@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} } @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{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{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{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{MeyerKuhnertGraef2011, author = {Meyer, Irene and Kuhnert, Oliver and Gr{\"a}f, Ralph}, title = {Functional analyses of lissencephaly-related proteins in Dictyostelium}, series = {Seminars in cell \& developmental biology}, volume = {22}, journal = {Seminars in cell \& developmental biology}, number = {1}, publisher = {Elsevier}, address = {London}, issn = {1084-9521}, doi = {10.1016/j.semcdb.2010.10.007}, pages = {89 -- 96}, year = {2011}, abstract = {Lissencephaly is a severe brain developmental disease in human infants, which is usually caused by mutations in either of two genes, LIS1 and DCX. These genes encode proteins interacting with both the microtubule and the actin systems. Here, we review the implications of data on Dictyostelium LIS1 for the elucidation of LIS1 function in higher cells and emphasize the role of LIS1 and nuclear envelope proteins in nuclear positioning, which is also important for coordinated cell migration during neocortical development. Furthermore, for the first time we characterize Dictyostelium DCX, the only bona fide orthologue of human DCX outside the animal kingdom. We show that DCX functionally interacts with LIS1 and that both proteins have a cytoskeleton-independent function in chemotactic signaling during development. Dictyostelium LIS1 is also required for proper attachment of the centrosome to the nucleus and, thus, nuclear positioning, where the association of these two organelles has turned out to be crucial. It involves not only dynein and dynein-associated proteins such as LIS1 but also SUN proteins of the nuclear envelope. Analyses of Dictyostelium SUN1 mutants have underscored the importance of these proteins for the linkage of centrosomes and nuclei and for the maintenance of chromatin integrity. Taken together, we show that Dictyostelium amoebae, which provide a well-established model to study the basic aspects of chemotaxis, cell migration and development, are well suited for the investigation of the molecular and cell biological basis of developmental diseases such as lissencephaly.}, language = {en} } @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{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{PutzlerMeyerGraef2016, author = {Putzler, Sascha and Meyer, Irene and Gr{\"a}f, Ralph}, title = {CP91 is a component of the Dictyostelium centrosome involved in centrosome biogenesis}, series = {European journal of cell biology}, volume = {95}, journal = {European journal of cell biology}, publisher = {Royal Society}, address = {Jena}, issn = {0171-9335}, doi = {10.1016/j.ejcb.2016.03.001}, pages = {124 -- 135}, year = {2016}, abstract = {The Dictyostelium centrosome is a model for acentriolar centrosomes and it consists of a three-layered core structure surrounded by a corona harboring microtubule nucleation complexes. Its core structure duplicates once per cell cycle at the G2/M transition. Through proteomic analysis of isolated centrosomes we have identified CP91, a 91-kDa coiled coil protein that was localized at the centrosomal core structure. While GFP-CP91 showed almost no mobility in FRAP experiments during interphase, both GFP-CP91 and endogenous CP91 dissociated during mitosis and were absent from spindle poles from late prophase to anaphase. Since this behavior correlates with the disappearance of the central layer upon centrosome duplication, CP91 is a putative component of this layer. When expressed as GFP-fusions, CP91 fragments corresponding to the central coiled coil domain and the preceding N-terminal part (GFP-CP91cc and GFP-CP91N, respectively) also localized to the centrosome but did not show the mitotic redistribution of the full length protein suggesting a regulatory role of the C-terminal domain. Expression of all GFP-fusion proteins suppressed expression of endogenous CP91 and elicited supernumerary centrosomes. This was also very prominent upon depletion of CP91 by RNAi. Additionally, CP91-RNAi cells exhibited heavily increased ploidy due to severe defects in chromosome segregation along with increased cell size and defects in the abscission process during cytokinesis. Our results indicate that CP91 is a central centrosomal core component required for centrosomal integrity, proper centrosome biogenesis and, independently, for abscission during cytokinesis. (c) 2016 Elsevier GmbH. All rights reserved.}, language = {en} }