Desigh Syntlesis and Biological Application of Fluorescence Probes

发色团辅助激光失活（CALI）是研究细胞过程中原位蛋白功能的有力方法。通过使用CALI，可以以前所未有的时空分辨率取消目标蛋白的功能。然而，CALI仍有一定的局限性，主要是由于其使用抗体进行靶标识别，限制了其在生物学上的广泛应用。CALI的一个不受欢迎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，我们开发了膜渗透合成小分子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CALI实验。为了实现基于小分子的CALI (smCALI)，我们瞄准了肌醇1,4,5-三磷酸受体（IP_3R），该受体调节细胞内Ca^<2+>动力学，从而在各种生理功能中发挥重要作用。我们合成了孔雀石绿偶联IP3类似物（MGIP_3/PM）作为smCALI探针，并检测了MGIP_3/PM在完整DT40鸡B细胞中的作用。我们证实了smCALI在细胞水平上是有效的，因此我们利用smCALI来阐明CCE的机制，其中IP_3R参与了CCE的作用。即使smCALI使IP_3R失活，thapsigargin诱导的CCE在DT40鸡B细胞中仍未受到影响，这表明IP_3R的激活对CCE并不是必需的。我们的结果表明，smCALI应该是研究活细胞中蛋白质功能时空差异的有效方法。

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.