Cep192, a Novel Missing Link between the Centrosomal Core and Corona in Dictyostelium Amoebae

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.

Partial Disassembly of the Nuclear Pore Complex Proteins during Semi-Closed Mitosis in Dictyostelium discoideum

“…Thus, it is not far-fetched to presume that NUP93 could also bind to the centrosomal fraction of NUP53 in Dictyostelium. Due to its central localization it can be assumed that NUP53 interacts with one of the core components, either with Cep192 [40] in the outer layers, or one of the central layer proteins CP39, CP75 or CP91 [16]. For CP75 it has been speculated to play a role in the insertion of the centrosome into the nuclear envelope at the onset of mitosis, and it is conceivable that NUP53 assists the centrosomal protein in this function by virtue of its membrane-shaping properties [41].…”

The Dictyostelium Centrosome

“…Three proteins of the core structure, CP39, CP91 and CP75, were attributed to the central layer since they all disappear from mitotic centrosomes [33,53]. This attribution was later confirmed by ExM [54]. All three appear to be essential, since their depletion caused severe phenotypes, and knockout attempts failed altogether.…”

“…As in case of CP148, depletion of CDK5RAP2 causes disintegration of the corona and the appearance of multiple, cytosolic microtubule nucleation complexes [71]. Superresolution microscopy indicated that it forms the interface between the corona and the layered core, since its localization closely matches that of the outer core layer component Cep192 [54].…”

“…We are aware that this categorization may be an over-simplification. Electron microscopy has shown that the corona contains nodules as a further substructure, and recent superresolution light microscopy data indicate that it can be subdivided in at least two distinct sheaths, one adjacent to the layered core and mainly consisting of CDK5RAP2, and another, distal sheath containing the majority of other corona proteins [54]. Also, sublayers exist within the three major layers of the core structure [27,28].…”

“…Cep192 was identified in the centrosomal proteome and when expressed as a GFP fusion protein it was found at the core structure, and at spindle poles during mitosis [52,64]. Only recently we analyzed Cep192 localization and function more closely [54]. Using expansion microscopy it could clearly be assigned to the outer core layers.…”