Abstract
The plasmalemma and hyaline ectoplasm together constitute the sensory and motor organ of macrophages. The purpose of this study was to isolate this cell fraction in order to analyze it biochemically and functionally. Brief sonification of warmed rabbit lung macrophages caused release of heterodisperse hyaline blebs and filopodia, which were easily collected by differential centrifugation. Viewed in the electron microscope, these structures consisted of membrane-bounded sacs principally containing actin filaments. Some contained secondary lysosomes. They were enriched threefold over whole cell homogenates in specific adenylate cyclase activity and in trichloroacetic-acid-precipitable (125)I when derived from cells labeled with 125(I) by means of a lactoperoxidase-catalyzed reaction. These markers were found to have identical isopycnic densitites when macrophage homogenates were subjected to sedimentation in a focusing sucrose density gradient system, and these markers had densities distinct from those of other cytoplasmic organelles. These markers were therefore assumed to be associated with macrophage plasma membranes. The specific β- glucuronidase activity of the bleb fraction was similar to that of homogenates, but the blebs had considerably lower specific succinic dehydrogenase activity and RNA content, and DNA was undetectable. Electrophoresis of blebs solubilized in sodium dodecyl sulfate on polyacrylamide gels revealed polypeptides co-migrating with macrophage actin-binding protein, myosin, and actin; blebs also had EDTA-activated adenosine triphosphatase activity characteristic of myosin. The concentrations of actin-binding protein and myosin were higher in blebs than in cells or cytoplasmic extracts, whereas actin concentrations were similar (relative to extracts) or only slightly greater (than in cells). Blebs and intact cells had high lactate dehydrogenase activities in the presence but not the absence of Triton X-100. Blebs and cells oxidased 1-[(14)C]glucose, and the rate of glucose oxidation was increased substantially in the presence of latex beads. We conclude that intact sacs of plasmalemma encasing contractile proteins and cytoplasmic enzymes can be isolated from macrophages. They are enriched in myosin and actin-binding protein, indicating that the contractile apparatus is regulated in the cell periphery. These structures have the capacity to respond to environmental signals. We suggest the name "podosomes" for them because of their resemblance to macrophage pseudopodia. We propose that podosome formation results from rapid dissolution of the cortical gel when the membrane is in an actively extended configuration.
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- Beaufay H., Jacques P., Baudhuin P., Sellinger O. Z., Berthet J., De Duve C. Tissue fractionation studies. 18. Resolution of mitochondrial fractions from rat liver into three distinct populations of cytoplasmic particles by means of density equilibration in various gradients. Biochem J. 1964 Jul;92(1):184–205. doi: 10.1042/bj0920184. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bhisey A. N., Freed J. J. Remnant motility of macrophages treated with cytochalasin B in the presence of colchicine. Exp Cell Res. 1975 Oct 15;95(2):376–384. doi: 10.1016/0014-4827(75)90563-7. [DOI] [PubMed] [Google Scholar]
- COOPERSTEIN S. J., LAZAROW A., KURFESS N. J. A microspectrophotometric method for the determination of succinic dehydrogenase. J Biol Chem. 1950 Sep;186(1):129–139. [PubMed] [Google Scholar]
- Cheng H., Farquhar M. G. Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. I. Biochemical determination. J Cell Biol. 1976 Sep;70(3):660–670. doi: 10.1083/jcb.70.3.660. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Debanne M. T., Bell R., Dolovich J. Characteristics of the macrophage uptake of proteinase-alpha-macroglobulin complexes. Biochim Biophys Acta. 1976 Apr 23;428(2):466–475. doi: 10.1016/0304-4165(76)90055-6. [DOI] [PubMed] [Google Scholar]
- Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
