A Rho GDP-dissociation inhibitor is involved in cytokinesis of Dictyostelium

Myotubularin-related genes define a novel highly conserved family of eukaryotic proteins of at least 11 human members. The hMTM1 gene that codes for myotubularin is mutated in X-linked myotubular myopathy, a severe congenital disease. Recently, we and others have characterized myotubularin as a potent and specific phosphatidylinositol 3-phosphate 3-phosphatase. In the present study we investigated the lipid phosphatase activity and the subcellular localization of two other members of the family, hMTMR2 protein that is mutated in the demyelinating neuropathy Charcot-Marie-Tooth type 4B and the FYVE-finger containing hMTMR3 protein. Our results show that both proteins are potent phosphatidylinositol 3-phosphate 3-phosphatases either in vitro or in yeast where they interfered with vesicular trafficking. Their localization is mainly cytoplasmic, with however strong labeling of Rac-inducible plasma membrane ruffles. The fact that the ubiquitously expressed hMTM1 and hMTMR2 genes are involved in different pathologies indicates that despite their shared enzymatic activity, they are not functionally redundant, at least in certain cell types. This might be explained by subtle differences in expression and/or in recruitment and regulation at their specific site of action.     

Re-expression of proteins involved in cytokinesis during cardiac hypertrophy

Cardiomyocytes stop dividing after birth and postnatal heart growth is only achieved by increase in cell volume. In some species, cardiomyocytes undergo an additional incomplete mitosis in the first postnatal week, where karyokinesis takes place in the absence of cytokinesis, leading to binucleation. Proteins that regulate the formation of the actomyosin ring are known to be important for cytokinesis. Here we demonstrate for the first time that small GTPases like RhoA along with their downstream effectors like ROCK I, ROCK II and Citron Kinase show a developmental stage specific expression in heart, with high levels being expressed in cardiomyocytes only at stages when cytokinesis still occurs (i.e. embryonic and perinatal). This suggests that downregulation of many regulatory and cytoskeletal components involved in the formation of the actomyosin ring may be responsible for the uncoupling of cytokinesis from karyokinesis in rodent cardiomyocytes after birth. Interestingly, when the myocardium tries to adapt to the increased workload during pathological hypertrophy a re-expression of proteins involved in DNA synthesis and cytokinesis can be detected. Nevertheless, the adult cardiomyocytes do not appear to divide despite this upregulation of the cytokinetic machinery. The inability to undergo complete division could be due to the presence of stable, highly ordered and functional sarcomeres in the adult myocardium or could be because of the inefficiency of degradation pathways, which facilitate the division of differentiated embryonic cardiomyocytes by disintegrating myofibrils.     

Rap1-dependent pathways coordinate cytokinesis in Dictyostelium

Cytokinesis is the final step of mitosis when a mother cell is separated into two daughter cells. Major cytoskeletal changes are essential for cytokinesis; it is, however, not well understood how the microtubules and actomyosin cytoskeleton are exactly regulated in time and space. In this paper, we show that during the early stages of cytokinesis, in rounded-up Dictyostelium discoideum cells, the small G-protein Rap1 is activated uniformly at the cell cortex. When cells begin to elongate, active Rap1 becomes restricted from the furrow region, where the myosin contractile ring is subsequently formed. In the final stages of cytokinesis, active Rap1 is only present at the cell poles. Mutant cells with decreased Rap1 activation at the poles showed strongly decreased growth rates. Hyperactivation of Rap1 results in severe growth delays and defective spindle formation in adherent cells and cell death in suspension. Furthermore, Rap mutants show aberrant regulation of the actomyosin cytoskeleton, resulting in extended furrow ingression times and asymmetrical cell division. We propose that Rap1 drives cytokinesis progression by coordinating the three major cytoskeletal components: microtubules, actin, and myosin II. Importantly, mutated forms of Rap also affect cytokinesis in other organisms, suggesting a conserved role for Rap in cell division.     

