Identification of novel centrosomal proteins in Dictyostelium discoideum by comparative proteomic approaches

The centrosome functions as the main microtubule-organization center of the cell and is of importance for all microtubule-dependent processes such as organelle transport and directionality of cell migration. One of the major model organisms in centrosome research is the slime mold Dictyostelium discoideum. Since only 10 centrosomal proteins are known so far in Dictyostelium discoideum, the elucidation of new centrosomal components may give a more comprehensive understanding of centrosomal function. To distinguish between centrosomal and contaminating proteins we established different separation and relative quantification strategies including techniques such as iTRAQ and DIGE. In this work, we present the identification of several known components as well as more than 70 new candidates--currently subject of further investigations--for the protein inventory of the Dictyostelium centrosome. Among these protein identifications, 44% represent hypothetical proteins of still unknown function associated with the centrosome.     

Cell cycle-dependent localization of monoclonal antibodies raised against isolated Dictyostelium centrosomes.

The ultrastructure of the Dictyostelium centrosome is markedly different from that of the well known yeast spindle pole body and vertebrate centriole-containing centrosome. It consists of a box-shaped, layered core structure surrounded by a corona with dense nodules embedded in an amorphous matrix. For further structural and biochemical analyses of this type of centrosome we used highly enriched isolated Dictyostelium centrosomes as an antigen to raise 14 new centrosomal monoclonal antibodies. Immunofluorescence microscopy and Western blot analysis revealed that at least 10 of them were directed against different antigens. Immunofluorescence microscopy also showed that the monoclonal antibodies fell into three different groups: A) antibodies localizing to the centrosome during the entire cell cycle; B) antibodies staining the centrosome mainly during mitosis; and C) antibodies labeling centrosome associated structures. All antibodies, except one, exhibited a cell cycle-dependent staining pattern underscoring the highly dynamic properties of the Dictyostelium centrosome.     

Centrosome-associated Chk1 prevents premature activation of cyclin-B-Cdk1 kinase

Entry into mitosis occurs after activation of Cdk1, resulting in chromosome condensation in the nucleus and centrosome separation, as well as increased microtubule nucleation activity in the cytoplasm. The active cyclin-B1-Cdk1 complex first appears at the centrosome, suggesting that the centrosome may facilitate the activation of mitotic regulators required for the commitment of cells to mitosis. However, the signalling pathways involved in controlling the initial activation of Cdk1 at the centrosome remain largely unknown. Here, we show that human Chk1 kinase localizes to interphase, but not mitotic, centrosomes. Chemical inhibition of Chk1 resulted in premature centrosome separation and activation of centrosome-associated Cdk1. Forced immobilization of kinase-inactive Chk1 to centrosomes also resulted in premature Cdk1 activation. Conversely, under such conditions wild-type Chk1 impaired activation of centrosome-associated Cdk1, thereby resulting in DNA endoreplication and centrosome amplification. Activation of centrosomal Cdk1 in late prophase seemed to be mediated by cytoplasmic Cdc25B, whose activity is controlled by centrosome-associated Chk1. These results suggest that centrosome-associated Chk1 shields centrosomal Cdk1 from unscheduled activation by cytoplasmic Cdc25B, thereby contributing to proper timing of the initial steps of cell division, including mitotic spindle formation.     

Characterization of microsome-associated tobacco BY-2 centrins

Centrin - higher plants - MTOCs - microtubules nucleation In most eukaryotic cells, the Ca(2+)-binding protein centrin is associated with structured microtubule-organizing centers (MTOCs) such as centrosomes. In these cells, centrin either forms centrosome-associated contractile fibers, or is involved in centrosome biogenesis. Our aim was to investigate the functions of centrin in higher plant cells which do not contain centrosome-like MTOCs. We have cloned two tobacco BY-2 centrin cDNAs and we show that higher plant centrins define a phylogenetic group of proteins distinct from centrosome-associated centrins. In addition, tobacco centrins were found primarily associated with microsomes and did not colocalize with gamma-tubulin, a known MTOC marker. While the overall level of centrin did not vary during the cell cycle, centrin was prominently detected at the cell plate during telophase. Our results suggest that in tobacco, the major portion of centrin is not MTOC-associated and could be involved in the formation of the cell plate during cytokinesis.     

