PROTEIN ABLATION MICROSCOPE

本申请的目的是建立一个最先进的 用于执行细胞和亚细胞“敲除”的设施 通过生色团辅助激光技术研究蛋白质功能 灭活（卡利）。在该技术中，发色团孔雀石绿色是 与抗体共价偶联。孔雀石绿-抗体缀合物 被显微注射到活细胞中。可控激光脉冲 氮泵浦染料激光器。在所使用的波长下，只有 发色团吸收激光能量。孔雀石结合蛋白质 绿色标记的抗体被自由基损伤灭活。因为 抗体被用作靶向探针，卡利技术提供了一种 高度分子特异性。因为失活只发生在 在激光照射时，该技术提供了极好的时间控制， 以及对细胞进行高分辨率成像的能力 失活最后，因为激光可以聚焦在整个细胞上 或一个小的分区，该技术提供了良好的空间 破坏细胞通路的解决方案。主要用户的成员 研究小组打算将这种方法应用于哺乳动物细胞四个领域 生物学：1）M期检查点的信号传导组分（Gorbsky），2） SCAMPS和其他囊泡运输蛋白的作用（Castle），3） 粘着斑的信号传导和组装，4）Map激酶的激活 G1和有丝分裂中的信号通路（Weber）。目前，各实验室已 将功能阻断抗体显微注射到活细胞中， 研究细胞信号和生理学。然而，大多数抗体 是中性的;即它们没有固有的功能阻断能力。卡利 这项技术使用这些中性抗体作为敲除试剂， 靶蛋白在体内的作用。蛋白质消融显微镜 工作站旨在补充其他敲除策略（抗 正义，小鼠基因敲除），并成为一个关键的资源， 一群研究人员正在寻找一种简单、廉价和有效的方法， 研究在活的哺乳动物中灭活特定蛋白质的效果， 细胞

The objective of this application is to establish a state-of-the-art facility for the execution of cellular and subcellular "knockouts" of protein function through the technique of Chromophore-Assisted Laser Inactivation (CALI). In this technique the chromophore Malachite green is covalently coupled to an antibody. The Malachite green-antibody conjugate is microinjected into living cells. Controlled laser pulses from a nitrogen pumped dye laser are applied. At the wavelengths used, only the chromophore absorbs the laser energy. Proteins bound by the Malachite green-labeled antibody are inactivated by free radical damage. Because antibodies are used as targeting probes, the CALI technique provides a high degree of molecular specificity. Because inactivation occurs only upon laser irradiation, the technique provides excellent temporal control and the ability to image cells at high resolution before and after inactivation. Finally, because the laser can be focused on the entire cell or onto a small subregion, the echnique provides excellent spatial resolution in disrupting cellular pathways. The members of the major user group intend to apply this approach in four areas of mammalian cell biology: 1) signaling components of the M phase checkpoint (Gorbsky), 2) the role of SCAMPS and other vesicle trafficking proteins (Castle), 3) signaling and assembly of focal adhesions, 4) activation of the Map kinase pathway in G1 and mitosis (Weber). Currentiy each laboratory has been microinjecting function-blocking antibodies into living cells as a means of studying cellular signaling and physiology. However, most antibodies are neutral; i.e they have no inherent function-blocking ability. The CALI technique uses these neutral antibodies as knockout reagents to examine the role of target proteins in vivo. The protein ablation microscope workstation is intended to complement other knockout strategies (anti- sense, mouse gene knockouts) and become a pivotal resource for the community of researchers seeking a simple, inexpensive, and effective way to study the effects of inactivating specific proteins in living mammalian cells.