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First published online March 16, 2007
Journal of Experimental Biology 210, 1275-1287 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02743

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Extracellular heat shock protein 70 has novel functional effects on sea urchin eggs and coelomocytes

Carole L. Browne^1,2,*, Justin B. Swan^1,2, Ellen E. Rankin^2,3, Hayes Calvert¹, Shylise Griffiths^2,4 and Michael Tytell^2,5

¹ Department of Biology, Wake Forest University, Winston-Salem, NC 27109, USA
² Marine Biological Laboratory, Woods Hole, MA 02543, USA
³ Department of Psychology, Colgate University, Hamilton, NY 13346, USA
⁴ Biology Department, University of North Carolina at Greensboro, Greensboro, NC 27402, USA
⁵ Department of Neurobiology and Anatomy, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA

View larger version (7K): [in this window] [in a new window]   Fig. 1. The effect of Hsp/Hsc70 on nuclear envelope breakdown (NEB) in fertilized sea urchin eggs. Fertilized sea urchin eggs were incubated in varying concentrations of Hsc70 and Hsc/Hsp70 ranging from 0.1–10 µg ml–1, in artificial sea water (ASW) (control) or in 1 µg ml–1 of bovine serum albumin (BSA) or ovalbumin, and the times to NEB and cleavage were observed. Eggs exposed to Hsp/Hsc70 reached NEB earlier than eggs incubated in ASW, ovalbumin or BSA. There were no significant differences in the time to NEB between untreated control eggs and BSA- and ovalbumin-treated eggs. The difference between Hsc/Hsp70-treated and control, BSA and ovalbumin-treated eggs was statistically significant at all concentrations, as indicated by the asterisks (P<0.05). Values are means ± s.e.m. N=6 replicate experiments in which at least 100 eggs were observed.

View larger version (6K): [in this window] [in a new window]   Fig. 2. The effect of rbHsc70 on nuclear envelope breakdown (NEB) in fertilized sea urchin eggs. Fertilized sea urchin eggs were incubated in varying concentrations of Hsc70, and the times to NEB and cleavage were observed. Exogenous Hsc70 reduced the time to NEB at all concentrations tested. The difference between Hsc-treated and control eggs was statistically significant at 5 µg ml–1 Hsc, as indicated by the asterisk (P<0.05). Values are means ± s.e.m. N=6 replicate experiments in which at least 100 eggs were observed.

View larger version (4K): [in this window] [in a new window]   Fig. 3. Renaturation of luciferase in the presence of Hsc/Hsp70 or Hsc70 as a measure of their refolding activities. 2 µg ml–1 of Hsp70 and of Hsc/Hsp70 was heated in the presence of luciferase to determine the ability of the Hsp preparations to prevent luciferase denaturation. The heated luciferase–Hsp mixture was combined with luciferin reagent and luminescence recorded as a percentage of unheated luciferase. The difference in phosphorescence between luciferase heated in the absence of Hsp and in the presence of Hsp was statistically significant as indicated by the asterisk (P<0.05). There was no difference in effectiveness between the two Hsp preparations. Values are means ± s.e.m. N=6 replicate experiments.

View larger version (72K): [in this window] [in a new window]   Fig. 4. Distribution of Hsc70 immunofluorescence in unfertilized eggs. These images are examples of two eggs out of 25 observed. (A,B) Examples of diffuse cytoplasmic distribution of Hsc70 and exclusion from the nucleus in two eggs incubated in 20 or 5 µg Hsc70, respectively. In each set of panels, 1, 2 and 3, and 1', 2' and 3' are corresponding DIC and fluorescence optical sections. Section 2' in each panel is through the center of the nucleus (arrow), whereas 1' and 3' are 1.5 µm above and below that, respectively. (C) An enlargement of one area of an egg treated with 5 µg Hsc70 for 30 min, illustrating the absence of correspondence between locations of higher Hsc70 immunofluorescence and vesicles. The left and right panels are, respectively, the DIC and fluorescence images of identical regions. The solid circles enclose examples of vesicles in the DIC image and the corresponding areas in the fluorescence image, showing that higher fluorescence intensity does not co-localize with vesicles. Conversely, two locations of bright fluorescence enclosed by the broken circles in the right panel do not co-localize with vesicles in the left DIC image. The center pseudocolored rendition of the fluorescence image was created to confirm that the circles correspond exactly to the same areas in the two images. In this image, blue represents low and red represents high fluorescence intensity. The gray oval is a portion of the DIC image rendered semitransparent and superimposed over the identical area in the pseudocolor image to confirm correspondence.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Effect of Hsc70 on immunoreactive phospho-cdc2 levels following fertilization. (A) Gray-scale image of one blot of immunoreactive phospho-cdc2. (B) The same image in pseudocolor to emphasize the relative differences in each lane. The color spectrum bar shows the grayscale-to-color correspondence, with violet representing black, and red representing white. Variation in background is what accounts for the blue color in the left half of the blot and the blue-green on the right. Lanes 1 and 2 are unfertilized eggs collected at 0 and 50 min after 5 µg ml–1 Hsc70. Lanes 3 and 4 are fertilized eggs in 5 µg ml–1 Hsc70 at 25 and 50 min after fertilization, respectively. Lanes 5 and 6 are fertilized eggs in the absence of Hsc70 at 35 and 50 min after fertilization, respectively. Phospho-cdc2 remained at a higher level in Hsc70-treated eggs for the first 25–35 min after fertilization (compare lanes 3 and 5), but was about the same in the two groups by 50 min after fertilization. This blot is one of two, each showing the same trends.