- Gonatas N. K., Gonatas J. O., Stieber A., Antoine J. C., Avrameas S. Quantitative ultrastructural autoradiographic studies of iodinated plasma membranes of lymphocytes during segregation and internalization of surface immunoglobulins. J Cell Biol. 1976 Sep;70(3):477–493. doi: 10.1083/jcb.70.3.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hartwig J. H., Stossel T. P. Interactions of actin, myosin, and an actin-binding protein of rabbit pulmonary macrophages. III. Effects of cytochalasin B. J Cell Biol. 1976 Oct;71(1):295–303. doi: 10.1083/jcb.71.1.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hubbard A. L., Cohn Z. A. Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells. J Cell Biol. 1975 Feb;64(2):438–460. doi: 10.1083/jcb.64.2.438. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Keller H. U., Bessis M. Migration and chemotaxis of anucleate cytoplasmic leukocyte fragments. Nature. 1975 Dec 25;258(5537):723–724. doi: 10.1038/258723a0. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- MOORE R. D., SCHOENBERG M. D. THE RESPONSE OF THE HISTIOCYTES AND MACROPHAGES IN THE LUNGS OF RABBITS INJECTED WITH FREUND'S ADJUVANT. Br J Exp Pathol. 1964 Oct;45:488–497. [PMC free article] [PubMed] [Google Scholar]
- MYRVIK Q., LEAKE E. S., FARISS B. Studies on pulmonary alveolar macrophages from the normal rabbit: a technique to procure them in a high state of purity. J Immunol. 1961 Feb;86:128–132. [PubMed] [Google Scholar]
- Miranda A. F., Godman G. C., Tanenbaum S. W. Action of cytochalasin D on cells of established lines. II. Cortex and microfilaments. J Cell Biol. 1974 Aug;62(2):406–423. doi: 10.1083/jcb.62.2.406. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Quinlan D. C., Hochstadt J. Group translocation of the ribose moiety of inosine by vesicles of plasma membrane from T(3 cells transformed by Simian virus 40. J Biol Chem. 1976 Jan 25;251(2):344–354. [PubMed] [Google Scholar]
- Ray T. K., Forte J. G. Adenyl cyclase of oxyntic cells. Its association with different cellular membranes. Biochim Biophys Acta. 1974 Sep 23;363(3):320–339. doi: 10.1016/0005-2736(74)90072-8. [DOI] [PubMed] [Google Scholar]
- Scott R. E. Plasma membrane vesiculation: a new technique for isolation of plasma membranes. Science. 1976 Nov 12;194(4266):743–745. doi: 10.1126/science.982044. [DOI] [PubMed] [Google Scholar]
- Stossel T. P., Hartwig J. H. Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. II. Role in cytoplasmic movement and phagocytosis. J Cell Biol. 1976 Mar;68(3):602–619. doi: 10.1083/jcb.68.3.602. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stossel T. P., Mason R. J., Pollard T. D., Vaughan M. Isolation and properties of phagocytic vesicles. II. Alveolar macrophages. J Clin Invest. 1972 Mar;51(3):604–614. doi: 10.1172/JCI106850. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stossel T. P., Murad F., Mason R. J., Vaughan M. Regulation of glycogen metabolism in polymorphonuclear leukocytes. J Biol Chem. 1970 Nov 25;245(22):6228–6234. [PubMed] [Google Scholar]
- ULMER D. D., VALLEE B. L., WACKER W. E. Metalloenzymes and myocardial infarction. II. Malic and lactic dehydrogenase activities and zinc concentrations in serum. N Engl J Med. 1956 Sep 6;255(10):450–456. doi: 10.1056/NEJM195609062551001. [DOI] [PubMed] [Google Scholar]
- White A. A., Zenser T. V. Separation of cyclic 3',5'-nucleoside monophosphates from other nucleotides on aluminum oxide columns. Application to the assay of adenyl cyclase and guanyl cyclase. Anal Biochem. 1971 Jun;41(2):372–396. doi: 10.1016/0003-2697(71)90156-4. [DOI] [PubMed] [Google Scholar]
- ZOLLINGER H. U. Cytologic studies with the phase Microscope; the formation of blisters on cells in suspension, photocytosis, with observations on the nature of the cellular membrane. Am J Pathol. 1948 May;24(3):545–567. [PMC free article] [PubMed] [Google Scholar]
- Zentgraf H., Deumling B., Jarasch E. D., Franke W. W. Nuclear membranes and plasma membranes from hen erythrocytes. I. Isolation, characterization, and comparison. J Biol Chem. 1971 May 10;246(9):2986–2995. [PubMed] [Google Scholar]