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内容記述 |
This thesis is divided into two parts. The first part deals with the development of a new probe to detect caspase activity in a living or ganism. The second part describes the characterization of the Drosophila homolog of Spred, a mammalian gene that negatively regulates Ras signaling. <br /> Caspase, the main subject in the first part, is a sequence-specific protease that is the final executioner of apoptosis. During animal development, many unnecessary cells are removed by apoptosis to give space for other cells. This is especially important for constructing a fine structure such as the central nervous system. However, compared with the current knowledge of apoptosis in vitro, little is known about naturally occurring apoptosis during development. This is partly due to difficulty in detecting apoptotic cells, because they are rapidly cleared by macrophages. Therefore, in order to gain a spatio-temporal map of apoptosis during development, it is necessary to detect dying cells at an early phase of apoptosis in real time. To this end, a novel probe was developed that allows the imaging of caspase activity in a living organism. It is a fusion protein of two fluorescent proteins, RFP and Venus, linked by a caspase-cleavable linker and tagged with a different subcellular localization signal at each terminus, so that the probe changes its "color pattern" upon cleavage by caspase. This probe was successfully applied to time lapse analysis of cultured cells. Transgenic flies harboring this probe were generated and several cases of apoptosis were successfully visualized in live samples. In addition to the detection of apoptosis, some results suggesting local activation of caspase without cell death were obtained in the developmentally regulated degeneration of axons during metamorphosis. <br /> In the second part, characterization of a molecule that regulates the Ras signaling pathway is described. Many intracellular events such as cell differentiation, cell division and cell survival are controlled by Ras signaling. Since improper activation of Ras signaling can result in misspecification of cell fates and cancer formation, cells are equipped with a number of negative regulators to prevent these unwanted consequences. Spred is an intracellular protein identified in mammalian cells as a suppressor of Ras signaling. The carboxyl terminal domain of Spred has homology to the well-known negative regulator of Ras signaling, Sprouty. <br /> In this study, the ortholog of Spred in Drosophila was identified and its molecular function was investigated in vivo and in vitro. Drosophila Spred could suppress Ras signaling when overexpressed in the developing eye and in cultured Cells. During development, Spred was expressed at sites of high Ras activity. The expression of Spred could be induced by ectopic activation of Ras signaling, suggesting the presence of negative feed back. To in vestigate the requirement of Spred during development, mutants in Spred were isolated. Surprisnngly, the Spred mutants were fully viable, showing no detectable anatomical abnormalities even in sensitive backgrounds where Ras signaling is either above or below the physiological level. The expresston of Spred was also detected in neurons, and its overexpression in all neurons caused locomotion defects. Hence, it was speculated that Spred may be involved in an aspect of Ras signaling where the loss of negative regulation does not result in anatomical abnormalities, such as neuronal physiology. |
要旨・審査要旨 / Abstract, Screening Result (322.3 kB)
