Ectopic expression of cyclase associated protein CAP restores the streaming and aggregation defects of adenylyl cyclase a deficient Dictyostelium discoideum cells

<p>Abstract</p> <p>Background</p> <p>Cell adhesion, an integral part of <it>D. discoideum </it>development, is important for morphogenesis and regulated gene expression in the multicellular context and is required to trigger cell-differentiation. G-protein linked adenylyl cyclase pathways are crucially involved and a mutant lacking the aggregation specific adenylyl cyclase ACA does not undergo multicellular development.</p> <p>Results</p> <p>Here, we have investigated the role of cyclase-associated protein (CAP), an important regulator of cell polarity and F-actin/G-actin ratio in the <it>aca^-</it>mutant. We show that ectopic expression of GFP-CAP improves cell polarization, streaming and aggregation in <it>aca^-</it>cells, but it fails to completely restore development. Our studies indicate a requirement of CAP in the ACA dependent signal transduction for progression of the development of unicellular amoebae into multicellular structures. The reduced expression of the cell adhesion molecule DdCAD1 together with csA is responsible for the defects in <it>aca^-</it>cells to initiate multicellular development. Early development was restored by the expression of GFP-CAP that enhanced the DdCAD1 transcript levels and to a lesser extent the csA mRNA levels.</p> <p>Conclusions</p> <p>Collectively, our data shows a novel role of CAP in regulating cell adhesion mechanisms during development that might be envisioned to unravel the functions of mammalian CAP during animal embryogenesis.</p

A Cytohesin Homolog in Dictyostelium Amoebae

Dictyostelium, an amoeboid motile cell, harbors several paralogous Sec7 genes that encode members of three distinct subfamilies of the Sec7 superfamily of Guanine nucleotide exchange factors. Among them are proteins of the GBF/BIG family present in all eukaryotes. The third subfamily represented with three members in D. discoideum is the cytohesin family that has been thought to be metazoan specific. Cytohesins are characterized by a Sec7 PH tandem domain and have roles in cell adhesion and migration. Dictyostelium SecG exhibits highest homologies to the cytohesins. It harbors at its amino terminus several ankyrin repeats that are followed by the Sec7 PH tandem domain. Mutants lacking SecG show reduced cell-substratum adhesion whereas cell-cell adhesion that is important for development is not affected. Accordingly, multicellular development proceeds normally in the mutant. During chemotaxis secG(-) cells elongate and migrate in a directed fashion towards cAMP, however speed is moderately reduced. The data indicate that SecG is a relevant factor for cell-substrate adhesion and reveal the basic function of a cytohesin in a lower eukaryote

The cytohesin paralog Sec7 of Dictyostelium discoideum is required for phagocytosis and cell motility

Background: Dictyostelium harbors several paralogous Sec7 genes that encode members of three subfamilies of the Sec7 superfamily of guanine nucleotide exchange factors. One of them is the cytohesin family represented by three members in D. discoideum, SecG, Sec7 and a further protein distinguished by several transmembrane domains. Cytohesins are characterized by a Sec7-PH tandem domain and have roles in cell adhesion and migration. Results: We study here Sec7. In vitro its PH domain bound preferentially to phosphatidylinositol 3,4-bisphosphate (PI(3,4) P-2), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P-3). When following the distribution of GFP-Sec7 in vivo we observed the protein in the cytosol and at the plasma membrane. Strikingly, when cells formed pseudopods, macropinosomes or phagosomes, GFP-Sec7 was conspicuously absent from areas of the plasma membrane which were involved in these processes. Mutant cells lacking Sec7 exhibited an impaired phagocytosis and showed significantly reduced speed and less persistence during migration. Cellular properties associated with mammalian cytohesins like cell-cell and cell-substratum adhesion were not altered. Proteins with roles in membrane trafficking and signal transduction have been identified as putative interaction partners consistent with the data obtained from mutant analysis. Conclusions: Sec7 is a cytosolic component and is associated with the plasma membrane in a pattern distinctly different from the accumulation of PI(3,4,5)P-3. Mutant analysis reveals that loss of the protein affects cellular processes that involve membrane flow and the actin cytoskeleton

