ROLE OF INTRACELLULAR FREE CALCIUM IN CELL PROLIFERATION

生物学的焦点是研究胞浆内游离钙离子([Ca2+]i)的作用 帮助触发静止细胞进入细胞周期的变化 并通过有丝分裂来控制它们的进展。重点将是 致力于解决不同物种间[Ca~(2+)]_i的差异 相邻细胞和不同区域之间的大有丝分裂和运动 细胞。在前一批款期内开发的方法提供了 需要前所未有的灵敏度和空间分辨率。新的类比 四羧酸盐钙离子指示剂Quin2的报价增加了约30倍 荧光的亮度，随着激发的变化，有时 响应于钙离子的发射波长。波长变化意味着 [Ca~(2+)]i的变化现在可以通过荧光比率为2 波长，不受染料含量或细胞变化的干扰 单个6微米细胞的大小，通过荧光显微镜或通过 流式细胞术。图像[钙]i的空间分辨率只需要 视频处理系统，其可行性已被证明为 与设备更好的实验室合作。这个系统将被使用 检测单个淋巴细胞处理后[Ca~(2+)]i的时程变化 有丝分裂的和一些非有丝分裂的凝集素、抗体和淋巴因子。 淋巴细胞表面标志物将通过常见的抗体进行评估 选择性地对细胞进行染色或将其附着在腔室地板上。流动 细胞学将为人口统计提供一个独立的视角。 该视频系统还将用于研究成纤维细胞的[Ca~(2+)]i和Phi 用生长因子刺激或由温度敏感型 致癌基因。目前的研究表明[Ca~(2+)]i升高与几个 海胆受精卵有丝分裂的重大事件将由 成像功能，以查看隆起是否位于 手机。将检查PtK1或CHO等细胞系，看看它们是否也 有[Ca~(2+)]i波动。[Ca~(2+)]i瞬变的重要性 有丝分裂应使用光反应性钙离子螯合剂进行测试，以产生 在空间和时间上定义的[Ca~(2+)]i上升或下降。 同时，化学方面的努力将致力于进一步改善 [Ca~(2+)]i指示剂，增加其工作波长。进一步 研究光反应性钙离子螯合剂的目的是增加量子 光解的效率、速度和波长。钠离子选择性原型 指示剂需要优化荧光性能和添加 羧酸基使它们在生理上有用。

The biological focus is to study the roles of cytosolic free Ca2+ ([Ca2+]i) changes in helping trigger the entry of quiescent cells into the cell cycle and in controlling their progression through mitosis. Emphasis will be placed on resolving differences in [Ca2+]i between heterogeneous neighboring cells and between different regions of large mitotic and motile cells. Methodology developed during the previous grant period offers the unprecedented sensitivity and spatial resolution needed. New analogs of the tetracarboxylate Ca2+ indicator quin2 offer about thirty-fold increased brightness of fluorescence, with change in excitation and sometimes emission wavelengths in response to Ca2+. Wavelength shifts mean that [Ca2+]i changes can now be detected by ratios of fluorescence at two wavelengths, without interference from variations in dye content or cell size, in individual 6 um cells studied by fluorescence microscopy or by flow cytrometry. Spatial resolution to image [Ca2+]i merely requires a video processing system whose feasibility has been demonstrated by collaboration with a better-equipped laboratory. This system will be used to examine the time course of [Ca2+]i in individual lymphocytes treated with mitogenic and some non-mitogenic lectins, antibodies, and lymphokines. Lymphocyte surface markers will be assessed by the usual antibodies to selectively stain cells or adhere them to the chamber floor. Flow cytometry will provide an independent view of the population statistics. The video system will also be used to study [Ca2+]i and pHi in fibroblasts stimulated with growth factors or transformed by a temperature-sensitive oncogene. Present studies showing [Ca2+]i rises correlated with several major events in mitosis of sea urchin zygotes will be extended by the imaging capability to see if and where the rises are localized in the cell. Cell lines such as PtK1 or CHO will be checked to see if they too have [Ca2+]i fluctuations. The importance of [Ca2+]i transients during mitosis should be tested by using photoreactive Ca2+ chelators to generate spatially and temporally defined rises or falls in [Ca2+]i. Meanwhile, chemical efforts will be devoted to yet further improvements of [Ca2+]i indicators by increasing their wavelengths of operation. Further work on photoreactive Ca2+ chelators is aimed at increasing the quantum efficiency, speed, and wavelengths of photolysis. Prototype Na+ selective indicators need optimization of fluorescent properties and addition of carboxylate groups to make them physiologically useful.