REAL-TIME FLUORESCENCE IMAGING OF SIRT1CYTOSOLIC TRANSLOCATION UNDER THETREATMENT OF GROWTH FACTORDEPRIVATION

摘要

Sirtuins comprise a family of enzymes implicated in the determination of organismal lifespan in yeast and the nematode. Human sirtuin SIRT1 has been shown to deacetylate several proteins in a NAD~+-dependent manner. It is reported that SIRT1 regulates physiological processes including senescence, fat metabolism, glucose homeostasis, apoptosis, and neurodegeneration. In general, SIRT1 has initially been thought to represent an exclusive nuclear protein. However, depending on the cell lines and organisms examined, a partial or temporary cytoplasmic localization was observed in murine pancreatic beta cells and neonatal rat cardiomyocytes. Since SIRT1 deace-tylates both histone and nonhistone-proteins, such as a number of transcription factors, changes in subcellular localization probably play a role in the regulation of its function. In the present studies, we investigated the subcellular localization of SIRT1 in response to growth factor deprivation in African green monkey SV40-transformed kidney fibroblast cells (COS-7). Using SIRT1-EGFP fluorescence reporter, we found that SIRT1 localized to nucleus in physiological conditions. We devised a model enabling cell senescence via growth factor deprivation and found that SIRT1 partially translocated to cytosol under the treatment, suggesting a reduced level of SIRT1 activity. We found PI3K/Akt pathway was involved in the inhibition of SIRT1's cytosolic translocation, because inhibition of these kinases significantly decreased the amount of SIRT1 maintained in nucleus. Taken together, we demonstrate that growth factor deprivation induces cytosolic translocation of SIRT1, which suggests a possible connection between cytoplasm-localized SIRT1 and the aging process and provides a new application of single molecule fluorescence imaging of the molecule events in living cells.