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.2010 Feb 23;5(2):e9378.
doi: 10.1371/journal.pone.0009378.

A cytohesin homolog in Dictyostelium amoebae

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A cytohesin homolog in Dictyostelium amoebae

Maria Christina Shina et al. PLoS One..

Abstract

Background: 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.

Principal findings: 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.

Significance: 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.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Homology of the Sec7 domain.
A. Phylogenetic tree of the Sec7 domains inD. discoideum (red),P. pallidum (green) andD. fasciculatum (blue) proteins. The Sec7 domain of human cytohesin-1 (NP_004753, GI:4758964) was used for comparison. The tree was generated from a CLUSTALX alignment with the TreeView program. The assignment of the different proteins to the three subfamilies of the Sec7 family of the Dictyostelia is indicated on the right. The bootstrap support of each node is provided. The scale bar indicates 10% divergence.B Alignment of the Sec7 domains. At the top, the Sec7 domain of human cytohesin-1 is shown. The alignment was generated with the program Multalin (http://bioinfo.genotoul.fr/multalin/multalin.html). The color code and the symbols represent: upper case  =  high homology (red); lower case  =  lower homology (blue); !  =  mostly I, V; $  =  mostly L, M; %  =  mostly F, Y; #  =  mostly D, E, N, Q. The invariable E involved in nucleotide exchange on Arf is indicated by an arrow.
Figure 2
Figure 2. Distribution of GFP-SecG in living cells.
Localization of GFP-tagged full length SecG analyzed by live-cell imaging. Three frames taken at the indicated time points after start of the analysis are shown.
Figure 3
Figure 3. Generation of secG deficient cells.
A. Upper part: SecG domain structure. The domain structure of Sec G aligned along the nucleotide and amino acid sequence is schematically depicted.A. Lower part: Generation of a gene replacement vector. The vector was constructed by replacing an internal segment of the secG gene extending from position 576 to 2083 with the blasticidin resistance cassette (bsr). The secG gene is located on a 16.5 kb NdeI genomic fragment. Location of relevant restriction enzyme sites and of the probe used for Southern blot analysis is given.B. Southern blot analysis of XhoI/NdeI digested genomic DNA of individual transformants 1, 2, 3, 5, and 9. After successful replacement a shift from 16.5 kb for the wild type band (transformants 2, 3) down to 3.5 kb (transformants 1, 5, 9) occurred as detected by a 3′ gene specific probe.C. Northern blot analysis. InsecG− transformants (1, 9) the ∼4 kb mRNA of AX2 wild type is no longer detected using the 350 bp 3′ probe. For control of loading, the blot was probed with a ddFLN specific probe recognizing the ∼3.5 kb mRNA .
Figure 4
Figure 4. Cell-substratum adhesion insecG− cells.
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.
Figure 5
Figure 5. Distribution of labelled AX2 cells in a chimeric developmental organism.
AX2 cells expressing GFP-LimD were mixed withsecG− cells in a ratio of 10∶90. The GFP-labelled wild type cells show a random distribution at the multicellular stage (culminant). The phase contrast (A) and the fluorescence (B) image are shown.
Figure 6
Figure 6. Chemotaxis of aggregation competent cells.
AX2 wild type andsecG− cell lines 1 and 5 were subjected to a chemotaxis assay. Cells were traced from the movies and analysed by DIAS software . Representative stacked images are shown. The star indicates the location of the cAMP source.
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