Molecular analysis of MTOC assembly - the role of ninein-like protein

Abstract

Despite decades of work, the molecular requirements underlying microtubule- organizing center (MTOC) formation at the centrosome remain obscure. Progress is hindered by the complexity of this organelle, which is composed of hundreds of proteins that are involved in a range of functions, many unrelated to microtubule organization. To avoid such intricacy, we studied MTOC formation at an ectopic site. Employing a domain previously identified in a splice variant of the centrosome protein Cnn in Drosophila, we targeted fusion proteins to the cytoplasmic surface of mitochondria in human U2OS cells. We tested several human candidate proteins based on their described relationship with MTOCs such as CDK5RAP2, CEP192, NIN, NINL and CKAP5. MTOC formation capacity was analysed by probing for recruitment of the nucleator γTuRC and for microtubule nucleation activity. Interestingly, multiple candidates were able to recruit γTuRC to the ectopic site. However, only an N-terminal fragment of NINL was able to also induce microtubule nucleation similar to the CM1 region of CDK5RAP2, which served as positive control. Noteworthy, CKAP5 induced microtubule formation at the ectopic site independently of γTuRC. Testing of NINL truncation mutants allowed mapping of separate regions required for γTuRC binding and nucleation activation. Biotin- proximity labelling of mitochondrial surface-targeted NINL fragments further identified additional candidate proteins involved in these functions. Using super resolution microscopy, we found endogenous NINL to associate with subdistal appendages (SDAs) of the mother centriole, pointing to a possible role of NINL in MTOC formation at SDAs.