@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{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} } @article{GraefBatsiosMeyer2015, author = {Gr{\"a}f, Ralph and Batsios, Petros and Meyer, Irene}, title = {Evolution of centrosomes and the nuclear lamina: Amoebozoan assets}, series = {European journal of cell biology}, volume = {94}, journal = {European journal of cell biology}, number = {6}, publisher = {Elsevier}, address = {Jena}, issn = {0171-9335}, doi = {10.1016/j.ejcb.2015.04.004}, pages = {249 -- 256}, year = {2015}, abstract = {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.}, 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} } @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{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} } @article{KuhnertBaumannMeyeretal.2012, author = {Kuhnert, Oliver and Baumann, Otto and Meyer, Irene and Gr{\"a}f, Ralph}, title = {CP55, a novel key component of centrosomal organization in dictyostelium}, series = {Cellular and molecular life sciences}, volume = {69}, journal = {Cellular and molecular life sciences}, number = {21}, publisher = {Springer}, address = {Basel}, issn = {1420-682X}, doi = {10.1007/s00018-012-1040-3}, pages = {3651 -- 3664}, year = {2012}, abstract = {Dictyostelium centrosomes consist of a layered core structure surrounded by a microtubule-nucleating corona. At the G2/M transition, the corona dissociates and the core structure duplicates, yielding two spindle pole bodies. Finally, in telophase, the spindle poles mature into two new, complete centrosomes. CP55 was identified in a centrosomal proteome analysis. It is a component of the centrosomal core structure, and persists at the centrosome throughout the entire cell cycle. FRAP experiments revealed that during interphase the majority of centrosomal GFP-CP55 is immobile, which indicates a structural task of CP55 at the centrosome. The CP55null mutant is characterized by increased ploidy, a less structured, slightly enlarged corona, and by supernumerary, cytosolic MTOCs, containing only corona proteins and lacking a core structure. Live cell imaging showed that supernumerary MTOCs arise in telophase. Lack of CP55 also caused premature recruitment of the corona organizer CP148 to mitotic spindle poles, already in metaphase instead of telophase. Forces transmitted through astral microtubules may expel prematurely acquired or loosely attached corona fragments into the cytosol, where they act as independent MTOCs. CP55null cells were also impaired in growth, most probably due to difficulties in centrosome splitting during prophase. Furthermore, although they were still capable of phagocytosis, they appeared unable to utilize phagocytosed nutrients. This inability may be attributed to their partially disorganized Golgi apparatus.}, 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} }