Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces.
Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces.
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DOI：
10.7554/elife.61170
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发表时间：
2021-01-22
期刊：
影响因子：
7.7
通讯作者：
Tolić IM
中科院分区：
文献类型：
--
作者：
Jagrić M;Risteski P;Martinčić J;Milas A;Tolić IM
在中期，染色体在纺锤体赤道处的位置受到着丝粒微管和极弹射力施加的力的调节。然而，姐妹动粒纤维与桥接纤维的机械耦合所产生的力在染色体排列中的作用尚不清楚。在这里，我们开发了一种光遗传学方法，用于急性去除 PRC1，以部分分解桥接纤维，并表明它们促进染色体排列。对正端蛋白 EB3 的追踪显示，PRC1 去除后，桥接微管的反向平行重叠更长，并伴有着丝粒错位和滞后。 Kif4A/kinesin-4 和 Kif18A/kinesin-8 在桥接纤维内发现，并且在 PRC1 去除后大量丢失，表明这些蛋白质调节桥接微管的重叠长度。我们提出，PRC1 介导的桥接微管的交联和驱动蛋白向桥接纤维的募集通过传递到相关动粒纤维的重叠长度依赖性力促进染色体排列。在细胞分裂以创建自身副本之前，它们需要复制其遗传物质。为了帮助均匀地分裂 DNA，他们建造了一台称为有丝分裂纺锤体的机器。有丝分裂纺锤体由称为微管的精细管状结构组成，它捕获包含遗传信息的染色体并将它们排列在纺锤体的中心。微管通过延长或缩短染色体的尖端来推动和拉动染色体。但目前尚不清楚微管如何知道染色体何时到达中心点。找到答案的一种方法是去除分裂过程中积累在纺锤体中部的蛋白质，例如蛋白质 PRC1，它有助于组装称为桥接纤维的微管子集，以及蛋白质 Kif4A 和 Kif18A，它们的作用类似于分子尺，可以缩短长微管。通常，科学家会删除其中一种蛋白质，看看它会产生什么影响。然而，这些实验需要数天时间，给细胞足够的时间来适应，因此很难研究每种蛋白质的作用。在这里，Jagrić、Risteski、Martinčić 等人。利用光在染色体排列的精确时刻操纵蛋白质，并将 PRC1 从纺锤体移动到细胞膜。因此，Kif4A 和 Kif18A 从主轴中心移除。这导致与连接染色体的微管重叠的桥接纤维变得更薄。贾格里奇等人。发现如果没有分子标尺蛋白，桥接纤维也会太长。这增加了纺锤体中心微管之间的重叠，导致染色体迁移远离中心。这表明染色体在纺锤体中部的排列取决于桥接微管，桥接微管需要具有一定的长度才能有效地移动并将染色体保持在中心。因此，移动染色体的力在微管的尖端和沿着微管壁产生。这些结果可能会启发其他研究人员重新评估桥接纤维在细胞分裂中的作用。这里描述的光遗传学技术也可以帮助确定其他蛋白质必须发挥的作用。最终，这可能使研究人员能够识别排列染色体所需的所有蛋白质。
During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here, we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promote chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers. Before cells divide to create copies of themselves, they need to duplicate their genetic material. To help split their DNA evenly, they build a machine called the mitotic spindle. The mitotic spindle is made of fine, tube-like structures called microtubules, which catch the chromosomes containing the genetic information and line them up at the center of the spindle. Microtubules push and pull the chromosomes by elongating or shortening their tips. But it remains unclear how the microtubules know when the chromosomes have reached center point. One way to find out is to remove proteins that accumulate in the middle of the spindle during division, such as the protein PRC1, which helps to assemble a subset of microtubules called bridging fibers, and the proteins Kif4A and Kif18A, which work like molecular rulers, shortening long microtubules. Usually, scientists would delete one of these proteins to see what impact this has. However, these experiments take days, giving the cell enough time to adapt and thus making it difficult to study the role of each of the proteins. Here, Jagrić, Risteski, Martinčić et al. used light to manipulate proteins at the exact moment of chromosome alignment and to move PRC1 from the spindle to the cell membrane. Consequently, Kif4A and Kif18A were removed from the spindle center. This caused the bridging fibers, which overlap with the microtubules that connect to the chromosomes, to become thinner. Jagrić et al. discovered that without the molecular ruler proteins, the bridging fibers were also too long. This increased the overlap between the microtubules in the center of the spindle, causing the chromosomes to migrate away from the center. This suggests that the alignment of chromosomes in the middle of the spindle depends on the bridging microtubules, which need to be of a certain length to effectively move and keep the chromosomes at the center. Thus, forces that move the chromosomes are generated both at the tips of the microtubules and along the wall of microtubules. These results might inspire other researchers to reassess the role of bridging fibers in cell division. The optogenetic technique described here could also help to determine the parts other proteins have to play. Ultimately, this might allow researchers to identify all the proteins needed to align the chromosomes.