View larger version (10K): [in this window] [in a new window]   Fig. 6. Inhibition by Hsp of the spreading of hypotonically shocked coelomocytes. Phagocytic coelomocytes were exposed to hypotonic shock in the presence or absence of 5 µg ml–1 recombinant Hsp70 or 5 µg ml–1 bovine Hsc70. Both Hsps dramatically inhibited hypotonic medium-induced spreading in coelomocytes, increasing the numbers of smaller diameter coelomocytes and decreasing the numbers of larger diameter ones. Significant differences (P<0.05) between Hsc70- or Hsp70-treated cells and controls are indicated by asterisks (determined by a two-factor ANOVA). Values are means ± s.e.m. N=5 replicate experiments in which at least 100 cells were observed.

View larger version (5K): [in this window] [in a new window]   Fig. 7. The effect of endotoxin on cell spreading in hypotonically shocked coelomocytes. Recombinant proteins may be contaminated with small amounts of bacterial endotoxin. To confirm that inhibition of coelomocyte spreading in the presence of recombinant Hsps was not a result of endotoxin, hypotonically shocked coelomocytes were exposed to endotoxin in concentrations similar to those measured in the recombinant Hsps. The endotoxin-treated cells were compared to cells hypotonically shocked (control) and hypotonically shocked in the presence of Hsp70 or Hsc70. Endotoxin-treated cells were not significantly different from control cells that had received only a hypotonic shock. The Hsp70- and Hsc70-treated cells were significantly different from both the endotoxin-treated and control cells, as indicated by the asterisk (P<0.05). Values are means ± s.e.m. N=5 replicate experiments in which at least 100 cells were observed.

View larger version (6K): [in this window] [in a new window]   Fig. 8. The inhibition of coelomocyte spreading in response to hypotonic shock is specific to Hsc70 and Hsp70. Coelomocytes exposed to 5 µg ml–1 of other proteins (lactoglobulin, casein and actin) during hypotonic shock spread normally, while those exposed to Hsp during hypotonic shock were inhibited. This result confirms that inhibition of spreading is Hsp-specific and not a result of the interaction of any protein with the cells during hypotonic shock. Values are means ± s.e.m. N=3 replicate experiments in which at least 100 cells were observed.

View larger version (11K): [in this window] [in a new window]   Fig. 9. The effect of heat shock on coelomocyte spreading. Isolated coelomocytes were heat-shocked in isotonic medium at 31°C for 30 min. Controls remained at room temperature. The percentages of cells on each slide that had petaloid, fibroblastic, or filopodial morphology were determined. Heat-shocked cells showed significantly more petaloid forms and fewer filopodial forms than unshocked or hypotonically shocked cells (asterisks, P<0.05). Thus, heat shock prior to hypotonic shock inhibited coelomocyte spreading to a similar extent as exogenous Hsp70 and Hsc70. Values are means ± s.e.m.

View larger version (26K): [in this window] [in a new window]   Fig. 10. The appearance of the actin cytoskeleton in Hsp70-treated coelomocytes. Coelomocytes exposed to hypotonic shock in the presence or absence of Hsp70 were fixed and stained with Texas Red phalloidin to visualize the actin cytoskeleton. The actin in coelomocytes incubated in hypotonic medium polymerized into long filaments that extend to the edges of the large, flattened cells (A–C). Hsp70-treated cells failed to spread or spread only partially and the actin appears as unpolymerized pools in the center of the rounded cells (D–F).