The first 10 minutes of bud site assembly and the initiation of bud growth in the yeast S.cerevisiae

最近两个资助期使我们能够研究蛋白质相互作用网络的体内形成和功能，这些网络驱动芽殖酵母的极性生长和胞质分裂。我们建立了新的网络的组成，并剖析其在体内的拓扑结构。通过我们的时间互动分析，我们能够描述这些网络的时间和本地化依赖的转换不同的相互作用状态。分析帮助我们识别关键的边缘和节点，并通过特定的消融，为这些网络分配特定的功能。新的建议建立在这种方法的基础上，但将重点转移到在酵母中建立新的芽位上。芽位的组装是一个显著的和难以理解的过程。由活性Cdc 42的局部脉冲启动，数百种不同的蛋白质、脂质和其他大分子被吸引到小于一微米的单个点上。10分钟后，芽部位开始扩展并生长成新的子细胞。芽位的结构太复杂，可能太无定形，不能通过单一的线性逐步过程组装。相互影响和相互通知的平行途径的收敛代表了另一种可能，并且可能是更现实的观点，即芽位是如何形成的，我建议应用我们所获得的知识，并调整我们的方法来解剖相互作用网络的芽位组装的特点。具体来说，我建议研究芽位组装的以下中心问题，以更深入地了解酵母中这一过程的逻辑和机制： 在到达感受态芽位的过程中是否存在可区分的、稳定的中间结构？ - 我们能否识别和剖析不同组件和步骤组装之间的反馈回路？- 我们能否确定一旦组装出错就停止或中止组装的检查点和调节机制？- 芽位组装是如何与细胞周期协调的？为此，该提案涵盖了艰苦的基础工作，包括建立不同途径的成员在芽位点的出现顺序，开发光遗传学工具来识别蛋白质，以及解剖可能在组装过程中施加秩序和串扰的反馈电路。对这种复合复杂结构的形成机理的研究尚处于起步阶段。我们希望发现新的自组织原理，这些原理也适用于其他生物体中复杂的高阶结构的形成。

The last two funding periods allowed us to investigate the in vivo formation and function of protein interaction networks that drive polar growth and cytokinesis in the budding yeast. We established the composition of novel networks and dissected their in vivo topologies. Through our temporal interaction analysis we were able to describe these networks as time- and localization-dependent conversions of different interaction states. The analysis helped us to identify the critical edges and nodes and, through their specific ablation, to assign specific functions to these networks. The new proposal builds on this approach but shifts its focus on the establishment of a new bud site in yeast. The assembly of the bud site is a remarkable and hardly understood process. Kick-started by a local pulse of active Cdc42, hundreds of different proteins, lipids, and other macromolecules are attracted to a single spot of less than one micron. After 10 minutes the bud site starts to expand and to grow into a new daughter cell. The structure of the bud site is too complex and probably too amorphous to be assembled by a single linear, step-wise process. The convergence of parallel pathways that influence and inform each other represents an alternative, and probably more realistic view how the bud site forms.I propose to apply our gained knowledge and adjust our methods of dissecting interaction networks to the peculiarities of bud site assembly. Specifically, I propose to study the following central questions of bud site assembly to gain a deeper understanding of the logic and mechanisms of this process in yeast:- Are there distinguishable, stable intermediate structures on the way to a competent bud site? - Can we identify and dissect feedback loops between the assembly of different components and steps? - Can we identify checkpoints and regulatory mechanism that halt or abort assembly once assembly goes wrong? - How is bud site assembly coordinated by the cell cycle?To this end the proposal covers painstaking groundwork by establishing the order of appearances of the members of different pathways at the bud site, the development of optogenetic tools to inactivate proteins, as well as the dissection of feedback circuits that might impose order and crosstalk during the assembly process. The research on the formation of such a composite and complex structure is still at its beginnings. We expect to discover new principles of self-organization that are applicable to the formation of complex higher-order-structures in other organisms as well.