| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2002: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Apoptosis is characterized by several morphological features, such as cell shrinkage and membrane blebbing. Some molecules have been suggested to be involvedin the apoptotic morphological changes, but its mechanism is not understood yet. In this project, we performed the following analysis to elucidate the mechanisms of the apoptotic morphological changes.
1. To elucidate the where signaling pathway for bleb formation is generated, we examined the effects of several apoptosis inhibitors on bleb formation. We found that Bcl-2 prevented Fas-mediated apopotic nuclear changes but not bleb formation of HeLa cells. Bcl-2 did not prevent the activation of caspase-8, but strongly reduced the activation of caspase-3. Previous analysis cannot distinguish factors involved in nuclear morphological changes and those involved in cellular morphological changes, because activated caspase-3 cleaves various substrates upon apoptotic stimuli. Using HeLa cells overexpresing Bcl-2, we can focus the signalin
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g pathwmiy of apoptotic cellular morphological changes in the absence of reactions leading to nuclear morphological changes.
2. Taking advantages that the pathway downstream from the mitochondria is silenced by using HeLa cells overexpresing Bcl-2, we perfonned the proteome analysis to identify proteins that change their nature during apoptosis. We identified syntaxin, tropomyosin, lamnin B1, keratin 17 (N-fragment), keratin 17(C-fragment), clathrin light chain-a, vimentin, lamin A/C as proteins that change their nature in the cells where the signaling pathway leading to apoptotic cellular morphological changes are mainly operating.
3. Among these proteins, clathrin light chain-a was cpmpletely cleaved upon apoptotic stimuli depending on the activation of caspases. Furthermore, reduction of the expression of clathrin light chain-a by siRNA enhanced apoptotic blebbing. Therefore, it is suggested that the depletion of clathrin light chain-a by cleavage of caspases is likely to stimulate apoptotic blebbing. Less
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