One of the defenses raised against LPS by the horseshoe crab,. Limulus, is a small, 101-amino acid, cationic proteinâLimulus anti-LPS factor (LALF) (4)âwhich ...
CELL BIOLOGY
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Reference: Biol. Bull. 203: 203–204. (October 2002)
Immunohistochemical Demonstration of a Lipopolysaccharide in the Cell Wall of a Eukaryote, the Green Alga, Chlorella Peter B. Armstrong (Department of Molecular and Cellular Biology, University of California, Davis, California), Margaret T. Armstrong1, R. L. Pardy2, Alice Child3, and Norman Wainwright3 The lipopolysaccharides (LPS) are ubiquitous components of the outer leaflet of the outer membrane of all gram-negative bacteria and are the principal toxic products of these organisms (1). The membrane anchor of LPS is lipid A, a central phosphodisaccharide unit that is attached to multiple -hydroxy fatty acid chains. LPS also contains the novel sugar, 3-deoxy-D-mannooctulosonic acid (KDO). Although generally believed to be restricted to prokaryotes, specifically the gram-negative eubacteria and the cyanobacteria (2), an LPS-like molecule has recently been reported from a eukaryote, the green alga Chlorella sp., strain NC64A. The algal molecule includes KDO, lipid A, and -hydroxy fatty acids and is thus chemically similar to bacterial LPS
1 Department of Molecular and Cellular Biology, University of California, Davis, CA 95616. 2 School of Biological Sciences, University of Nebraska, Lincoln, NE 68583. 3 Marine Biological Laboratory, Woods Hole, MA 02543.
(3). The subject of this study is the localization of the LPS-like molecule in the algal cell. One of the defenses raised against LPS by the horseshoe crab, Limulus, is a small, 101-amino acid, cationic protein—Limulus anti-LPS factor (LALF) (4)—which is released from the secretory granules of the blood cells during their exocytosis response to LPS challenge (5). LALF binds and neutralizes bacterial LPS (6). In the present study, we use the specific LPS-binding activity of LALF to localize the LPS-like molecule in eukaryotic and prokaryote cells. The reactivity of LALF is apparently specific for LPS, with an amphipathic loop of LALF serving as the LPS-binding motif (7). A standard assay for bacterial LPS is the Limulus amebocyte lysate (LAL) test. This test (Charles River Endosafe, used according to accompanying instructions) reported 7.3 U/ml for 50 g/ml of algal LPS. This activity was diminished to 0.08 U/ml in the presence of 50 g/ml of LALF. For immunohistochemical investigation, Chlorella cells, strain NC64A, were grown under bacteria-free conditions, then were freeze-dried and fixed in freshly-prepared 4% paraformaldehyde
Figure 1. Immunohistochemical demonstration of LPS at the surfaces of the Gram-negative bacterium, Escherichia coli (Fig. 1A, B), and the green alga, Chlorella strain NC64A (Fig. 1C, D). Cells were exposed to the LPS-binding protein, LALF, then immunostained with a rabbit anti-LALF antibody and a FITC-labeled second antibody. Fluorescence in Figure 1B and 1D shows the location of binding of LALF and, thus, the localization of highest concentrations of LPS. Figures 1A and 1C are phase contrast views of the fields shown in Figures 1B and 1D, respectively. Scale: 20 m.
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dissolved in phosphate-buffered saline for 10 min, then blocked successively with 0.1 M glycine in Tris-buffered saline (TBS) and with 5 mg/ml bovine serum albumin (BSA) in the same buffer. The cells were then exposed to 0.1 mg/ml LALF in TBS, then were treated with an anti-LALF antiserum (rabbit) in TBS ⫹ BSA, followed by exposure to a fluorescein-labeled goat anti-rabbit IgG second antibody in the same buffer. Finally, the cells were examined with a Zeiss Axiophot 2 fluorescence microscope. The LALF labeling procedure stained the outer surface of aldehyde-fixed Escherichia coli cells, which served as our positive control for the method (Fig. 1A, 1B). Cells of the alga Chlorella showed a similar staining pattern, with LALF staining the outer surface of the cell, the cell wall (Fig. 1C, 1D). The cells failed to stain when LALF was omitted from the reaction scheme. This observation indicates that the LPS-like molecule of Chlorella is positioned at the appropriate location for a true lipopolysaccharide and adds substance to the still controversial claim that LPS is not confined to the prokaryotes, but is also present in certain eukaryotes. This research was supported by NSF Grant No. MCB 26771.
