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. 2014 Oct 17;9(10):e110544.
doi: 10.1371/journal.pone.0110544. eCollection 2014.

Loss of Cln3 function in the social amoeba Dictyostelium discoideum causes pleiotropic effects that are rescued by human CLN3

Robert J Huber  1 , Michael A Myre  1 , Susan L Cotman  1
Affiliations

Loss of Cln3 function in the social amoeba Dictyostelium discoideum causes pleiotropic effects that are rescued by human CLN3

Robert J Huber et al. PLoS One. .

Abstract

The neuronal ceroid lipofuscinoses (NCL) are a group of inherited, severe neurodegenerative disorders also known as Batten disease. Juvenile NCL (JNCL) is caused by recessive loss-of-function mutations in CLN3, which encodes a transmembrane protein that regulates endocytic pathway trafficking, though its primary function is not yet known. The social amoeba Dictyostelium discoideum is increasingly utilized for neurological disease research and is particularly suited for investigation of protein function in trafficking. Therefore, here we establish new overexpression and knockout Dictyostelium cell lines for JNCL research. Dictyostelium Cln3 fused to GFP localized to the contractile vacuole system and to compartments of the endocytic pathway. cln3- cells displayed increased rates of proliferation and an associated reduction in the extracellular levels and cleavage of the autocrine proliferation repressor, AprA. Mid- and late development of cln3- cells was precocious and cln3- slugs displayed increased migration. Expression of either Dictyostelium Cln3 or human CLN3 in cln3- cells suppressed the precocious development and aberrant slug migration, which were also suppressed by calcium chelation. Taken together, our results show that Cln3 is a pleiotropic protein that negatively regulates proliferation and development in Dictyostelium. This new model system, which allows for the study of Cln3 function in both single cells and a multicellular organism, together with the observation that expression of human CLN3 restores abnormalities in Dictyostelium cln3- cells, strongly supports the use of this new model for JNCL research.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. Bioinformatic analysis of Dictyostelium Cln3.
(A) Alignment of human CLN3 and the Dictyostelium 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 (<) , , –. Dictyostelium Cln3 also contains a putative prenylation motif (i.e., CFIL; underlined). (B) Phylogenetic tree showing the relationship of Dictyostelium Cln3 to CLN3 orthologs from 20 different organisms (i.e., mammals and NIH model systems).
Figure 2
Figure 2. Intracellular localization of Dictyostelium GFP-Cln3 using epifluorescence microscopy.
(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.
Figure 3
Figure 3. Intracellular localization of Dictyostelium GFP-Cln3 using confocal microscopy.
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-GFP (rabbit polyclonal anti-GFP for anti-VatM and anti-p80 co-staining and mouse monoclonal anti-GFP for anti-Rh50 co-staining) followed by anti-rabbit or anti-mouse Alexa 488. Cells were then probed with one of anti-VatM, anti-Rh50, or anti-p80 followed by the appropriate secondary antibody linked to Alexa 555. Two z-sections are shown for each cell. Z-sections 1 and 2 are approximately 1 µm and 3 µm, respectively, from the bottom of each cell. VC, vacuolar-shaped structures; VS, cytoplasmic vesicles; T, tubular-like structures within the cytoplasm; P, punctate distributions within the cytoplasm. Scale bars = 2.5 µm.
Figure 4
Figure 4. Generation of a Dictyostelium cln3 knockout mutant.
(A) Creation of a Dictyostelium cln3 knockout mutant by homologous recombination. The pLPBLP targeting vector and sites of recombination are shown. (B) Validation of cln3 knockout by PCR analysis. Primers are denoted by Roman numerals and arrows. The Dictyostelium gene denoted DDB_G0291155 lies downstream of cln3 and was amplified to confirm that the insertion of the bsr cassette did not affect this gene. (C) Validation of cln3 knockout by Southern blotting. DNA ladder (in bp) is shown to the left of the blot.
Figure 5
Figure 5. Effect of cln3 knockout on cell proliferation and pinocytosis.
(A) Axenic growth of AX3 cln3−, cln3−/[act15]:Cln3:GFP, and AX3/[act15]: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 cln3− cells in FM medium. Data presented as mean concentration (×106 cells/ml) ± s.e.m (n = 8). (C) Effect of cln3 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.
Figure 6
Figure 6. Effect of cln3 knockout on the intra- and extracellular levels of AprA and CfaD.
AX3 and cln3− 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).
Figure 7
Figure 7. Effect of cln3 knockout on the formation of tipped mounds and slugs.
(A) AX3, cln3−, or cln3− cells overexpressing GFP-Cln3 or expressing GFP-Cln3 or GFP-CLN3 under the control of the cln3 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.
Figure 8
Figure 8. Effect of cln3 knockout on slug migration and fruiting body formation.
(A) AX3, cln3−, or cln3− cells overexpressing GFP-Cln3 or expressing GFP-Cln3 or GFP-CLN3 under control of the cln3 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.
Figure 9
Figure 9. Effect of calcium chelation on AX3 and cln3− slug formation and migration.
(A) AX3 and cln3− 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 cln3− 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.
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