ligand is present, and inserted a circularly permuted marker (the GFP variant T-Sapphire or YFP). In both cases, Rex-NADH interactions induce an increase in fluorescence. The NADH biosensor from Hung et al., called Peredox, is relatively resistant to pH change; Frex, the chimeric protein from Zhao et al., is pH sensitive and the fluorescence must be corrected by a circularly permuted YFP (equally sensitive to pH) measured in parallel. Peredox's properties make the sensor unsuitable for mitochondrial applications, whereas Frex can function in both the cytosol, and, when appropriately targeted, mitochondria. By exposing cells to metabolic substrates or agents that perturb these pathways, the two groups provide strong evidence that both Peredox and Frex probe the cellular NADH-NAD+ redox state, with Frex highly selective for NADH, and Peredox sensing the NADH:NAD+ ratio. While these biosensors may not be as fast as autofluorescence detection, they are much more sensitive and specific, and are compatible with high-content image analysis of cellular bioenergetics.
Zhao et al. 2011. (Genetically encoded fluorescent sensors for intracellular NADH detection) and Hung et al. (Imaging cytosolic NADH-NAD+ redox state with a genetically encoded fluorescent biosensor). Cell Metab. 14(4):545-566.
Stressed Out?People don't always deal with stress productively, but cells have a very practical response. Oxidative damage or exposure to toxins can damage or denature proteins, which may harm the cell through reduced functionality or aggregation. In response, cells ramp up quality-control mechanisms to dispose of faulty proteins. One such pathway is chaperone-mediated autophagy (CMA), in which proteins are individually translocated into the lysosomal lumen by chaperone-induced unfolding. Besides removing damaged proteins, CMA may also recycle amino acids from nonessential proteins during extended periods of nutritional deprivation. CMA is of interest because it appears to diminish as organisms age, and may help to explain the poorer response to stress in older cells. Unfortunately, techniques to monitor CMA depend on in vitro assays of isolated lysosomes. Preferable would be studying dynamics in intact, individual cells, particularly for nondividing cell types such as neurons that cannot be grown to quantities sufficient for biochemical analysis. But Koga et al., writing in Nature Communications, describe a photoconvertible fluorescent reporter that circumvents these limitations. The authors took a photoswitchable cyan fluorescent protein and added a KFERQ tag recognized by hsc70, the chaperone that directs CMA substrates to the lysosome. Upon UV excitation, the reporter is irreversibly converted from cyan to green. Because any newly synthesized reporter will be blue, it does not interfere with ongoing tracking of the green reporter. When cells are serum deprived for 10 hours to stimulate CMA, diffuse green fluorescence coalesces to a punctate pattern, consistent with targeting to the lysosomes. Also, as expected, the amount of green fluorescence in cells decreases over time, corresponding to CMA-mediated degradation of the reporter. Users must be cautious to check that UV exposure is not toxic to the cells, and that the puncta colocalize with lysosomal markers. Moreover, the putative CMA signal must be distinguished from other types of autophagy by showing lack of colocalization with autophagic vacuoles and no effects on puncta formation with an inhibitor of macroautophagy, as well as demonstrating no puncta in cells deficient for the lysosomal protein that recognizes hsc70. Autofluorescence could also be a confounder because aging cells are known to collect such deposits. To combat this, the authors recommend using a second KFERQ-bearing construct they created, an mCherry for which UV irradiation stimulates fluorescence. This marker is compatible with flow cytometry, an option suited for cells undergoing morphological changes that make counting puncta difficult. Together, these new reporters should help probe the significance of CMA in aging and age-related disorders such as Parkinson disease and diabetes.
Koga et al. 2011. A photoconvertible fluorescent reporter to track chaper-one-mediated autophagy. Nat Commun. 2:386.
