Abstract
The Dictyostelium centrosome is a nucleus-associated body with a diameter of approx. 500 nm. It contains no centrioles but consists of a cylindrical layered core structure surrounded by a microtubule-nucleating corona. At the onset of mitosis, the corona disassembles and the core structure duplicates through growth, splitting, and reorganization of the outer core layers. During the last decades our research group has characterized the majority of the 42 known centrosomal proteins. In this work we focus on the conserved, previously uncharacterized Cep192 protein. We use superresolution expansion microscopy (ExM) to show that Cep192 is a component of the outer core layers. Furthermore, ExM with centrosomal marker proteins nicely mirrored all ultrastructurally known centrosomal substructures. Furthermore, we improved the proximity-dependent biotin identification assay (BioID) by adapting the biotinylase BioID2 for expression in Dictyostelium and applying a knock-in strategy for the expression of BioID2-tagged centrosomal fusion proteins. Thus, we were able to identify various centrosomal Cep192 interaction partners, including CDK5RAP2, which was previously allocated to the inner corona structure, and several core components. Studies employing overexpression of GFP-Cep192 as well as depletion of endogenous Cep192 revealed that Cep192 is a key protein for the recruitment of corona components during centrosome biogenesis and is required to maintain a stable corona structure.
Highlights
Centrosomes are best known for their function as main microtubule organizing centers (MTOCs) [1]
In an early survey of the light microscopic localization of GFP-fusion proteins derived from all centrosomal candidates found in this proteomic approach, we were unsure whether Cep192 should be designated to the core structure, or to a previously uncharacterized inner part of the corona directly attached to the core layers [40]
We have presented evidence that Dictyostelium Cep192 is the major component of the outer core layers and is required both for centrosome biogenesis and for integrity of the corona
Summary
Centrosomes are best known for their function as main microtubule organizing centers (MTOCs) [1]. Despite their occasional absence in some subgroups such as higher plants, there is no doubt that they were already part of the inventory of the last eukaryotic common ancestor (LECA) [2,3]. The centrosome is the largest known protein complex of the cell and consists of more than one hundred different proteins (depending on the species) that form several functional groups [4]. It can be distinguished between centriole-containing and acentriolar centrosomes. The mother centriole is embedded in a pericentriolar matrix, which contains the majority of the microtubule-nucleation complexes
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