Abstract
Actin is of fundamental importance to all eukaryotic cells. Of the six mammalian actins, beta (beta) and gamma (gamma) cytoplasmic are the isoforms found in all nonmuscle cells and differ by only four amino acids at the amino-terminal region. Both genes are regulated temporally and spatially, though no differences in protein function have been described. Using fluorescent double in situ hybridization we describe the simultaneous intracellular localization of both beta and gamma actin mRNA. This study shows that myoblasts differentially segregate the beta and gamma actin mRNAs. The distribution of gamma actin mRNA, only to perinuclear and nearby cytoplasm, suggests a distribution based on diffusion or restriction to nearby cytoplasm. The distribution of beta actin mRNA, perinuclear and at the cell periphery, implicates a peripheral localizing signal which is unique to the beta isoform. The peripheral beta actin mRNA corresponded to cellular morphologies, extending processes, and ruffling edges that reflect cell movement. Total actin and gamma actin protein steady-state distributions were identified by specific antibodies. gamma actin protein was found in both stress fibers and at the cell plasma membrane and does not correspond to its mRNA distribution. We suggest that localized protein synthesis rather than steady-state distribution functionally differentiates between the actin isoforms.
Full Text
The Full Text of this article is available as a PDF (3.8 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Burgin K. E., Waxham M. N., Rickling S., Westgate S. A., Mobley W. C., Kelly P. T. In situ hybridization histochemistry of Ca2+/calmodulin-dependent protein kinase in developing rat brain. J Neurosci. 1990 Jun;10(6):1788–1798. doi: 10.1523/JNEUROSCI.10-06-01788.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carter K. C., Taneja K. L., Lawrence J. B. Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus. J Cell Biol. 1991 Dec;115(5):1191–1202. doi: 10.1083/jcb.115.5.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Colman D. R., Kreibich G., Frey A. B., Sabatini D. D. Synthesis and incorporation of myelin polypeptides into CNS myelin. J Cell Biol. 1982 Nov;95(2 Pt 1):598–608. doi: 10.1083/jcb.95.2.598. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DeNofrio D., Hoock T. C., Herman I. M. Functional sorting of actin isoforms in microvascular pericytes. J Cell Biol. 1989 Jul;109(1):191–202. doi: 10.1083/jcb.109.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Erba H. P., Eddy R., Shows T., Kedes L., Gunning P. Structure, chromosome location, and expression of the human gamma-actin gene: differential evolution, location, and expression of the cytoskeletal beta- and gamma-actin genes. Mol Cell Biol. 1988 Apr;8(4):1775–1789. doi: 10.1128/mcb.8.4.1775. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Erba H. P., Gunning P., Kedes L. Nucleotide sequence of the human gamma cytoskeletal actin mRNA: anomalous evolution of vertebrate non-muscle actin genes. Nucleic Acids Res. 1986 Jul 11;14(13):5275–5294. doi: 10.1093/nar/14.13.5275. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Garner C. C., Tucker R. P., Matus A. Selective localization of messenger RNA for cytoskeletal protein MAP2 in dendrites. Nature. 1988 Dec 15;336(6200):674–677. doi: 10.1038/336674a0. [DOI] [PubMed] [Google Scholar]
- Gavis E. R., Lehmann R. Localization of nanos RNA controls embryonic polarity. Cell. 1992 Oct 16;71(2):301–313. doi: 10.1016/0092-8674(92)90358-j. [DOI] [PubMed] [Google Scholar]
