@article{PitzenSanderBaumannetal.2021, author = {Pitzen, Valentin and Sander, Sophia and Baumann, Otto and Gr{\"a}f, Ralph and Meyer, Irene}, title = {Cep192, a novel missing link between the centrosomal core and corona in Dictyostelium amoebae}, series = {Cells : open access journal}, volume = {10}, journal = {Cells : open access journal}, number = {9}, publisher = {MDPI}, address = {Basel}, issn = {2073-4409}, doi = {10.3390/cells10092384}, pages = {19}, year = {2021}, 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.}, language = {en} } @article{PitzenAskarzadaGraefetal.2018, author = {Pitzen, Valentin and Askarzada, Sophie and Gr{\"a}f, Ralph and Meyer, Irene}, title = {CDK5RAP2 Is an Essential Scaffolding Protein of the Corona of the Dictyostelium Centrosome}, series = {Cells}, volume = {7}, journal = {Cells}, number = {4}, publisher = {MDPI}, address = {Basel}, issn = {2073-4409}, doi = {10.3390/cells7040032}, pages = {17}, year = {2018}, abstract = {Dictyostelium centrosomes consist of a nucleus-associated cylindrical, three-layered core structure surrounded by a corona consisting of microtubule-nucleation complexes embedded in a scaffold of large coiled-coil proteins. One of them is the conserved CDK5RAP2 protein. Here we focus on the role of Dictyostelium CDK5RAP2 for maintenance of centrosome integrity, its interaction partners and its dynamic behavior during interphase and mitosis. GFP-CDK5RAP2 is present at the centrosome during the entire cell cycle except from a short period during prophase, correlating with the normal dissociation of the corona at this stage. RNAi depletion of CDK5RAP2 results in complete disorganization of centrosomes and microtubules suggesting that CDK5RAP2 is required for organization of the corona and its association to the core structure. This is in line with the observation that overexpressed GFP-CDK5RAP2 elicited supernumerary cytosolic MTOCs. The phenotype of CDK5RAP2 depletion was very reminiscent of that observed upon depletion of CP148, another scaffolding protein of the corona. BioID interaction assays revealed an interaction of CDK5RAP2 not only with the corona markers CP148, gamma-tubulin, and CP248, but also with the core components Cep192, CP75, and CP91. Furthermore, protein localization studies in both depletion strains revealed that CP148 and CDK5RAP2 cooperate in corona organization.}, language = {en} } @phdthesis{Pitzen2022, author = {Pitzen, Valentin}, title = {Weitergef{\"u}hrte funktionelle Charakterisierung des centrosomalen Proteins Cep192 und Untersuchung der Topologie des Centrosoms in Dictyostelium Am{\"o}ben}, doi = {10.25932/publishup-54889}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-548891}, school = {Universit{\"a}t Potsdam}, pages = {XI, 104}, year = {2022}, abstract = {Das Centrosom von Dictyostelium ist acentriol{\"a}r aufgebaut, misst ca. 500 nm und besteht aus einer dreischichten Core-Struktur mit umgebender Corona, an der Mikrotubuli nukleieren. In dieser Arbeit wurden das centrosomale Protein Cep192 und m{\"o}gliche Interaktionspartner am Centrosom eingehend untersucht. Die einleitende Lokalisationsuntersuchung von Cep192 ergab, dass es w{\"a}hrend der gesamten Mitose an den Spindelpolen lokalisiert und im Vergleich zu den anderen Strukturproteinen der Core-Struktur am st{\"a}rksten exprimiert ist. Die dauerhafte Lokalisation an den Spindelpolen w{\"a}hrend der Mitose wird f{\"u}r Proteine angenommen, die in den beiden identisch aufgebauten {\"a}ußeren Core-Schichten lokalisieren, die das mitotische Centrosom formen. Ein Knockdown von Cep192 f{\"u}hrte zur Ausbildung von {\"u}berz{\"a}hligen Mikrotubuli-organisierenden Zentren (MTOC) sowie zu einer leicht erh{\"o}hten Ploidie. Deshalb wird eine Destabilisierung des Centrosoms durch die verminderte Cep192-Expression angenommen. An Cep192 wurden zwei kleine Tags, der SpotH6- und BioH6-Tag, etabliert, die mit kleinen fluoreszierenden Nachweiskonjugaten markiert werden konnten. Mit den so getagten Proteinen konnte die hochaufl{\"o}sende Expansion Microscopy f{\"u}r das Centrosom optimiert werden und die Core-Struktur erstmals proteinspezifisch in der Fluoreszenzmikroskopie dargestellt werden. Cep192 lokalisiert dabei in den {\"a}ußeren Core-Schichten. Die kombinierte Markierung von Cep192 und den centrosomalen Proteinen CP39 und CP91 in der Expansion Microscopy erlaubte die Darstellung des dreischichtigen Aufbaus der centrosomalen Core-Struktur, wobei CP39 und CP91 zwischen Cep192 in der inneren Core-Schicht lokalisieren. Auch die Corona wurde in der Expansion Microscopy untersucht: Das Corona-Protein CDK5RAP2 lokalisiert in r{\"a}umlicher N{\"a}he zu Cep192 in der inneren Corona. Ein Vergleich der Corona-Proteine CDK5RAP2, CP148 und CP224 in der Expansion Microscopy ergab unterscheidbare Sublokalisationen der Proteine innerhalb der Corona und relativ zur Core-Struktur. In Biotinylierungsassays mit den centrosomalen Core-Proteinen CP39 und CP91 sowie des Corona-Proteins CDK5RAP2 konnte Cep192 als m{\"o}glicher Interaktionspartner identifiziert werden. Die Ergebnisse dieser Arbeit zeigen die wichtige Funktion des Proteins Cep192 im Dictyostelium-Centrosom und erm{\"o}glichen durch die Kombination aus Biotinylierungsassays und Expansion Microscopy der untersuchten Proteine ein verbessertes Verst{\"a}ndnis der Topologie des Centrosoms.}, language = {de} } @article{GraefGrafeMeyeretal.2021, author = {Gr{\"a}f, Ralph and Grafe, Marianne and Meyer, Irene and Mitic, Kristina and Pitzen, Valentin}, title = {The dictyostelium centrosome}, series = {Cells : open access journal}, volume = {10}, journal = {Cells : open access journal}, number = {10}, publisher = {MDPI}, address = {Basel}, issn = {2073-4409}, doi = {10.3390/cells10102657}, pages = {26}, year = {2021}, abstract = {The centrosome of Dictyostelium amoebae contains no centrioles and consists of a cylindrical layered core structure surrounded by a corona harboring microtubule-nucleating gamma-tubulin complexes. It is the major centrosomal model beyond animals and yeasts. Proteomics, protein interaction studies by BioID and superresolution microscopy methods led to considerable progress in our understanding of the composition, structure and function of this centrosome type. We discuss all currently known components of the Dictyostelium centrosome in comparison to other centrosomes of animals and yeasts.}, language = {en} }