Refine
Year of publication
Document Type
- Article (31)
- Postprint (4)
- Conference Proceeding (2)
- Other (1)
- Part of Periodical (1)
- Review (1)
Keywords
- Dictyostelium (20)
- lamin (9)
- Centrosome (8)
- centrosome (7)
- nuclear lamina (7)
- nuclear envelope (6)
- Microtubules (5)
- NE81 (5)
- Nucleus (4)
- centriole (3)
The centrosome is the main microtubule-organizing center and constitutes the largest protein complex in a eukaryotic cell. The Dictyostelium centrosome is an established model for acentriolar centrosomes and it consists of a layered core structure Surrounded by a so-called corona, which harbors microtubule nucleation complexes. We have identified 34 new centrosomal candidate proteins through mass spectrometrical analysis of the proteome of isolated Dictyostelium centrosomes. Here we present a characterization of 12 centrosomal candidate proteins all featuring coiled coil regions and low expression levels, which are the most common attributes of centrosomal proteins. We used GFP fusion proteins to localize the candidate proteins in whole cells and on microtubule-free, isolated centrosomes. Thus we were able to identify nine new genuine centrosomal proteins including a putative orthologue of Cep192, an interaction partner of polo-like kinase 4 in human centriole biogenesis. In this respect, centrosomal localization of the only polo-like kinase in Dictyostelium, Pik, is also shown in this work. Using confocal deconvolution microscopy, four components, CP39, CP55, CP75, and CP91 could be clearly assigned to the so far almost uncharacterized centrosomal core structure, while CP148 and Cep192 localized to a zone between that of corona marker and core proteins. Finally, CP103 and CP248 were constituents of the corona. In contrast, NE81 was localized at the nuclear envelope and three others, an orthologue of the spindle checkpoint component Mad1, the novel Cenp68, and the centrosomal CP248 were observed at the centromeres, which are clustered and linked to the centrosome throughout the entire cell cycle. Cell Motil. Cytoskeleton 66: 915-928, 2009.
Centrins are a family of proteins within the calcium-binding EF-hand superfamily. In addition to their archetypical role at the microtubule organizing center (MTOC), centrins have acquired multiple functionalities throughout the course of evolution. For example, centrins have been linked to different nuclear activities, including mRNA export and DNA repair. Dictyostelium discoideum centrin B is a divergent member of the centrin family. At the amino acid level, DdCenB shows 51% identity with its closest relative and only paralog, DdCenA. Phylogenetic analysis revealed that DdCenB and DdCenA form a well-supported monophyletic and divergent group within the centrin family of proteins. Interestingly, fluorescently tagged versions of DdCenB were not found at the centrosome (in whole cells or in isolated centrosomes). Instead, DdCenB localized to the nuclei of interphase cells. This localization disappeared as the cells entered mitosis, although Dictyostelium cells undergo a closed mitosis in which the nuclear envelope (NE) does not break down. DdCenB knockout cells exhibited aberrant nuclear architecture, characterized by enlarged and deformed nuclei and loss of proper centrosome-nucleus anchoring (observed as NE protrusions). At the centrosome, loss of DdCenB resulted in defects in the organization and morphology of the MTOC and supernumerary centrosomes and centrosome-related bodies. The multiple defects that the loss of DdCenB generated at the centrosome can be explained by its atypical division cycle, transitioning into the NE as it divides at mitosis. On the basis of these findings, we propose that DdCenB is required at interphase to maintain proper nuclear architecture, and before delocalizing from the nucleus, DdCenB is part of the centrosome duplication machinery.
Centrosomal attachment to nuclei is crucial for proper mitosis and nuclear positioning in various organisms, and generally involves Sun-family proteins located at the inner nuclear envelope. There is still no common scheme for the outer nuclear membrane proteins interacting with Sun I in centrosome/nucleus attachment. Here we propose a model in which Sun1 mediates a physical link between centrosomes and clustered centromeres through both nuclear membranes in Dictyostelium. For the first time we provide a detailed microscopic analysis of the centrosomal and nuclear envelope localization of endogenous Dictyostelium Sun1 during interphase and mitosis. By immunogold electron microscopy we show that Sun1 is a resident of both nuclear membranes. Disruption of Sun1 function by overexpression of full-length GFP-Sun1 or a GFP-Sun-domain deletion construct revealed not only the established function in centrosome/nucleus attachment and maintenance of ploidy, but also a requirement of Sun1 for the association of the centromere cluster with the centrosome. Live-cell imaging visualized the occurrence of mitotic defects, and demonstrated the requirement of microtubules for dynamic distance changes between centrosomes and nuclei. FRAP analysis revealed at least two populations of Sun1, with an immobile fraction associated with the centrosome, and a mobile fraction in the nuclear envelope.
