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Sphingosine-1-phosphate lyase has a central role in the development of Dictyostelium discoideum

Guochun Li^*,, Christopher Foote^*, Stephen Alexander and Hannah Alexander

Present address: Department of Biology, University of California, San Diego, LaJolla, CA 92093-0568, USA
Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400, USA
^* These authors contributed equally to this study

View larger version (8K): [in a new window]   Fig. 1. Pathway of sphingomyelin degradation. The pathway reflects studies in animal cells. To date the genes for the S-1-P lyase (Accession Number, AF233610), two sphingosine kinases (cDNA clone SLG787 – Accession Numbers, AU061963 and AU039939, and cDNA clone SLE414 – Accession Number, AU061484), two sphingosine-1-P phosphatases (cDNA clone SSE389 – Accession Numbers, AU072386 and AU037870, and cDNA clone SSC687 – Accession Number, C85040) and a ceramidase (Accession Number, AAB69633) have been identified in Dictyostelium.

View larger version (87K): [in a new window]   Fig. 2. Sequence homology of S-1-P lyase gene product. The sequence information of the S-1-P lyase contig was assembled by combining sequence information from the various Dictyostelium sequencing project databases (see Materials and Methods). The predicted amino acid sequence of Dictyostelium S-1-P lyase was aligned with the human, mouse and Saccharomyces cerevisiae S-1-P lyase gene products (NCBI Accession Numbers, AAD44755, NP033189 and NP010580, respectively). Alignment was performed using the MegAlign program of DNASTAR.

View larger version (34K): [in a new window]   Fig. 3. Expression of sglA gene during growth and development. Ax4 wild-type cells were allowed to develop synchronously on filters. Total RNA samples were prepared at the indicated time points and analyzed for sglA mRNA levels. The band intensities on the northern blots were quantified with a FUJIFILM FLA-2000 PhosphorImager. The mRNA levels are depicted as -fold increase over the mRNA level at 0 hours of development.

View larger version (39K): [in a new window]   Fig. 4. Molecular analyses of the S-1-P lyase disruption mutant. (A) A schematic presentation of the parental gene, the disruption vector and the resultant mutant. (B) Southern analysis. Genomic DNA (20 µg) of wild-type Ax4 (W) and the S-1-P lyase null mutant (M) were digested with NdeI, ClaI and HindIII, separated on 0.8% agarose gel, and transferred to a nylon membrane. The membrane was probed with the sglA probe, stripped and then re-probed with the bsr probe. The asterisk represents a 0.1 kb ClaI fragment in which the insertion has occurred, to yield the 1.5 kb fragment. (C) Northern analysis of the S-1-P lyase disruption mutant. Total RNA samples (10 µg) of Ax4 and S-1-P lyase null cells were separated on 1% agarose/7.5% formamide gel, blotted onto nitrocellulose membrane and probed with sglA probe.

View larger version (18K): [in a new window]   Fig. 5. Disruption of the S-1-P lyase gene affects viability of cells in stationary phase. (A) Wild-type and S-1-P lyase-null cells were inoculated into HL-5 medium at 2x105 cells per ml for axenic growth and the cell numbers were followed. Triplicate cultures of each were examined. Each point is the mean of duplicate counts. (B) Wild-type and S-1-P lyase cells were harvested at a density of 3x106 cells per ml and deposited at a standard density of 8x106 cells/cm2 for development. At the indicated times, duplicate filters were harvested into 20 ml of SS buffer containing 10 mM EDTA and disaggregated by vortexing until there was a uniform suspension of single cells. The cells from each filter were counted in duplicate and the bars in the figure indicate the standard deviation.

View larger version (143K): [in a new window]   Fig. 6. The S-1-P lyase null strain has abnormal development. Axenically grown wild-type Ax4 and S-1-P lyase null cells were allowed to develop on filters and were photographed at the indicated times. 1 cm=4.2 mm.

View larger version (11K): [in a new window]   Fig. 7. Sporulation is reduced in the S-1-P lyase-null strain. Wild-type Ax4 and S-1-P lyase-null cells were allowed to develop synchronously on filters. Samples were harvested at indicated time points and analyzed for the percentage of spores out of total number of cells counted.

View larger version (33K): [in a new window]   Fig. 8. Migration is impaired in S-1-P lyase null slugs. The wild-type Ax4 (A) and S-1-P lyase null (B) cells were grown axenically and harvested for the slug migration assay as described in the Materials and Methods. Gray lines represent the trails of slime sheath, which are left behind during the migration of the slug during the 2 day period; dark dots indicate the final position of the migrating slug. The position of the light source is indicated by the arrowhead.

View larger version (53K): [in a new window]   Fig. 9. Aggregating S-1-P lyase mutant cells have altered shape and aberrant cell F-actin localization. Aggregating wild-type Ax4 (A) and S-1-P lyase null (B) cells were prepared as described in the Materials and Methods, and stained with rhodamine-conjugated phalloidin. Scale bars: 10 mm.

View larger version (47K): [in a new window]   Fig. 10. Late developmental gene expression is altered in the S-1-P lyase-null strain. Wild-type Ax4 and S-1-P lyase null cells developing synchronously on filters were harvested. Northern blots of total RNA prepared from each time point were probed for the expression of different developmental marker genes using gene-specific probes.

View larger version (136K): [in a new window]   Fig. 11. S-1-P treatment of wild-type cells during development produces a phenocopy of the S-1-P lyase null mutant. Wild-type Ax4 cells were allowed to develop synchronously on filters (A,B). S-1-P was added at 5 (C,D), 8 (E,F) and 12 (G,H) hours of development, as described in Materials and Methods. The photographs were taken at 24 (A,C,E,G) and 60 (B,D,F,H) hours of development. 1 cm=2 mm.