Directed assembly of artificial mitotic spindles

描述(申请人提供)：1在每一轮细胞分裂中，染色体的成功分裂是由有丝分裂纺锤体完成的，有丝分裂纺锤体是一种化学机械机器，由微管、分子马达和相关的调节蛋白组成。由于它在细胞生物学中的核心作用，以及它是抗有丝分裂癌症治疗的靶点，人们正在进行大量的研究工作，以了解纺锤体形态发生和维持的潜在机制。在细胞中的实验已经确定了一些参与有丝分裂的分子马达和其他蛋白质。然而，由于涉及到大量的分子和内置的功能冗余来防止染色体错误分离，关于驱动有丝分裂的确切分子相互作用和物理机制仍有许多悬而未决的问题。特别是，对分离蛋白质的单分子研究与在细胞复杂环境中观察到的行为之间存在差距。该项目的目标是在体外将微管组装成人工有丝分裂纺锤体，并应用这一新的实验工具来研究特定的运动和微管结合蛋白的性能，其几何形状类似于分裂细胞中的发现。人造纺锤将使用微型制造的腔室、交流电场和表面图案化的马达蛋白质进行组装，所产生的结构将使用荧光显微镜进行表征。将进行具体的实验，以检验现有的纺锤体形态发生和维护模型的充分性。前两个实验将测试马达将自己集中在人造纺锤体上并重组纺锤体微管的能力。第三个实验将测试C端动蛋白马达将微管末端聚焦到细胞中的致密簇中的能力。通过在体外重建有丝分裂纺锤体的复杂细胞内结构，这种方法将揭示运动蛋白和微管之间的纳米级动态相互作用，以一种目前的单分子测量或完整细胞测量无法实现的方式。 相关性：这项工作的目标是开发一种新的方法来研究动物细胞分裂的分子机制。由于癌症是由不受控制的细胞分裂引起的，这项工作是朝着开发新的抗肿瘤疗法迈出的重要一步。

DESCRIPTION (provided by applicant): 1 during each round of cell division, the successful partitioning of chromosomes is carried out by the mitotic spindle, a chemomechanical machine that is comprised of microtubules, molecular motors, and associated regulatory proteins. Due to its central role in cell biology and because it is a target for antimitotic cancer therapies, there is a large research effort underway to understand mechanisms underlying spindle morphogenesis and maintenance. Experiments in cells have defined a number of molecular motors and other proteins that are involved in mitosis. However, because of the large number of molecules involved and the built-in functional redundancy employed to prevent chromosome missegregation, there are many unresolved questions regarding the precise molecular interactions and physical mechanisms driving mitosis. In particular, there is a gap between single-molecule investigations on isolated proteins and the behavior observed in the complex environment of the cell. The goal of this project is to assemble microtubules into artificial mitotic spindles in vitro, and to apply this novel experimental tool to study the performance of specific motor and microtubule binding proteins in a geometry resembling that found in dividing cells. The artificial spindles will be assembled using microfabricated chambers, AC electric fields and surface patterned motor proteins, and the resulting structures will be characterized using fluorescence microscopy. Specific experiments will be carried out to test the sufficiency of current models of spindle morphogenesis and maintenance. The first two experiments will test the ability of motors to center themselves on the artificial spindle and reorganize the spindle microtubules. The third experiment will test the ability of a C-terminal kinesin motor to focus the microtubule ends into the tight clusters found in cells. By recreating the complex intracellular architecture of the mitotic spindle in vitro, this approach will uncover the nanoscale dynamic interactions of motor proteins and microtubules underlying mitosis in a way that is not possible using current single-molecule measurements or measurements in intact cells. Relevance: The goal of this work is to develop a new approach to studying the molecular mechanisms underlying cell division in animal cells. Because cancer results from uncontrolled cell division, this work is an important step towards developing novel anti-tumor therapies.