A versatile platform for reconstructing the spatial organization of intracellular signaling during cell-division

摘要 细胞生物学中的一个中心问题是微米尺度上的空间信息是如何 在细胞质中编码。这对于维持基因组的完整性至关重要， 在有丝分裂中，细胞分裂平面必须精确地定位在~ 10-15的中心。 在胞质分裂过程中，现在已经确定微管细胞骨架 在此过程中起着关键作用，它在细胞位点附近形成一种特殊的结构， 乳沟这种结构，称为纺锤体中间区，作为一个微米尺度的“标记”， 细胞中心然后，这些微米级的标记被信号蛋白“读取”，这些信号蛋白产生自我- 组织活动模式，进一步精确定义细胞裂解位点。随后 该信息被传递到细胞皮层以组织收缩环。我们 现在有一个几乎完整的蛋白质清单，调节这些过程。但 分子机制，通过它的中间区模板的空间活动模式的形成 细胞质和细胞皮层中的调节蛋白是完全不完整的。这里我们将 开发使用微管、重构双层、单分子的体外重构试验 FRET传感器和DNA折纸为基础的纳米模板，以破译空间组织的 在微管模板上实现信号传导活性以及这些反应是如何组织的 在膜-微管界面。这些研究将提供一个保守的见解 在人类癌症中，细胞分裂及其错误调节的关键信号级联。 重要的是，这里开发的创新方法将广泛适用于解剖 空间活动模式被编码和破译的其他信号机制， 细胞分裂、生长和发育过程中的细胞质。

ABSTRACT A central question in cell biology is how spatial information on the micron-length scale is encoded within the cellular cytoplasm. This is essential for maintaining genome integrity during mitosis where the cell-cleavage plane must be precisely positioned at the center of a ~ 10-15 um long cell during cytokinesis. It is now well established that the microtubule cytoskeleton plays a critical role in this process by forming a specialized structure proximal to the site of cell cleavage. This structure, known as the spindle midzone, acts as a micron scale `mark' for the cell center. These micron-scale marks are then `read' by signaling proteins that generate self- organizing activity patterns that further precisely define the site of cell-cleavage. Subsequently this information is transmitted to the cell cortex for the organization of the contractile ring. We now have a near complete parts list of the proteins that regulate these processes. However, the molecular mechanism by which the midzone templates the formation of spatial activity patterns of regulatory proteins within the cytoplasm and the cell cortex is starkly incomplete. Here we will develop in vitro reconstitution assays using microtubules, reconstituted bilayer, singe molecule FRET sensors and DNA origami-based nanotemplates to decipher how spatial organization of signaling activity is achieved on microtubule templates and how these reactions are organized at membrane-microtubule interface. These studies will provide insights into a conserved signaling cascade that is critical for cell division and its mis-regulation in human cancer. Importantly, the innovative methodologies developed here will be widely applicable to dissecting other signaling mechanisms by which spatial activity patterns are encoded and deciphered in the cellular cytoplasm during cell division, growth and development.