Ursula Euteneuer

Dynamin 2 binds gamma-tubulin and participates in centrosome cohesion

Dynamin 2 (Dyn2) is a large GTPase involved in vesicle formation and actin reorganization. In this study, we report a novel role for Dyn2 as a component of the centrosome that is involved in centrosome cohesion. By light microscopy, Dyn2 localized aside centrin and colocalized with γ-tubulin at the centrosome; by immunoelectron microscopy, however, Dyn2 was detected in the pericentriolar material as well as on centrioles. Exogenously expressed green fluorescent protein (GFP)-tagged Dyn2 also localized to the centrosome, whereas glutathione S-transferase (GST)-tagged Dyn2 pulled down a protein complex(es) containing actin, α-tubulin and γ-tubulin from liver homogenate. Furthermore, gel overlay and immunoprecipitation indicated a direct interaction between γ-tubulin and a 219-amino-acid middle domain of Dyn2. Reduction of Dyn2 protein levels with small-interfering RNA (siRNA) resulted in centrosome splitting, whereas microtubule nucleation from centrosomes was not affected, suggesting a role for Dyn2 in centrosome cohesion. Finally, fluorescence recovery after photobleaching (FRAP) analysis of a GFP-tagged Dyn2 middle domain indicated that Dyn2 is a dynamic exchangeable component of the centrosome. These findings suggest a novel function for Dyn2 as a participant in centrosome cohesion.

The Large GTPase Dynamin Associates with the Spindle Midzone and Is Required for Cytokinesis

Cytokinesis involves the concerted efforts of the microtubule and actin cytoskeletons as well as vesicle trafficking and membrane remodeling to form the cleavage furrow and complete daughter cell separation. The exact mechanisms that support membrane remodeling during cytokinesis remain largely undefined. In this study, we report that the large GTPase dynamin, a protein involved in membrane tubulation and vesiculation, is essential for successful cytokinesis. Using biochemical and morphological methods, we demonstrate that dynamin localizes to the spindle midzone and the subsequent intercellular bridge in mammalian cells and is also enriched in spindle midbody extracts. In Caenorhabditis elegans, dynamin localized to newly formed cleavage furrow membranes and accumulated at the midbody of dividing embryos in a manner similar to dynamin localization in mammalian cells. Further, dynamin function appears necessary for cytokinesis, as C. elegans embryos from a dyn-1 ts strain, as well as dynamin RNAi-treated embryos, showed a marked defect in the late stages of cytokinesis. These findings indicate that, during mitosis, conventional dynamin is recruited to the spindle midzone and the subsequent intercellular bridge, where it plays an essential role in the final separation of dividing cells.

hPOC5 is a centrin-binding protein required for assembly of full-length centrioles

Centrin has been shown to be involved in centrosome biogenesis in a variety of eukaryotes. In this study, we characterize hPOC5, a conserved centrin-binding protein that contains Sfi1p-like repeats. hPOC5 is localized, like centrin, in the distal portion of human centrioles. hPOC5 recruitment to procentrioles occurs during G2/M, a process that continues up to the full maturation of the centriole during the next cell cycle and is correlated with hyperphosphorylation of the protein. In the absence of hPOC5, RPE1 cells arrest in G1 phase, whereas HeLa cells show an extended S phase followed by cell death. We show that hPOC5 is not required for the initiation of procentriole assembly but is essential for building the distal half of centrioles. Interestingly, the hPOC5 family reveals an evolutionary divergence between vertebrates and organisms like Drosophila melanogaster or Caenorhabditis elegans, in which the loss of hPOC5 may correlate with the conspicuous differences in centriolar structure.

Dictyostelium Sun-1 connects the centrosome to chromatin and ensures genome stability.

