@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{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{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{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{KoonceTikhonenkoGraef2020,
  author    = {Koonce, Michael and Tikhonenko, Irina and Gr{\"a}f, Ralph},
  title     = {Dictyostelium cell fixation},
  series = {Methods and protocols},
  volume    = {3},
  journal   = {Methods and protocols},
  number    = {3},
  publisher = {MDPI},
  address   = {Basel},
  issn      = {2409-9279},
  doi       = {10.3390/mps3030047},
  pages     = {6},
  year      = {2020},
  abstract  = {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.},
  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}
}
@misc{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 = {Potsprint der Universit{\"a}t Potsdam Mathematisch-Naturwissenschaftliche Reihe},
  journal   = {Potsprint der Universit{\"a}t Potsdam Mathematisch-Naturwissenschaftliche Reihe},
  number    = {682},
  issn      = {1866-8372},
  doi       = {10.25932/publishup-42597},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-425976},
  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{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}
}