cDNA sequence of cyclophilin from Dictyostelium discoideum

A cDNA encoding a protein homologous to cyclophilins from other species has been isolated from a Dictyostelium discoideum cDNA library. From the deduced amino acid sequence a protein with a molecular mass of 19 kD and 64% identity with human cyclophilin is predicted. Southern blot analysis indicates that there is one cyclophilin gene in the D. discoideum genome. The mRNA is present in all developmental stages.     

The centrosome in human genetic disease

The centrosome is an indispensable component of the cell-cycle machinery of eukaryotic cells, and the perturbation of core centrosomal or centrosome-associated proteins is linked to cell-cycle misregulation and cancer. Recent work has expanded our understanding of the functional complexity and importance of this organelle. The centrosomal localization of proteins that are involved in human genetic disease, and the identification of novel centrosome-associated proteins, has shown that numerous, seemingly unrelated, cellular processes can be perturbed by centrosomal dysfunction. Here, we review the mechanistic relationship between human disease phenotypes and the function of the centrosome, and describe some of the newly-appreciated functions of this organelle in animal cells.     

Mapping the cytochrome c553 interacting site using 1H and 15N NMR

The cDNA encoding the centrosomal protein CCD41 is identical with the cDNA for the endothelial cell protein C receptor. This finding is not due to an artefact, e.g. caused by selection of false positive clones. The segment of the CCD41 cDNA encoding the protein originally termed CCD41 and deletion mutants of it were fused with the nucleotide sequence encoding the enhanced green fluorescent protein (EGFP). Transfection and expression of the full length construct produces a fusion protein mainly located in cell membranes reflecting the receptor-type protein. Deletion mutants, e.g. those where the signal sequence is deleted, result in fusion proteins which are exclusively incorporated into a small perinuclear structure which is the site of the centrosome. This result suggests that post-translational modification, namely deletion of the signal sequence, is decisive for the centrosomal location of the resulting centrosomal protein while the unprocessed protein is incorporated into cell membranes.     

The cell cycle-dependent localization of the CP190 centrosomal protein is determined by the coordinate action of two separable domains

CP190, a protein of 1,096 amino acids from Drosophila melanogaster, oscillates in a cell cycle-specific manner between the nucleus during interphase, and the centrosome during mitosis. To characterize the regions of CP190 responsible for its dynamic behavior, we injected rhodamine-labeled fusion proteins spanning most of CP190 into early Drosophila embryos, where their localizations were characterized using time-lapse fluorescence confocal microscopy. A single bipartite 19- amino acid nuclear localization signal was detected that causes nuclear localization. Robust centrosomal localization is conferred by a separate region of 124 amino acids; two adjacent, nonoverlapping fusion proteins containing distinct portions of this region show weaker centrosomal localization. Fusion proteins that contain both nuclear and centrosomal localization sequences oscillate between the nucleus and the centrosome in a manner identical to native CP190. Fusion proteins containing only the centrosome localization sequence are found at centrosomes throughout the cell cycle, suggesting that CP190 is actively recruited away from the centrosome by its movement into the nucleus during interphase. Both native and bacterially expressed CP190 cosediment with microtubules in vitro. Tests with fusion proteins show that the domain responsible for microtubule binding overlaps the domain required for centrosomal localization. CP60, a protein identified by its association with CP190, also localizes to centrosomes and to nuclei in a cell cycle-dependent manner. Experiments in which colchicine is used to depolymerize microtubules in the early Drosophila embryo demonstrate that both CP190 and CP60 are able to attain and maintain their centrosomal localization in the absence of microtubules.     

Identification of the single gene for calmodulin in Dictyostelium discoideum.

We report the isolation and sequence determination of a cDNA containing most of the coding sequence for Dictyostelium discoideum calmodulin. The cloned cDNA was used as a probe to examine the complexity of D. discoideum genomic DNA. These studies indicated that D. discoideum cells possess a single calmodulin gene.     

