Three-dimensional imaging of the yeast actin cytoskeleton through the budding cell cycle
Three-dimensional imaging of the yeast actin cytoskeleton through the budding cell cycle
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DOI：
10.1091/mbc.9.12.3259
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发表时间：
1998-12-01
影响因子：
3.3
通讯作者：
Amberg, DC
中科院分区：
文献类型：
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作者：
Amberg, DC
酿酒酵母的肌动蛋白细胞骨架是由一个单一的，传统的肌动蛋白亚型，是86%相同的哺乳动物肌动蛋白除了电池的类似保守的相关蛋白。这种组分及其功能的保守性使得酵母遗传学家和细胞生物学家使用酵母的实验能力来研究肌动蛋白细胞骨架组装的调节和肌动蛋白细胞骨架的基本管家功能（对于全面的综述，参见Botstein et al.，1997年）。对这些研究很重要的是通过细胞周期的肌动蛋白细胞骨架的可视化，以及酵母细胞骨架的调节因子和组分的突变如何影响这种组织。酵母肌动蛋白细胞骨架的第一张图像显示，它由两种基于顺应性的结构组成：肌动蛋白皮质片和肌动蛋白索（亚当斯和普林格尔，1984;基尔马丁和亚当斯，1984）。肌动蛋白皮层补丁显示极化分布，在细胞周期中的变化：首先，他们出现在初期的芽网站，建议在芽出现的作用，此后不久，他们也发现在生长芽，芽生长的作用，并在细胞周期的后期，他们重组成两个环的颈部，在那里他们被认为是参与分隔和胞质分裂。通过电子显微镜，肌动蛋白皮质斑块已经显示为质膜的内陷，肌动蛋白丝和肌动蛋白相关蛋白围绕质膜组织（Mulholland等人，1994年）。最近已经表明，肌动蛋白皮质斑的子集可以以高达1 μm/s的速度移动（Doyle和Botstein，1996; Waddle等人，1996年）。由成束的肌动蛋白丝组成的肌动蛋白索通常沿着出芽细胞的长轴延伸。这种组织结构与肌动蛋白参与极化细胞生长、细胞皮层动态重组、细胞皮层膜运输和细胞分裂时细胞器分离的理解非常吻合。由于酵母肌动蛋白细胞骨架组织结构的某些方面无法通过传统的二维显微镜很好地解决或记录，我已经研究了使用DeltaVision去卷积显微镜可视化的酵母肌动蛋白细胞骨架的三维。该仪器数字地捕获Z平面中的焦点部分，并且通过在称为迭代去卷积的过程中从每个部分检查相邻焦点部分来数学地去除失焦光（Agard等人，1989; Scalettar等人，1996年）的报告。然后可以将澄清的焦点部分组装起来以产生高分辨率的三维图像。我发现这项技术对于酵母细胞解剖学的研究非常强大。关于肌动蛋白细胞骨架的组织，我已经证实，肌动蛋白索（就像斑块一样）在它们的排列中是皮质的，可以观察到它们的末端和侧面都附着在皮质斑块上。在某些情况下，可以看到多个电缆连接到单个皮质补片。此外，我观察了芽萌发的最早阶段，发现芽首先通过一圈皮层斑块出现，只有在这个阶段之后，皮层斑块才迁移到芽中。在我的菌株背景（S288 C），我没有观察到一个极化分布的皮层补丁在芽在任何时候在芽生长。
The actin cytoskeleton of Saccharomyces cerevisiae is composed of a single, conventional actin isoform that is 86% identical to mammalian actins in addition to a battery of similarly conserved associated proteins. This conservation of components and their functions has led yeast geneticists and cell biologists to use the experimental power of yeast toward the study of the regulation of actin cytoskeleton assembly and the basic, housekeeping functions of the actin cytoskeleton (for a comprehensive review, see Botstein et al., 1997). Important to these studies has been the visualization of the actin cytoskeleton through the cell cycle and how mutations in regulators and components of the yeast cytoskeleton affect this organization. The first images of the yeast actin cytoskeleton showed that it consists of two filament-based structures: the actin cortical patch and the actin cables (Adams and Pringle, 1984; Kilmartin and Adams, 1984). The actin cortical patches show a polarized distribution that changes during the cell cycle: first they appear at the incipient bud site, suggesting a role in bud emergence; soon thereafter they are also found within the growing bud, indicating a role in bud growth; and late in the cell cycle they reorganize into two rings in the neck, where they are believed to be involved in septation and cytokinesis. By electron microscopy, the actin cortical patches have been shown to be invaginations of the plasma membrane around which actin filaments and actin-associated proteins are organized (Mulholland et al., 1994). Recently it has been shown that subsets of actin cortical patches can move at speeds of up to 1 μm/s (Doyle and Botstein, 1996; Waddle et al., 1996). The actin cables, which consist of bundled actin filaments, were observed to generally run along the long axis of budding cells. This organization fits well with the understanding that actin is involved in polarized cell growth, dynamic reorganization of the cell cortex, membrane trafficking at the cell cortex, and organelle segregation at cell division.Because certain aspects of the organization of the yeast actin cytoskeleton cannot be well addressed or documented by conventional two-dimensional microscopy, I have investigated the use of a DeltaVision deconvolution microscope for visualization of the yeast actin cytoskeleton in three dimensions. This instrument digitally captures focal sections in the Z plane and mathematically removes out-of-focus light by examining neighboring focal sections, from each section in a process called iterative deconvolution (Agard et al., 1989; Scalettar et al., 1996). The clarified focal sections can then be assembled to produce highresolution three-dimensional images. I have found that this technology is extremely powerful for the study of yeast cell anatomy. With respect to the organization of the actin cytoskeleton, I have confirmed that the actin cables (like the patches) are cortical in their arrangement and can be observed to attach both at their ends and laterally to the cortical patches. In some cases, multiple cables can be seen to attach to single cortical patches. In addition, I have observed the earliest stages of bud emergence and have found that buds first emerge through a ring of cortical patches, and only after this stage do the cortical patches migrate into the bud. In my strain background (S288C), I do not observe a polarized distribution of cortical patches in the bud at any time during bud growth.