4D RECONSTRUCTIONS OF ACTIN CYTOSKELETON IN LIVE YEAST CELLS

肌动蛋白细胞骨架动力学的研究是必要的 了解肌动蛋白细胞骨架在酵母中的功能。 的 这些研究的结果不仅适用于酵母， 由于两种系统的同源性， 具体目标 这个项目的目的是研究肌动蛋白细胞骨架的动力学， 通过4-1）显微镜观察活酵母细胞。 以前我们能够 用GFP标记酵母肌动蛋白斑块，并证明它们 动 我们还进行了初步的跟踪移动补丁在2-D 我们能够确定它们的速度（Waddle等人，1996年）。 （4-1） 重建应该可以让我们追踪单个肌动蛋白斑块 通过多个焦平面并且持续很长时间。 4-1)跟踪 补丁程序应该提醒我们， 补丁和移动出焦平面。 4-D跟踪还应 允许我们明确地记录片分割的实例， 在活细胞中的融合、组装和拆卸。 我们也想学习 基因型和环境对转基因植株生长速度和性状的影响 斑块运动 我们希望按照个人补丁 通过光漂白和光活化标记， 蛋白质在斑块中的周转以及肌动蛋白 组装到贴片移动。 我们目前的电脑视频系统 不允许必要的采集速度来执行快速 跟踪4-1中的单个补丁所需的Z扫描）。 在我们的系统中， 高放大率下的长期观察受到以下因素的限制： GFP标记的光漂白和对细胞的光损伤。 多光子激光扫描荧光显微镜似乎是最好的 适合于长期观察而没有光漂白， 光损伤 我们希望HAR高质量的视频carneras将 这有助于我们快速捕获连续焦平面。 我们 打算使用IMR软件对补丁和4-D进行大规模跟踪， 重建。

Studies of actin cytoskeleton dynamics are the necessary to understand the function of the actin cytoskeleton in yeast. The results of these studies should be applicable not only to yeasts but to higher organisms due to homology of both systems. The specific aim of this project is to study the dynamics of the actin cytoskeleton in live yeast cells by means of 4-1) microscopy. Previously we were able to label the yeast actin patches with GFP and to demonstrate that they move. We also performed preliminary tracking of moving patches in 2-D and we were able to determine their speed (Waddle et al., 1996). 4-1) reconstructions should permit us to track the individual actin patches through multiple focal planes and for a long time. 4-1) tracking of patches should p6nnit us to distinguish between the disassembly of patches and movement out of the focal plane. 4-D tracking should also permit us to unambiguously document the instances of patch division, fusion, assembly and disassembly in live cell. We also want to study the impact of genotype and environment on the speed and character of patch movement. We would like to follow the individual patches labeled by photobleaching and photoactivation and establish the rate of protein turnover in the patches and a possible connection of actin assembly to patch movement. Our current computerized video-system does not permit the necessary speed of acquisition to perform the fast Z-scans necessary to track individual patches in 4-1). In our system, long-term observations at high magnification are limited by photobleaching of the GFP label and photodamage to the cells. Multi-photon laser-scanning fluorescence microscopy appears to be best suitable for a long-term observation without photobleaching and photodamaging. We hope that HAR high-quality video carneras will peffnit us to perfonn fast capturing of sequential focal planes. We intend to use IMR software for large-scale tracking of patches and 4-D reconstructions.