GTR · Dissecting gamma-TuRC composition and activity by Single Molecule Pull-down

Dissecting gamma-TuRC composition and activity by Single Molecule Pull-down

The main purpose of this project was to dissect the composition and activity of gamma-tubulin ring complexes (γTuRCs), the main nucleator of microtubule (MT) polymerization. MTs are filamentous structures, composed of α- and β-tubulin dimers, that mediate essential processes such as intracellular transport, cell migration or chromosome segregation during cell division. MT polymerization in vivo requires specific MT nucleators which regulate MT assembly at MT organizing centers (MTOCs). The major MT nucleator is the γTuRC, a multi-protein complex assembled from γ-tubulin and gamma complex proteins (GCPs) 2-6. According to the current model, activated γTuRCs nucleate MTs by providing a template for MT assembly: the helical arrangement of ~13 γ-tubulin molecules in the γTuRC resembles the arrangement α-β-tubulin subunits in a MT, thereby serving as a platform for the addition of alpha-beta-tubulin dimers. Throughout the cell cycle, γTuRC localizes to the centrosome, the major MTOC in animal somatic cells. Apart from residing in the pericentriolar material (PCM), where γTuRC nucleates MTs that extend into the cytoplasm during interphase or are incorporated into the mitotic spindle during mitosis, γ-tubulin also localizes specifically to centrioles, the core structures of the centrosome. Here, γTuRC might acts as the nucleator of the MTs that constitute the centriolar cylinder. However, convincing data to support this hypothesis is still missing and it remains possible that γTuRC has functions beyond MT nucleation. It is conceivable that both γTuRC activity and its mode of action can be tuned to allow for the formation of distinct MT-based structures in a cell site- and/or cell cycle specific manner. This might be achieved through interactions with selective factors or through alterations in γTuRC subunit composition. In this project, we set out to test these hypotheses. We established conditions to analyse γTuRC by SiMPull, an innovative in vitro approach that is based on the immobilization of single γTuRCs immunoprecipitated from crude cell extract on a PEG-passivated glass surface and imaging of these complexes by high-resolution microscopy. Complementary, we dissected γTuRC activity and composition in a relevant context, namely during centriole assembly. We were able to identify γTuRC interactors that target the complex to centrioles and, intriguingly, we could show that γTuRC might be required for stabilizing the centriolar cylinder when centrioles have already reached their final size. This novel function of γTuRC might be important to guarantee the stable inheritance of centrioles to the daughter cells during cell division and to stably maintain centrioles in non-cycling cells, to allow formation of cilia, cell protrusions that are templated by “mother” centrioles in resting or post-mitotic cells and that are required for cell signalling among other functions. Overall, our findings provide novel insight into γTuRC activity, especially in the context of centrioles, which frequently show structural and numerical abnormalities in a broad range of diseases such as cancer, microcephaly and a range of complex disorders collectively termed ciliopathies.

Data: CORDIS, © European Union