ABC transporters in Dictyostelium discoideum development

Abstract

ATP-binding cassette (ABC) transporters can translocate a broad spectrum of molecules across the cell membrane including physiological cargo and toxins. ABC transporters are known for the role they play in resistance towards anticancer agents in chemotherapy of cancer patients. There are 68 ABC transporters annotated in the genome of the social amoeba Dictyostelium discoideum. We have characterized more than half of these ABC transporters through a systematic study of mutations in their genes. We have analyzed morphological and transcriptional phenotypes for these mutants during growth and development and found that most of the mutants exhibited rather subtle phenotypes. A few of the genes may share physiological functions, as reflected in their transcriptional phenotypes. Since most of the abc-transporter mutants showed subtle morphological phenotypes, we utilized these transcriptional phenotypes to identify genes that are important for development by looking for transcripts whose abundance was unperturbed in most of the mutants. We found a set of 668 genes that includes many validated D. discoideum developmental genes. We have also found that abcG6 and abcG18 may have potential roles in intercellular signaling during terminal differentiation of spores and stalks.

Figure 1. Transcriptional phenotypes of abc-transporter mutants.

We determined the transcriptional phenotypes of the wild-type AX4 strain (WT) and 35 abc-transporter mutant strains at 0-, 6-, 12- and 18-hours of development. The relations between the transcriptional phenotypes are illustrated using multidimensional scaling, where the strains appear close together if their phenotype distance is small, or appear apart from each other if the phenotype distance is large. We constructed multidimensional scaling plots by considering transcriptional profiles for all the time points together where transcriptional profiles of each mutant at 0-, 6-, 12- and 18-hours were concatenated prior to distance computation (A) and for individual time points, 0-hours (B), 6-hours (C), 12-hours (D), and 18-hours (E). The axes are dimensionless. Each circle represents a strain, with an abbreviated strain name inside (e.g. G19 stands for abcG19 ⁻). The colors indicate classes of ABC transporters. Arrows point to specific strains that are similar: tan – mutants showing delayed development, blue – abcB4⁻ and abcC13⁻, green – wild type.

Figure 2. Sorting preference of the abcB4⁻ and abcC13⁻ mutants in chimeric slugs.

We co-developed unlabeled mutant cells with GFP-labeled wild-type cells. Pictures were taken at 16-hours of development. In chimeric organisms, the wild-type cells (A) were evenly distributed throughout the slug; the abcB4⁻ (B) and abcC13⁻ (C) mutant cells preferentially sorted to the prestalk area and formed most of the pstO cells (arrowheads). Panels A and B are representative images whereas panel C represents fewer than half of the chimeric slugs observed.

Figure 3. Identification of important developmental genes.

The method used to identify developmental genes is depicted as a flow chart (A). Blue boxes represent the wild type (AX4) and mutant strains with normal or delayed development as indicated. Red boxes represent transcripts whose abundance was not significantly altered by the mutations. Green boxes represent selected developmental genes. Ellipses represent the genomes of D. discoideum (yellow) and D. purpureum (purple) and the overlapping orthologs (not to scale). The volcano plot (B) shows the false discovery rate (FDR) of each transcript (y-axis, −log[FDR]) as a function of the difference in mRNA abundance between the median of all the normally developing mutants and the wild type (x-axis, log(mutant/WT) at 12-hours, when the gene expression was most variable across mutants. Each dot represents a gene, yellow dots represent genes that were identified as important for development and conserved between D. discoideum and D. purpureum (Gene set D), red dots represent genes that show little or no perturbation in expression during development (Gene set C – Gene set D), and black dots represent the rest of the genes. We validated the approach by examining the enrichment of developmental and non-developmental genes in gene sets C and D (C).

Figure 4. Cell type enriched abc-transporter transcripts and their effect on cell-type differentiation.

The bar graph shows the fraction (%) of prespore- or prestalk- enriched transcripts that were altered in each of the mutants compared to the wild type. The names of the mutated abc genes are indicated below the bars. The table below the graph summarizes properties of the abc-transcripts in wild-type cells (first three rows) and the phenotypes of the respective mutant strains (last three rows). Sporulation efficiency is shown as the fraction (%) of the cells that made spores +/− S.E.M.; * indicates statistically significant difference from AX4 (Student's t-test, two sided, n = 3, p-value<0.005).