Chemotaxis-mediated scission contributes to efficient cytokinesis in Dictyostelium

Interphase amoeba of Entamoeba invadens are attracted to the furrowing region of a neighboring dividing cell to assist with the division. A seemingly similar behavior has been observed in Dictyostelium discoideum, but in this case, it has not been shown whether the movements were truly directed toward the furrowing region or whether they have any relevance. We thus used myosin II-null cells, which spend more time than wild type cells in cytokinesis, and successfully demonstrated that nearly half of the division events involve the attraction of a neighbor cell to the furrowing region. Cells lacking the beta subunit of the trimeric G protein (Gbeta), which are incapable of chemotaxis, did not show such midwifery. Culturing wild type cells flattened under agarose sheets also slowed the cytokinesis process, and this allowed us to demonstrate that phosphatidylinositol trisphosphate was enriched in the anterior region of midwifing cells, consistent with the view that midwifery in D. discoideum is also chemotaxis. On substrates, while only 3.6% of wild type cells were multinucleate, 8.1% of Gbeta-null cells were multinucleate, and this was reduced to 3.4% when they were surrounded by wild type cells. Conversely, multinucleated wild type cells increased to 6.8% when they were surrounded by Gbeta-null cells. Thus, Gbeta-null cells frequently fail to divide because they cannot assist each other's division and midwifery ensures successful cytokinesis in Dictyostelium discoideum.     

Identification of multiple cyclic AMP-binding proteins in developing Dictyostelium discoideum cells

We have purified two cAMP-binding proteins from developing Dictyostelium discoideum cells, which we designate as CABP-1 and CABP-2. Purified CABP-1 consists of two polypeptides of Mr 41,000 and 36,000, which we refer to as CABP-1A and CABP-1B, respectively. Although CABP-1 exhibited specificity for cAMP, it was not labeled at a detectable level when mixed with 8-azidoadenosine 3':5'-monophosphate (8-N3[3H]cAMP). Unlike CABP-1, CABP-2 was labeled efficiently with 8-N3[3H]cAMP. Purified CABP-2 has a molecular weight of 41,000 and an isoelectric point of 5.8-6.0. The physical and biochemical properties of CABP-2 suggest that it is the regulatory subunit of cAMP-dependent protein kinase described by others (de Gunzburg, J., Part, D., Guiso, N., and Veron, M. (1984) Biochemistry 23, 3805-3812; Majerfeld, J. H., Leichtling, B. H., Maligeni, J. A., Spitz, E., and Rickenberg, H. V. (1984) J. Biol. Chem. 259, 654-661). Although CABP-1A and CABP-2 have the same molecular weight, they appear to be encoded by different genes. Two-dimensional gel electrophoresis revealed that the two polypeptides had different isoelectric points. Moreover, monoclonal antibodies raised against CABP-1 did not cross-react with CABP-2. Also, in vitro translation followed by immunoprecipitation showed that these two polypeptides were derived from primary translation products. Our finding of a novel cAMP-binding protein, CABP-1, suggests that cAMP-dependent protein kinase may not be the only intracellular regulator mediating the effects of cAMP in developing D. discoideum cells.     

Identification of proteins involved in microglial endocytosis of alpha-synuclein

Aggregated alpha-synuclein, a protein playing pivotal roles in the pathogenesis of Parkinson disease (PD) and related synucleinopathy, has been shown to activate microglia, the key cells in neuroinflammation. However, the mechanisms by which aggregated alpha-synuclein enters microglia remain uncharacterized. In this study, we first replicated our previous results with a modified protocol that generated aggregated alpha-synuclein more efficiently. Next, using two recently developed proteomic techniques, SILAC (Stable Isotope Labeling of Amino acid in Cell cultures) and PROCEED (PROteome of Cell Exposed Extracellular Domains), we studied the plasma membrane proteins of primary cultured microglia that might be interacting with aggregated alpha-synuclein and mediating its internalization. The results demonstrated that 250 nM alpha-synuclein, aged for 6 h with a magnetic stir bar, was just as potent in activating microglia as the aggregated alpha-synuclein produced by aging without constant agitation for 7 days. The proteomic analysis identified 111 membrane proteins; of these, 46 proteins were altered in relative abundance in the membrane compartment after treatment with aggregated alpha-synuclein for 3 h. Two of these proteins, clathrin and calnexin, were further evaluated with Western blotting, demonstrating good agreement with quantitative proteomics. Finally, immunocytochemical as well as co-immunoprecipitation studies indicated that clathrin was indeed co-localized with internalized alpha-synuclein in microglia. These results suggest for the first time that microglial activation secondary to internalization of aggregated alpha-synuclein likely requires participation of clathrin, which is an essential protein of the polyhedral coat of coated pits and vesicles that play major roles in endocytosis and vesicular trafficking.     

