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

最后两个资助期使我们能够研究蛋白质相互作用网络在体内的形成和功能，这些相互作用网络驱动萌芽酵母的极地生长和胞质分裂。我们确定了新型网络的组成，并剖析了它们的体内拓扑结构。通过我们的时间相互作用分析，我们能够将这些网络描述为不同相互作用状态的依赖于时间和位置的转换。这一分析帮助我们确定了关键的边缘和节点，并通过它们的特定消融，为这些网络分配了特定的功能。新的提案建立在这种方法的基础上，但将重点转移到在酵母中建立一个新的芽位。芽位的组装是一个非凡而又难以理解的过程。在活跃的Cdc42局部脉冲的启动下，数百种不同的蛋白质、脂类和其他大分子被吸引到一个不到一微米的单一斑点。10分钟后，芽位开始扩张，并长成新的子细胞。芽位的结构太复杂，也可能太无定形，不能通过单一的线性、分步过程来组装。相互影响和信息的平行路径的收敛代表了一种替代的，也可能是更现实的观点，即芽点是如何形成的。我建议应用我们获得的知识，调整我们解剖相互作用网络的方法，以适应芽点组装的特点。具体地说，我建议研究芽位组装的以下核心问题，以更深入地了解酵母中这一过程的逻辑和机制：-在通往合格芽位的道路上是否存在可区分的、稳定的中间结构？-我们能否识别并剖析不同组件和步骤的组装之间的反馈循环？-我们能否识别检查点和调控机制，一旦组装出错，就停止或中止组装？-芽位组装如何由细胞周期协调？为此，该提案包括通过建立芽位不同途径成员在芽位出现的顺序，开发光遗传工具来灭活蛋白质，以及在组装过程中可能强加秩序和串扰的反馈电路的剖析。对于这种复合复杂结构的形成机理的研究还处于起步阶段。我们希望发现新的自组织原理，同样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.