- Gottlieb E. The 3' untranslated region of localized maternal messages contains a conserved motif involved in mRNA localization. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7164–7168. doi: 10.1073/pnas.89.15.7164. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gunning P., Ponte P., Kedes L., Hickey R. J., Skoultchi A. I. Expression of human cardiac actin in mouse L cells: a sarcomeric actin associates with a nonmuscle cytoskeleton. Cell. 1984 Mar;36(3):709–715. doi: 10.1016/0092-8674(84)90351-9. [DOI] [PubMed] [Google Scholar]
- Hesketh J. E., Pryme I. F. Interaction between mRNA, ribosomes and the cytoskeleton. Biochem J. 1991 Jul 1;277(Pt 1):1–10. doi: 10.1042/bj2770001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoock T. C., Newcomb P. M., Herman I. M. Beta actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during the cellular response to injury. J Cell Biol. 1991 Feb;112(4):653–664. doi: 10.1083/jcb.112.4.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lawrence J. B., Singer R. H. Intracellular localization of messenger RNAs for cytoskeletal proteins. Cell. 1986 May 9;45(3):407–415. doi: 10.1016/0092-8674(86)90326-0. [DOI] [PubMed] [Google Scholar]
- Lawrence J. B., Singer R. H. Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Res. 1985 Mar 11;13(5):1777–1799. doi: 10.1093/nar/13.5.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lloyd C., Gunning P. Noncoding regions of the gamma-actin gene influence the impact of the gene on myoblast morphology. J Cell Biol. 1993 Apr;121(1):73–82. doi: 10.1083/jcb.121.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lloyd C., Schevzov G., Gunning P. Transfection of nonmuscle beta- and gamma-actin genes into myoblasts elicits different feedback regulatory responses from endogenous actin genes. J Cell Biol. 1992 May;117(4):787–797. doi: 10.1083/jcb.117.4.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Macdonald P. M., Struhl G. cis-acting sequences responsible for anterior localization of bicoid mRNA in Drosophila embryos. Nature. 1988 Dec 8;336(6199):595–598. doi: 10.1038/336595a0. [DOI] [PubMed] [Google Scholar]
- Martin P., Lewis J. Actin cables and epidermal movement in embryonic wound healing. Nature. 1992 Nov 12;360(6400):179–183. doi: 10.1038/360179a0. [DOI] [PubMed] [Google Scholar]
- Masibay A. S., Qasba P. K., Sengupta D. N., Damewood G. P., Sreevalsan T. Cell-cycle-specific and serum-dependent expression of gamma-actin mRNA in Swiss mouse 3T3 cells. Mol Cell Biol. 1988 Jun;8(6):2288–2294. doi: 10.1128/mcb.8.6.2288. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McCairns E., Fahey D., Muscat G. E., Murray M., Rowe P. B. Changes in levels of actin and tubulin mRNAs upon the lectin activation of lymphocytes. Mol Cell Biol. 1984 Sep;4(9):1754–1760. doi: 10.1128/mcb.4.9.1754. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McKenna N., Meigs J. B., Wang Y. L. Identical distribution of fluorescently labeled brain and muscle actins in living cardiac fibroblasts and myocytes. J Cell Biol. 1985 Jan;100(1):292–296. doi: 10.1083/jcb.100.1.292. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Merlie J. P., Sanes J. R. Concentration of acetylcholine receptor mRNA in synaptic regions of adult muscle fibres. Nature. 1985 Sep 5;317(6032):66–68. doi: 10.1038/317066a0. [DOI] [PubMed] [Google Scholar]
- Mowry K. L., Melton D. A. Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes. Science. 1992 Feb 21;255(5047):991–994. doi: 10.1126/science.1546297. [DOI] [PubMed] [Google Scholar]
- Ng S. Y., Gunning P., Eddy R., Ponte P., Leavitt J., Shows T., Kedes L. Evolution of the functional human beta-actin gene and its multi-pseudogene family: conservation of noncoding regions and chromosomal dispersion of pseudogenes. Mol Cell Biol. 1985 Oct;5(10):2720–2732. doi: 10.1128/mcb.5.10.2720. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Otey C. A., Kalnoski M. H., Bulinski J. C. Identification and quantification of actin isoforms in vertebrate cells and tissues. J Cell Biochem. 1987 Jun;34(2):113–124. doi: 10.1002/jcb.240340205. [DOI] [PubMed] [Google Scholar]
