Directed assembly of artificial mitotic spindles

描述（由申请人提供）：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进行的，有丝分裂纺锤体是由微管、分子马达和相关调节蛋白组成的化学机械机器。由于其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机制。细胞实验已经确定了许多参与有丝分裂的分子马达和其他蛋白质。然而，由于涉及大量分子和内置的功能冗余用于防止染色体错分离，关于驱动有丝分裂的精确分子相互作用和物理机制还有许多未解决的问题。特别是，在孤立蛋白质的单分子研究和在复杂的细胞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，并应用这种新的实验工具来研究特定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结构。人工纺锤体将使用微加工室、交流电场和表面图案马达蛋白进行组装，并使用荧光显微镜对所得结构进行表征。具体的实验将进行，以测试纺锤体形态发生和维持的现有模型的充分性。前两个实验将测试马达在人造主轴上居中的能力，以及重组主轴微管的能力。第三个实验将测试c端运动蛋白马达将微管末端聚焦成细胞内紧密簇的能力。通过在体外重建有丝分裂纺锤体的复杂细胞内结构，该方法将揭示运动蛋白和微管在有丝分裂基础上的纳米级动态相互作用，这是目前使用单分子测量或完整细胞测量无法实现的。

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.