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Desigh Syntlesis and Biological Application of Fluorescence Probes

荧光探针的设计合成及生物学应用

基本信息

批准号：
13672321
负责人：
KIKUCHI Kazuya
金额：
$ 2.3万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

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.

发色团辅助激光失活（卡利）是研究细胞过程中原位蛋白质功能的有效方法。通过使用卡利，有可能以前所未有的时空分辨率消除靶蛋白的功能。但卡利也存在一定的局限性，主要是由于其主要依靠抗体来识别靶点，从而限制了其在生物学上的广泛应用。卡利的一个不希望的限制是必须使用侵入性方法将抗体引入细胞。为了解决这个问题，我们开发了膜渗透性的合成小分子来代替抗体进行分子识别，这样我们就可以在生理条件下进行卡利实验。为了实现基于小分子的卡利（smCALI），我们靶向肌醇1，4，5-三磷酸受体（IP_3R），其调节细胞内Ca^2+动态，从而在各种生理功能中起重要作用。我们合成了一种与孔雀石绿偶联的IP 3类似物（MGIP_3/PM）作为smCALI探针，并在完整的DT 40鸡B细胞中检测了基于MGIP_3/PM的卡利的效果。在细胞水平上证实了smCALI的有效性，因此我们应用smCALI来阐明容量性Ca^<2+>内流（CCE）的机制，其中可能涉及IP_3 R。在DT 40鸡B细胞中，即使用smCALI灭活IP_3 R，毒胡萝卜素诱导的CCE仍不受影响，这表明IP_3 R的激活不是CCE所必需的。我们的研究结果表明，smCALI应该是一个有用的方法来研究蛋白质的功能在活细胞中的时空差异。