Cell cycle-dependent localization of novel centrosomal and centromeric proteins in Dictyostelium
(2009)
Covalently crosslinked PEI hydrogels are efficient templates for calcium phosphate mineralization in SBF. In contrast to the PEI hydrogels, non-crosslinked PEI does not lead to calcium phosphate nucleation and growth in SBF. The precipitate is a mixture of brushite and hydroxyapatite. The PEI/calcium phosphate composite material exhibits a sponge like morphology and a chemical composition that is interesting for implants. Cytotoxicity tests using Dictyostelium discoideum amoebae show that both the non-mineralized and mineralized hydrogels have a very low cytotoxicity. This suggests that next generation PEI hydrogels, where also the degradation products are non-toxic, could be interesting for biomedical applications.
The centromeric histone H3 variant (CenH3) serves to target the kinetochore to the centromeres and thus ensures correct chromosome segregation during mitosis and meiosis. The Dictyostelium H3-like variant H3v1 was identified as the CenH3 ortholog. Dictyostelium CenH3 has an extended N-terminal domain with no similarity to any other known proteins and a histone fold domain at its C-terminus. Within the histone fold, alpha-helix 2 (alpha 2) and an extended loop 1 (L1) have been shown to be required for targeting CenH3 to centromeres. Compared to other known and putative CenH3 histones, Dictyostelium CenH3 has a shorter L1, suggesting that the extension is not an obligatory feature. Through ChIP analysis and fluorescence microscopy of live and fixed cells, we provide here the first survey of centromere structure in amoebozoa. The six telocentric centromeres were found to mostly consist of all the DIRS-1 elements and to associate with H3K9me3. During interphase, the centromeres remain attached to the centrosome forming a single CenH3-containing cluster. Loading of Dictyostelium CenH3 onto centromeres occurs at the G2/prophase transition, in contrast to the anaphase/ telophase loading of CenH3 observed in metazoans. This suggests that loading during G2/prophase is the ancestral eukaryotic mechanism and that anaphase/telophase loading of CenH3 has evolved more recently after the amoebozoa diverged from the animal linage.
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.
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.
A lamin in lower eukaryotes?
(2012)
Lamins are the major components of the nuclear lamina and serve not only as a mechanical support, but are also involved in chromatin organization, epigenetic regulation, transcription and mitotic events. Despite these universal tasks, lamins have so far been found only in metazoans. Yet, recently we have identified Dictyostelium NE81 as the first lamin-like protein in a lower eukaryote. Based on the current knowledge, we draw a model for nuclear envelope organization in Dictyostelium in this Extra View and we review the experimental data that justified this classification. Furthermore we provide unpublished data underscoring the requirement of posttranslational CaaX-box processing for proper protein localization at the nuclear envelope. Sequence comparison of NE81 sequences from four Dictyostelia with bona fide lamins illustrates the evolutional relationship between these proteins. Under certain conditions these usually unicellular social amoebae congregate to form a multicellular body. We propose that the evolution of the lamin-like NE81 went along with the invention of multicellularity.
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.
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.
Characterization of NE81, the first lamin-like nucleoskeleton protein in a unicellular organism
(2012)
Lamins build the nuclear lamina and are required for chromatin organization, gene expression, cell cycle progression, and mechanical stabilization. Despite these universal functions, lamins have so far been found only in metazoans. We have identified protein NE81 in Dictyostelium, which has properties that justify its denomination as a lamin-like protein in a lower eukaryote. This is based on its primary structure, subcellular localization, and regulation during mitosis, and its requirement of the C-terminal CaaX box as a posttranslational processing signal for proper localization. Our knockout and overexpression mutants revealed an important role for NE81 in nuclear integrity, chromatin organization, and mechanical stability of cells. All our results are in agreement with a role for NE81 in formation of a nuclear lamina. This function is corroborated by localization of Dictyostelium NE81 at the nuclear envelope in human cells. The discovery of a lamin-like protein in a unicellular organism is not only intriguing in light of evolution, it may also provide a simple experimental platform for studies of the molecular basis of laminopathies.
Portal Wissen = Raum
(2012)
Mit „Portal Wissen“ laden wir Sie ein, die Forschung an der Universität Potsdam zu entdecken und in ihrer Vielfalt kennenzulernen. In der ersten Ausgabe dreht sich alles um „Räume“. Räume, in denen geforscht wird, solche, die es zu erforschen gilt, andere, die durch Wissenschaft zugänglich oder erschlossen werden, aber auch Räume, die Wissenschaft braucht, um sich entfalten zu können. Forschung vermisst Räume: „Wissenschaft wird von Menschen gemacht“, schrieb der Physiker Werner Heisenberg. Umgekehrt lässt sich sagen: Wissenschaft macht Menschen, widmet sich ihnen, beeinflusst sie. Dieser Beziehung ist „Portal Wissen“ nachgegangen. Wir haben Wissenschaftler getroffen, sie gefragt, wie aus ihren Fragen Projekte entstehen, haben sie auf dem oft verschlungenen Weg zum Ziel begleitet. Ein besonderes Augenmerk dieses Heftes gilt den „Kulturellen Begegnungsräumen“, denen ein eigener Profilbereich der Forschung an der Universität Potsdam gewidmet ist.