Literature Cited 1. Levin, J. 1988. Pp. 3–15 in The Horseshoe Crab: A Model for Gram-Negative Sepsis in Marine Organisms and Humans, J. Levin, H. R. Buller, J. W. Ten Cate, S. J. H. VanDeventer, and A. Sturk, eds. Alan R. Liss, New York. 2. Mikheyskaya, L. V., R. G. Ovodova, and Y. S. Ovodov. 1977. J. Bacteriol. 130: 1–3. 3. Royce, C. L., and R. L. Pardy, 1996. J. Endotoxin Res. 3: 437– 444. 4. Aketagawa, J., T. Miyata, S. Ohtsubo, T. Nakamura, T. Morita, H. Hayashida, S. Iwanaga, T. Takao, and Y. Shimonishi. 1986. J. Biol. Chem. 261: 7357–7365. 5. Armstrong, P. B., and F. R. Rickles. 1982. Exp. Cell Res. 140: 15–24. 6. Wainwright, N. R., R. J. Miller, E. Paus, T. J. Novitsky, M. A. Fletcher, T. M. McKenna, and T. Williams. 1990. Pp. 315–325 in Cellular and Molecular Aspects of Endotoxin Reactions, A. Nowotmy, J. J. Spitzer, and E. J. Ziegler, eds. Elsevier Science Publishers B.V., New York. 7. Hoess, A., S. Watson, G. R. Siber, and R. Liddington. 1993. EMBO J. 12: 3351–3356.
Reference: Biol. Bull. 203: 204 –206. (October 2002)
Rapid Visualization of Microtubules in Blood Cells and Other Cell Types in Marine Model Organisms K-G. Lee (CUNY Graduate Center, New York), A. Braun1, I. Chaikhoutdinov1, J. DeNobile1, M. Conrad1, and W. Cohen1 Although specific proteins in living cells can now be labeled routinely with Green Fluorescent Protein, indirect immunofluorescence (IIF) methods for fixed material remain in widespread use (e.g., 1). While relatively easy to apply, the standard IIF procedure is lengthy and, for blood cells and other cell types in suspension, the required attachment of the material to a glass substrate can result in differential adhesion or losses. In addition, the nonmammalian erythrocytes and clotting cells (2–5) studied in our laboratory undergo a variety of naturally occurring or experimentally induced alterations to cell morphology. For these cell types, the fixation and permeabilization methods that have produced our best IIF cytoskeletal labeling to date have not preserved the morphology of the living cells very well. This work had three initial objectives: (a) developing improved methods for morphological preservation and permeabilization of non-mammalian erythrocytes and clotting cells prior to IIF; (b) combining such methods with rapid fluorescence pre-labeling of a mouse primary antibody (use of Zenon™; 6) to eliminate steps including substrate attachment; and (c) testing the combined approach on cells studied by others, or previously unstudied. For objectives (a) and (b) we employed dogfish erythrocytes and thrombocytes (Mustelus canis), blood ark erythrocytes (Anadara ovalis), and horseshoe crab amebocytes (Limulus polyphemus), all of which contain a marginal band (MB) of microtubules. For (c) we tested sea urchin sperm (Arbacia punctulata) and dividing 1
Hunter College of the City University of New York.
zygotes (Lytechinus pictus) with known microtubule organization, plus spider crab hemocytes (Libinia emarginata) not studied previously. Dogfish erythrocytes were first employed in an experimental survey of variables to develop both sequential and simultaneous methods of rapid fixation and permeabilization (objective a). Standard aldehyde or methanol fixation had produced cross-linked hemoglobin (Hb) that blocked antibody access in our earlier studies, and complete detergent lysis prior to fixation distorted cell morphology. Our experiments produced a major advance: brief formaldehyde prefixation (1%, ⬍ 7 min) and detergent extraction (0.4% Triton X-100, 10 min) yielded partial Hb retention and superior preservation of erythrocyte morphology, yet also allowed IIF labeling. Similar results were obtained with erythrocytes treated simultaneously with 4% formaldehyde and 0.6% Brij 58 (10 min). The slow extraction rate observed with Brij (compared with Triton) minimized morphological distortion when cells were not pre-fixed. These methods were then tested on erythrocytes, other blood cells, and additional cell types in combination with Zenon™ labeling (objectives b and c). Thrombocytes were pre-fixed in 1% formaldehyde in 3% non-pyrogenic NaCl (”saline,” ⬃7 min), then extracted with 0.4% Triton X-100 in PEM (100 mM PIPES, 5 mM EGTA, 1 mM MgCl2, pH 6.8, 10 min). Other cell types were permeabilized and fixed simultaneously in PEM containing 4% formaldehyde, 0.6% Brij 58, plus precautionary protease inhibitors (Sigma P8340 cocktail ⫹ 10 mM TAME; amebocytes: TAME only). All preparations were washed in phosphate buffered saline,
