| Budget Amount *help |
¥49,010,000 (Direct Cost: ¥37,700,000、Indirect Cost: ¥11,310,000)
Fiscal Year 2008: ¥14,300,000 (Direct Cost: ¥11,000,000、Indirect Cost: ¥3,300,000)
Fiscal Year 2007: ¥14,300,000 (Direct Cost: ¥11,000,000、Indirect Cost: ¥3,300,000)
Fiscal Year 2006: ¥20,410,000 (Direct Cost: ¥15,700,000、Indirect Cost: ¥4,710,000)
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| Research Abstract |
ERK signaling networks can regulate various cellular processes. One of the remarkable features of signaling networks is that the same signaling networks can regulate multiple cellular functions. In PC12 cells, EGF and NGF induces transient and sustained ERK activation, leading to cell proliferation and differentiation, respectively. In this study, we analyzed how ERK signaling networks can regulate cell proliferation and differentiation, depending on the distinct temporal activation patterns by use of systems biology approach. First, we tried to analyze the differentiation processes. We performed a discontinuous stimulation assay consisting of a first transient stimulation followed by an interval and then a second sustained stimulation and quantified the neurite extension level (Chung, J. et al, 2010). Consequently, we observed a timing-dependent action of NGF on cell differentiation, and discontinuous NGF stimulation similarly induced differentiation. The first stimulation did not ind
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uce neurite extension, whereas the second stimulation induced fast neurite extension ; therefore, the first stimulation is likely required as a prerequisite condition. These observations indicate that the action of NGF can be divided into two processes : an initial stimulation-driven latent process and a second stimulation-driven extension process. The latent process appears to require the activities of ERK and transcription, but not PI3K, whereas the extension-process requires the activities of ERK and PI3K, but not transcription. We screened the possible genes involved in the latent process, and identified 3 genes as a possible candidates. We developed a fully automatable assay technique, termed quantitative image cytometry, which integrates a quantitative immunostaining technique and a high precision image-processing algorithm for cell identification (Ozaki, Y. et al, 2010). With the aid of an automated sample preparation system, this device can quantify protein expression, phosphorylation and localization with subcellular resolution at one-minute intervals. We will investigate the signaling dynamics of the ERK pathway in PC12 cells, demonstrating using QIC. Less
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