PROTEIN ABLATION MICROSCOPE

这个应用程序的目标是建立一个最先进的 用于执行细胞和亚细胞“击倒”的设施 利用发色团辅助激光技术实现蛋白质功能 失活(加州)。在这项技术中，生色团孔雀石绿是 与抗体共价偶联。孔雀石绿-抗体结合物 被显微注射到活细胞中。受控制的激光脉冲来自一个 采用氮气泵浦的染料激光器。在使用的波长下，只有 发色团吸收激光能量。孔雀石结合的蛋白质 绿色标记的抗体因自由基损伤而失活。因为 抗体用作靶向探针，CALI技术提供了一种 高度的分子特异性。因为停用仅发生在 在激光照射后，该技术提供了出色的时间控制 以及以高分辨率对细胞进行前后成像的能力 失活。最后，因为激光可以聚焦在整个细胞上 或扩展到一个小区域，这项技术提供了极好的空间 在扰乱细胞通路方面的分辨率。主要用户的成员 该小组打算将这种方法应用于哺乳动物细胞的四个领域 生物学：1)M期检查点的信号成分(戈尔布斯基)，2) SCAMPS和其他囊泡运输蛋白的作用(Castle)，3) 局灶性粘连的信号和组装，4)MAPK的激活 G1期途径和有丝分裂(Weber)。目前，每个实验室都已经 将功能阻断抗体微量注射到活细胞中作为一种手段 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。加州 技术使用这些中性抗体作为敲除试剂来检查 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淘汰策略(反 感觉，小鼠基因敲除)，并成为关键的资源 寻求一种简单、廉价和有效的方法的研究人员社区 研究活体哺乳动物体内特定蛋白失活的效果 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.