Structured illumination with particle averaging reveals novel roles for yeast centrosome components during duplication.
Structured illumination with particle averaging reveals novel roles for yeast centrosome components during duplication.
复制标题
DOI：
10.7554/elife.08586
复制
发表时间：
2015-09-15
期刊：
影响因子：
7.7
通讯作者：
Jaspersen SL
中科院分区：
文献类型：
--
作者：
Burns S;Avena JS;Unruh JR;Yu Z;Smith SE;Slaughter BD;Winey M;Jaspersen SL
酵母中心体（称为纺锤体极体，SPB）的复制被认为是通过一系列离散的步骤进行的，最终新的SPB插入核膜（NE）。为了更好地理解这一过程，我们开发了一种新的单粒子平均双色结构照明显微术（SPA - SIM）方法，利用内源性表达的荧光蛋白衍生物研究复制过程中所有18种SPB组分的定位。使用这种方法获得的更高分辨率和定量强度信息使我们能够证明，SPB复制始于有丝分裂末期不对称Sfi1丝的形成，随后在其顶端进行依赖于Mps1的Spc29和Spc42依赖复合物的组装。我们观察到参与膜插入的蛋白质，如Mps2、Bbp1和Ndc1，在复制早期也在新的SPB处积累，这表明在野生型细胞中SPB形成过程中，SPB组装和核膜插入是耦合事件。
DOI: http://dx.doi.org/10.7554/eLife.08586.001
细胞分裂产生两个新的子细胞，每个子细胞都包含相同的遗传物质。首先，亲代细胞的DNA被复制，然后它必须通过一种由称为微管的细丝构成的结构在物理上分离到子细胞中。为了确保DNA被分成两个相等的部分，微管必须从细胞中的两个点发出，这两个点被称为纺锤体极。
每个纺锤体极由一组（或“复合物”）蛋白质组成，在细胞分裂之前这些蛋白质必须被复制。虽然我们了解DNA是如何被复制的，但我们不知道细胞是如何复制蛋白质的。酵母中的纺锤体极——被称为纺锤体极体——是研究这个问题的理想模型，因为构成它的蛋白质已经被确定，而且在实验室中研究酵母很容易。
伯恩斯等人开发了一种新方法，利用荧光蛋白标签和一种复杂的显微技术研究纺锤体极体。实验绘制了纺锤体极体复制过程中18种蛋白质的位置图谱。其中一些蛋白质使纺锤体极能够插入围绕细胞核的膜中。出乎意料的是，伯恩斯等人观察到这组蛋白质在新的纺锤体极形成时与其相互作用，而不是像之前认为的那样在之后相互作用。
伯恩斯等人的研究结果表明，纺锤体极体在被复制的同时组装到围绕细胞核的膜中。接下来的挑战是了解这一过程的细节，并使用相同的方法研究细胞中的其他大型蛋白质复合物。到目前为止，对蛋白质结构的高度详细的研究仅限于少数蛋白质和条件。伯恩斯等人开发的方法使得在细胞分裂过程中研究整个蛋白质复合物的运动成为可能。
DOI: http://dx.doi.org/10.7554/eLife.08586.002
Duplication of the yeast centrosome (called the spindle pole body, SPB) is thought to occur through a series of discrete steps that culminate in insertion of the new SPB into the nuclear envelope (NE). To better understand this process, we developed a novel two-color structured illumination microscopy with single-particle averaging (SPA-SIM) approach to study the localization of all 18 SPB components during duplication using endogenously expressed fluorescent protein derivatives. The increased resolution and quantitative intensity information obtained using this method allowed us to demonstrate that SPB duplication begins by formation of an asymmetric Sfi1 filament at mitotic exit followed by Mps1-dependent assembly of a Spc29- and Spc42-dependent complex at its tip. Our observation that proteins involved in membrane insertion, such as Mps2, Bbp1, and Ndc1, also accumulate at the new SPB early in duplication suggests that SPB assembly and NE insertion are coupled events during SPB formation in wild-type cells. DOI: http://dx.doi.org/10.7554/eLife.08586.001 Cells divide to produce two new daughter cells that each contain the same genetic material. First, the DNA of the parent cell is copied, then it must be physically separated into the daughter cells by a structure made of filaments called microtubules. To ensure that the DNA is separated into two equal parts, the microtubules must emerge from two points in the cell, known as spindle poles. Each spindle pole is made of a group (or ‘complex’) of proteins and these have to be copied before the cell can divide. While we understand how DNA is copied, we do not know how cells copy proteins. The spindle pole in yeast—known as the spindle pole body—is an ideal model to study this problem because the proteins that form it have already been identified and it is easy to study yeast in the laboratory. Burns et al. developed a new method to study the spindle pole body using fluorescent protein tags and a sophisticated microscopy technique. The experiments mapped the positions of 18 proteins within the spindle pole body during its duplication. Some of these proteins enable the spindle pole to insert into the membrane that surrounds the cell's nucleus. Unexpectedly, Burns et al. observed that this set of proteins interact with the new spindle pole as it forms, instead of afterwards as was previously believed. Burns et al.'s findings suggest that the spindle pole body assembles into the membrane surrounding the nucleus at the same time as it is copied. The next challenges are to understand the details of how this works and to use the same method to study other large protein complexes in cells. Until now, highly detailed surveys of protein structures have been limited to a handful of proteins and conditions. The method developed by Burns et al. makes it possible to carry out studies that examine the movements of whole protein complexes during cell division. DOI: http://dx.doi.org/10.7554/eLife.08586.002