DYNAMICS OF CALMODULIN IN NEURONS

Cat+介导的信号转导的一个基本问题是，通过可扩散的第二信使钙的变化如何激活多种酶，导致时间和空间限制效应。本研究的目的是进一步研究猫+结合蛋白钙调素的空间和时间动态，通过测量其扩散利用生物化学和成像方法的组合。初步实验和先前的工作（Persechini和Cronk，1999; Luby-Phelps等人，1995; Tansey等人，1994）表明钙调蛋白在细胞中是限制性的。如果发现对神经元是正确的，这一发现对神经元钙调素依赖性酶如何被激活提供了重要的限制，并对钙调素作为Ca 2+瞬变检测系统调节细胞过程的作用产生了深远的影响：本研究项目的具体目标是：1）使用生物化学方法测定不同Ca 2+浓度下PC 12细胞中可扩散和结合的钙调蛋白的量;（2）用免疫细胞化学和共聚焦显微镜观察钙调素在PC 12细胞和神经元中的区域化和定位;（3）用多光子荧光光漂白恢复（MPFPR）和荧光显微镜观察PC 12细胞和神经元中钙调素的扩散。和荧光相关光谱（MPFCS）。通过定量分析钙调素在不同Cat+水平下的扩散和空间定位，将阐明神经元中Ca 2+信号传导和钙调素可用性之间的更完整的理解。这些研究将为理解神经元如何通过Ca 2 +/钙调素依赖性信号通路处理信息提供重要信息。

One of the fundamental questions of Cat+-mediated signal transduction is how activation of multiple enzymes by changes in a diffusible second messenger, calcium, leads to temporally and spatially restricted effects. The objective of this research is to examine further the spatial and temporal dynamics of the Cat+-binding protein calmodulin by measuring its diffusion utilizing a combination of biochemical and imaging approaches. Preliminary experiments and previous work (Persechini & Cronk, 1999; Luby-Phelps et al., 1995; Tansey et al., 1994) suggest that calmodulin is limiting in cells. If found true for neurons, this discovery imparts important constraints on how neuronal calmodulin-dependent enzymes are activated and poses profound implications on the role of calmodulin in acting as the detection system for Ca2+ transients to regulate cellular processes: The specific aims of this research project are: 1) to determine the amount of diffusible and bound calmodulin in PC12 cells at differing Ca2+ concentrations using a biochemical approach; 2) to characterize the compartmentalization and localization of calmodulin in PC12 cells and neurons using immunocytochemistry and confocal microscopy; and 3) to determine the diffusion of calmodulin in PC12 cells and neurons using both multiphoton fluorescence photobleaching recovery (MPFPR) and fluorescence correlation spectroscopy (MPFCS). Through the quantitative analysis of diffusion and spatial localization of calmodulin at different levels of Cat+, a more complete understanding between Ca2+ signaling and calmodulin availability in neurons will be elucidated. These studies will provide essential information for understanding how neurons process information through the Ca2+/calmodulin-dependent signaling pathway.