Development of a New Fluorescent Protein that Enables Detailed Observation of Living Cells Laboratory for Cell Function Dynamics 1. Background Cellular phenomena such as differentiation, locomotion, and division manifest along specific reaction paths inside of cells. A large number of the functional molecules involved in transmitting signals inside of cells have been biochemically or genetically identified. However, in order to gain a comprehensive understanding of matters such as the signal transmission system, it is necessary to observe phenomena inside of individual cells. To achieve this, fluorescent labeling of targeted genes and other intracellular sites is carried out using various substances, and it is necessary to design various techniques in order to enable observations. However, green fluorescent protein (GFP), which is one of the conventional fluorescent labeling substances, is of low effectiveness in forming chromophores, and although it is a protein, it is not capable of luminescence when immature. There is a pressing need to observe subtle changes in fluorescence that occur locally in areas such as intracellular organelles. Finding a GFP that effectively forms chromophores is also required. 2. Regarding a new, modified GFP We introduced random amino acid substitutions into modified GFP (approximately 240 amino acids) from Aequorea coerulescens. We paid particular attention to the oxidation reaction, which is thought to be the most important of the reactions associated with chromophore formation. At 37, the optimum culturing temperature for mammalian cells, we found that the chromophore formation reaction hastened dramatically when we substituted leucine for the 46th phenylalanine. We also identified amino acid substitutions that increase the effectiveness of protein folding (Note 1), and ascertained that these accelerate the maturation of GFP. We developed a new, modified GFP from these amino acids, and named it Venus, after the planet, since it is the brightest GFP in the world. Venus is capable of emitting fluorescence even in small amounts, because of the extremely high efficiency of its maturation. It achieves luminescence 30-100 times that of conventional modified GFP in Escherichia coli, and 3-100 times in mammalian cells, and is capable of providing fluorescence that it is possible to detect even with ordinary microscopic equipment. Accordingly, it permits more effective observation of fluorescence, without bringing about intracellular toxicity. The amount of time from the introduction of GFP genes until the manifestation of luminescence is also shortened from a range of 1/2-1 day to within several hours. Brain slices that have just been prepared may be promptly fluorescent-labeled and observed. 3. Results obtained with Venus We confirmed that when we had labeled secretory granules present in neuroblast lineage PC12, using Venus that we had prepared, nearly 100% of the secretory granules were accurately labeled, with at least 10 times the luminescence achieved previously. As a result, it became possible to observe in real time elementary processes in secretory granules resulting from depolarization and cell stimulation. We were also able to quantify the amount of secretion from cells by the fluorescence emitted into the culture solution. 4. Future expectations We expect that labeling technology using Venus will further deepen understanding of intracellular phenomena, particularly signal transmission, and also provide a major key to the elucidation of developmental processes, brain functions, and the mechanisms of diseases, etc. Venus may also be expected to play a role as a genetic reporter, faithfully reflecting the activation of targeted genes, because of the rapidity of its maturation. Note 1: Folding The process whereby proteins are folded three-dimensionally from peptide chains. Folding is necessary, first of all, in order for GFP to form chromophores, and mature. Ca2+ imaging in a Purkinje neuron of a mouse cerebellar slice. (A) A low-magnification fluorescence photograph merged with its dark-field image. (B) A confocal fluorescence micrograph of a Purkinje cell indicated by a box in (A). The region monitored for Ca2+ change is boxed. (C) Ratio images from the dendrite indicated in (B) before and after depolarization stimulation. Takeharu Nagai, Keiji Ibata, Eun Sun Park, Mie Kubota, Katsuhiko Mikoshiba & Atsushi Miyawaki Nature Biotechnology January 2002 Vol. 20-1, 87-90