The centrosome‐nucleus attachment is a prerequisite for faithful chromosome segregation during mitosis. We addressed the function of the nuclear envelope (NE) protein Sun‐1 in centrosome‐nucleus connection and the maintenance of genome stability in Dictyostelium discoideum. We provide evidence that Sun‐1 requires direct chromatin binding for its inner nuclear membrane targeting. Truncation of the cryptic N‐terminal chromatin‐binding domain of Sun‐1 induces dramatic separation of the inner from the outer nuclear membrane and deformations in nuclear morphology, which are also observed using a Sun‐1 RNAi construct. Thus, chromatin binding of Sun‐1 defines the integrity of the nuclear architecture. In addition to its role as a NE scaffold, we find that abrogation of the chromatin binding of Sun‐1 dissociates the centrosome‐nucleus connection, demonstrating that Sun‐1 provides an essential link between the chromatin and the centrosome. Moreover, loss of the centrosome‐nucleus connection causes severe centrosome hyperamplification and defective spindle formation, which enhances aneuploidy and cell death significantly. We highlight an important new aspect for Sun‐1 in coupling the centrosome and nuclear division during mitosis to ensure faithful chromosome segregation.

Isolation of nucleation-competent centrosomes from Dictyostelium discoideum

The centrosome of Dictyostelium discoideum is a box-shaped, layered core structure surrounded by a corona which is made up of dense nodules embedded in amorphous material. It is also known as nucleus-associated body. Because of its tight association with the nucleus the centrosome has resisted so far all attempts for isolation in sufficient purity and quantity for biochemical analysis. Here we report on the large-scale isolation of D. discoideum centrosomes after treatment of nucleus-centrosome complexes with a buffer containing sodium pyrophosphate. Following heparin treatment and a filtration step, centrosomes were further purified by density gradient centrifugation. Immunofluorescence analysis of the isolated centrosomes revealed the presence of the D. discoideum 350-kDa antigen, a centrosomal marker protein, gamma-tubulin, and the D. discoideum homologues of pericentrin, Spc110p, and Cdc31p. The structural integrity of the isolated centrosomes was demonstrated by confocal laser microscopy and electron microscopy. Microtubule nucleation assays with purified pig brain tubulin showed that the isolation procedure did not only preserve the structure but also the functionality of the isolated centrosomes. D. discoideum centrosomes should now become an attractive new model system in addition to, and for comparison with, centriolar centrosomes and yeast spindle pole bodies.

Structural transformation of epidermal tonofilaments upon cold treatment

Abstract Treatment of filament-containing epidermal cells of several teleost species with low temperature (0 °C) causes a profound structural rearrangement of their tonofilaments. Dense amorphous or granular aggregates formed from tonofilament material appear throughout the cytoplasm and in association with desmosomes at the cell periphery. This so far unique response of one class of intermediate-sized filaments to a physical agent is readily reversed, within minutes, by rewarming the cells. Recovery from cold treatment is characterized by a decrease in density of tonofilament aggregates and a concomitant increase in tonofilaments in their vicinity. Aggregate formation is unrelated to the depolymerization of microtubules also caused by cold treatment, since neither colchicine nor vinblastine elicit a similar effect. Tonofilament aggregates do not form in Triton X-100 extracted cells cooled after lysis, while lysis of cooled intact cells has no influence on the aggregates thus formed. It is hypothesized that the structural transformation of tonofilaments upon cold treatment may be restricted to the class of intermediate-sized filaments present in cells of the teleost epidermis.

Differential Effects of Heterochromatin Protein 1 Isoforms on Mitotic Chromosome Distribution and Growth in Dictyostelium discoideum

ABSTRACT Heterochromatin protein 1 (HP1) is a well-characterized heterochromatin component conserved from fission yeast to humans. We identified three HP1-like genes (hcpA, hcpB, and hcpC) in the Dictyostelium discoideum genome. Two of these (hcpA and hcpB) are expressed, and the proteins colocalized as green fluorescent protein (GFP) fusion proteins in one major cluster at the nuclear periphery that was also characterized by histone H3 lysine 9 dimethylation, a histone modification so far not described for Dictyostelium. The data strongly suggest that this cluster represents the centromeres. Both single-knockout strains displayed only subtle phenotypes, suggesting that both isoforms have largely overlapping functions. In contrast, disruption of both isoforms appeared to be lethal. Furthermore, overexpression of a C-terminally truncated form of HcpA resulted in phenotypically distinct growth defects that were characterized by a strong decrease in cell viability. Although genetic evidence implies functional redundancy, overexpression of GFP-HcpA, but not GFP-HcpB, caused growth defects that were accompanied by an increase in the frequency of atypic anaphase bridges. Our data indicate that Dictyostelium discoideum cells are sensitive to changes in HcpA and HcpB protein levels and that the two isoforms display different in vivo and in vitro affinities for each other. Since the RNA interference (RNAi) machinery is frequently involved in chromatin remodeling, we analyzed if knockouts of RNAi components influenced the localization of H3K9 dimethylation and HP1 isoforms in Dictyostelium. Interestingly, heterochromatin organization appeared to be independent of functional RNAi.