Myosin II-independent cytokinesis in Dictyostelium: its mechanism and implications

Similar to higher animal cells, ameba cells of the cellular slime mold Dictyostelium discoideum form contractile rings containing filaments of myosin II during mitosis, and it is generally believed that contraction of these rings bisects the cells both on substrates and in suspension. In suspension, mutant cells lacking the single myosin II heavy chain gene cannot carry out cytokinesis, become large and multinucleate, and eventually lyze, supporting the idea that myosin II plays critical roles in cytokinesis. These mutant cells are however viable on substrates. Detailed analyses of these mutant cells on substrates revealed that, in addition to "classic" cytokinesis which depends on myosin II ("cytokinesis A"), Dictyostelium has two distinct, novel methods of cytokinesis, 1) attachment-assisted mitotic cleavage employed by myosin II null cells on substrates ("cytokinesis B"), and 2) cytofission, a cell cycle-independent division of adherent cells ("cytokinesis C"). Cytokinesis A, B, and C lose their function and demand fewer protein factors in this order. Cytokinesis B is of particular importance for future studies. Similar to cytokinesis A, cytokinesis B involves formation of a cleavage furrow in the equatorial region, and it may be a primitive but basic mechanism of efficiently bisecting a cell in a cell cycle-coupled manner. Analysis of large, multinucleate myosin II null cells suggested that interactions between astral microtubules and cortices positively induce polar protrusive activities in telophase. A model is proposed to explain how such polar activities drive cytokinesis B, and how cytokinesis B is coordinated with cytokinesis A in wild type cells.     

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.     

Identification of the cell surface molecule involved in sexual cell fusion of Dictyostelium discoideum

Dictyostelium discoideum was used as a model system for elucidating the molecular mechanism of sexual cell fusion. In heterothallic strains NC4 and HM1 of D. discoideum, complements in mating type, amoeboid cells acquire fusion competence only under certain environmental conditions, such as the presence of excess water and a certain period of darkness, to fuse sexually. The surface of cells which acquired fusion competence was found to possess specific antigens. Monovalent antibodies prepared from rabbit antiserum against fusion-competent NC4 cells inhibit the sexual cell fusion of these cells completely. Two specific antigenic proteins, 39 and 138 k Da in relative molecular mass and specific for fusion-competent cells, were detected. Only one, the 138-k Da protein, was capable of neutralizing the fusion-inhibitory activity of the monovalent antibody. These results show that the 139-k Da protein is the one involved in the sexual cell fusion of NC4 and HM1 strains in D. discoideum.     

Identification of key proteins involved in the anammox reaction

Bacteria performing anaerobic ammonium oxidation (anammox) are key players in the global nitrogen cycle due to their inherent ability to convert biologically available nitrogen to N₂. Anammox is increasingly being exploited during wastewater treatment worldwide, and about 50% of the total N₂ production in marine environments is estimated to proceed by the anammox pathway. To fully understand the microbial functionality and mechanisms that control environmental feedbacks of the anammox reaction, key proteins involved in the reaction must be identified. In this study we have utilized an analytical protocol that facilitates detection of proteins associated with the anammoxosome, an intracellular membrane compartment within the anammox bacterium. The protocol enabled us to identify several key proteins of the anammox reaction including a hydrazine hydrolase producing hydrazine, a hydrazine-oxidizing enzyme converting hydrazine to N₂ and a membrane-bound ATP synthase generating ATP from the gradients of protons formed in the anammox reaction. We also performed immunogold labelling electron microscopy to determine the subcellular location of the hydrazine hydrolase. The results from our study support the hypothesis that proteins associated with the anammoxosome host the complete suite of reactions during anammox.     

