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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 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.
Cell cycle-dependent localization of novel centrosomal and centromeric proteins in Dictyostelium
(2009)
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.
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 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.
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.
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.