Structure-function analysis of γ-TuRC regulation and targeting by NEDD1-CEP192, NME7 and the CM1 motif

• Applicants: Dr. Stefan Pfeffer; Professor Dr. Elmar Schiebel
• Subject Area: Cell Biology ; Structural Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 547269389

Microtubules (MTs) are dynamic tubulin polymers with essential functions in chromosome segregation, cell polarity and intra-cellular transport. The assembly of MTs from tubulin subunits at defined cellular locations is regulated by the gamma-tubulin ring complex (gamma-TuRC). Despite central importance for cell function and viability, the molecular mechanisms of how the MT assembly activity of the gamma-TuRC is controlled by regulators and targeting factors are largely unknown. Based on extensive preliminary data and our well-documented expertise in structural biology of gamma-tubulin complexes, we here propose experiments that will close this gap. NEDD1 is an essential targeting factor of the γ-TuRC that is required for γ-TuRC recruitment to centrosomes and MT branching during spindle formation. Our preliminary cryo-EM data indicate that NEDD1 docks to the tip of the γ-TuRC cone as a tetramer. We will refine and test this model by resolving the interaction at high-resolution and by biochemically reconstituting and analyzing central NEDD1-γ-TuRC interactions. We will further test the functional consequences of perturbing individual interfaces of the γ-TuRC-NEDD1 interaction by studying how mutant γ-TuRC and NEDD1 versions behave in cells. NME7 is a core component and regulator of the γ-TuRC. Our preliminary cryo-EM data indicate that NME7 binds and stabilizes a previously unresolved insertion domain in the γ-TuRC subunit GCP6 and thereby allosterically modulates γ-TuRC conformation. To elucidate the underlying mechanism, we will improve resolution for cryo-EM structures of the γ-TuRC in the presence and absence of NME7 and analyze the MT nucleation activity of mutant NME7-lacking γ-TuRC in vitro and in vivo. The CM1 motif-containing protein CDK5RAP2 is an important activator of the γ-TuRC. Our preliminary cryo-EM tomography data indicate that the structure of γ-TuRCs with a native set of interaction partners can be analyzed at molecular resolution in purified human centrosomes. This provides an opportunity to structurally characterize the impact of CDK5RAP2-derived CM1 motifs and CEP192-NEDD1 on regulation and centrosomal targeting of the γ-TuRC. We will deplete CM1 motif-containing proteins, NEDD1 and CEP192, and analyze how this impacts the structure, concentration, activity and function of the γ-TuRC in purified centrosomes and in vivo. Cumulatively, using a combination of biochemistry, cell biology and structural biology, this proposal will address central outstanding questions regarding the structure, function and integration of key regulatory proteins of γ-TuRC-mediated MT nucleation. Thus, it addresses how MTs are formed in cells, which is a fundamental aspect of life.

• DFG Programme: Research Grants