Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

Sirtuin 1 (SIRT1) is an NAD⁺-dependent deacetylase that functions as metabolic sensor of cellular energy and modulates biochemical pathways in the adaptation to changes in the environment. SIRT1 substrates include histones and proteins related to enhancement of mitochondrial function as well as antioxidant protection. Fluctuations in intracellular NAD⁺ levels regulate SIRT1 activity, but how SIRT1 enzymatic activity impacts on NAD⁺ levels and its intracellular distribution remains unclear. Here, we show that SIRT1 determines the nuclear organization of protein-bound NADH. Using multiphoton microscopy in live cells, we show that free and bound NADH are compartmentalized inside of the nucleus, and its subnuclear distribution depends on SIRT1. Importantly, SIRT6, a chromatin-bound deacetylase of the same class, does not influence NADH nuclear localization. In addition, using fluorescence fluctuation spectroscopy in single living cells, we reveal that NAD⁺ metabolism in the nucleus is linked to subnuclear dynamics of active SIRT1. These results reveal a connection between NAD⁺ metabolism, NADH distribution, and SIRT1 activity in the nucleus of live cells and pave the way to decipher links between nuclear organization and metabolism.