The interactome and function of the PP1γ interactor RepoMan in mitotic spindle assembly

The role of RepoMan in chromosome-dependent microtubule nucleation

The assembly of a mitotic spindle that makes stable contacts with chromosomes is crucial for accurate cell division. Both the centrosomal and chromosomal pathway of microtubule nucleation contribute to the efficient build-up of the bipolar spindle. The growth of microtubules starts at the γ-tubulin ring complex (γ-TuRC), where γ-tubulin serves as a template for α-/β-tubulin dimers to polymerize. Here, we investigated the role of RepoMan, a chromosome associated protein, in spindle assembly. Using immunofluorescence, we show that a RepoMan knockdown leads to less spread chromosomes in prometaphase. This effect is dependent on the stability of microtubules as shown by nocodazole washout and taxol stabilization assays. This effect is due to the misregulation of the chromosomal pathway of microtubule nucleation since a knockdown of RepoMan leads to a decrease in the size of chromosomal microtubule asters. Using BioID and the newly developed Split-BioID technique, we found that RepoMan interacts with several regulators of spindle stability and α-, β- and γ-tubulin itself. Furthermore, we found that RepoMan co-immunoprecipitates with importin in a RanGTP dependent manner, just like spindle assembly factors such as TPX2, NuSAP1 and Kid. We propose a model where RepoMan regulates the activity or localization of one or more of these newly identified interactors. This can explain RepoMan’s stabilizing effect on chromosome-dependent microtubules in prometaphase.