A SH3 domain-based protein interaction network drives cytokinesis from contraction into abscission.

• Applicant: Professor Dr. Nils Johnsson
• Subject Area: Cell Biology
• Term: from 2016 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 329438330

A SH3 domain-based protein interaction network drives cytokinesis from contraction into abscission.The transitions during the cell cylce are caused by changes in the composition and organization of the cytosol and its underlying protein networks. These differences are often affected by changes in comparatively weak and temporal protein interactions. Through their cooperative and synergistic nature these networks are still able to precisely determine the when and where of the enzymes and protein machines actions. The in-depth characterization of these dynamic protein matrices is thus a prerequisite for understanding the exact timing and spatial activation of cellular processes.Abscission during cell separation is achieved in the budding yeast by the synthesis of the secondary septum that immediately follows AMR constriction and PS synthesis. As central hypothesis of this proposal I postulate that the transition from AMR contraction to abscission is accompanied by the dissolution of the central cytocinetic complex and the simultaneous formation of the Nba1-complex. Both complexes are built by small SH3-domain interactions networks and share with Hof1p and Cyk3p two common subunits. The proposal aims at describing the cellular formation and dissolution of both complexes in time and spatial resolution, and to correlate these events with the other simultaneously occurring activities during cytokinesis.The identities of its subunits let us assume that the Nba1-complex supports exocytosis during cytokinesis and fine-tunes the balance between GDP- and GTP-loaded Rho-GTPases. We will establish methods to follow single secretory vesicles during cytokinesis and monitor the activities of the Rho-GTPases. Using our Split-Ubiquitin based yeast arrays we will additionally search for binding partners of the RhoGAPS, GEFs and the critical Rho-dependent effectors. The obtained hits will be tested for their impact on localization and activities of theses proteins during cytokinesis.Central for the proposed analysis of the CCC- and the Nba1-complex and the functional annotations of their individual interactions are the creation of alleles where only one interaction is selectively eliminated. We will harness our recently established Split-ubiquitin based life/death strategy that enriches these alleles through a selection for non-binding. The desired mutations will be identified by NGS and subsequently engineered into the genome of the yeast to test their impact onto the transition from AMR contraction to abscission.The aim of the proposal is to understand and visualize cytokinesis as sequential progression of collaborating protein interaction states. Our analysis will identify the nodes within this network that serve as points of regulation. These nodes of intervention are important to coordinate chromosome separation with abscission. We are convinced that these nodes and the mechanism of their regulation are conserved through evolution.

• DFG Programme: Research Grants