The centrosomal component CEP161 of Dictyostelium discoideum interacts with the Hippo signaling pathway

CEP161 is a novel component of the Dictyostelium discoideum centrosome which was identified as binding partner of the pericentriolar component CP250. Here we show that the amino acids 1-763 of the 1381 amino acids CEP161 are sufficient for CP250 binding, centrosomal targeting and centrosome association. Analysis of AX2 cells over-expressing truncated and full length CEP161 proteins revealed defects in growth and development. By immunoprecipitation experiments we identified the Hippo related kinase SvkA (Hrk-svk) as binding partner for CEP161. Both proteins colocalize at the centrosome. In in vitro kinase assays the N-terminal domain of CEP161 (residues 1-763) inhibited the kinase activity of Hrk-svk. A comparison of D. discoideum Hippo kinase mutants with mutants overexpressing CEP161 polypeptides revealed similar defects. We propose that the centrosomal component CEP161 is a novel player in the Hippo signaling pathway and affects various cellular properties through this interaction

The Cyclase-associated Protein CAP as Regulator of Cell Polarity and cAMP Signaling in Dictyostelium

Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton

Analysis of chemotactic cell motility of <i>secG⁻</i> cells.

<p>Time-lapse image series were captured and stored on a computer hard drive at 30-second intervals. Images were taken at magnifications of 10X and 40X every 6 s. The DIAS software was used to trace individual cells along image series and calculate motility parameters. Objects whose speed was <2 µm/min were excluded from the analysis. Speed refers to the speed of the cell's centroid movement along the total path; directionality indicates migration straightness; direction change refers to the number and frequency of turns; persistence is an estimation of movement in the direction of the path; and roundness indicates the cell polarity. Values are mean ± standard deviation of >30 cells from three or more independent experiments. The difference in speed was statistically significant (P value 0.0416* and 0.012**).</p

Cell-substratum adhesion in <i>secG⁻</i> cells.

<p>Adhesion of vegetative cells was measured. After a 4 hour incubation period the cells were subjected to rotation at 65 rpm on a gyratory shaker. The number of detached cells was determined after one hour of shaking and set in relation to the total number of cells. AX2 wild type and three different mutant cell lines were examined. The results are from 23 independent experiments for AX2 and a total of 39 experiments for the mutant cell lines. Shown are the mean values and the mean deviations. The P values (secG-1, 0.0721; secG-5, 0.0903; secG-9; 0.0964) were not quite statistically significant.</p

The Sec7 family of the <i>Dictyostelia</i>.

<p>The Sec7 family members in three taxonomic divisions of the <i>Dictyostelia</i> are listed. The <i>D. discoideum</i> genome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009378#pone.0009378-Eichinger1" target="_blank">[11]</a> was searched for proteins containing a Sec7 domain (dictyBase, <a href="http://dictybase.org/index.html" target="_blank">http://dictybase.org/index.html</a>). The families were classified according to Casanova <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009378#pone.0009378-Casanova1" target="_blank">[46]</a>. The domains listed were identified by Blast searches (<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a>). The <i>D. discoideum</i> (<i>D. d.</i>) proteins were then used to search for homologues in the <i>D. falciparum</i> (<i>D. f.</i>) and <i>P. pallidum</i> (<i>P. p.</i>) genomes at <a href="http://sacgb.fli-leibniz.de/cgi/index.pl" target="_blank">http://sacgb.fli-leibniz.de/cgi/index.pl</a>. <i>D. falciparum</i> belongs to group 1, <i>P. pallidum</i> is a member of group 2 and <i>D. discoideum</i> (<i>D. d.</i>) is a member of group 4. The homology search was done by Blast. DUF1981, DUF2339, domains of unknown function, present in predicted membrane proteins. MFS, Major-Facilitator-Superfamily, group of membrane proteins; TM, transmembrane; DUF1981, <u>d</u>omain of <u>u</u>nknown <u>f</u>unction, present in predicted membrane proteins, PH, PH-domain.</p