| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
Chromophore-assisted laser inactivation (CALI) is a powerful method for the study of in situ protein function in cellular processes. By using CALI, it is possible to abrogate the function of a target protein with unprecedented spatiotemporal resolution. However, CALI has some limitations, which restrict wider biological application, owing mainly to the use of antibody for target recognition. One of the undesirable limitations of CALI is the necessity to use invasive methods to introduce antibodies into cells. To solve this problem, we have developed membrane-permeant synthetic small molecules instead of antibodies for molecular recognition, so that we can carry out CALI experiments under physiological conditions. For the implementation of small molecule-based CALI (smCALI), we targeted inositol 1,4,5-trisphosphate receptor (IP_3R), which regulates intracellular Ca^<2+> dynamics and thereby plays an important role in various physiological functions. We synthesized a malachite green-conjugated IP3 analog (MGIP_3/PM) as a smCALI probe and examined the effect of MGIP_3/PM-based CALI in intact DT40 chicken B cells. It was confirmed that smCALI was effective at the cellular level, so we then applied smCALI to clarify the mechanism of capacitative Ca^<2+> entry (CCE), in which involvement of IP_3R was suggested. The thapsigargin-induced CCE remained unaffected in DT40 chicken B cells even after inactivation of IP_3R by smCALI, suggesting that activation of IP_3R is not essential for CCE. Our results demonstrate that smCALI should be a useful method to study spatiotemporal differences of protein function in living cells.
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