An early stage in the establishment of cell polarity during chemotaxis of Dictyostelium dicoideum has been identified by a recent study; the new results also show that the development of cell polarity does not rely upon cytoskeletal rearrangement, and may use a spatial sensing mechanism. (+info)
Myosin II-independent F-actin flow contributes to cell locomotion in dictyostelium.
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While the treadmilling and retrograde flow of F-actin are believed to be responsible for the protrusion of leading edges, little is known about the mechanism that brings the posterior cell body forward. To elucidate the mechanism for global cell locomotion, we examined the organizational changes of filamentous (F-) actin in live Dictyostelium discoideum. We labeled F-actin with a trace amount of fluorescent phalloidin and analyzed its dynamics in nearly two-dimensional cells by using a sensitive, high-resolution charge-coupled device. We optically resolved a cyclic mode of tightening and loosening of fibrous cortical F-actin and quantitated its flow by measuring temporal and spatial intensity changes. The rate of F-actin flow was evaluated with respect to migration velocity and morphometric changes. In migrating monopodial cells, the cortical F-actin encircling the posterior cell body gradually accumulated into the tail end at a speed of 0.35 microm/minute. We show qualitatively and quantitatively that the F-actin flow is closely associated with cell migration. Similarly, in dividing cells, the cortical F-actin accumulated into the cleavage furrow. Although five times slower than the wild type, the F-actin also flows rearward in migrating mhcA- cells demonstrating that myosin II ('conventional' myosin) is not absolutely required for the observed dynamics of F-actin. Yet consistent with the reported transportation of ConA-beads, the direction of observed F-actin flow in Dictyostelium is conceptually opposite from a barbed-end binding to the plasma membrane. This study suggests that the posterior end of the cell has a unique motif that tugs the cortical actin layer rearward by means of a mechanism independent from myosin II; this mechanism may be also involved in cleavage furrow formation. (+info)
The Dictyostelium developmental cDNA project: generation and analysis of expressed sequence tags from the first-finger stage of development.
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In an effort to identify and characterize genes expressed during multicellular development ill Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism. (+info)
Interaction of 5-lipoxygenase with cellular proteins.
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5-Lipoxygenase (5LO) plays a pivotal role in cellular leukotriene synthesis. To identify proteins interacting with human 5LO, we used a two-hybrid approach to screen a human lung cDNA library. From a total of 1.5 x 10(7) yeast transformants, nine independent clones representing three different proteins were isolated and found to specifically interact with 5LO. Four 1.7- to 1.8-kb clones represented a 16-kDa protein named coactosin-like protein for its significant homology with coactosin, a protein found to be associated with actin in Dictyostelium discoideum. Coactosin-like protein thus may provide a link between 5LO and the cytoskeleton. Two other yeast clones of 1.5 kb encoded transforming growth factor (TGF) type beta receptor-I-associated protein 1 partial cDNA. TGF type beta receptor-I-associated protein 1 recently has been reported to associate with the activated form of the TGF beta receptor I and may be involved in the TGF beta-induced up-regulation of 5LO expression and activity observed in HL-60 and Mono Mac 6 cells. Finally, three identical 2.1-kb clones contained the partial cDNA of a human protein with high homology to a hypothetical helicase K12H4. 8 from Caenorhabditis elegans and consequently was named DeltaK12H4. 8 homologue. Analysis of the predicted amino acid sequence revealed the presence of a RNase III motif and a double-stranded RNA binding domain, indicative of a protein of nuclear origin. The identification of these 5LO-interacting proteins provides additional approaches to studies of the cellular functions of 5LO. (+info)
Comparison of base specificity and other enzymatic properties of two protozoan ribonucleases from Physarum polycephalum and Dictyostelium discoideum.
