Abstract
We localized myosin in vertebrate nonmuscle cells by electron microscopy using purified antibodies coupled with ferritin. Native and formaldehyde-fixed filaments of purified platelet myosin filaments each consisting of approximately 30 myosin molecules bound an equivalent number of ferritin-antimyosin conjugates. In preparations of crude platelet actomyosin, the ferritin-antimyosin bound exclusively to similar short, 10-15 nm wide filaments. In both cases, binding of the ferritin-antimyosin to the myosin filaments was blocked by preincubation with unlabeled antimyosin. With indirect fluorescent antibody staining at the light microscope level, we found that the ferritin-antimyosin and unlabeled antimyosin stained HeLa cells identically, with the antibodies concentrated in 0.5-microns spots along stress fibers. By electron microscopy, we found that the concentration of ferritin-antimyosin in the dense regions of stress fibers was five to six times that in the intervening less dense regions, 20 times that in the cytoplasmic matrix, and 100 times that in the nucleus. These concentration differences may account for the light microscope antibody staining pattern of spread interphase cells. Some, but certainly not all, of the ferritin-antimyosin was associated with 10-15-nm filaments. In mouse intestinal epithelial cells, ferritin- antimyosin was located almost exclusively in the terminal web. In isolated brush borders exposed to 5 mM MgCl2, ferritin-antimyosin was also concentrated in the terminal web associated with 10-15-nm filaments.
Full Text
The Full Text of this article is available as a PDF (830.7 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bretscher A., Weber K. Localization of actin and microfilament-associated proteins in the microvilli and terminal web of the intestinal brush border by immunofluorescence microscopy. J Cell Biol. 1978 Dec;79(3):839–845. doi: 10.1083/jcb.79.3.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Drenckhahn D., Gröschel-Stewart U. Localization of myosin, actin, and tropomyosin in rat intestinal epithelium: immunohistochemical studies at the light and electron microscope levels. J Cell Biol. 1980 Aug;86(2):475–482. doi: 10.1083/jcb.86.2.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fujiwara K., Pollard T. D. Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells. J Cell Biol. 1976 Dec;71(3):848–875. doi: 10.1083/jcb.71.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldman R. D., Chojnacki B., Yerna M. J. Ultrastructure of microfilament bundles in baby hamster kidney (BHK-21) cells. The use of tannic acid. J Cell Biol. 1979 Mar;80(3):759–766. doi: 10.1083/jcb.80.3.759. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Herman I. M., Pollard T. D. Actin localization in fixed dividing cells stained with fluorescent heavy meromyosin. Exp Cell Res. 1978 Jun;114(1):15–25. doi: 10.1016/0014-4827(78)90030-7. [DOI] [PubMed] [Google Scholar]
- Herman I. M., Pollard T. D. Comparison of purified anti-actin and fluorescent-heavy meromyosin staining patterns in dividing cells. J Cell Biol. 1979 Mar;80(3):509–520. doi: 10.1083/jcb.80.3.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kishida Y., Olsen B. R., Berg R. A., Prockop D. J. Two improved methods for preparing ferritin-protein conjugates for electron microscopy. J Cell Biol. 1975 Feb;64(2):331–339. doi: 10.1083/jcb.64.2.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuczmarski E. R., Rosenbaum J. L. Studies on the organization and localization of actin and myosin in neurons. J Cell Biol. 1979 Feb;80(2):356–371. doi: 10.1083/jcb.80.2.356. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mooseker M. S., Tilney L. G. Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells. J Cell Biol. 1975 Dec;67(3):725–743. doi: 10.1083/jcb.67.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Niederman R., Pollard T. D. Human platelet myosin. II. In vitro assembly and structure of myosin filaments. J Cell Biol. 1975 Oct;67(1):72–92. doi: 10.1083/jcb.67.1.72. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pepe F. A. The myosin filament. II. Interaction between myosin and actin filaments observed using antibody staining in fluorescent and electron microscopy. J Mol Biol. 1967 Jul 28;27(2):227–236. doi: 10.1016/0022-2836(67)90017-4. [DOI] [PubMed] [Google Scholar]
- Pollard T. D., Thomas S. M., Niederman R. Human platelet myosin. I. Purification by a rapid method applicable to other nonmuscle cells. Anal Biochem. 1974 Jul;60(1):258–266. doi: 10.1016/0003-2697(74)90152-3. [DOI] [PubMed] [Google Scholar]
- Pollard T. D., Weihing R. R. Actin and myosin and cell movement. CRC Crit Rev Biochem. 1974 Jan;2(1):1–65. doi: 10.3109/10409237409105443. [DOI] [PubMed] [Google Scholar]
- Roisen F., Inczedy-Marcsek M., Hsu L., Yorke W. Myosin: immunofluorescent localization in neuronal and glial cultures. Science. 1978 Mar 31;199(4336):1445–1448. doi: 10.1126/science.343252. [DOI] [PubMed] [Google Scholar]
- Stendahl O. I., Hartwig J. H., Brotschi E. A., Stossel T. P. Distribution of actin-binding protein and myosin in macrophages during spreading and phagocytosis. J Cell Biol. 1980 Feb;84(2):215–224. doi: 10.1083/jcb.84.2.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Unsicker K., Drenckhahn D., Grüschel-Stewart U. Further immunofluorescence-microscopic evidence for myosin in various peripheral nerves. Cell Tissue Res. 1978 Apr 17;188(2):341–344. doi: 10.1007/BF00222643. [DOI] [PubMed] [Google Scholar]