TY - JOUR A1 - Schweigel, Ulrike A1 - Batsios, Petros A1 - Müller-Taubenberger, Annette A1 - Gräf, Ralph A1 - Grafe, Marianne T1 - Dictyostelium spastin is involved in nuclear envelope dynamics during semi-closed mitosis JF - Nucleus N2 - Dictyostelium amoebae perform a semi-closed mitosis, in which the nuclear envelope is fenestrated at the insertion sites of the mitotic centrosomes and around the central spindle during karyokinesis. During late telophase the centrosome relocates to the cytoplasmic side of the nucleus, the central spindle disassembles and the nuclear fenestrae become closed. Our data indicate that Dictyostelium spastin (DdSpastin) is a microtubule-binding and severing type I membrane protein that plays a role in this process. Its mitotic localization is in agreement with a requirement for the removal of microtubules that would hinder closure of the fenestrae. Furthermore, DdSpastin interacts with the HeH/ LEM-family protein Src1 in BioID analyses as well as the inner nuclear membrane protein Sun1, and shows subcellular co-localizations with Src1, Sun1, the ESCRT component CHMP7 and the IST1-like protein filactin, suggesting that the principal pathway of mitotic nuclear envelope remodeling is conserved between animals and Dictyostelium amoebae. KW - Spastin KW - LEM-domain KW - ESCRT KW - sun1 KW - dictyostelium KW - nuclear envelope KW - mitosis Y1 - 2022 U6 - https://doi.org/10.1080/19491034.2022.2047289 SN - 1949-1034 SN - 1949-1042 VL - 13 IS - 1 SP - 144 EP - 154 PB - Taylor & Francis Group CY - Philadelphia 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 - 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 - 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 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 - JOUR A1 - Grafe, Marianne A1 - Batsios, Petros A1 - Meyer, Irene A1 - Lisin, Daria A1 - Baumann, Otto A1 - Goldberg, Martin W. A1 - Gräf, Ralph T1 - Supramolecular Structures of the Dictyostelium Lamin NE81 JF - Cells N2 - 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. KW - lamin KW - NE81 KW - Dictyostelium KW - nuclear envelope KW - nuclear lamina KW - expansion microscopy Y1 - 2019 U6 - https://doi.org/10.3390/cells8020162 SN - 2073-4409 VL - 8 IS - 2 PB - Molecular Diversity Preservation International CY - Basel ER - TY - JOUR A1 - Batsios, Petros A1 - Gräf, Ralph A1 - Koonce, Michael P. A1 - Larochelle, Denis A. A1 - Meyer, Irene T1 - Nuclear envelope organization in Dictyostelium discoideum JF - The international journal of developmental biology N2 - 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. KW - nuclear envelop KW - Dictyostelium KW - lamin KW - NET KW - centrosome KW - centromere Y1 - 2019 U6 - https://doi.org/10.1387/ijdb.190184rg SN - 0214-6282 SN - 1696-3547 VL - 63 IS - 8-10 SP - 509 EP - 519 PB - UBC Pr CY - Bilbao 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 - Mai, Tobias A1 - Wolski, Karol A1 - Puciul-Malinowska, Agnieszka A1 - Kopyshev, Alexey A1 - Gräf, Ralph A1 - Bruns, Michael A1 - Zapotoczny, Szczepan A1 - Taubert, Andreas T1 - Anionic polymer brushes for biomimetic calcium phosphate mineralization BT - A surface with application potential in biomaterials JF - Polymers N2 - 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. KW - polymer brushes KW - calcium phosphate KW - hydroxyapatite KW - carbonated apatite KW - bone mimic KW - biocompatibility KW - Dictyostelium discoideum Y1 - 2018 U6 - https://doi.org/10.3390/polym10101165 SN - 2073-4360 VL - 10 IS - 10 PB - MDPI CY - Basel ER -