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.     

The sudden recruitment of gamma-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules.

gamma-Tubulin is a centrosomal component involved in microtubule nucleation. To determine how this molecule behaves during the cell cycle, we have established several vertebrate somatic cell lines that constitutively express a gamma-tubulin/green fluorescent protein fusion protein. Near simultaneous fluorescence and DIC light microscopy reveals that the amount of gamma-tubulin associated with the centrosome remains relatively constant throughout interphase, suddenly increases during prophase, and then decreases to interphase levels as the cell exits mitosis. This mitosis-specific recruitment of gamma-tubulin does not require microtubules. Fluorescence recovery after photobleaching (FRAP) studies reveal that the centrosome possesses two populations of gamma-tubulin: one that turns over rapidly and another that is more tightly bound. The dynamic exchange of centrosome-associated gamma-tubulin occurs throughout the cell cycle, including mitosis, and it does not require microtubules. These data are the first to characterize the dynamics of centrosome-associated gamma-tubulin in vertebrate cells in vivo and to demonstrate the microtubule-independent nature of these dynamics. They reveal that the additional gamma-tubulin required for spindle formation does not accumulate progressively at the centrosome during interphase. Rather, at the onset of mitosis, the centrosome suddenly gains the ability to bind greater than three times the amount of gamma-tubulin than during interphase.     

Expression and properties of the regulatory subunit of Dictyostelium cAMP-dependent protein kinase encoded by lambda gt11 cDNA clones

lambda gt11 phages harboring five different cDNA fragments for the regulatory (R) subunit of Dictyostelium discoideum cAMP-dependent protein kinase (CAK) directed the synthesis of this protein in Escherichia coli cells. Crude bacterial extracts were probed with an antiserum against the Dictyostelium R subunit. The presence of specific epitopes for the R subunit in a given extract was compared with high-affinity cAMP-binding activity and with the ability to inhibit the catalytic (C) subunit through protein-protein interaction. The expression and the biochemical properties of these proteins were correlated with their cDNA nucleotide sequence. The results show that the Dictyostelium R subunit can be functionally expressed in E. coli cells either as a fusion protein with beta-galactosidase or as a nonfusion protein. In both cases, the products of cDNA clones containing the entire coding sequence retained high-affinity cAMP-binding activity and the capacity to interact with the catalytic subunit. One of the fusions, lacking the 94 N-terminal residues, failed to inhibit catalytic activity, although it bound cAMP with an affinity similar to that of the native R protein from D. discoideum.     

A second functional delta5 fatty acid desaturase in the cellular slime mould Dictyostelium discoideum.

A cDNA with homology to fatty acid desaturases was selected by searching the cDNA data bank of Dictyostelium discoideum (http://www. csm.biol.tsukuba.ac.jp/cDNAproject.html) with conserved histidine box motifs. Using this sequence, genomic DNA encoding the Delta5 desaturase was amplified from the genomic DNA of D. discoideum, and its desaturase activity was confirmed by the overexpression mutation in D. discoideum and the gain-of-function mutation in yeast. The cloned cDNA is 1565 nucleotides in length, and the deduced amino-acid sequence comprised 467 amino-acid residues containing an N-terminal cytochrome b5 domain that shared 43% identity with cytochrome b5 of Oryza sativa. The whole sequence was 42% identical to the Delta5 desaturase of Mortierella alpina. This desaturase is a novel member of the cytochrome b5-containing Delta5 fatty acid desaturase. As we have already reported one other Delta5 desaturase in Dictyostelium, this organism is the first to be confirmed as having two functional Delta5 fatty acid desaturase genes. The substrate specificities of the two functional Delta5 desaturases of D. discoideum were also examined.     