Forschung hat Räume: Labore, Bibliotheken, Gewächshäuser oder Archive – hier ist Wissenschaft zu Liebe Leserinnen und Leser, Hause. All diese Orte sind so einzigartig wie die Wissenschaftler, die in ihnen arbeiten, oder die Untersuchungen, die hier stattfinden. Erst die Vision davon, wie ein Problem zu lösen ist, macht aus einfachen Zimmern „Laborräume“. Wir haben ihre Türen geöffnet, um zu zeigen, was – und wer – sich dahinter befindet.
Forschung eröffnet Räume: Wenn Wissenschaft erfolgreich ist, bewegt sie uns, bringt uns voran. Auf dem Weg einer wissenschaftlichen Erkenntnis aus dem Labor in den Alltag stehen mitunter Hürden, die meist nicht auf den ersten Blick zu erkennen sind. Auf jeden Fall aber ist ihre Anwendung erster Ausgangspunkt von Wissenschaft, Antrieb und Motivation jedes Forschers. „Portal Wissen“ zeigt, welche „Praxisräume“ sich aus der Übersetzung von Forschungsresultaten ergeben. Dort, wo wir es unbedingt erwarten, und dort, wo vielleicht nicht.
Forschung erschließt Räume: Bei Expeditionen, Feldversuchen und Exkursionen wird nahezu jede Umgebung zum mobilen Labor. So eröffnet Wissenschaft Zugänge auch zu Orten, die auf vielfach andere Weise verschlossen oder unzugänglich scheinen. Wir haben uns in Forscher- Reisetaschen gemogelt, um bei Entdeckungsreisen dabei zu sein, die weit weg – vor allem nach Afrika – führen. Zugleich haben wir beobachtet, wie „Entwicklungsräume“ sich auch von Potsdam aus erschließen lassen oder zumindest ihre Vermessung in Potsdam beginnen kann.
Forschung braucht Räume: Wissenschaft hat zwei Geschlechter, endlich. Noch nie waren so viele Frauen in der Forschung tätig wie derzeit. Ein Grund zum Ausruhen ist dies gleichwohl nicht. Deutschlandweit ist aktuell nur jede fünfte Professur von einer Frau besetzt. „Portal Wissen“ schaut, welche „Entwicklungsräume“ Frauen sich in der Wissenschaft, aber auch darüber hinaus geschaffen haben. Und wo sie ihnen verwehrt werden. Wir wünschen Ihnen eine anregende Lektüre und dass auch Sie einen Raum finden, der Sie inspiriert.
Prof. Dr. Robert Seckler
Vizepräsident für Forschung und wissenschaftlichen Nachwuchs
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.
Diaphanous-related formins (DRFs) drive the nucleation and elongation of linear actin filaments downstream of Rho GTPase signalling pathways. Dictyostelium formin C (ForC) resembles a DRF, except that it lacks a genuine formin homology domain 1 (FH1), raising the questions whether or not ForC can nucleate and elongate actin filaments. We found that a recombinant ForC-FH2 fragment does not nucleate actin polymerization, but moderately decreases the rate of spontaneous actin assembly and disassembly, although the barbed-end elongation rate in the presence of the formin was not markedly changed. However, the protein bound to and crosslinked actin filaments into loose bundles of mixed polarity. Furthermore, ForC is an important regulator of morphogenesis since ForC-null cells are severely impaired in development resulting in the formation of aberrant fruiting bodies. Immunoblotting revealed that ForC is absent during growth, but becomes detectable at the onset of early aggregation when cells chemotactically stream together to form a multicellular organism, and peaks around the culmination stage. Fluorescence microscopy of cells ectopically expressing a GFP-tagged, N-terminal ForC fragment showed its prominent accumulation in the leading edge, suggesting that ForC may play a role in cell migration. In agreement with its expression profile, no defects were observed in random migration of vegetative mutant cells. Notably, chemotaxis of starved cells towards a source of cAMP was severely impaired as opposed to control. This was, however, largely due to a marked developmental delay of the mutant, as evidenced by the expression profile of the early developmental marker csA. In line with this, chemotaxis was almost restored to wild type levels after prolonged starvation. Finally, we observed a complete failure of phototaxis due to abolished slug formation and a massive reduction of spores consistent with forC promoter-driven expression of beta-galactosidase in prespore cells. Together, these findings demonstrate ForC to be critically involved in signalling of the cytoskeleton during various stages of development.
Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in tRNA(Asp(GUC)) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified tRNA(Glu(CUC/UUC)) and tRNA(Gly(GCC)) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.
Evolution of the nucleus
(2014)
The nucleus represents a major evolutionary transition. As a consequence of separating translation from transcription many new functions arose, which likely contributed to the remarkable success of eukaryotic cells. Here we will consider what has recently emerged on the evolutionary histories of several key aspects of nuclear biology; the nuclear pore complex, the lamina, centrosomes and evidence for prokaryotic origins of relevant players.
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.