Effect of <i>cln3</i> knockout on the intra- and extracellular levels of AprA and CfaD.

<p>AX3 and <i>cln3⁻</i> cells grown axenically in HL5 were harvested and lysed after 48 and 72 hours of growth. Whole cell lysates (20 µg) (i.e., intracellular) and samples of conditioned growth media (i.e., extracellular) were separated by SDS-PAGE and analyzed by western blotting with anti-AprA, anti-CfaD, anti-tubulin, and anti-actin. Molecular weight markers (in kDa) are shown to the right of each blot. (A) Intra- and extracellular protein levels of AprA. Immunoblots that were exposed for a longer period of time (i.e., longer exposure) are included to show the 55-kDa and 37-kDa protein bands detected by anti-AprA. Note that the 37-kDa protein was detected in samples of conditioned growth media, but not in whole cell lysates. (B) Intra- and extracellular protein levels of CfaD. Data in all plots presented as mean amount of protein relative to AX3 48 hour sample (%) ± s.e.m (n = 4 independent experimental means, from 2 replicates in each experiment). Statistical significance was determined using a one-sample t-test (mean, 100; two-tailed) vs. the AX3 48 hour sample. *p-value<0.05. **p-value<0.01. (C) Detection of tubulin and actin in whole cell lysates (WC; lanes 1–2), but not in samples of conditioned growth media (lanes 3–6).</p

Effect of <i>cln3</i> knockout on the formation of tipped mounds and slugs.

<p>(A) AX3, <i>cln3⁻</i>, or <i>cln3⁻</i> cells overexpressing GFP-Cln3 or expressing GFP-Cln3 or GFP-CLN3 under the control of the <i>cln3</i> upstream element imaged after 12 and 15 hours of development. Images are a top-view of developing cells. (B) Quantification of the number of tipped mounds observed after 12 hours of development. Data presented as mean % tipped mounds ± s.e.m (n = 10–19). (C) Quantification of the number of fingers and slugs observed after 15 hours of development. Data presented as mean % fingers and slugs ± s.e.m (n = 10–33). Statistical significance was assessed using the Kruskal-Wallis test followed by the Dunn multiple comparison test (***p-value<0.001 vs. AX3). Scale bars = 1 mm. M, mound; TM, tipped-mound; F, finger; S, slug.</p

Effect of <i>cln3</i> knockout on cell proliferation and pinocytosis.

<p>(A) Axenic growth of AX3 <i>cln3⁻, cln3⁻</i>/[<i>act15</i>]:Cln3:GFP, and AX3/[<i>act15</i>]:Cln3:GFP cells in HL5 medium. Data presented as mean concentration (×10⁶ cells/ml) ± s.e.m (n = 10–20). (B) Axenic growth of AX3 and <i>cln3⁻</i> cells in FM medium. Data presented as mean concentration (×10⁶ cells/ml) ± s.e.m (n = 8). (C) Effect of <i>cln3</i> knockout on the intracellular accumulation of FITC-dextran. Data presented as mean % fluorescence change ± s.e.m (n = 10). Statistical significance was assessed using two-way ANOVA followed by Bonferroni post-hoc analysis. Two-way ANOVA revealed a significant effect of genotype on the growth curves shown in panels A and B (p<0.001 and p<0.01, respectively). **p-value<0.01 and ****p-value<0.0001 vs. AX3 as determined from Bonferroni post-hoc analysis at the indicated time points.</p

Effect of calcium chelation on AX3 and <i>cln3⁻</i> slug formation and migration.

<p>(A) AX3 and <i>cln3⁻</i> cells developed in the presence of KK2± EGTA and imaged after 15 hours of development. Scale bar = 1 mm. (B) Quantification of the number of fingers and slugs observed after 15 hours of development. Data presented as mean % fingers and slugs ± s.e.m (n≥4). (C) AX3 and <i>cln3⁻</i> cells developed in the presence of KK2± EGTA and imaged after 18 hours of development. Scale bar = 1 mm. (D) Quantification of the number of slugs that migrated outside the spot of deposition after 18 hours. Data presented as mean outside structures/total structures (%) ± s.e.m (n≥5). Images in A and C are a top-view of developing cells. Statistical significance in B was assessed using the Kruskal-Wallis test followed by the Dunn multiple comparison test (*p-value<0.05 vs. AX3). Statistical significance in D was assessed using one-way ANOVA (p<0.0001) followed by the Bonferroni multiple comparison test (**p-value<0.01 vs. AX3). F, finger; S, slug.</p

Effect of <i>cln3</i> knockout on slug migration and fruiting body formation.

