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

Chemotaxis of aggregation competent cells.

<p>AX2 wild type and <i>secG⁻</i> cell lines 1 and 5 were subjected to a chemotaxis assay. Cells were traced from the movies and analysed by DIAS software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009378#pone.0009378-Wessels1" target="_blank">[41]</a>. Representative stacked images are shown. The star indicates the location of the cAMP source.</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

Generation of secG deficient cells.

<p><b>A</b>. Upper part: SecG domain structure. The domain structure of Sec G aligned along the nucleotide and amino acid sequence is schematically depicted. <b>A.</b> 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>B.</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. <b>C.</b> Northern blot analysis. In <i>secG⁻</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009378#pone.0009378-Brink1" target="_blank">[45]</a>.</p

Distribution of GFP-SecG in living cells.

<p>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.</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

Putative nuclear export signals.

<p>The signals according to Wada et al. (1998) have been identified manually. Sequences in brackets had too weak characteristics to define them as explicit nuclear export signals.</p

The <i>D. discoideum</i> actinome.

<p>41 members of the actinome were identified according to their actin sequence profile. 17 conventional actins share identical amino acid sequences but are encoded by 17 distinct genes (Act8 group, right panel). 17 actins share high homologies to conventional actin but are different in their protein sequences (left panel, #1, 3–17). Nine members of the actinome are conserved actin-related proteins (Arps, #18–25).</p

Occurrence of Arps in model organisms. (* assembly).

<p>Occurrence of Arps in model organisms. (* assembly).</p

Actin pseudogenes in the <i>D. discoideum</i> genome (aa: amino acids).

<p>Actin pseudogenes in the <i>D. discoideum</i> genome (aa: amino acids).</p