Abstract
The time course and pattern of incorporation of rhodamine-labeled actin microinjected into cultured fibroblastic cells were examined by fluorescence microscopy. Following microinjection, the fluorescent probe was incorporated rapidly into ruffling membranes, and within 5 min faintly fluorescent stress fibers were observed. Levels of fluorescence in ruffling membranes then tended to remain constant while fluorescence of the stress fibers continued to increase until approximately 20-min postinjection. Small, discrete regions of some microinjected cells displayed high levels of fluorescence that appeared initially approximately 5-10 min postinjection. I observed these small areas of intense fluorescence frequently near the cell periphery, which corresponded to focal contacts when examined with interference reflection optics. The results of this study show that a relationship exists between patterns of fluorescent actin incorporation in these cells and cellular areas or structures presumed to play a role in cell movement. These findings suggest that actin within stress fibers and the microfilament network of ruffling membranes undergoes a rapid turnover that may relate directly to the motility of the cell.
Full Text
The Full Text of this article is available as a PDF (2.5 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Abercrombie M., Dunn G. A. Adhesions of fibroblasts to substratum during contact inhibition observed by interference reflection microscopy. Exp Cell Res. 1975 Apr;92(1):57–62. doi: 10.1016/0014-4827(75)90636-9. [DOI] [PubMed] [Google Scholar]
- Birchmeier C., Kreis T. E., Eppenberger H. M., Winterhalter K. H., Birchmeier W. Corrugated attachment membrane in WI-38 fibroblasts: alternating fibronectin fibers and actin-containing focal contacts. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4108–4112. doi: 10.1073/pnas.77.7.4108. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blikstad I., Carlsson L. On the dynamics of the microfilament system in HeLa cells. J Cell Biol. 1982 Apr;93(1):122–128. doi: 10.1083/jcb.93.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bray D., Thomas C. The actin content of fibroblasts. Biochem J. 1975 May;147(2):221–228. doi: 10.1042/bj1470221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burridge K., Feramisco J. R. Microinjection and localization of a 130K protein in living fibroblasts: a relationship to actin and fibronectin. Cell. 1980 Mar;19(3):587–595. doi: 10.1016/s0092-8674(80)80035-3. [DOI] [PubMed] [Google Scholar]
- Diacumakos E. G. Methods for micromanipulation of human somatic cells in culture. Methods Cell Biol. 1973;7:287–311. doi: 10.1016/s0091-679x(08)61783-5. [DOI] [PubMed] [Google Scholar]
- Feramisco J. R., Blose S. H. Distribution of fluorescently labeled alpha-actinin in living and fixed fibroblasts. J Cell Biol. 1980 Aug;86(2):608–615. doi: 10.1083/jcb.86.2.608. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Feramisco J. R. Microinjection of fluorescently labeled alpha-actinin into living fibroblasts. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3967–3971. doi: 10.1073/pnas.76.8.3967. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gawlitta W., Stockem W., Wehland J., Weber K. Organization and spatial arrangement of fluorescein-labeled native actin microinjected into normal locomoting and experimentally influenced Amoeba proteus. Cell Tissue Res. 1980;206(2):181–191. doi: 10.1007/BF00232762. [DOI] [PubMed] [Google Scholar]
- Gawlitta W., Stockem W., Wehland J., Weber K. Pinocytosis and locomotion of amoebae. XV. Visualization of Ca++-dynamics by chlorotetracycline (CTC) fluorescence during induced pinocytosis in living Amoeba proteus. Cell Tissue Res. 1980;213(1):9–20. doi: 10.1007/BF00236916. [DOI] [PubMed] [Google Scholar]
- Geiger B. A 130K protein from chicken gizzard: its localization at the termini of microfilament bundles in cultured chicken cells. Cell. 1979 Sep;18(1):193–205. doi: 10.1016/0092-8674(79)90368-4. [DOI] [PubMed] [Google Scholar]
