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First published online 15 January 2008
doi: 10.1242/jcs.012096

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Localization and diversity of 185/333 proteins from the purple sea urchin – unexpected protein-size range and protein expression in a new coelomocyte type

¹ George Washington University, Department of Biological Sciences, Washington, DC 20052, USA
² Dickinson College, Department of Biology, Carlisle, PA 17013, USA
³ Macquarie University, Department of Biological Sciences, North Ryde, NSW, 2109, Australia

View larger version (69K): [in this window] [in a new window]   Fig. 1. 185/333 proteins are expressed by small phagocytes and polygonal cells. Coelomocytes were labeled for actin (A, green), 185/333 proteins (B, red), and DNA (C, blue) and only the subset of small phagocytes (labeled S in A) are strongly labeled for 185/333 proteins. Small phagocytes have different filopodial morphology and actin organization than the type 1 (discoidal) phagocytes (labeled 1 in A) or type 2 (polygonal) phagocytes (labeled 2 in A). The larger of the type 2 cells has perinuclear vesicles that are 185/333+ (B, arrow; D). Some type 2 cells show more extensive localization of 185/333 proteins (E). Bar, 10 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 2. Confocal images of 185/333 proteins and actin in coelomocytes. Coelomocytes labeled for 185/333 proteins (A,D,G) and actin (B,E,H) shows the spectrum of morphologies of 185/333+ cells. Small phagocytes (S) range from those that are slightly larger and more spread (A), to others that are smaller and more filopodial (D,G). These cells have an extensive actin cytoskeleton (B,E,H), which is distinct from the radial cytoskeleton in type 1 (discoidal) cells (labeled 1 in B,H) or the long axis oriented actin cables in type 2 (polygonal) cells (labeled 2 in B,E,H). The 185/333+ cells have intense labeling of the plasma membrane as well as vesicles distributed in a perinuclear array (A,D,G). Bars, 10 µm.

View larger version (74K): [in this window] [in a new window]   Fig. 3. The organization of microtubules in 185/333+ cells is distinct. Merged confocal images show the localization of 185/333 proteins (red) and microtubules (green) in coelomocytes. The 185/333+ cells have an unusual pattern of microtubule bundles that are associated with filopodia (A) and cytoplasm (B) that contrasts with the perinuclear microtubules seen in type 1 (discoidal) cells (labeled 1) and the extensive cytoplasmic array of single microtubules present in type 2 (polygonal) cells (labeled 2). Bars, 10 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 4. Identification of living cells that express the 185/333 proteins. Digitally enhanced phase-contrast microscopy of living cells (A,B) demonstrates the presence of small filopodial cells in populations of coelomocytes that appear similar to 185/333+ small phagocytes (Figs 1, 2, 3), but quite different from type 2 (polygonal; labeled 2 in A) or type 1 (discoidal; labeled 1 in B) coelomocytes. A type 1 coelomocyte with a characteristic radial cytoskeleton (labeled 1 in C) is shown adjacent to a smaller, filopodial small phagocyte in C. The 185/333 antibodies stain the entire small phagocyte and appear concentrated in punctate perinuclear structures (D). A type 2 coelomocyte (labeled 2 in E) with a characteristic distributed array of cytoplasmic granules is shown adjacent to a smaller, filopodial small phagocyte (E,F). Bars, 10 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 5. The 185/333 proteins are on the surface of small phagocytes. Confocal images of 185/333+ coelomocytes that were labeled for 185/333 proteins prior to fixation show spherical structures present at the edge of the cell (A, arrows) and on filopodia (B, arrow; C, arrows) that appear as knobs on the membrane. The cell in A has a spread morphology in contrast with those in B and C with a more highly filopodial morphology. The 185/333 proteins are not present in the perinuclear region, and anti-actin labeling was negative (not shown), both of which are consistent with a lack of membrane permeabilization. This result suggests that some of the 185/333 epitopes are present on the extracellular face of the plasma membrane. Bars, 10 µm.

View larger version (107K): [in this window] [in a new window]   Fig. 6. 185/333+ cells appear in small aggregations of coelomocytes. (A) Small aggregations of live cells were imaged with digitally enhanced phase-contrast microscopy (arrow) which shows that these aggregates are present in the living state. In addition to the phagocytes in A, there are red and colorless spherule cells and vibratile cells. (B) Aggregates labeled for 185/333 proteins (red), actin (green) and DNA (blue) shows that most of the cells in the aggregate are 185/333+. In this case, the aggregate is surrounded by type 2 (polygonal) coelomocytes, of which one has 185/333+ perinuclear vesicles (arrow). Bars, 10 µm.

View larger version (24K): [in this window] [in a new window]   Fig. 7. Numbers of coelomocytes and the frequency of 185/333+ cells increase after challenge with LPS. (A) Total coelomocyte counts were taken at various times after sea urchins were injected with LPS (n=4, bars indicate s.d.). (B) Injection of LPS increased the relative frequency of all 185/333+ coelomocytes (% of total coelomocytes) in CF collected at various times after LPS injection (n=4, bars indicate s.d.). (C) Coelomocytes and 185/333+ coelomocytes from adult sea urchins (n=7) were counted separately before (white bar) and after (black bar) administering two separate injections of LPS (1 µg LPS/ml CF) at 0 hours and 24 hours. Small and polygonal phagocytes are shown as the percentage of total coelomocytes. The 185/333+ cells are shown as the percentage of small or polygonal phagocytes. Asterisks indicate significant differences (see text) between pre-challenged and LPS challenged sea urchins.

View larger version (36K): [in this window] [in a new window]   Fig. 8. 185/333 proteins are a complex mixture of sizes. Coelomocytes were collected from two sea urchins (animals 7 and 20) and analyzed by western blot with the anti-185/333 antisera. (A) Individual sea urchins show differences in the range of 185/333 sizes. Standards are shown to the left. (B) The amount of 185/333 proteins in coelomic fluid increased after injection with LPS. Coelomocytes were collected from animal 7 before injection with LPS (lane 1) and after injection at 1 hour (lane 2), 6 hours (lane 3) and at 24 hours (lane 4).

View larger version (34K): [in this window] [in a new window]   Fig. 9. Expression of recombinant 185/333 proteins. (A) Expression in E. coli. Western blot analysis of recombinant 185/333 protein, Sp0032 (GenBank acc. no. DQ183168; E1 pattern) expressed in E. coli (M15 strain). Bacteria were induced with 1 mM IPTG for 4 hours at 25°C prior to collection. Lane 1, pelleted E. coli without 1 mM IPTG induction. Lane 2, pelleted E. coli with 1 mM IPTG induction. Lane 3, pelleted E. coli with 1 mM IPTG and analyzed with pre-immune serum. Pelleted cells were lysed and separated into pellet and supernatant. Lane 4, sonicated pellet. Lane 5, sonicated supernatant. Standards are shown to the left. (B) Expression in insect cells. Drosophila S2 cells were transfected with Sp0296, a cDNA of the E2 pattern (GenBank acc. no. DQ183121) (see Terwilliger et al., 2006) and induced with 100 mM CuSO4. Recombinant 185/333 proteins were secreted into the culture medium, which was collected and analyzed by western blot with the anti-185/333 antisera. The expected monomer of 31.5 kDa (without post-translational modifications), putative dimers of 65 kDa, and multimers of 150 kDa (lane 2 only) were detected. Lane 1, 1 day after induction. Lane 2, 2 days after induction. Size standards are shown to the left.