Department of Bioengineering, The Pennsylvania State University, University Park, 16802, USA.
Integr Biol (Camb). 2011 Jan;3(1):57-64. doi: 10.1039/c0ib00065e. Epub 2010 Oct 29.
The mitotic spindle is a dynamic assembly of microtubules and microtubule-associated proteins that controls the directed movement of chromosomes during cell division. Because proper segregation of the duplicated genome requires that each daughter cell receives precisely one copy of each chromosome, numerous overlapping mechanisms have evolved to ensure that every chromosome is transported to the cell equator during metaphase. However, due to the inherent redundancy in this system, cellular studies using gene knockdowns or small molecule inhibitors have an inherent limit in defining the sufficiency of precise molecular mechanisms as well as quantifying aspects of their mechanical performance. Thus, there exists a need for novel experimental approaches that reconstitute important aspects of the mitotic spindle in vitro. Here, we show that by microfabricating Cr electrodes on quartz substrates and micropatterning proteins on the electrode surfaces, AC electric fields can be used to assemble opposed bundles of aligned and uniformly oriented microtubules as found in the mitotic spindle. By immobilizing microtubule ends on each electrode, analogous to anchoring at centrosomes, solutions of motor or microtubule binding proteins can be introduced and their resulting dynamics analyzed. Using this "artificial mitotic spindle" we show that beads functionalized with plus-end kinesin motors move in an oscillatory manner analogous to the movements of chromosomes and severed chromosome arms during metaphase. Hence, features of directional instability, an established characteristic of metaphase chromosome dynamics, can be reconstituted in vitro using a pair of uniformly oriented microtubule bundles and a plus-end kinesin functionalized bead.
有丝分裂纺锤体是微管和微管相关蛋白的动态组装体，它控制着染色体在细胞分裂过程中的定向运动。由于复制基因组的正确分离要求每个子细胞恰好收到每个染色体的一份拷贝，因此已经进化出许多重叠的机制来确保在中期将每个染色体运送到细胞赤道。然而，由于该系统内在的冗余性，使用基因敲低或小分子抑制剂的细胞研究在定义精确分子机制的充分性以及量化其机械性能方面存在固有限制。因此，需要新的实验方法来在体外重建有丝分裂纺锤体的重要方面。在这里，我们展示了通过在石英基板上微制造 Cr 电极并在电极表面上微图案化蛋白质，可以使用交流电场来组装在有丝分裂纺锤体中发现的对向束排列和均匀取向的微管。通过将微管末端固定在每个电极上，类似于在中心体处的锚定，可以引入微管结合蛋白或马达蛋白的溶液，并分析其结果动力学。使用这种“人工有丝分裂纺锤体”，我们表明用正极向驱动蛋白功能化的珠子以类似于中期染色体和切断的染色体臂的运动方式进行振荡运动。因此，可以使用一对均匀取向的微管束和正极向驱动蛋白功能化的珠子在体外重建定向不稳定性的特征，这是中期染色体动力学的一个既定特征。
