Three-dimensional motion and torque generation of mitotic motors

在有丝分裂中，细胞使用纺锤体来分离染色体。正确的分离是至关重要的，因为错误可能导致非整倍体，并最终导致癌症。在分离过程中，纺锤体纤维(即在细胞分裂期间移动染色体的微管束)需要被组织起来，从而决定纺锤体的形状。分子马达，如运动蛋白-5、运动蛋白-14和细胞质动力蛋白，存在于纺锤体中，并被证明弯曲微管束，从而扭曲纺锤体。尽管马达在二维(2D)上得到了很好的研究，并且被证明是向前和向侧的方向，但由于结构的三维(3D)形状，它们在纺锤体纤维中的功能仍然难以捉摸。在这个项目中，我们的目标是确定人激动素-5(KIF11)、激动素-14(HSET)和细胞质动力蛋白集合的个人3D运动参数，以及它们的集体、抵消行为。此外，我们计划阐明运动伸展在微管滑动中的作用。在力的产生方面，我们将用新的体外重建分析、光钳和数学模型来研究马达产生的3D扭矩。这将阐明前进和侧向运动以及力的产生是如何相互耦合的。综上所述，该项目的结果将提供有丝分裂马达功能的详细图景，并将阐明它们如何有助于纺锤体结构和染色体分离。我们期望我们的发现将有助于解决在有丝分裂纺锤体中观察到的扭曲现象，其目的可能是机械稳定性，通过释放储存的弯曲能量或提供相邻束的物理分离，在后期有效地传递力。

In mitosis, the cell is using the spindle apparatus to segregate the chromosomes. Correct segregation is essential because errors can lead to aneuploidy and ultimately cancer. During segregation, spindle fibers (i.e., bundles of microtubules that move chromosomes during cell division) need to be organized, determining the spindle shape. Molecular motors, such as kinesin-5, kinesin-14 and cytoplasmic dynein, are present in the spindle and were shown to bend microtubule bundles, which twists the spindle. Although the motors are well studied in two dimension (2D) and were shown to step in forward and sideward direction, their functioning in spindle fibers remains elusive due to the three-dimensional (3D) shape of the structure. In this project, we aim to determine the individual 3D motility parameters of human kinesin-5 (KIF11), kinesin-14 (HSET) and cytoplasmic dynein ensembles, as well as, their collective, counteracting behavior. Moreover, we plan to elucidate the role of motor extension during microtubule sliding. With regard to force generation, we will investigate the 3D torques generated by the motors with novel in vitro reconstitution assays, optical tweezers and mathematical modeling. This will clarify how forward and sideward motion as well as force production are coupled to each other. Taken together, the results of this project shall provide a detailed picture of the functioning of mitotic motors and will clarify how they contribute to spindle architecture and chromosome segregation. We expect that our findings will help to address the observed phenomenon of twist in the mitotic spindle, whose purpose could be mechanical robustness, efficient force transmission in late anaphase through release of stored bending energy or providing physical separation of adjacent bundles.