<p>(A) AX3, <i>cln3⁻</i>, or <i>cln3⁻</i> cells overexpressing GFP-Cln3 or expressing GFP-Cln3 or GFP-CLN3 under control of the <i>cln3</i> upstream element imaged after 18 and 21 hours of development. (B) Quantification of the number of slugs that migrated outside the spot of deposition after 18 hours. Data presented as mean outside structures/total structures (%) ± s.e.m (n = 10–28). (C) Quantification of the number of fruiting bodies observed after 18–21 hours of development. Data presented as mean % fruiting bodies ± s.e.m (n = 10–32). (D) Fruiting bodies formed after 24 hours of development. Images in A and D are a top-view of developing cells. Statistical significance in B was assessed using one-way ANOVA (p<0.0001) followed by the Bonferroni multiple comparison test (****p-value<0.0001 vs. AX3). Statistical significance in C was assessed using the Kruskal-Wallis test followed by the Dunn multiple comparison test (**p-value<0.01 vs. AX3). Scale bars = 1 mm. S, slug; FB, fruiting body.</p

Intracellular localization of <i>Dictyostelium</i> GFP-Cln3 using epifluorescence microscopy.

<p>(A) AX3 cells overexpressing GFP-Cln3 imaged live in water. Scale bar = 5 µm. (B) AX3 cells overexpressing GFP-Cln3 were fixed in either ultra-cold methanol (for VatM and Rh50 immunostaining) or 4% paraformaldehyde (for p80 immunostaining) and then probed with anti-VatM, anti-Rh50, or anti-p80, followed by the appropriate secondary antibody linked to Alexa 555. Cells were stained with DAPI to reveal nuclei (blue). Images were merged with ImageJ/Fiji. VC, vacuolar-shaped structures; VS, cytoplasmic vesicles; T, tubular-like structures within the cytoplasm; P, punctate distributions within the cytoplasm. Scale bars (B, C) = 2.5 µm.</p

Bioinformatic analysis of <i>Dictyostelium</i> Cln3.

<p>(A) Alignment of human CLN3 and the <i>Dictyostelium</i> ortholog. The following residues are conserved; N-linked glycosylation sites (*), sites of missense point mutations (;), sites of nonsense point mutations (:), target for myristoylation (#), sites that when mutated cause a slower disease progression in compound heterozygosity with the common 1.02 kb deletion mutation (∧), sites that when mutated cause a slower disease progression in homozygosity (<) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110544#pone.0110544-TheInternationalBattenDisease1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110544#pone.0110544-Cotman1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110544#pone.0110544-Bause1" target="_blank">[85]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110544#pone.0110544-Haskell2" target="_blank">[87]</a>. <i>Dictyostelium</i> Cln3 also contains a putative prenylation motif (i.e., CFIL; underlined). (B) Phylogenetic tree showing the relationship of <i>Dictyostelium</i> Cln3 to CLN3 orthologs from 20 different organisms (i.e., mammals and NIH model systems).</p

Generation of a <i>Dictyostelium cln3</i> knockout mutant.

<p>(A) Creation of a <i>Dictyostelium cln3</i> knockout mutant by homologous recombination. The pLPBLP targeting vector and sites of recombination are shown. (B) Validation of <i>cln3</i> knockout by PCR analysis. Primers are denoted by Roman numerals and arrows. The <i>Dictyostelium</i> gene denoted DDB_G0291155 lies downstream of <i>cln3</i> and was amplified to confirm that the insertion of the <i>bsr</i> cassette did not affect this gene. (C) Validation of <i>cln3</i> knockout by Southern blotting. DNA ladder (in bp) is shown to the left of the blot.</p