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Figure 1. An adult California purple sea urchin, Strongylocentrotus purpuratus. Image kindly provided by Hung-Yen Chou and Yen-Lin Kuo.
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Figure 2. Sea urchin coelomocytes. Coelomocytes from the green sea urchin, Strongylocentrotus droebachiensis, were settled onto a glass coverslip and imaged live. Cell types that are typical of echinoids include discoidal phagocytes (1), polygonal phagocytes (2), red spherule cells (3), colorless spherule cells (4), vibratile cells (5; the lower cell has lost the prominent flagellum that is present in the upper cell), and small phagocytes (6, inset). Bar = 10 μm. This is Figure 2 from Smith et al. (2010), reproduced with permission from Landes Bioscience and Springer Science + Business Media.
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Figure 3. Sp185/333 proteins are expressed by subsets of small phagocytes and polygonal phagocytes. Phagocytes are labeled for actin (green), Sp185/333 proteins (red), and DNA (blue). Small phagocytes (S) have different filopodial morphology and actin organization than the discoidal phagocytes (D) or polygonal phagocytes (P). A subset of small phagocytes are strongly labeled for Sp185/333 proteins. The larger polygonal cell has perinuclear vesicles that are Sp185/333+. Bar = 10 μm. This is Figure 1D reproduced from Brockton et al. (2008).
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Figure 4. An Sp185/333+ Small Phagocyte. Confocal image of a Sp185/333+ small phagocyte labeled with anti-Sp185/333 sera prior to fixation shows the filopodial morphology of the cell and indicates the presence of Sp185/333 proteins on the cell surface. This includes knobs on the filopodia that are strongly positive for Sp185/333 (arrows). The dark area in the center of the cell is the location of the nucleus. Bar = 10 μm. This is Figure 5B reproduced from Brockton et al. (2008).
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Figure 5. Genome blot of Sp185/333 genes indicates that the genes are small. Probes for the 5′ end and the 3′ end of the second exon hybridize to the same bands. This includes bands that are 1.4–1.55 kb (double headed arrows), demonstrating that the genes are small. Genomic DNA from three sea urchins (1, 2, 3) was digested to completion with PstI and separated by electrophoresis. Duplicate gels were blotted onto nylon membranes and analyzed with 32P-labeled riboprobes according to Terwilliger et al. (2006). Cloned templates used to generate the riboprobes were amplified by PCR from genomic DNA using primers that hybridized to elements 1 and 7 (5′ probe), and elements 7 and 25 (3′ probe). See Figure 6A for element positions. Size standards in kilobase are shown to the right. (Terwilliger and Smith, unpublished).
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Figure 6. Two different alignments for Sp185/333 sequences are equally optimal. (A) The cDNA alignment was initially done with ESTs and full-length cDNA sequences (Terwilliger et al., 2006, 2007). (B) The Repeat-based alignment optimizes correspondence between repeats and elements whenever possible (Buckley and Smith, 2007). Optimal alignments require the insertion of artificial gaps (horizontal black lines) that delineate individual elements shown as colored blocks (the consensus of all elements are numbered across the top of each alignment; L, leader). Different element patterns are based on the variable presence or absence of elements. Designations of element patterns are listed to the left of each alignment. There are three types of element 25 in (A) (a, b, and c), which are defined by the location of three possible stop codons. A common, single nucleotide RNA edit in element pattern E2 alters a glycine codon to a stop (X) in element 13 and is denoted as an E2.1 pattern. Repeats, shown at the bottom of (A,B), are shows as different colors and occur as tandem repeats and interspersed tandem repeats. The genes have two exons (brackets at the top of each alignment), of which the first encodes the leader and the second encodes the mature protein including all of the elements. A single intron of ∼400 nt is positioned between the leader and the first element and is not shown. Modified from Ghosh et al. (2010) with permission from Elsevier.
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Figure 7. Sp185/333 gene sequences are not shared among sea urchins but element sequences are shared among genes. Full-length genes were cloned and sequenced from three sea urchins; 38 unique genes from animal 10 (blue circle), 64 unique genes from animal 2 (red circle), 30 unique genes from animal 4 (green circle). Nucleotide sequences were compared among full-length genes and among individual elements, and the numbers of shared unique sequences are shown in the intersections of the circles. Unpublished figure provided by Katherine Buckley.