Identification of carbohydrate moieties involved in EDTA-stable or EDTA-sensitive cell contact of Dictyostelium discoideum

Antisera against purified contact site A glycoprotein, with an apparent molecular weight of 80 X 10(3) (80 kDa), from Dictyostelium discoideum were raised by using Freund's adjuvant (antiserum-A) and by using Alu-Gel-S (antiserum-B) as immunoadjuvants. They were converted into Fab fragments for the cell agglutination assay. Fab fragments of antiserum-B inhibited only EDTA-stable cell contact, whereas Fab fragments of antiserum-A (Fab-A) inhibited EDTA-sensitive cell contact as well as EDTA-stable cell contact. We prepared several cell types in order to identify target antigens for the adhesion-blocking Fab-A in EDTA-sensitive cell contact or EDTA-stable cell contact. One of these cell types produced contact site A without N-glycosidically-linked carbohydrate chains. It is known that contact site A contains two kinds of N-glycosidically-linked carbohydrate chains (carbohydrates I and II, Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984) EMBO J. 3, 2653-2670). When growth-phase cells were treated with tunicamycin (TM) at a final concentration of 2 micrograms/ml in nutrient medium (TM-pretreated cells), the cells produced contact site A without N-glycosidically-linked carbohydrate chains (53 kDa) at the normal developmental stage. These cells lacked EDTA-sensitive cell contact as well as EDTA-stable cell contact. The neutralization of the adhesion-blocking Fab-A was done by using particulate fractions from each cell type. The blocking activity in EDTA-stable cell contact was neutralized by the cell type with carbohydrate II. Taking these results into consideration, EDTA-stable cell contact may be formed by the interaction between protein moieties of contact site A and carbohydrate II. Concerning EDTA-sensitive cell contact, the blocking activity was neutralized by each cell type irrespective of TM treatment. This suggests that O-glycosidically-linked carbohydrate chains play a role in EDTA-sensitive cell contact. Moreover, the biological activity in EDTA-sensitive cell contact of TM-pretreated cells suggests that N-glycosidically-linked carbohydrate chains may also be involved in this contact.     

Identification of serum proteins involved in pancreatic cancer cachexia

AimsTreatment of cachexia requires pharmacological intervention which, in turn, requires knowledge of the mediators and processes. Cachexia markers that are specifically expressed in pancreatic cancer and secreted into the blood circulation have yet to be identified. The aim of our study was to investigate the serum protein profiles and protein alterations associated with cachexia and to identify potential disease protein biomarkers indicative for this syndrome.Main methodsSerum samples from cachectic and non-cachectic patients undergoing pancreatic cancer (PaCa) surgery and controls were investigated by Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). The identity of detected discriminatory markers was determined by a combination of protein fractionation, chromatographic purification steps, gel electrophoresis, and mass spectrometry.Key findingsUsing Cu-IMAC array and CM-10 array based SELDI-TOF-MS. we identified eleven up- and four down-regulated proteins associated with cachexia. CiphergenExpress analysis revealed four disease-associated protein features (38559 Da, 9138 Da, 8925 Da and 3358 Da) that were elevated by a factor of 2.3, 1.7, 1.4 and 1.4, respectively. Zinc-α2-glycoprotein (ZAG), apolipoproteins apo C-II and apo C-III and glucagon-like peptide-1 (GLP-1) were identified as markers for PaCa-associated cachexia syndrome. ZAG levels were additionally evaluated in serum and tissue samples by ELISA and immunohistochemistry and the obtained data confirmed the SELDI-TOF-MS results.SignificanceThe identified proteins could be routinely and reliably measured in the serum of patients and provide an elegant non-invasive approach for early diagnosis of cachectic pancreatic cancer patients. Controlling ZAG and GLP-1 activity could be beneficial in the management of cancers and cachexia-induced conditions.     

