Dependency on CEP192 and AKAP9 for the organization of the microtubule network

Abstract

The microtubule network is organized by microtubule organizing centers (MTOCs). These organize the network by nucleating new microtubules and binding their minus ends. In many types of animal cells, the centrosome functions as the major MTOC, with the Golgi functioning as a second lesser MTOC. The MTOCs rely heavily on the γ-tubulin ring complex (γ-TuRC). This complex serves as a microtubule nucleation catalyst by forming a template and protects the minus ends of these newly formed microtubules. The centrosome utilizes multiple proteins to recruit, anchor and activate the γ-TuRCs. The centrosomal protein CEP192 plays a role in the recruitment of NEDD1 bound γ-TuRC and in the duplication of the centrosome. Thus far it was unclear which domains of CEP192 are responsible for the recruitment of NEDD1 bound y-TuRC. To investigate this, we performed relocalisation experiments and pulldown assays using CEP192 truncations. We found that CEP192 768-2537aa is capable of recruiting NEDD1 and γ-tubulin in a cellular environment and identified a NEDD1/γ-tubulin binding domain spanning 768-930aa. We also tested whether relocalisation of CEP192 768-2537aa could recruit pericentrin and found this not to be the case. In addition to researching CEP192, we also have generated a cell line that lost all MTOC functionality, as this would allow for studying the balance between positive and negative nucleation regulators within the cell. We achieved this by knocking out AKAP9’s C-terminal domain (1921-3908aa) in the CEP295/p53 double knockout cell line, which already lost its centrosome. The resulting AKAP9-C/CEP295/p53 triple knockout cell line has lost the ability to recruit CDK5RAP2 and CAMPSAP2 to the Golgi. Since pericentrin colocalises with the Golgi in the CEP295/p53 double knockout, we also investigated pericentrin localisation in AKAP9-C/CEP295/p53 triple knockout and found it to no longer colocalise with the Golgi. In summary, we have identified CEP192 768-930aa to be a NEDD1/γ-tubulin binding domain and generated a MTOC-less cell line.