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Studies on cell death and survival signals via connexin channels during cell injury

细胞损伤期间通过连接蛋白通道的细胞死亡和存活信号的研究

基本信息

批准号：
17390118
负责人：
OYAMADA Masahito
金额：
$ 9.42万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17390118/
关键词：
connexin gap functions cell death cell survival CALI multi-photon microscopy GFP intercellular communication 細胞障害心筋梗塞生体分子

项目摘要

Gap junctions are considered to play an important role in moderating cell death and survival. Genetic approaches to the connexins (Cxs) such as gene knockouts and RNA interference, which ultimately reduce Cx levels in the entire cell, have provided direct evidence that gap junctional intercellular communication is essential for cell survival. However, it is possible that one kind of Cx performs different functions depending on its location and/or a particular stage of temporal events. Such spatiotemporal differences in protein function are difficult to analyze with conventional genetic approaches. In this study, we have developed multiphoton excitation-evoked chromophore-assisted laser inactivation (MP-CALI),that specific gap junctions made of Cx-enhanced green fluorescent protein(EGFP)can be spatiotemporally inactivated by brief laser irradiation. Experiments were performed on a pair of HeLa cells connected via a small gap junction plaque (length, <1 μm)consisting of Cx43-EGFP. While … More junctional currents were measured using the dual whole-cell voltage-clamp -method, a single point of the Cx43-EGFP plaque was irradiated with 850-nm femtosecond laser light with a laser irradiance of 2.7 MW/cm^2for 380 ms. We found that during the laser irradiation, the gap junction current decreased "○80%. However the same irradiation did not change gap junctional currents between HeLa cell pairs expressing Cx43 tagged with monomeric red fluorescent protein(mRFP),indicating that MP-CALI at a wavelength of 850 nm specifically inactivates gap junctions made of Cx-EGFP. We also found that MPCALI can inhibit dye coupling between specifically selected cell pairs. MP-CALI of Cx43-EGFP showed a laser power dependent decrease of gap junction current i.e., the threshold laser power for current reduction is between 1.0 and 1.3 MW/cm2. Reactive oxygen species (ROS) scavengers, edaravone and sodium azide, impaired the MP-CALI response, indicating involvement of ROS in MP-CALI. Thus, MP-CALI could be useful for the elucidation of new Cx functions. Less

缝隙连接被认为在调节细胞死亡和存活方面发挥着重要作用。连接蛋白(CXs)的遗传途径，如基因敲除和RNA干扰，最终降低整个细胞中Cx的水平，已经提供了直接证据表明缝隙连接细胞间的通讯对细胞生存是必不可少的。然而，一种CX可能根据其位置和/或时间事件的特定阶段而执行不同的功能。这种蛋白质功能的时空差异很难用传统的遗传方法进行分析。在这项研究中，我们发展了多光子激发诱导发色团辅助激光失活(MP-CALI)，即Cx增强的绿色荧光蛋白(EGFP)所形成的特定缝隙连接可以在短暂的激光照射下时空失活。实验在一对HeLa细胞上进行，通过由Cx43-EGFP组成的小间隙连接斑块(长度为1μm)连接。而…用双全细胞电压钳方法测量更多的结点电流，用波长为850 nm、激光强度为2.7 mW/cm2的飞秒激光照射Cx43-EGFP斑块单点380ms。我们发现，在激光辐照过程中，缝隙结电流下降了“080%”。然而，同样的照射并没有改变表达Cx43的单体红色荧光蛋白(MRFP)标记的HeLa细胞对之间的缝隙连接电流，这表明850 nm的MP-CALI特异性地灭活了由Cx-EGFP组成的缝隙连接。我们还发现，MPCALI可以抑制特定选择的细胞对之间的染料偶联。Cx43-EGFP的MP-CALI显示出间隙结电流随激光功率的减小，即减小电流的阈值激光功率在1.0~1.3 mW/cm2之间。活性氧(ROS)清除剂依达拉奉和叠氮化钠可抑制MP-CALI反应，表明ROS参与了MP-CALI。因此，MP-CALI可能有助于阐明新的Cx功能。较少