NSF-MCB/BSF Exploring Kinesin-5 Evolutionary Adaptations for Mitotic Spindle Dynamics

细胞分裂是所有生命的一个特征，每个细胞通过分裂复制成两个子细胞。真核细胞(如酵母和人类)通过形成有丝分裂纺锤体进行分裂，有丝分裂纺锤体是由微管组成的两极结构。微管的动态伸长和反平行组织将每个复制的染色体向细胞的相反侧分离，最终导致新的子细胞的产生。在整个真核生物生命中，有丝分裂纺锤体的组装方式存在差异。在酵母中，有丝分裂的纺锤体在细胞核内组装，而在人类和果蝇中，细胞核被分解。组装和组织过程是由马达分子驱动的，马达分子是细胞分裂的关键，在真核生物中非常相似。其中，Kinesin-5马达具有独特的哑铃形状，在相对的两端有两组活动区域，它们同时沿着从有丝分裂纺锤体的相反两极发出的两根细丝移动。通过这种活动，Kinesin-5发动机促进了细胞分裂过程中微管的滑动分离，导致纺锤体的延长和染色体分离。当Kinesin-5功能受损时，分裂细胞会停滞并最终死亡，因为细胞无法分离复制的染色体。我们将研究在真核生物中发现的不同版本的Kinesin-5中进化的独特机制和适应，以及它们在组织有丝分裂纺锤体中的作用。我们假设，这种独特的适应指定了在真核生物中观察到的有丝分裂纺锤体组装的差异的特征。美国和以色列研究小组的成功合作揭示了具有独特细胞分裂形式的生物体中Kinesin-5分子的显着进化。在低等真核生物中，与果蝇等高等真核生物中发现的运动蛋白-5马达相比，运动蛋白-5马达向有丝分裂纺锤体的另一端移动。然而，较低的真核生物Kinesin-5马达的方向相反，因为它们以一种独特的方式聚集在细丝末端，一旦捕捉到第二丝；而在动物中，Kinesin-5分子不具备这些特征。在这里，我们应用包括生化分析、酵母遗传学和单马达荧光成像在内的跨学科方法来阐明这些独特的适应是如何在Kinesin-5马达中进化到影响在不同生物体中观察到的有丝分裂纺锤体的不同组装和组织状态的。首先，我们将确定在酵母人和果蝇中发现的Kinesin-5分子之间聚集的起源，以及导致分子如何在纺锤体细丝的不同末端保持不变。其次，我们将确定导致一个马达进行方向切换的特征(在酵母中，但在人类和果蝇中不是)。第三，我们将确定哑铃形Kinesin-5分子的物理组织如何指定微管滑动。这些研究将有助于深入理解Kinesin-5产生的运动性与真核生物进化的关系。这项研究是由以色列和美国的一个联合项目资助的，其中NSF为位于加州大学戴维斯分校的美国实验室提供资金，而以色列BSF为位于内盖夫的本-古里安大学的以色列实验室提供资金。

Cell division is a feature of all life by which each cell duplicates into two daughter cells. Eukaryotic cells (such as yeast and humans) divide by forming a mitotic spindle, which is a bipolar structure composed of microtubules. The dynamic elongation and antiparallel organization of microtubules segregates each duplicated chromosome towards opposite sides of the cell, eventually leading to the creation of new daughter cells. Across eukaryotic life, there are variations in how mitotic spindles are assembled. In yeast, the mitotic spindle assembles inside the nucleus, while in humans and fruit flies, the nucleus is disassembled. The assembly and organization processes are driven by motor molecules, which are essential for cell division, and are highly similar in eukaryotes. Among them, Kinesin-5 motors possess a unique dumbbell shape, with two sets of motile regions located at opposite ends, which simultaneously move along two filaments emanating from opposite poles of the mitotic spindle. Through this activity, the Kinesin-5 motors promote the sliding-apart of microtubules during cell division, leading to elongation of spindles and chromosome segregation. When Kinesin-5 function is impaired, dividing cells stall and eventually die, because cells cannot segregate duplicated chromosomes. We will investigate the unique mechanisms and adaptations that have evolved in different versions of Kinesin-5 found in eukaryotes and their role in organizing mitotic spindles. We hypothesize that such unique adaptations specify features of the observed differences in mitotic spindle assembly found among eukaryotes.A successful collaboration between US and Israeli research groups revealed the remarkable evolution of Kinesin-5 molecules from organisms with unique forms of cell division. In lower eukaryotes, Kinesin-5 motors move towards the opposite end of mitotic spindles compared to those found in higher eukaryotes such as fruit fly kinesin-5 motors. Yet the lower eukaryotic kinesin-5 motors reverse direction as they cluster in a unique way near the ends of filaments, once capturing second filaments; while in animals the kinesin-5 molecules do not possess these features. Here, we apply an interdisciplinary approach including biochemical analyses, yeast genetics and single motor fluorescence imaging, to elucidate how these unique adaptations evolved in Kinesin-5 motors to influence the diverse assembly and organization states of mitotic spindles observed in different organisms. First, we will determine the origin of clustering among kinesin-5 molecules found in both yeast humans and fruit fly versions and how that leads molecules persist at different ends of spindle filaments. Second, we will determine the features that lead one motor to direction switch (in yeast, but not in the humans and fruit flies). Third, we will determine how the physical organization of the dumbbell-shaped kinesin-5 molecules specifies microtubule sliding. These studies will provide a deep understanding of kinesin-5 generated motility in relation to the evolution of eukaryotes. This research was funded by a joint Israeli and United State program in which the NSF provides funding for the US laboratory, located at UC Davis, while the Israeli BSF provides funds for the Israeli lab located at Ben-Gurion University in Negev.