Electrophysiology of nuclear membrane InsP3 receptor

总结 R37项目采用生物物理学（电生理学，光学成像，光度学），生物化学， 药理学，遗传学和细胞生物学方法，以了解一个重要的新范式， 调节细胞生物能量学，包括内质网的组成性Ca 2+输送 通过InsP3R Ca2 +-释放通道的Ca2+释放和线粒体的Ca2+摄取介导的线粒体 线粒体Ca~（2+）单向转运体复合体。在我们的目标中，每天都采用的一个关键实验方法是 单个活细胞和细胞内细胞器的定量低光水平荧光显微镜。最近， 这台有18年历史的电脑控制着图像采集、外围设备和图像分析， 无法挽回尽管我们很幸运有一台18年前的备份电脑， 重新启动硬件，软件的主要功能在这台计算机上不再起作用。该系统是如此 旧的，它不再支持原来的供应商。因此，我们的大部分研究都是迫在眉睫的。 计算机崩溃的危险，这将使我们无法执行我们提出的研究。我们要求 资金，以取代受损的18岁的显微镜和成像系统。我们计划将指示染料成像 和遗传编码的指标，在细胞质，线粒体和内质网在单细胞超过 延长期限。这需要高分辨率和低毒性。旋转圆盘共聚焦显微镜是理想的 我们的需求因此，本申请是为购买BioVision Technologies，Inc.定制纺纱 圆盘共聚焦显微镜我们预计，该仪器将每天由多名研究人员使用， 实验室在几年的时间里进行了研究，与R37的延长资助同时进行。

SUMMARY The parent R37 project employs biophysical (electrophysiology, optical imaging, photometry), biochemical, pharmacological, genetic and cell biological approaches to understand an important new paradigm regarding the regulation of cellular bioenergetics that involves constitutive Ca2+ delivery from the endoplasmic reticulum to mitochondria mediated by Ca2+ release through the InsP3R Ca2+-release channel and Ca2+ uptake by the mitochondrial Ca2+-uniporter complex. A key experimental approach in our Aims that is employed daily is quantitative low-light level fluorescence microscopy of single living cells and intracellular organelles. Recently, the 18-year old computer that controls image acquisition, peripheral devices and image analysis, crashed irretrievably. Although we were fortunate to have an 18-year old backup computer that has enabled us to reboot the hardware, key features of the software are no longer functional in this computer. The system is so old that it is no longer supported by the original vendor. Accordingly, much of our research is in imminent jeopardy of a computer crash that will leave us unable to perform our proposed studies. We are requesting funds to replace the crippled 18-year old microscope and imaging system. We plan to image indicator dyes and genetically-encoded indicators in cytoplasm, mitochondria and endoplasmic reticulum in single cells over extended periods. This requires high resolution and low toxicity. Spinning-disk confocal microscopy is ideal for our needs. Accordingly, this request is for funds to purchase a BioVision Technologies, Inc. custom spinning disk confocal microscope. We anticipate that the instrument will be used daily by multiple investigators in the lab over a several year period, coincident with the extended funding of the R37.