The role of actin in the γ-TuRC

• Applicants: Dr. Stefan Pfeffer; Professor Dr. Elmar Schiebel
• Subject Area: Structural Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 506671472

In vertebrates, the γ-tubulin ring complex (γ-TuRC) functions as the principal microtubule (MT) assembly factor. The γ-TuRC mainly consists of five paralogous GCP proteins (GCP2 to GCP6), γ-tubulin, actin, and the two small proteins MZT1 (mitotic spindle organizing protein 1) and MZT2. Recently, we have published the high-resolution cryo-EM structure of the vertebrate γ-TuRC. GCP subunits arranged in a defined order and stoichiometry form an open left-handed spiral of 14 γ-tubulin/GCP heterodimers. γ-tubulin subunits at positions 1 and 14 overlap along the MT axis, so only 13 γ-tubulin molecules are exposed to nucleate MTs with exactly 13 protofilaments by a template-based mechanism. A structural scaffold, the ‘lumenal bridge’, is located inside of the γ-TuRC and consists of two molecules of MZT1 bound to the N-termini of GCP6 and one copy of GCP3, as well as – surprisingly - one molecule of actin. Although actin is firmly embedded in the γ-TuRC structure, interfaces are still partially exposed, opening up the possibility that proteins recognize γ-TuRC-associated actin and regulate γ-TuRC localisation and/or activity.In this proposal, we aim to analyse the function of actin within the γ-TuRC. 1) Structural analysis of an actin binding-deficient γ-TuRC that we have constructed indicates a more open conformation with 14 exposed γ-tubulin molecules. We will therefore address whether actin binding-deficient γ-TuRC organizes MTs with a differing protofilament number. 2) Combining light microscopy with high-resolution information from cellular cryo-electron tomography will allow us to locate wild-type and actin binding-deficient γ-TuRC within cells at single molecule precision and to analyse their concentration, distribution and structures at different cellular locations. 3) Using mass spectrometry, we will analyze whether γ-TuRC with and without actin interacts with a distinct subset of proteins. In addition, we will study whether γ-TuRC–associated actin is exchanging within the free actin pool and whether ATP hydrolysis by actin is important for the function of the γ-TuRC. 4) Finally, we will analyze when actin binding-capability of the γ-TuRC emerged (or was lost) during evolution by bioinformatic analysis and by studying the interaction of actin with recombinantly expressed MZT1-N-GCP6 from yeast, over plants to human cells. Actin binding to the γ-TuRC of Drosophila is of particular interest, because MZT1 is expressed only in a tissue-specific manner in this organism, raising the question of whether and how actin is bound to the γ-TuRC in MZT1 proficient and deficient tissues. Expression and purification of recombinant Drosophila γ-TuRC followed by functional and cryo-EM analysis will address this open question. Cumulatively, this proposal will provide important insights into key outstanding questions regarding the function of actin in the γ-TuRC by using a combination of biochemistry, cell biology and cutting-edge structural biology approaches.

• DFG Programme: Research Grants