Identification and characterization of developmentally regulated mRNP proteins of Dictyostelium discoideum

The isolation of poly(A)+ polysomal and nonpolysomal RNPs by oligo(dT)-cellulose chromatography has led to the identification of more than 20 polypeptides that bind to the poly(A)+ mRNA in growing Dictyostelium cells. Most of these polypeptides were identified in experiments using short-wave UV light (254 nm) to crosslink specifically bound proteins to the RNA. Digestion of the RNPs with ribonucleases A and T1 prior to their application to oligo(dT)-cellulose permitted the isolation of the 3' poly(A)-protein complexes. In polysomal RNPs, two major polypeptides, with molecular weights of 31,000 (p31) and 31,500 (p31.5), are bound to poly(A). These proteins can also be purified from cytoplasmic extracts by affinity chromatography on poly(A)-Sepharose. Partial proteolytic digestion of p31 and p31.5 indicates that they are closely related. The UV-crosslinking experiments established that p31 and p31.5 bind to the non-poly(A) segments of mRNA as well. In nonpolysomal RNPs, p31 and a polypeptide with a molecular weight of 29,500 (p29.5) are the major species associated with poly(A). Partial proteolytic digestion of p29.5 indicates that it is closely related to p31 and p31.5. Only small amounts of p29.5 were observed in the polysomal poly(A)-protein complexes. Early in Dictyostelium development, when cellular translation activity is sharply reduced, most of the p29.5, p31 and p31.5 present is selectively degraded. These observations are consistent with a translational role for these proteins.     

裂殖酵母Cnb1参与胞质分裂过程

丝/苏氨酸特异性钙调磷酸酶(Calcineurin, CN)是一种在真核生物中广泛存在的蛋白, 是参与转录调控的重要分子。裂殖酵母中的CN是由催化亚基Ppb1和调节亚基Cnb1组成的异源二聚体。文章报道了裂殖酵母中cnb1+的缺失引起细胞生长速度缓慢, 产生多隔膜现象, 胞质分裂受阻滞。胞质分裂过程中, Cnb1与Ppb1组成CN复合物, 与收缩环在分裂平面上共定位, 并与收缩环一起收缩。cnb1Δ菌株的隔膜成熟过程存在缺陷, 微管出现纵穿隔膜的现象。上述结果说明Cnb1可能参与隔膜的成熟过程。此外, 还检测了cnb1D菌株中胞裂蛋白的信号。胞裂蛋白包括Spn1、Spn2、Spn3和Spn4, 它们是引导隔膜降解的重要分子。结果显示, 在cnb1D菌株中, 80%左右的细胞在隔膜处缺失Spn2和Spn3的信号, 20%左右的细胞缺失Spn1和Spn4的信号。由于胞裂蛋白的蛋白表达量在cnb1D中没有降低, 因此胞裂蛋白信号的消失不是转录缺陷引起的, 这暗示Cnb1可能采用了不依赖转录的方式来调控胞裂蛋白环的稳定性。以上结果提示, Cnb1可能通过影响隔膜的成熟及胞裂蛋白环的稳定性参与调节裂殖酵母的胞质分裂过程。     

Identification and localization of proteins encoded by two DIF-inducible genes of Dictyostelium

We show that pDd56 and pDd63, two related DIF-inducible genes of Dictyostelium, respectively encode the ST310 and ST430 polypeptides identified by Morrissey, Devine, and Loomis (1984, Dev. Biol. 103, 414-424). We localize the two proteins by immunoelectron microscopy to the extracellular matrix surrounding the stalk cells and the stalk tube. Coupled with their predicted amino acid sequence and biochemical properties, this suggests that they are structural proteins of the stalk.     

Actin-associated proteins in Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum is becoming the premier system for the explication of the biochemical and cellular events that occur during motile processes. Proteins associated with the actin cytoskeleton, in particular, appear to play key roles in cellular responses to many external stimuli. This review summarizes our present understanding of the actin-associated proteins in Dictyostelium, including their in vitro activities and their structural and/or functional analogues in mammalian cells.     