Part of Ran is associated with AKAP450 at the centrosome: involvement in microtubule-organizing activity

The small Ran GTPase, a key regulator of nucleocytoplasmic transport, is also involved in microtubule assembly and nuclear membrane formation. Herein, we show by immunofluorescence, immunoelectron microscopy, and biochemical analysis that a fraction of Ran is tightly associated with the centrosome throughout the cell cycle. Ran interaction with the centrosome is mediated by the centrosomal matrix A kinase anchoring protein (AKAP450). Accordingly, when AKAP450 is delocalized from the centrosome, Ran is also delocalized, and as a consequence, microtubule regrowth or anchoring is altered, despite the persisting association of gamma-tubulin with the centrosome. Moreover, Ran is recruited to Xenopus sperm centrosome during its activation for microtubule nucleation. We also demonstrate that centrosomal proteins such as centrin and pericentrin, but not gamma-tubulin, AKAP450, or ninein, undertake a nucleocytoplasmic exchange as they concentrate in the nucleus upon export inhibition by leptomycin B. Together, these results suggest a challenging possibility, namely, that centrosome activity could depend upon nucleocytoplasmic exchange of centrosomal proteins and local Ran-dependent concentration at the centrosome.     

A protein with homology to the C-terminal repeat sequence of Octopus rhodopsin and synaptophysin is a member of a multigene family in Dictyostelium discoideum

Monoclonal antibodies were raised against a protein with a molecular mass of 24 kDa that has been described as a membrane-associated, actin binding protein from Dictyostelium discoideum [( 1985) J. Cell Biol. 100, 727-735]. Using these monoclonal antibodies we isolated from a lambda gt11 expression library cDNA clones coding for this protein. The cDNA deduced amino acid sequence revealed the presence of an unusual carboxy-terminus which has homologies to the C-termini of Octopus rhodopsin and synaptophysin. This part of the protein sequence contains 5 direct repeats with the motif GYP (P)Q(P). Southern and Northern blots showed that this sequence is present in a series of Dictyostelium genes transcribed in all stages of development.     

Dictyostelium annexin VII (synexin). cDNA sequence and isolation of a gene disruption mutant.

By cloning the cDNA coding for the membrane associated actin-binding protein p24, we identified a repetitive sequence motif consisting of the amino acids Gly, Tyr, Pro, Gln which is characteristic for a gene family in Dictyostelium discoideum. Using a cDNA probe corresponding to this motif, we isolated cDNA clones coding for a protein of the annexin family. On the basis of a long NH2-terminal sequence encompassing the Gly/Tyr/Pro/Gln motifs, the Dictyostelium annexin was identified as a homolog of vertebrate annexin VII (synexin). The mRNA coding for the Dictyostelium annexin VII has a size of 1.6 kilobases and is present during all developmental stages. Annexin VII is coded for by a single gene in Dictyostelium. A mutant deficient in annexin VII was isolated using a vector which carried the amino-terminal third of the Dictyostelium annexin VII cDNA followed by a viral epitope specific for a monoclonal antibody and a stop codon. Using this approach, homologous recombination in the annexin VII gene led to an expression of the viral epitope under the control of the endogenous annexin VII promoter. Lack of annexin VII is not a lethal event for D. discoideum, and the cells are able to undergo development on agar plates.     

Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits.

A soluble actin binding protein of Dictyostelium discoideum cells has been extracted and purified from precipitated actin-myosin complexes. This protein with a relative molecular mass of 55 kDa has been named coronin because of its association with crown-shaped cell surface projections of growth-phase D. discoideum cells. In aggregating cells, which respond most sensitively to the chemoattractant cyclic AMP, coronin is accumulated at the front where surface projections are directed towards a cAMP source. Since these cells can quickly change shape and polarity, it follows that coronin is rapidly reshuffled within the cells during motion and chemotactic orientation. The cDNA derived sequence of coronin indicates a protein of 49 kDa, consisting of an amino-terminal domain with similarities to the beta subunits of G proteins and a carboxy-terminal domain with a high tendency for alpha-helical structure. It is hypothesized that coronin is implicated in the transmission of chemotactic signals from cAMP receptors in the plasma membrane through G proteins to the cortical cytoskeleton, whose structure and activity is locally modulated.