| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1999: ¥1,700,000 (Direct Cost: ¥1,700,000)
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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 involved in 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. We examined the change in actin cytoskeleton during apoptosis. We expressed EGFP-labeled actin in the cell, and analyzed them by time lapse video microscopy after Fas-treatment to induce apoptosis. We found that actin stress fibers gradually form a ring-shaped actin structure and blebs seem to be formed near this ring, suggesting that cellular shrinkage and bleb formation are associated with reorganization (ring formation) of actin fibers at the bottom of cell.
2. 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 apoptotic 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. Because bleb formation was dependent of activation of caspases, signals to induce bleb formation could be generated between caspase-8 activation and Bcl-2 functional step.
3. We performed the proteome analysis to identify proteins that change their nature during apoptosis. Taking advantage that the pathway downstream from the mitochondria is silenced in the cells overexpressing Bcl-2, we identified lamin A/C, Lamin B, and Vimentin as proteins that change their nature in the pathway started between caspase-8 activation and mitochondria activation
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