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 - 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 - 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 - GEN A1 - Gräf, Ralph T1 - Comparative Biology of Centrosomal Structures in Eukaryotes T2 - Cells N2 - The centrosome is not only the largest and most sophisticated protein complex within a eukaryotic cell, in the light of evolution, it is also one of its most ancient organelles. This special issue of "Cells" features representatives of three main, structurally divergent centrosome types, i.e., centriole-containing centrosomes, yeast spindle pole bodies (SPBs), and amoebozoan nucleus-associated bodies (NABs). Here, I discuss their evolution and their key-functions in microtubule organization, mitosis, and cytokinesis. Furthermore, I provide a brief history of centrosome research and highlight recently emerged topics, such as the role of centrioles in ciliogenesis, the relationship of centrosomes and centriolar satellites, the integration of centrosomal structures into the nuclear envelope and the involvement of centrosomal components in non-centrosomal microtubule organization. KW - centrosome KW - centriole KW - cilium KW - basal body KW - spindle pole body KW - SPB KW - nucleus-associated body KW - NAB KW - microtubules Y1 - 2018 U6 - https://doi.org/10.3390/cells7110202 SN - 2073-4409 VL - 7 IS - 11 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 - Krämer, Nadine A1 - Ravindran, Ethiraj A1 - Zaqout, Sami A1 - Neubert, Gerda A1 - Schindler, Detlev A1 - Ninnemann, Olaf A1 - Gräf, Ralph A1 - Seiler, Andrea E. M. A1 - Kaindl, Angela M. T1 - Loss of CDK5RAP2 affects neural but not non-neural mESC differentiation into cardiomyocytes JF - Cell cycle N2 - Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors. KW - CDK5RAP2 KW - MCPH KW - mental retardation KW - neural differentiation KW - primary microcephaly KW - stem cell Y1 - 2015 U6 - https://doi.org/10.1080/15384101.2015.1044169 SN - 1538-4101 SN - 1551-4005 VL - 14 IS - 13 SP - 2044 EP - 2057 PB - Taylor & Francis Group CY - Philadelphia ER - TY - JOUR A1 - Meyer, Irene A1 - Kuhnert, Oliver A1 - Gräf, Ralph T1 - Functional analyses of lissencephaly-related proteins in Dictyostelium JF - Seminars in cell & developmental biology N2 - 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. KW - Dictyostelium KW - Lissencephaly KW - LIS1 KW - DCX KW - SUN1 KW - Centrosome Y1 - 2011 U6 - https://doi.org/10.1016/j.semcdb.2010.10.007 SN - 1084-9521 VL - 22 IS - 1 SP - 89 EP - 96 PB - Elsevier CY - London ER - TY - JOUR A1 - Gräf, Ralph A1 - Batsios, Petros A1 - Meyer, Irene T1 - Evolution of centrosomes and the nuclear lamina: Amoebozoan assets JF - European journal of cell biology N2 - The current eukaryotic tree of life groups most eukaryotes into one of five supergroups, the Opisthokonta, Amoebozoa, Archaeplastida, Excavata and SAR (Stramenopile, Alveolata, Rhizaria). Molecular and comparative morphological analyses revealed that the last eukaryotic common ancestor (LECA) already contained a rather sophisticated equipment of organelles including a mitochondrion, an endomembrane system, a nucleus with a lamina, a microtubule-organizing center (MTOC), and a flagellar apparatus. Recent studies of MTOCs, basal bodies/centrioles, and nuclear envelope organization of organisms in different supergroups have clarified our picture of how the nucleus and MTOCs co-evolved from LECA to extant eukaryotes. In this review we summarize these findings with special emphasis on valuable contributions of research on a lamin-like protein, nuclear envelope proteins, and the MTOC in the amoebozoan model organism Dictyostelium discoideum. (C) 2015 Elsevier GmbH. All rights reserved. KW - LINC complex KW - Sun1 KW - Nuclear lamina KW - Lamin KW - Nuclear envelope KW - Centrosome KW - Basal body KW - Centriole KW - LEM-domain Y1 - 2015 U6 - https://doi.org/10.1016/j.ejcb.2015.04.004 SN - 0171-9335 SN - 1618-1298 VL - 94 IS - 6 SP - 249 EP - 256 PB - Elsevier CY - Jena ER -