@article{TikhonenkoMagidsonGraefetal.2013, author = {Tikhonenko, Irina and Magidson, Valentin and Gr{\"a}f, Ralph and Khodjakov, Alexey and Koonce, Michael P.}, title = {A kinesin-mediated mechanism that couples centrosomes to nuclei}, series = {Cellular and molecular life sciences}, volume = {70}, journal = {Cellular and molecular life sciences}, number = {7}, publisher = {Springer}, address = {Basel}, issn = {1420-682X}, doi = {10.1007/s00018-012-1205-0}, pages = {1285 -- 1296}, year = {2013}, abstract = {The M-type kinesin isoform, Kif9, has recently been implicated in maintaining a physical connection between the centrosome and nucleus in Dictyostelium discoideum. However, the mechanism by which Kif9 functions to link these two organelles remains obscure. Here we demonstrate that the Kif9 protein is localized to the nuclear envelope and is concentrated in the region underlying the centrosome point of attachment. Nuclear anchorage appears mediated through a specialized transmembrane domain located in the carboxyl terminus. Kif9 interacts with microtubules in in vitro binding assays and effects an endwise depolymerization of the polymer. These results suggest a model whereby Kif9 is anchored to the nucleus and generates a pulling force that reels the centrosome up against the nucleus. This is a novel activity for a kinesin motor, one important for progression of cells into mitosis and to ensure centrosome-nuclear parity in a multinuclear environment.}, language = {en} } @article{BatsiosPeterBaumannetal.2012, author = {Batsios, Petros and Peter, Tatjana and Baumann, Otto and Stick, Reimer and Meyer, Irene and Gr{\"a}f, Ralph}, title = {A lamin in lower eukaryotes?}, series = {Nucleus}, volume = {3}, journal = {Nucleus}, number = {3}, publisher = {Landes Bioscience}, address = {Austin}, issn = {1949-1034}, doi = {10.4161/nucl.20149}, pages = {237 -- 243}, year = {2012}, abstract = {Lamins are the major components of the nuclear lamina and serve not only as a mechanical support, but are also involved in chromatin organization, epigenetic regulation, transcription and mitotic events. Despite these universal tasks, lamins have so far been found only in metazoans. Yet, recently we have identified Dictyostelium NE81 as the first lamin-like protein in a lower eukaryote. Based on the current knowledge, we draw a model for nuclear envelope organization in Dictyostelium in this Extra View and we review the experimental data that justified this classification. Furthermore we provide unpublished data underscoring the requirement of posttranslational CaaX-box processing for proper protein localization at the nuclear envelope. Sequence comparison of NE81 sequences from four Dictyostelia with bona fide lamins illustrates the evolutional relationship between these proteins. Under certain conditions these usually unicellular social amoebae congregate to form a multicellular body. We propose that the evolution of the lamin-like NE81 went along with the invention of multicellularity.}, language = {en} } @article{SamereierBaumannMeyeretal.2011, author = {Samereier, Matthias and Baumann, Otto and Meyer, Irene and Gr{\"a}f, Ralph}, title = {Analysis of dictyostelium TACC reveals differential interactions with CP224 and unusual dynamics of dictyostelium microtubules}, series = {Cellular and molecular life sciences}, volume = {68}, journal = {Cellular and molecular life sciences}, number = {2}, publisher = {Springer}, address = {Basel}, issn = {1420-682X}, doi = {10.1007/s00018-010-0453-0}, pages = {275 -- 287}, year = {2011}, abstract = {We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores. Moreover, we have now a marker to study dynamics and behavior of microtubule plus ends in living Dictyostelium cells. In a combination of live cell imaging of microtubule plus ends and fluorescence recovery after photobleaching (FRAP) experiments of GFP-alpha-tubulin cells we show that Dictyostelium microtubules are dynamic only in the cell periphery, while they remain stable at the centrosome, which also appears to harbor a dynamic pool of tubulin dimers.}, language = {en} } @article{MaiWolskiPuciulMalinowskaetal.2018, author = {Mai, Tobias and Wolski, Karol and Puciul-Malinowska, Agnieszka and Kopyshev, Alexey and Gr{\"a}f, Ralph and Bruns, Michael and Zapotoczny, Szczepan and Taubert, Andreas}, title = {Anionic polymer brushes for biomimetic calcium phosphate mineralization}, series = {Polymers}, volume = {10}, journal = {Polymers}, number = {10}, publisher = {MDPI}, address = {Basel}, issn = {2073-4360}, doi = {10.3390/polym10101165}, pages = {17}, year = {2018}, abstract = {This article describes the synthesis of anionic polymer brushes and their mineralization with calcium phosphate. The brushes are based on poly(3-sulfopropyl methacrylate potassium salt) providing a highly charged polymer brush surface. Homogeneous brushes with reproducible thicknesses are obtained via surface-initiated atom transfer radical polymerization. Mineralization with doubly concentrated simulated body fluid yields polymer/inorganic hybrid films containing AB-Type carbonated hydroxyapatite (CHAP), a material resembling the inorganic component of bone. Moreover, growth experiments using Dictyostelium discoideum amoebae demonstrate that the mineral-free and the mineral-containing polymer brushes have a good biocompatibility suggesting their use as biocompatible surfaces in implantology or related fields.