The Genome of the Foraminiferan Reticulomyxa filosa

BACKGROUND Rhizaria are a major branch of eukaryote evolution with an extensive microfossil record, but only scarce molecular data are available. The rhizarian species Reticulomyxa filosa, belonging to the Foraminifera, is free-living in freshwater environments. In culture, it thrives only as a plasmodium with thousands of haploid nuclei in one cell. The R. filosa genome is the first foraminiferal genome to be deciphered. RESULTS The genome is extremely repetitive, and the large amounts of identical sequences hint at frequent amplifications and homologous recombination events. Presumably, these mechanisms are employed to provide more gene copies for higher transcriptional activity and to build up a reservoir of gene diversification in certain gene families, such as the kinesin family. The gene repertoire indicates that it is able to switch to a single-celled, flagellated sexual state never observed in culture. Comparison to another rhizarian, the chlorarachniophyte alga Bigelowiella natans, reveals that proteins involved in signaling were likely drivers in establishing the Rhizaria lineage. Compared to some other protists, horizontal gene transfer is limited, but we found evidence of bacterial-to-eukaryote and eukaryote-to-eukaryote transfer events. CONCLUSIONS The R. filosa genome exhibits a unique architecture with extensive repeat homogenization and gene amplification, which highlights its potential for diverse life-cycle stages. The ability of R. filosa to rapidly transport matter from the pseudopodia to the cell body may be supported by the high diversification of actin and kinesin gene family members.

Comparative structural, molecular, and functional aspects of the Dictyostelium discoideum centrosome.

Publisher Summary The cellular slime mold Dictyostelium discoideum is a well-established model organism for the study of basic aspects of development, cell motility, and phagocytosis. It shows a simple form of development and exhibits fast ameboid motility. This chapter discusses the comparative morphology and molecular biology of the Dictyostelium centrosome, the Saccharomyces cerevisiae ( S. cerevisiae ) spindle pole body (SPB), and the animal centriole-containing centrosome. The best-known function of centrosomes is the organization of the interphase microtubule array and the mitotic spindle apparatus. Most fundamental centrosomal functions are shared among all eukaryotic cells, but the morphology of their centrosomes varies considerably. Centrosomal structure and their duplication are diagrammatically represented in the chapter. The unusual mode of centrosome duplication in Dictyostelium is best illustrated when compared to the modes of centrosome duplication in animal and yeast cells. In Dictyostelium , the entire duplication procedure takes place during mitosis. The Dictyostelium centrosome is unique with regard to the splitting process of the core structure and the timing of duplication events within the cell cycle. The Dictyostelium centrosome and the S. cerevisiae SPB share some morphological features, but in Dictyostelium , the centrosomal core appears to be built symmetrically, and its outer layers most likely consist of the same proteins. The progress and future perspectives in Dictyostelium centrosome research are also discussed.

CP250, a Novel Acidic Coiled Coil Protein of the Dictyostelium centrosome, Affects Growth, Chemotaxis, and the Nuclear Envelope

The Dictyostelium centrosome is a nucleus associated body consisting of a box-shaped core surrounded by the corona, an amorphous matrix functionally equivalent to the pericentriolar material of animal centrosomes which is responsible for the nucleation and anchoring of microtubules. Here we describe CP250 a component of the corona, an acidic coiled coil protein that is present at the centrosome throughout interphase while disappearing during prophase and reappearing at the end of late telophase. Amino acids 756-1148 of the 2110 amino acids are sufficient for centrosomal targeting and cell cycle-dependent centrosome association. Mutant cells lacking CP250 are smaller in size, growth on bacteria is delayed, chemotaxis is altered, and development is affected, which, in general, are defects observed in cytoskeletal mutants. Furthermore, loss of CP250 affected the nuclear envelope and led to reduced amounts and altered distribution of Sun-1, a conserved nuclear envelope protein that connects the centrosome to chromatin.