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

• Applicant: Professor Dr. Nils Johnsson
• Subject Area: Cell Biology
• Term: from 2008 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 88450366

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.

• DFG Programme: Research Grants