- Geiger B. The association of rhodamine - labelled alpha-actinin with actin bundles in demembranated cells. Cell Biol Int Rep. 1981 Jun;5(6):627–634. doi: 10.1016/s0309-1651(81)80015-x. [DOI] [PubMed] [Google Scholar]
- Glacy S. D. Pattern and time course of rhodamine-actin incorporation in cardiac myocytes. J Cell Biol. 1983 Apr;96(4):1164–1167. doi: 10.1083/jcb.96.4.1164. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gordon W. E., 3rd, Bushnell A. Immunofluorescent and ultrastructural studies of polygonal microfilament networks in respreading non-muscle cells. Exp Cell Res. 1979 May;120(2):335–348. doi: 10.1016/0014-4827(79)90393-8. [DOI] [PubMed] [Google Scholar]
- Herman I. M., Crisona N. J., Pollard T. D. Relation between cell activity and the distribution of cytoplasmic actin and myosin. J Cell Biol. 1981 Jul;90(1):84–91. doi: 10.1083/jcb.90.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Keith C. H., Feramisco J. R., Shelanski M. Direct visualization of fluorescein-labeled microtubules in vitro and in microinjected fibroblasts. J Cell Biol. 1981 Jan;88(1):234–240. doi: 10.1083/jcb.88.1.234. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kiehart D. P., Mabuchi I., Inoué S. Evidence that myosin does not contribute to force production in chromosome movement. J Cell Biol. 1982 Jul;94(1):165–178. doi: 10.1083/jcb.94.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klymkowsky M. W. Intermediate filaments in 3T3 cells collapse after intracellular injection of a monoclonal anti-intermediate filament antibody. Nature. 1981 May 21;291(5812):249–251. doi: 10.1038/291249a0. [DOI] [PubMed] [Google Scholar]
- Korn E. D. Biochemistry of actomyosin-dependent cell motility (a review). Proc Natl Acad Sci U S A. 1978 Feb;75(2):588–599. doi: 10.1073/pnas.75.2.588. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kreis T. E., Birchmeier W. Stress fiber sarcomeres of fibroblasts are contractile. Cell. 1980 Nov;22(2 Pt 2):555–561. doi: 10.1016/0092-8674(80)90365-7. [DOI] [PubMed] [Google Scholar]
- Kreis T. E., Geiger B., Schlessinger J. Mobility of microinjected rhodamine actin within living chicken gizzard cells determined by fluorescence photobleaching recovery. Cell. 1982 Jul;29(3):835–845. doi: 10.1016/0092-8674(82)90445-7. [DOI] [PubMed] [Google Scholar]
- Kreis T. E., Winterhalter K. H., Birchmeier W. In vivo distribution and turnover of fluorescently labeled actin microinjected into human fibroblasts. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3814–3818. doi: 10.1073/pnas.76.8.3814. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin J. J., Feramisco J. R. Disruption of the in vivo distribution of the intermediate filaments in fibroblasts through the microinjection of a specific monoclonal antibody. Cell. 1981 Apr;24(1):185–193. doi: 10.1016/0092-8674(81)90514-6. [DOI] [PubMed] [Google Scholar]
- Mabuchi I., Okuno M. The effect of myosin antibody on the division of starfish blastomeres. J Cell Biol. 1977 Jul;74(1):251–263. doi: 10.1083/jcb.74.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meeusen R. L., Bennett J., Cande W. Z. Effect of microinjected N-ethylmaleimide-modified heavy meromyosin on cell division in amphibian eggs. J Cell Biol. 1980 Sep;86(3):858–865. doi: 10.1083/jcb.86.3.858. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pollard T. D. Cytoplasmic contractile proteins. J Cell Biol. 1981 Dec;91(3 Pt 2):156s–165s. doi: 10.1083/jcb.91.3.156s. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rubinstein N., Chi J., Holtzer H. Coordinated synthesis and degradation of actin and myosin in a variety of myogenic and non-myogenic cells. Exp Cell Res. 1976 Feb;97(2):387–393. doi: 10.1016/0014-4827(76)90630-3. [DOI] [PubMed] [Google Scholar]
- Rungger D., Rungger-Brändle E., Chaponnier C., Gabbiani G. Intranuclear injection of anti-actin antibodies into Xenopus oocytes blocks chromosome condensation. Nature. 1979 Nov 15;282(5736):320–321. doi: 10.1038/282320a0. [DOI] [PubMed] [Google Scholar]