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Base specificity and other enzymatic properties of two protozoan RNases, RNase Phyb from a true slime mold (Physarum polycephalum) and RNase DdI from a cellular slime mold (Dictyostelium discoideum), were compared. These two RNases have high amino acid sequence similarity (83 amino acid residues, 46%). The base specificities of two base recognition sites, The B1 site (base recognition site for the base at 5'-side of scissile phosphodiester bond) and the B2 site (base recognition site for the base at 3'-side of the scissile bond) of the both enzymes were estimated by the rates of hydrolysis of 16 dinucleoside phosphates. The base specificities estimated of B1 and B2 sites of RNase Phyb and RNase DdI were A, G, U > C and A > or = G > C > U, and A > or = G, U > C and G > U > A, C, respectively. The base specificities estimated from the depolymerization of homopolynucleotides and those from the releases of four mononucleotides upon digestion of RNA coincided well with those of the B2 sites of both enzymes. Thus, in these enzymes, the contribution of the B2 site to base specificity seems to be larger than that of the B1 site. pH-stability, optimum temperature, and temperature stability, of both enzymes are discussed considering that RNase Phyb has one disulfide bridge deleted, compared to the RNase DdI with four disulfide bridges. (+info)
Multiple developmental roles for CRAC, a cytosolic regulator of adenylyl cyclase.
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Receptor-mediated activation of adenylyl cyclase (ACA) in Dictyostelium requires CRAC protein. Upon translocation to the membrane, this pleckstrin homology (PH) domain protein stimulates ACA and thereby mediates developmental aggregation. CRAC may also have roles later in development since CRAC-null cells can respond to chemotactic signals and participate in developmental aggregation when admixed with wild-type cells, but they do not complete development within such chimeras. To test whether the role of CRAC in postaggregative development is related to the activation of ACA, chemotactic aggregation was bypassed in CRAC-null cells by activating the cAMP-dependent protein kinase (PKA). While such strains formed mounds, they did not complete fruiting body morphogenesis or form spores. Expression of CRAC in the prespore cells of these strains rescued sporulation and fruiting body formation. This later function of CRAC does not appear to require its PH domain since the C-terminal portion of the protein (CRAC-DeltaPH) can substitute for full-length CRAC in promoting spore cell formation and morphogenesis. No detectable ACA activation was observed in any of the CRAC-null strains rescued by PKA activation and expression of CRAC-DeltaPH. Finally, we found that the development of CRAC-null ACA-null double mutants could be rescued by the activation of PKA together with the expression of CRAC-DeltaPH. Thus, there appears to be a required function for CRAC in postaggregative development that is independent of its previously described function in the ACA activation pathway. (+info)
The mitochondrial adenine nucleotide translocator from Dictyostelium discoideum. Functional characterization and DNA sequencing.
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The mitochondrial adenine nucleotide translocator (ANT) catalyses the exchange of ATP and ADP between the mitochondria and the cytosol. We have cloned and sequenced the gene encoding the Dictyostelium discoideum ANT (DdANT) and analysed its transcriptional regulation. The single copy D. discoideum ant gene encodes a protein of 309 amino acid residues with a predicted molecular mass of 33,469 Da and a pI of 9.85. These values are comparable to those of ANTs from mammals, insects and fungi. The long N-terminal extension characteristic of plant ANT is absent in DdANT. The protein coding region of the D. discoideum ant gene is interrupted by three introns. Polyclonal antibodies directed against the beef heart mitochondrial ANT or its C-terminal peptide recognized the D. discoideum protein. Northern blot analysis revealed that the expression of the D. discoideum ant gene decreased rapidly during the first hours of multicellular development but the amount of protein remained stable throughout differentiation. (+info)
Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins in Dictyostelium discoideum.
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The soil amoeba Dictyostelium discoideum is a host cell that provides simple genetics in combination with complex protein synthesis. We show that the complex human heterodimeric gonadotropins can be produced and secreted by this organism. Furthermore, both follicle stimulation hormone and choriogonadotropin produced by D. dictyostelium bind to their human receptors and elicit a biological response comparable to the wild-type hormones. We also show that structure-function analysis using random mutagenesis and screening of recombinant glycoprotein hormones is feasible. Thus, expression of gonadotropins in D. dictyostelium opens the way to the engineering of potential new therapeutic analogues. (+info)