TY - JOUR A1 - Koonce, Michael A1 - Tikhonenko, Irina A1 - Gräf, Ralph T1 - Dictyostelium cell fixation BT - two simple tricks JF - Methods and protocols N2 - We share two simple modifications to enhance the fixation and imaging of relatively small, motile, and rounded model cells. These include cell centrifugation and the addition of trace amounts of glutaraldehyde to existing fixation methods. Though they need to be carefully considered in each context, they have been useful to our studies of the spatial relationships of the microtubule cytoskeletal system. KW - Dictyostelium KW - cell fixation KW - microscopy KW - microtubule KW - cytoskeleton Y1 - 2020 U6 - https://doi.org/10.3390/mps3030047 SN - 2409-9279 VL - 3 IS - 3 PB - MDPI CY - Basel ER - TY - JOUR A1 - Grafe, Marianne A1 - Hofmann, Phillip A1 - Batsios, Petros A1 - Meyer, Irene A1 - Gräf, Ralph T1 - In vivo assembly of a Dictyostelium lamin mutant induced by light, mechanical stress, and pH JF - Cells : open access journal N2 - We expressedDictyosteliumlamin (NE81) lacking both a functional nuclear localization signal and a CAAX-box for C-terminal lipid modification. This lamin mutant assembled into supramolecular, three-dimensional clusters in the cytosol that disassembled at the onset of mitosis and re-assembled in late telophase, thus mimicking the behavior of the endogenous protein. As disassembly is regulated by CDK1-mediated phosphorylation at serine 122, we generated a phosphomimetic S122E mutant called GFP-NE81-S122E-Delta NLS Delta CLIM. Surprisingly, during imaging, the fusion protein assembled into cytosolic clusters, similar to the protein lacking the phosphomimetic mutation. Clusters disassembled again in the darkness. Assembly could be induced with blue but not green or near ultraviolet light, and it was independent of the fusion tag. Assembly similarly occurred upon cell flattening. Earlier reports and own observations suggested that both blue light and cell flattening could result in a decrease of intracellular pH. Indeed, keeping the cells at low pH also reversibly induced cluster formation. Our results indicate that lamin assembly can be induced by various stress factors and that these are transduced via intracellular acidification. Although these effects have been shown in a phosphomimetic CDK1 mutant of theDictyosteliumlamin, they are likely relevant also for wild-type lamin. KW - lamin KW - NE81 KW - Dictyostelium KW - nuclear envelope KW - nuclear lamina Y1 - 2020 U6 - https://doi.org/10.3390/cells9081834 SN - 2073-4409 VL - 9 IS - 8 PB - MDPI CY - Basel ER - TY - JOUR A1 - Gräf, Ralph A1 - Grafe, Marianne A1 - Meyer, Irene A1 - Mitic, Kristina A1 - Pitzen, Valentin T1 - The dictyostelium centrosome JF - Cells : open access journal N2 - 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. KW - microtubule-organizing center KW - microtubule-organization KW - centrosome KW - Dictyostelium KW - mitosis Y1 - 2021 U6 - https://doi.org/10.3390/cells10102657 SN - 2073-4409 VL - 10 IS - 10 PB - MDPI CY - Basel ER - TY - JOUR A1 - Pitzen, Valentin A1 - Sander, Sophia A1 - Baumann, Otto A1 - Gräf, Ralph A1 - Meyer, Irene T1 - Cep192, a novel missing link between the centrosomal core and corona in Dictyostelium amoebae JF - Cells : open access journal N2 - 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. KW - Cep192 KW - SPD-2 KW - centrosome KW - Dictyostelium KW - microtubule-organization KW - MTOC Y1 - 2021 U6 - https://doi.org/10.3390/cells10092384 SN - 2073-4409 VL - 10 IS - 9 PB - MDPI CY - Basel ER - TY - JOUR A1 - Meyer, Irene A1 - Peter, Tatjana A1 - Batsios, Petros A1 - Kuhnert, Oliver A1 - Krueger-Genge, Anne A1 - Camurca, Carl A1 - Gräf, Ralph T1 - CP39, CP75 and CP91 are major structural components of the Dictyostelium JF - European journal of cell biology N2 - 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. KW - Dictyostelium KW - Mitosis KW - Microtubules KW - Centrosome KW - Nucleus Y1 - 2017 U6 - https://doi.org/10.1016/j.eicb.2017.01.004 SN - 0171-9335 SN - 1618-1298 VL - 96 SP - 119 EP - 130 PB - Elsevier CY - Jena ER - TY - JOUR A1 - Mitic, Kristina A1 - Grafe, Marianne A1 - Batsios, Petros A1 - Meyer, Irene T1 - Partial Disassembly of the Nuclear Pore Complex Proteins during Semi-Closed Mitosis in Dictyostelium discoideum JF - Cells N2 - Dictyostelium cells undergo a semi-closed mitosis, during which the nuclear envelope (NE) persists; however, free diffusion between the cytoplasm and the nucleus takes place. To permit the formation of the mitotic spindle, the nuclear envelope must be permeabilized in order to allow diffusion of tubulin dimers and spindle assembly factors into the nucleus. In Aspergillus, free diffusion of proteins between the cytoplasm and the nucleus is achieved by a partial disassembly of the nuclear pore complexes (NPCs) prior to spindle assembly. In order to determine whether this is also the case in Dictyostelium, we analysed components of the NPC by immunofluorescence microscopy and live cell imaging and studied their behaviour during interphase and mitosis. We observed that the NPCs are absent from the contact area of the nucleoli and that some nucleoporins also localize to the centrosome and the spindle poles. In addition, we could show that, during mitosis, the central FG protein NUP62, two inner ring components and Gle1 depart from the NPCs, while all other tested NUPs remained at the NE. This leads to the conclusion that indeed a partial disassembly of the NPCs takes place, which contributes to permeabilisation of the NE during semi-closed mitosis. KW - nuclear pore complex KW - nucleoporins KW - semi-closed mitosis KW - centrosome KW - Dictyostelium Y1 - 2021 U6 - https://doi.org/10.3390/cells11030407 SN - 2073-4409 VL - 11 IS - 3 PB - MDPI CY - Basel ER - TY - GEN A1 - Mitic, Kristina A1 - Grafe, Marianne A1 - Batsios, Petros A1 - Meyer, Irene T1 - Partial Disassembly of the Nuclear Pore Complex Proteins during Semi-Closed Mitosis in Dictyostelium discoideum T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Dictyostelium cells undergo a semi-closed mitosis, during which the nuclear envelope (NE) persists; however, free diffusion between the cytoplasm and the nucleus takes place. To permit the formation of the mitotic spindle, the nuclear envelope must be permeabilized in order to allow diffusion of tubulin dimers and spindle assembly factors into the nucleus. In Aspergillus, free diffusion of proteins between the cytoplasm and the nucleus is achieved by a partial disassembly of the nuclear pore complexes (NPCs) prior to spindle assembly. In order to determine whether this is also the case in Dictyostelium, we analysed components of the NPC by immunofluorescence microscopy and live cell imaging and studied their behaviour during interphase and mitosis. We observed that the NPCs are absent from the contact area of the nucleoli and that some nucleoporins also localize to the centrosome and the spindle poles. In addition, we could show that, during mitosis, the central FG protein NUP62, two inner ring components and Gle1 depart from the NPCs, while all other tested NUPs remained at the NE. This leads to the conclusion that indeed a partial disassembly of the NPCs takes place, which contributes to permeabilisation of the NE during semi-closed mitosis. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1233 KW - nuclear pore complex KW - nucleoporins KW - semi-closed mitosis KW - centrosome KW - Dictyostelium Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-545341 SN - 1866-8372 IS - 3 ER - TY - JOUR A1 - Pitzen, Valentin A1 - Askarzada, Sophie A1 - Gräf, Ralph A1 - Meyer, Irene T1 - CDK5RAP2 Is an Essential Scaffolding Protein of the Corona of the Dictyostelium Centrosome JF - Cells N2 - 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. KW - centrosome KW - centriole KW - Dictyostelium KW - microtubules KW - mitosis Y1 - 2018 U6 - https://doi.org/10.3390/cells7040032 SN - 2073-4409 VL - 7 IS - 4 PB - MDPI CY - Basel ER - TY - JOUR A1 - Grafe, Marianne A1 - Hofmann, Phillip A1 - Batsios, Petros A1 - Meyer, Irene A1 - Gräf, Ralph T1 - In vivo assembly of a Dictyostelium lamin mutant induced by light, mechanical stress, and pH JF - Cells N2 - We expressed Dictyostelium lamin (NE81) lacking both a functional nuclear localization signal and a CAAX-box for C-terminal lipid modification. This lamin mutant assembled into supramolecular, three-dimensional clusters in the cytosol that disassembled at the onset of mitosis and re-assembled in late telophase, thus mimicking the behavior of the endogenous protein. As disassembly is regulated by CDK1-mediated phosphorylation at serine 122, we generated a phosphomimetic S122E mutant called GFP-NE81-S122E-∆NLS∆CLIM. Surprisingly, during imaging, the fusion protein assembled into cytosolic clusters, similar to the protein lacking the phosphomimetic mutation. Clusters disassembled again in the darkness. Assembly could be induced with blue but not green or near ultraviolet light, and it was independent of the fusion tag. Assembly similarly occurred upon cell flattening. Earlier reports and own observations suggested that both blue light and cell flattening could result in a decrease of intracellular pH. Indeed, keeping the cells at low pH also reversibly induced cluster formation. Our results indicate that lamin assembly can be induced by various stress factors and that these are transduced via intracellular acidification. Although these effects have been shown in a phosphomimetic CDK1 mutant of the Dictyostelium lamin, they are likely relevant also for wild-type lamin. KW - lamin KW - NE81 KW - Dictyostelium KW - nuclear envelope KW - nuclear lamina Y1 - 2020 VL - 9 IS - 8 PB - MDPI CY - Basel ER - TY - GEN A1 - Grafe, Marianne A1 - Hofmann, Phillip A1 - Batsios, Petros A1 - Meyer, Irene A1 - Gräf, Ralph T1 - In vivo assembly of a Dictyostelium lamin mutant induced by light, mechanical stress, and pH T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - We expressed Dictyostelium lamin (NE81) lacking both a functional nuclear localization signal and a CAAX-box for C-terminal lipid modification. This lamin mutant assembled into supramolecular, three-dimensional clusters in the cytosol that disassembled at the onset of mitosis and re-assembled in late telophase, thus mimicking the behavior of the endogenous protein. As disassembly is regulated by CDK1-mediated phosphorylation at serine 122, we generated a phosphomimetic S122E mutant called GFP-NE81-S122E-∆NLS∆CLIM. Surprisingly, during imaging, the fusion protein assembled into cytosolic clusters, similar to the protein lacking the phosphomimetic mutation. Clusters disassembled again in the darkness. Assembly could be induced with blue but not green or near ultraviolet light, and it was independent of the fusion tag. Assembly similarly occurred upon cell flattening. Earlier reports and own observations suggested that both blue light and cell flattening could result in a decrease of intracellular pH. Indeed, keeping the cells at low pH also reversibly induced cluster formation. Our results indicate that lamin assembly can be induced by various stress factors and that these are transduced via intracellular acidification. Although these effects have been shown in a phosphomimetic CDK1 mutant of the Dictyostelium lamin, they are likely relevant also for wild-type lamin. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1213 KW - lamin KW - NE81 KW - Dictyostelium KW - nuclear envelope KW - nuclear lamina Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-525075 SN - 1866-8372 IS - 8 ER -