- Sanger J. W., Sanger J. M., Kreis T. E., Jockusch B. M. Reversible translocation of cytoplasmic actin into the nucleus caused by dimethyl sulfoxide. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5268–5272. doi: 10.1073/pnas.77.9.5268. [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]
- Small J. V., Isenberg G., Celis J. E. Polarity of actin at the leading edge of cultured cells. Nature. 1978 Apr 13;272(5654):638–639. doi: 10.1038/272638a0. [DOI] [PubMed] [Google Scholar]
- Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
- Stockem W., Weber K., Wehland J. The influence of microinjected phalloidin on locomotion, protoplasmic streaming and cytoplasmic organization in Amoeba proteus and Physarum polycephalum. Cytobiologie. 1978 Oct;18(1):114–131. [PubMed] [Google Scholar]
- Taylor D. L., Blinks J. R., Reynolds G. Contractile basis of ameboid movement. VII. Aequorin luminescence during ameboid movement, endocytosis, and capping. J Cell Biol. 1980 Aug;86(2):599–607. doi: 10.1083/jcb.86.2.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Taylor D. L., Wang Y. L. Fluorescently labelled molecules as probes of the structure and function of living cells. Nature. 1980 Apr 3;284(5755):405–410. doi: 10.1038/284405a0. [DOI] [PubMed] [Google Scholar]
- Taylor D. L., Wang Y. L., Heiple J. M. Contractile basis of ameboid movement. VII. The distribution of fluorescently labeled actin in living amebas. J Cell Biol. 1980 Aug;86(2):590–598. doi: 10.1083/jcb.86.2.590. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang Y. L., Heiple J. M., Taylor D. L. Fluorescent analog cytochemistry of contractile proteins. Methods Cell Biol. 1982;25(Pt B):1–11. [PubMed] [Google Scholar]
- Wang Y. L., Taylor D. L. Distribution of fluorescently labeled actin in living sea urchin eggs during early development. J Cell Biol. 1979 Jun;81(3):672–679. doi: 10.1083/jcb.81.3.672. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang Y. L., Taylor D. L. Preparation and characterization of a new molecular cytochemical probe: 5-iodoacetamidofluorescein-labeled actin. J Histochem Cytochem. 1980 Nov;28(11):1198–1206. doi: 10.1177/28.11.6107318. [DOI] [PubMed] [Google Scholar]
- Weeds A. Actin-binding proteins--regulators of cell architecture and motility. Nature. 1982 Apr 29;296(5860):811–816. doi: 10.1038/296811a0. [DOI] [PubMed] [Google Scholar]
- Wehland J., Osborn M., Weber K. Phalloidin associates with microfilaments after microinjection into tissue culture cells. Eur J Cell Biol. 1980 Jun;21(2):188–194. [PubMed] [Google Scholar]
- Wehland J., Osborn M., Weber K. Phalloidin-induced actin polymerization in the cytoplasm of cultured cells interferes with cell locomotion and growth. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5613–5617. doi: 10.1073/pnas.74.12.5613. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wehland J., Weber K. Actin rearrangement in living cells revealed by microinjection of a fluorescent phalloidin derivative. Eur J Cell Biol. 1981 Jun;24(2):176–183. [PubMed] [Google Scholar]
- Wehland J., Weber K. Distribution of fluorescently labeled actin and tropomyosin after microinjection in living tissue culture cells as observed with TV image intensification. Exp Cell Res. 1980 Jun;127(2):397–408. doi: 10.1016/0014-4827(80)90444-9. [DOI] [PubMed] [Google Scholar]
- Willingham M. C., Yamada S. S., Davies P. J., Rutherford A. V., Gallo M. G., Pastan I. Intracellular localization of actin in cultured fibroblasts by electron microscopic immunocytochemistry. J Histochem Cytochem. 1981 Jan;29(1):17–37. doi: 10.1177/29.1.7009728. [DOI] [PubMed] [Google Scholar]
- von Olenhusen K. G., Wohlfarth-Bottermann K. E. Evidence for actin transformation during the contraction-relaxation cycle of cytoplasmic actomyosin: cycle blockade by phalloidin injection. Cell Tissue Res. 1979 Feb 28;196(3):455–470. doi: 10.1007/BF00234740. [DOI] [PubMed] [Google Scholar]