@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} } @article{GrafeBatsiosMeyeretal.2019, author = {Grafe, Marianne and Batsios, Petros and Meyer, Irene and Lisin, Daria and Baumann, Otto and Goldberg, Martin W. and Gr{\"a}f, Ralph}, title = {Supramolecular Structures of the Dictyostelium Lamin NE81}, series = {Cells}, volume = {8}, journal = {Cells}, number = {2}, publisher = {Molecular Diversity Preservation International}, address = {Basel}, issn = {2073-4409}, doi = {10.3390/cells8020162}, pages = {17}, year = {2019}, abstract = {Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells.}, language = {en} } @article{MiticGrafeBatsiosetal.2022, author = {Mitic, Kristina and Grafe, Marianne and Batsios, Petros and Meyer, Irene}, title = {Partial Disassembly of the Nuclear Pore Complex Proteins during Semi-Closed Mitosis in Dictyostelium discoideum}, series = {Cells}, volume = {11}, journal = {Cells}, number = {3}, publisher = {MDPI}, address = {Basel}, issn = {2073-4409}, doi = {10.3390/cells11030407}, pages = {14}, year = {2022}, abstract = {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.}, language = {en} } @misc{MiticGrafeBatsiosetal.2022, author = {Mitic, Kristina and Grafe, Marianne and Batsios, Petros and Meyer, Irene}, title = {Partial Disassembly of the Nuclear Pore Complex Proteins during Semi-Closed Mitosis in Dictyostelium discoideum}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {3}, issn = {1866-8372}, doi = {10.25932/publishup-54534}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-545341}, pages = {16}, year = {2022}, abstract = {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.}, language = {en} } @article{BatsiosGraefKoonceetal.2019, author = {Batsios, Petros and Gr{\"a}f, Ralph and Koonce, Michael P. and Larochelle, Denis A. and Meyer, Irene}, title = {Nuclear envelope organization in Dictyostelium discoideum}, series = {The international journal of developmental biology}, volume = {63}, journal = {The international journal of developmental biology}, number = {8-10}, publisher = {UBC Pr}, address = {Bilbao}, issn = {0214-6282}, doi = {10.1387/ijdb.190184rg}, pages = {509 -- 519}, year = {2019}, abstract = {The nuclear envelope consists of the outer and the inner nuclear membrane, the nuclear lamina and the nuclear pore complexes, which regulate nuclear import and export.The major constituent of the nuclear lamina of Dictyostelium is the lamin NE81. It can form filaments like B-type lamins and it interacts with Sun 1, as well as with the LEM/HeH-family protein Src1. Sun 1 and Src1 are nuclear envelope transmembrane proteins involved in the centrosome-nucleus connection and nuclear envelope stability at the nucleolar regions, respectively. In conjunction with a KASH-domain protein, Sun 1 usually forms a so-called LINC complex.Two proteins with functions reminiscent of KASH-domain proteins at the outer nuclear membrane of Dictyostelium are known; interaptin which serves as an actin connector and the kinesin Kif9 which plays a role in the microtubule-centrosome connector. However, both of these lack the conserved KASH-domain. The link of the centrosome to the nuclear envelope is essential for the insertion of the centrosome into the nuclear envelope and the appropriate spindle formation. Moreover, centrosome insertion is involved in perm eabilization of the mitotic nucleus, which ensures access of tubulin dimers and spindle assembly factors. Our recent progress in identifying key molecular players at the nuclear envelope of Dictyostelium promises further insights into the mechanisms of nuclear envelope dynamics.}, language = {en} } @article{GrafeHofmannBatsiosetal.2020, author = {Grafe, Marianne and Hofmann, Phillip and Batsios, Petros and Meyer, Irene and Gr{\"a}f, Ralph}, title = {In vivo assembly of a Dictyostelium lamin mutant induced by light, mechanical stress, and pH}, series = {Cells : open access journal}, volume = {9}, journal = {Cells : open access journal}, number = {8}, publisher = {MDPI}, address = {Basel}, issn = {2073-4409}, doi = {10.3390/cells9081834}, pages = {14}, year = {2020}, abstract = {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.}, language = {en} } @article{GrafeHofmannBatsiosetal.2020, author = {Grafe, Marianne and Hofmann, Phillip and Batsios, Petros and Meyer, Irene and Gr{\"a}f, Ralph}, title = {In vivo assembly of a Dictyostelium lamin mutant induced by light, mechanical stress, and pH}, series = {Cells}, volume = {9}, journal = {Cells}, number = {8}, publisher = {MDPI}, address = {Basel}, pages = {14}, year = {2020}, abstract = {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.}, language = {en} } @misc{GrafeHofmannBatsiosetal.2020, author = {Grafe, Marianne and Hofmann, Phillip and Batsios, Petros and Meyer, Irene and Gr{\"a}f, Ralph}, title = {In vivo assembly of a Dictyostelium lamin mutant induced by light, mechanical stress, and pH}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {8}, issn = {1866-8372}, doi = {10.25932/publishup-52507}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-525075}, pages = {16}, year = {2020}, abstract = {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.}, language = {en} } @inproceedings{KuhnertBaumannMeyeretal.2011, author = {Kuhnert, Oliver and Baumann, Otto and Meyer, Irene and Gr{\"a}f, Ralph}, title = {Functional characterization of CP148, a novel key component for centrosome integrity in Dictyostelium}, series = {Molecular biology of the cell : the official publication of the American Society for Cell Biology}, volume = {22}, booktitle = {Molecular biology of the cell : the official publication of the American Society for Cell Biology}, publisher = {American Society for Cell Biology}, address = {Bethesda}, issn = {1059-1524}, pages = {2}, year = {2011}, language = {en} } @article{KuhnertBaumannMeyeretal.2012, author = {Kuhnert, Oliver and Baumann, Otto and Meyer, Irene and Gr{\"a}f, Ralph}, title = {Functional characterization of CP148, a novel key component for centrosome integrity in Dictyostelium}, series = {Cellular and molecular life sciences}, volume = {69}, journal = {Cellular and molecular life sciences}, number = {11}, publisher = {Springer}, address = {Basel}, issn = {1420-682X}, doi = {10.1007/s00018-011-0904-2}, pages = {1875 -- 1888}, year = {2012}, abstract = {The centrosome consists of a layered core structure surrounded by a microtubule-nucleating corona. A tight linkage through the nuclear envelope connects the cytosolic centrosome with the clustered centromeres within the nuclear matrix. At G2/M the corona dissociates, and the core structure duplicates, yielding two spindle poles. CP148 is a novel coiled coil protein of the centrosomal corona. GFP-CP148 exhibited cell cycle-dependent presence and absence at the centrosome, which correlates with dissociation of the corona in prophase and its reformation in late telophase. During telophase, GFP-CP148 formed cytosolic foci, which coalesced and joined the centrosome. This explains the hypertrophic appearance of the corona upon strong overexpression of GFP-CP148. Depletion of CP148 by RNAi caused virtual loss of the corona and disorganization of interphase microtubules. Surprisingly, formation of the mitotic spindle and astral microtubules was unaffected. Thus, microtubule nucleation complexes associate with centrosomal core components through different means during interphase and mitosis. Furthermore, CP148 RNAi caused dispersal of centromeres and altered Sun1 distribution at the nuclear envelope, suggesting a role of CP148 in the linkage between centrosomes and centromeres. Taken together, CP148 is an essential factor for the formation of the centrosomal corona, which in turn is required for centrosome/centromere linkage.}, language = {en} }