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doi: 10.1242/10.1242/dev.00523

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TagA, a putative serine protease/ABC transporter of Dictyostelium that is required for cell fate determination at the onset of development

J. Randall Good^*,1,3,, Matthew Cabral^*,2, Sujata Sharma², Jun Yang¹, Nancy Van Driessche^2,3, Chad A. Shaw², Gad Shaulsky^2,3 and Adam Kuspa^1,2,3,

¹ Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, 77030, USA
² Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, 77030, USA
³ Program in Developmental Biology, Baylor College of Medicine, Houston, Texas, 77030, USA
Present address: Science and Technology Division, Institute for Defense Analyses, 4850 Mark Center Drive, Alexandria, VA 22311-1882, USA

View larger version (110K): [in a new window]   Fig. 1. The predicted domain structure of TagA Similarity searches of the predicted amino acid sequence of the tagA gene suggests an amino-terminal serine protease domain and a carboxyl-terminal ABC transporter domain. A. Amino acid residues known to be required for protease activity are indicated by their single letter code and position. PR1 and PR2 represent regions of the gene that were used as probes for library screening and RNase protection assays. The position of a blasticidin resistance cassette (Bsr) insertion at the nucleotide binding site is also shown. B. Kyte-Doolittle hydropathy plot showing location of probable membrane-spanning regions within the ABC transporter domain (black bar). C. Sequence alignment of the deduced amino acid sequences for the predicted serine protease domains TagA and TagC. D. Sequence alignment of the deduced amino acid sequences for the predicted ABC transporter domains of Dictyostelium TagA, TagC, human ABCB.3 (Tap2) and human ABCB.1 (Hmdr1).

View larger version (101K): [in a new window]   Fig. 2. Timing of TagA expression. (A) RNase protection assays were performed with total RNA collected from wild-type (upper panel) or tagA mutant (lower panel) cells. Lanes are: P, riboprobe without RNase treatment (1/10 the input for other lanes), Y, RNase digestion of probe incubated with yeast RNA, and (R) RNase digestion of the probe without RNA added, or (0-24 h) after hybridization to RNA samples collected across the 24 hours of development. (B) Western blot stained with a TagA antibody detects a protein of an apparent molecular mass of 190 kDa (arrow). Equal amounts of protein (10 µg) from vegetative (0 hours) or developing (2-24 hours) wild-type cells (Ax4) were loaded in each lane, along with molecular mass standards (MM). The tagA mutant and rescued mutant (tagA-[tagA/tagA]) samples were mixtures of all vegetative and developmental time points. The amount of protein loaded in these lanes were equal (1x) or twofold (2x) the amounts of the developmental samples.

View larger version (31K): [in a new window]   Fig. 3. Development of tagA mutants. Cells were allowed to develop on filters as pure populations for 14 hours (A), for 16-18 hours to form slugs (B), or 24 for hours to form fruiting bodies (C). Wild-type (Ax4[ecmA/GFP]) or mutant (tagA-[ecmA/GFP]) cells expressing green fluorescent protein (GFP) under the control of the ecmA promoter were used to visualize prestalk cells in slugs (B) and spore heads (C) during development. The arrow indicates the lower cup of tagA mutants that appear to contain an excess number of cells. (D) Developing cells were scraped from filters, dissociated into single cells and observed by bright-field and fluorescence microscopy to determine percentage of ecmA/GFP-positive cells. Similar results were obtained at 14 and 18 hours of development whether an entire filter of cells was harvested for counting (D), or 10 individual developing structures were picked from filters, disrupted and counted. (E) Wild-type (Ax4) or tagA- cells were washed, plated at low density (1x104 cells/cm2) in 24-well plates and incubated with 5 mM cAMP in stalk buffer for 24 hours. Cells were then washed free of cAMP and incubated with DIF or DIF + 5 µM cerulenin for another 24 hours and examined by fluorescence microscopy for the expression of prestalk-specific expression of GFP (ecmA/GFP). Three independent determinations were carried out for each condition and results are given as the mean±s.e.m.

View larger version (46K): [in a new window]   Fig. 4. Expression of ecmB during development. (A) Northern analysis of the developmental expression of the prestalk/stalk-specific ecmB gene. The upper band in both panels represents hybridization to the highly similar ecmA gene. (B) An ecmB/lacZ reporter gene was used to visualize ecmB expression at a cellular level. Structures were fixed at 16 hours (upper panels) and 24 hours (lower panels) of development and stained for ß-galactosidase activity to visualize expression of the reporter gene. More cells appear to express ecmB in the tagA mutant, and many of the additional cells accumulate in the anterior of the slug, but their localization within fruiting bodies appears relatively normal.

View larger version (39K): [in a new window]   Fig. 5. Transcriptional profiling of tagA mutant cells. Wild-type (AX4) and mutant (tagA-) cells were developed for 24 hours. RNA samples were collected at 2-hour intervals and analyzed with a microarray of about 6,000 genes. The data from a selected set of 2,021 developmentally regulated genes were plotted to indicate the level of gene expression where the color scale represents the standardized log2 of the ratio between the test sample and the standard relative to the mean for each gene. Blue indicates lower than average level of gene expression for that gene and yellow indicates higher than average level of expression (Van Driessche et al., 2002). Each column represents a time point and each row represents a gene. (A) RNA from wild-type (Ax4) cells where the data are normalized with the gene means from the wild-type data (self-normalized). (B) RNA from tagA- cells where the data from every gene are self-normalized. (C) RNA from tagA- cells where the data from every gene are normalized to the corresponding gene mean in the wild-type (AX4) dataset. (D) The similarity (Pearson correlation) between all the genes at each time point in the AX4 dataset (y axis) and all the genes at each time point in the tagA- dataset (x axis) was calculated. For each time of tagA- development the most similar wild-type time point is plotted (solid line) in comparison with a theoretical plot between two identical time courses (broken line). The AX4 data set was published previously (Van Driessche et al., 2002) and was reanalyzed in the context of this experiment.

View larger version (40K): [in a new window]   Fig. 6. Cell-type specific gene expression in tagA mutants. (A) Cells expressing a lacZ reporter gene under the control of the tagA promoter in a wild-type or tagA mutant background were plated as pure populations and stained with X-gal. The arrows indicate the stained regions in the mutant. Scale bars: 0.1 mm. (B) RNase protection analysis of tagA mRNA in purified spores and stalks reveals a reproducible enrichment of tagA mRNA in the stalk RNA of the tagA mutant. Controls are 2- and 4-hour developing wild-type cells and 10% of the input probe, not treated with RNase. (C) A cotB/lacZ reporter was used to visualize prespore/spore gene expression. An unstained wild-type stalk (left panel) is in stark contrast to the stained stalk cells of the tagA mutant (3 right panels). A portion of the cotB-positive sorus is shown above the tagA mutant stalk (middle). D. Northern analyses of spore and stalk RNAs with probes for the spiA (spore-specific) and ecmB (prestalk/stalk-specific) genes.