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Figure 8. Six Sp185/333 genes are linked on the 7096 BAC insert. The finished-level assembly of the region containing the Sp185/333 genes was experimentally confirmed by PCR, pulsed field gel electrophoresis, AseI digests, and subclone sequences. The six Sp185/333 genes include one gene with the A2 element pattern (red), one B8 gene (orange), three D1 genes (yellow, green, blue), and one E2 gene (purple; see Figure for element patterns). All are located near the 3′ end of the BAC insert. Gene orientations are indicated and spacing is to scale unless otherwise noted. GA microsatellites flank each gene and GAT microsatellites flank segmental duplications that are positioned on 5′ side of B8 and include the three D1 genes. This is Figure 4 from Miller et al. (2010) reproduced with permission from BioMed Central.
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Figure 9. Representation of Sp185/333 protein structure showing all possible elements. The deduced protein contains a leader (L), a glycine-rich region (orange line), and a histidine-rich region (blue line). Symbols indicate the presence of an RGD motif within element 7 (red star with a yellow center), N-linked glycosylation sites (green heptagons), an O-linked glycosylation site (pink heptagon), patches of acidic amino acids (red arrows), and histidines (purple arrows). Modified from Terwilliger et al. (2006).
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Figure 10. Truncated Sp185/333 proteins are present in the coelomic fluid. Enlarged regions of three different two dimensional Western blots of coelomic fluid proteins from the same sea urchin were analyzed with different anti-Sp185/333 sera; anti-66, anti-68, or anti-71. Anti-66 recognizes AHAQRDFNERRGKENDTER from element 1; anti-68 recognizes GGRRGDGEEETDAAQQIGDGLC from element 7; anti-71 recognizes TEEGSPRRDGQRRPYGNR from element 25 (see Figure 6A for element positions). Decreasing numbers of spots in blots analyzed with antisera that recognize peptides in more C-terminal regions of the proteins suggests that many are either truncated or have missense sequence toward the C-terminus. Reprinted from Dheilly et al. (2009) with permission from the American Association of Immunologists, Inc., copyright 2009.
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Figure 11. A single sea urchin can have as many as 264 spots that are Sp185/333+. Coelomic fluid proteins (200 μg) from sea urchin 12 (see Figure 12) were separated by 2D electrophoresis and transferred to a filter. The filter was immunostained with an equal mixture of the three anti-Sp185/333 sera (see legend to Figure 10) and exposed to autoradiographic film for 1, 5, or 10 min. The different exposures were merged to give a final composite image. Isoelectric points (pI) are shown at the top and the molecular weight standards (kDa) are shown to the left. Reprinted from Dheilly et al. (2009) with permission from the American Association of Immunologists, Inc., copyright 2009.
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Figure 12. Different sea urchins express different arrays of Sp185/333 proteins. Western blots of coelomic fluid from 13 different sea urchins sampled 96 h after challenge with LPS show different arrays of Sp185/333 proteins. The blots were immunostained with an equal mixture of the three different anti-Sp185/333 sera (see legend for Figure 10). Reprinted from Dheilly et al. (2009) with permission from the American Association of Immunologists, Inc., copyright 2009.
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Figure 13. Native Sp185/333 proteins bind Vibrio diazotrophicus (Vd), a gram negative marine bacterial species. Whole coelomic fluid (wCF) lysate from a sea urchin was incubated with Vd. Bacteria were pelleted, washed, and analyzed by Western blot using equal amounts of all three anti-Sp185/333 sera (see legend for Figure 10). wCF and Vd alone are shown for comparison. Lane 1, wCF; lane 2, wCF proteins bound to Vd; lane 3, Vd. Protein standard is to the left. Unpublished figure provided by Catherine Schrankel.
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Figure 14. A recombinant Sp185/333 protein (rSp0032) binds Vibrio diazotrophicus (Vd). Biotinylated rSp0032 incubated with 109
Vd cells and post-labeled with Neutravidin–FITC shows increased binding with increased protein concentration within the gate area (R2). Binding plateaus at about 400 mM of rSp0032 indicating the saturation point. MFI, mean fluorescence intensity. Results from flow cytometry (inserted images) show fluorescence (X-axis; fluorescent events measuring FITC) associated with bacterial cells (Y-axis; side scatter or cell counts) for increasing concentrations of rSp0032. Unpublished figure provided by Catherine Schrankel.
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Figure 15. Diversification in the Sp185/333 system in the purple sea urchin. Putative diversification mechanisms based on genomic instability may function to increase gene sequence diversity and may also vary the size of the gene family among individuals. mRNA sequence diversity imparted by the genes is expanded by editing some of the mRNAs in addition to possible low fidelity transcription. The mRNAs are translated to generate a broad array of proteins that may multimerize and be post-translationally modified. The end result is protein diversity that is much broader than what is encoded by the genes.
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