Cell division in Dictyostelium with special emphasis on actomyosin organization in cytokinesis

This study focuses on the dynamic reorganization of actin and myosin ("conventional" myosin, or myosin-II) during cytokinesis in D. discoideum. This is the first study identifying the birefringence of the spindle microtubules as well as three sets of microfilamentous structure in Dictyostelium. The change of organization in these fibrillar structures was followed in real-time with video microscopy, using a Universal Polarizing Microscope equipped with polarized-light (POL) and differential interference contrast (DIC) optics combined with digital image processing. High-frequency mitotic cells were obtained by semi-synchronous culture, and high-resolution observations were made by utilizing the agar-overlay method (Yumura et al.: Journal of Cell Biology 99:894-899, 1984). The molecular identity of the birefringent structures was determined by fluorescence microscopy. Through-focus observations were performed with an axial resolution of 0.3 micron depth of field. The actomyosin fibrils show a dramatic reorganization throughout mitosis. The fibrils at the leading lamellipodia disappear, and there is a striking assembly of the cortical actomyosin in pro-metaphase, which is accompanied by a decrease in cell volume. The cortical actomyosin gradually increases through anaphase. After late anaphase, very active polar lamellipodia, with an average life of less than 1 minute, are formed. We confirmed that the polar lamellipodia include actin, but not myosin-II. At the cleavage furrow, the microfilaments form two distinctive structures: circular contractile ring at the equator, and a cortical filament array parallel to the polar axis. Myosin is localized in the contractile ring, but not associated with the axial array of F-actin. Actomyosin in the contractile ring gradually transforms into cortical network at the posterior region of daughter cells. The constriction of the furrow is accompanied by a drastic efflux of water as evidenced by highly active contractile vacuole formation and turbulent motion of minute vesicles connected to the furrow. This study demonstrates the presence of a new microfilament structure, as well as the dynamic property of the contractile ring, and sheds new light on the contractile mechanisms underlying cytokinesis.     

Genes involved in Dictyostelium discoideum sexual reproduction

Macrocyst formation in the cellular slime moulds is a sexual process induced under dark and humid conditions. Normal development life cycle in these organisms involves proliferation by cell division and, upon starvation, formation of multicellular aggregates and fruiting bodies, consisting of spores and stalk cells. Macrocyst formation, cell division by binary fission and spore formation are thus three alternative modes of reproduction, for which it is of interest to understand how a choice is made. The genetic basis of asexual development and fruiting body formation is well known, by contrast information on the genetic control of sexual reproduction during macrocyst formation is scarce. In Dictyostelium discoideum, the most widely used species, several cell-surface proteins relevant to sexual cell fusion have been identified using cell fusion-blocking antibodies, but isolation of the relevant genes has been unsuccessful. Analysis of sexually deficient mutants, some of which are normal for asexual development, has shown that sexual reproduction is regulated by both specific genes and genes that are also involved in asexual development. Reverse genetic analysis of 24 genes highly enriched in a gamete-specific subtraction library has revealed four genes involved in the regulation of sexual cell interactions. One of them was found to be a novel regulator of the cAMP signalling pathway specific to sexual development. Studies on the molecular genetic control of the sexual cycle will be reviewed and their contribution to our understanding of the organization and function of the D. discoideum genome as a whole discussed.     

The KEULE gene is involved in cytokinesis in Arabidopsis

We present evidence to show that the KEULE gene of Arabidopsis is involved in cytokinesis. Mutant keule embryos have large multinucleate cells with gapped or incomplete cross walls, as well as cell wall stubs that are very similar to those observed upon caffeine inhibition of cytokinesis in plants. These defects are observed in all populations of dividing cells in the mutant, including calli, but less frequently in mature cells. Cell division appears to be slowed down, and the planes of cell division are often misoriented. In late embryos and seedlings, cross-wall formation usually appears complete, suggesting that the requirement for KEULE during cytokinesis is not absolute. Nonetheless, keule mutants die as seedlings with large polyploid cells. The bloated surface layer of keule seedlings does not uniformly behave like wild-type epidermis, and patches of this layer assume characteristics of the underlying ground tissue. The cytokinesis defect of keule mutants may influence aspects of cellular differentiation.