Protein topology at the Dictyostelium centrosome

• Applicant: Dr. Irene Meyer
• Subject Area: Cell Biology ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2017 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 394020894

• Final Report Year: 2023

Final Report Abstract

The core structure consists of five known major components. In normal light microscopy studies we could so far, due to resolution limits, only identify these proteins as part of the core structure, but not specifically which layer. This was only possible through observation of behaviour of the proteins during mitosis for it has been observed in electron microscopy that the central layer vanishes prior to spindle assembly. In this study we wanted to use superresolution light microscopy to improve our knowledge of the protein topology at the Dictyostelium centrosome. We ended up using Expansion microscopy, which turned out to be very suitable for the analysis of centrosomal component of Dictyostelium. This revealed that our previous view with a subdivision of the Dictyostelium centrosome into a threelayered core structure surrounded by a corona was oversimplified. In fact, the corona should be subdivided into two distinct sheaths, one adjacent to the layered core and mainly consisting of CDK5RAP2, and another, distal sheath, containing the majority of the microtubule-nucleating corona proteins. Moreover, within the layered core structure, we should distinguish five layers, since both outer layers are flanked by a CP91 layer between them and the central layer. 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 interaction partners of Cep192, including CDK5RAP2. This corona protein shows interaction with components of the gamma-Tubulin ring complex, and with that links microtubule nucleation to the centrosomal core structure.

Publications

• CDK5RAP2 Is an Essential Scaffolding Protein of the Corona of the Dictyostelium Centrosome. Cells, 7(4), 32.
  Pitzen, Valentin; Askarzada, Sophie; Gräf, Ralph & Meyer, Irene
  
• Cep192, a Novel Missing Link between the Centrosomal Core and Corona in Dictyostelium Amoebae. Cells, 10(9), 2384.
  Pitzen, Valentin; Sander, Sophia; Baumann, Otto; Gräf, Ralph & Meyer, Irene
  
• The Dictyostelium Centrosome. Cells, 10(10), 2657.
  Gräf, Ralph; Grafe, Marianne; Meyer, Irene; Mitic, Kristina & Pitzen, Valentin