}, 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} } @article{SchulzErleGraefetal.2009, author = {Schulz, Irene and Erle, Alexander and Gr{\"a}f, Ralph and Krueger, Anne and Putzler, Sascha and Samereier, Matthias and Weidenthaler, Sebastian}, title = {Cell cycle-dependent localization of novel centrosomal and centromeric proteins in Dictyostelium}, issn = {0171-9335}, doi = {10.1016/S0171-9335(09)00023-5}, year = {2009}, language = {en} } @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{KruegerBatsiosBaumannetal.2012, author = {Kr{\"u}ger, Anne and Batsios, Petros and Baumann, Otto and Luckert, Eva and Schwarz, Heinz and Stick, Reimer and Meyer, Irene and Gr{\"a}f, Ralph}, title = {Characterization of NE81, the first lamin-like nucleoskeleton protein in a unicellular organism}, series = {Molecular biology of the cell : the official publication of the American Society for Cell Biology}, volume = {23}, journal = {Molecular biology of the cell : the official publication of the American Society for Cell Biology}, number = {2}, publisher = {American Society for Cell Biology}, address = {Bethesda}, issn = {1059-1524}, doi = {10.1091/mbc.E11-07-0595}, pages = {360 -- 370}, year = {2012}, abstract = {Lamins build the nuclear lamina and are required for chromatin organization, gene expression, cell cycle progression, and mechanical stabilization. Despite these universal functions, lamins have so far been found only in metazoans. We have identified protein NE81 in Dictyostelium, which has properties that justify its denomination as a lamin-like protein in a lower eukaryote. This is based on its primary structure, subcellular localization, and regulation during mitosis, and its requirement of the C-terminal CaaX box as a posttranslational processing signal for proper localization. Our knockout and overexpression mutants revealed an important role for NE81 in nuclear integrity, chromatin organization, and mechanical stability of cells. All our results are in agreement with a role for NE81 in formation of a nuclear lamina. This function is corroborated by localization of Dictyostelium NE81 at the nuclear envelope in human cells. The discovery of a lamin-like protein in a unicellular organism is not only intriguing in light of evolution, it may also provide a simple experimental platform for studies of the molecular basis of laminopathies.}, language = {en} } @article{MeyerPeterBatsiosetal.2017, author = {Meyer, Irene and Peter, Tatjana and Batsios, Petros and Kuhnert, Oliver and Krueger-Genge, Anne and Camurca, Carl and Gr{\"a}f, Ralph}, title = {CP39, CP75 and CP91 are major structural components of the Dictyostelium}, series = {European journal of cell biology}, volume = {96}, journal = {European journal of cell biology}, publisher = {Elsevier}, address = {Jena}, issn = {0171-9335}, doi = {10.1016/j.eicb.2017.01.004}, pages = {119 -- 130}, year = {2017}, abstract = {The acentriolar Dictyostelium centrosome is a nucleus-associated body consisting of a core structure with three plaque-like layers, which are surrounded by a microtubule-nucleating corona. The core duplicates once per cell cycle at the G2/M transition, whereby its central layer disappears and the two outer layers form the mitotic spindle poles. Through proteomic analysis of isolated centrosomes, we have identified CP39 and CP75, two essential components of the core structure. Both proteins can be assigned to the central core layer as their centrosomal presence is correlated to the disappearance and reappearance of the central core layer in the course of centrosome duplication. Both proteins contain domains with centrosome-binding activity in their N- and C-terminal halves, whereby the respective N-terminal half is required for cell cycle-dependent regulation. CP39 is capable of self-interaction and GFP-CP39 overexpression elicited supernumerary microtubule-organizing centers and pre-centrosomal cytosolic clusters. Underexpression stopped cell growth and reversed the MTOC amplification phenotype. In contrast, in case of CP75 underexpression of the protein by RNAi treatment elicited supernumerary MTOCs. In addition, CP75RNAi affects correct chromosome segregation and causes co-depletion of CP39 and CP91, another central core layer component. CP39 and CP75 interact with each other directly in a yeast two-hybrid assay. Furthermore, CP39, CP75 and CP91 mutually interact in a proximity-dependent biotin identification (BioID) assay. Our data indicate that these three proteins are all required for proper centrosome biogenesis and make up the major structural components of core structure's central layer.}, language = {en} } @article{KuhnertBaumannMeyeretal.2012, author = {Kuhnert, Oliver and Baumann, Otto and Meyer, Irene and Gr{\"a}f, Ralph}, title = {CP55, a novel key component of centrosomal organization in dictyostelium}, series = {Cellular and molecular life sciences}, volume = {69}, journal = {Cellular and molecular life sciences}, number = {21}, publisher = {Springer}, address = {Basel}, issn = {1420-682X}, doi = {10.1007/s00018-012-1040-3}, pages = {3651 -- 3664}, year = {2012}, abstract = {Dictyostelium centrosomes consist of a layered core structure surrounded by a microtubule-nucleating corona. At the G2/M transition, the corona dissociates and the core structure duplicates, yielding two spindle pole bodies. Finally, in telophase, the spindle poles mature into two new, complete centrosomes. CP55 was identified in a centrosomal proteome analysis. It is a component of the centrosomal core structure, and persists at the centrosome throughout the entire cell cycle. FRAP experiments revealed that during interphase the majority of centrosomal GFP-CP55 is immobile, which indicates a structural task of CP55 at the centrosome. The CP55null mutant is characterized by increased ploidy, a less structured, slightly enlarged corona, and by supernumerary, cytosolic MTOCs, containing only corona proteins and lacking a core structure. Live cell imaging showed that supernumerary MTOCs arise in telophase. Lack of CP55 also caused premature recruitment of the corona organizer CP148 to mitotic spindle poles, already in metaphase instead of telophase. Forces transmitted through astral microtubules may expel prematurely acquired or loosely attached corona fragments into the cytosol, where they act as independent MTOCs. CP55null cells were also impaired in growth, most probably due to difficulties in centrosome splitting during prophase. Furthermore, although they were still capable of phagocytosis, they appeared unable to utilize phagocytosed nutrients. This inability may be attributed to their partially disorganized Golgi apparatus.}, language = {en} }