Abstract
We purified actin antibodies by affinity chromatography from the serum of rabbits immunized with glutaraldehyde-fixed chicken gizzard actin filaments and used this anti-actin to localize actin in myofibrils and fixed cultured cells at each stage of the cell cycle. By double immunodiffusion the anti-actin reacted with both smooth and skeletal muscle actin. Several blocking and absorption experiments demonstrated that the antibodies also bound specifically to actin in nonmuscle cells. The same structures stained using either the direct or the indirect fluorescent antibody technique; and, while the indirect method was more sensitive, the direct method was superior because there was no detectable nonspecific staining. As expected, anti-actin stained the I- band of myofibrils. It also stained stress fibers and membrane ruffles in HeLa cells. Some PtK-2 cells have straight stress fibers which stained with anti-actin, but in confluent cultures all PtK-2 cells have, instead, sinuous phase-dense fibers which stained with antibody. At prophase the whole cytoplasm stained uniformly with anti-actin. During metaphase and anaphase, anti-actin staining was concentrated diffusely in the mitotic spindle. In contrast, fluorescent heavy meromyosin stained discrete fine spindle fibers in these fixed cells. During cytokinesis, anti-actin stained the whole cytoplasm uniformly and was not concentrated in the cleavage furrow.
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Selected References
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- 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]
- Gröshel-Stewart U., Unsicker K. Direct visualization of contractile proteins in pertubular cells of the guinea-pig testis using antibodies against highly purified actin and myosin. Histochemistry. 1977 Apr 4;51(4):315–319. doi: 10.1007/BF00494367. [DOI] [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]
- Hinkley R., Telser A. Heavy meromyosin-binding filaments in the mitotic apparatus of mammaliam cells. Exp Cell Res. 1974 May;86(1):161–164. doi: 10.1016/0014-4827(74)90662-4. [DOI] [PubMed] [Google Scholar]
- Ishikawa H., Bischoff R., Holtzer H. Formation of arrowhead complexes with heavy meromyosin in a variety of cell types. J Cell Biol. 1969 Nov;43(2):312–328. [PMC free article] [PubMed] [Google Scholar]
- Lazarides E. Immunofluorescence studies on the structure of actin filaments in tissue culture cells. J Histochem Cytochem. 1975 Jul;23(7):507–528. doi: 10.1177/23.7.1095651. [DOI] [PubMed] [Google Scholar]
- Osborn M., Franke W. W., Weber K. Visualization of a system of filaments 7-10 nm thick in cultured cells of an epithelioid line (Pt K2) by immunofluorescence microscopy. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2490–2494. doi: 10.1073/pnas.74.6.2490. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schroeder T. E. Actin in dividing cells: contractile ring filaments bind heavy meromyosin. Proc Natl Acad Sci U S A. 1973 Jun;70(6):1688–1692. doi: 10.1073/pnas.70.6.1688. [DOI] [PMC free article] [PubMed] [Google Scholar]