DEVELOPMENT OF SYSTEMS FOR IMAGING INTRACELLULAR SIGNAL TRANSDUCTIONS BY FLUOROPROBES

使用荧光指示剂的细胞内Ca^2+动力学成像技术的可用性促使我们尝试可视化活细胞中特定分子的动力学特征，因为它将为我们提供新的信息，允许直接分析特定生物功能中给定功能分子的作用。本课题的目的是研制一种能直接显示激酶活性动态特征的指示剂，并建立一个微弱荧光图像的检测和分析系统。为了监测CaMKII的活性，我们通过将荧光探针丙烯酰二氢吡喃与合成肽2（syntide 2）（CaMKII的特异性底物）结合来合成试剂AS 2，并显示其是在无细胞条件下由Ca^2+激活钙调蛋白和随后激活CaMKII的有效指示剂。为了可视化PKA活性的动态，我们构建了PKA的荧光底物，该底物由荧光探针acrylodan和PKA的两种同种型之一的调节结构域的部分氨基酸序列组成，所述调节结构域II（RII）含有特定的自磷酸化位点，丝氨酸残基（ARII）。发现ARII被PKA磷酸化，其荧光强度响应于磷酸化而降低。我们研制了一种新型的成像系统，该系统包括一个冷却的CCD器件和一个图像分析器。将新开发的荧光探针和成像系统应用于原代培养的神经细胞，并取得了理想的结果，显示了活细胞中激酶活性的动态特征。我们进一步对这些探针进行了改进，使其更加稳定和灵敏，并得到了几种有前途的染料。

The availability of imaging techniques for intracellular Ca^<2+> dynamics using fluorescent indicators prompted us to try to visualize the dynamic features of specific molecules in living cells, since it would provide us with new information permitting direct analysis of the role of given functional molecules in specific biological functions. Our aim in this project was to prepare reagents which could be used as indicators to direct visualize dynamic features of kinase activities and also to consist a system to detect and analyze faint fluorescence images. To monitor CaMKII activity, we synthesized a reagent, AS2, by conjugating a fluoroprobe, acrylodan to syntide 2, a specific substrate of CaMKII and showed it to be an effective indicator of the activation of calmodulin by Ca^<2+> and subsequent activation of CaMKII in cell-free conditions. In order to visualize dynamics of PKA activity, we constructed a fluorescence substrate for PKA consisting of a fluorescence probe acrylodan and a partial amino acid sequence of the one of the two isotypes of regulatory domain, regulatory domain II (RII) of PKA which contains a specific autophosphorylation site, a serine residue (ARII). ARII were found to be phosphorylated by PKA and its fluorescence intensity decreased in response to the phosphorylation. We developed a new imaging system with a cooled CCD device and an image analyzer. The newly developed fluoroprobes and imaging systems were applied to neuronal cells in primary culture and had desirable results to show the dynamic features of kinase activities in living cells. We further tried to improve these probes to make them much stable and sensitive, and got several promising dyes.