- Otey C. A., Kalnoski M. H., Bulinski J. C. Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle. Cell Motil Cytoskeleton. 1988;9(4):337–348. doi: 10.1002/cm.970090406. [DOI] [PubMed] [Google Scholar]
- Otey C. A., Kalnoski M. H., Lessard J. L., Bulinski J. C. Immunolocalization of the gamma isoform of nonmuscle actin in cultured cells. J Cell Biol. 1986 May;102(5):1726–1737. doi: 10.1083/jcb.102.5.1726. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pardo J. V., Pittenger M. F., Craig S. W. Subcellular sorting of isoactins: selective association of gamma actin with skeletal muscle mitochondria. Cell. 1983 Apr;32(4):1093–1103. doi: 10.1016/0092-8674(83)90293-3. [DOI] [PubMed] [Google Scholar]
- Peng I., Fischman D. A. Post-translational incorporation of actin into myofibrils in vitro: evidence for isoform specificity. Cell Motil Cytoskeleton. 1991;20(2):158–168. doi: 10.1002/cm.970200208. [DOI] [PubMed] [Google Scholar]
- Phillips L. L., Steward O. Increases in mRNA for cytoskeletal proteins in the denervated neuropil of the dentate gyrus: an in situ hybridization study using riboprobes for beta-actin and beta-tubulin. Brain Res Mol Brain Res. 1990 Aug;8(3):249–257. doi: 10.1016/0169-328x(90)90024-8. [DOI] [PubMed] [Google Scholar]
- Poirier J., Dea D., Baccichet A., Gauthier S. Modulation of gamma-actin and alpha 1-tubulin expression by corticosterone during neuronal plasticity in the hippocampus. Brain Res Mol Brain Res. 1992 Oct;15(3-4):263–268. doi: 10.1016/0169-328x(92)90117-t. [DOI] [PubMed] [Google Scholar]
- Pollard T. D., Cooper J. A. Actin and actin-binding proteins. A critical evaluation of mechanisms and functions. Annu Rev Biochem. 1986;55:987–1035. doi: 10.1146/annurev.bi.55.070186.005011. [DOI] [PubMed] [Google Scholar]
- Schevzov G., Lloyd C., Gunning P. High level expression of transfected beta- and gamma-actin genes differentially impacts on myoblast cytoarchitecture. J Cell Biol. 1992 May;117(4):775–785. doi: 10.1083/jcb.117.4.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Simon A. M., Hoppe P., Burden S. J. Spatial restriction of AChR gene expression to subsynaptic nuclei. Development. 1992 Mar;114(3):545–553. doi: 10.1242/dev.114.3.545. [DOI] [PubMed] [Google Scholar]
- Steward O., Banker G. A. Getting the message from the gene to the synapse: sorting and intracellular transport of RNA in neurons. Trends Neurosci. 1992 May;15(5):180–186. doi: 10.1016/0166-2236(92)90170-d. [DOI] [PubMed] [Google Scholar]
- Sundell C. L., Singer R. H. Actin mRNA localizes in the absence of protein synthesis. J Cell Biol. 1990 Dec;111(6 Pt 1):2397–2403. doi: 10.1083/jcb.111.6.2397. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sundell C. L., Singer R. H. Requirement of microfilaments in sorting of actin messenger RNA. Science. 1991 Sep 13;253(5025):1275–1277. doi: 10.1126/science.1891715. [DOI] [PubMed] [Google Scholar]
- Taneja K. L., Lifshitz L. M., Fay F. S., Singer R. H. Poly(A) RNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy. J Cell Biol. 1992 Dec;119(5):1245–1260. doi: 10.1083/jcb.119.5.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tokunaga K., Takeda K., Kamiyama K., Kageyama H., Takenaga K., Sakiyama S. Isolation of cDNA clones for mouse cytoskeletal gamma-actin and differential expression of cytoskeletal actin mRNAs in mouse cells. Mol Cell Biol. 1988 Sep;8(9):3929–3933. doi: 10.1128/mcb.8.9.3929. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vandekerckhove J., Weber K. At least six different actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino-terminal tryptic peptide. J Mol Biol. 1978 Dec 25;126(4):783–802. doi: 10.1016/0022-2836(78)90020-7. [DOI] [PubMed] [Google Scholar]
- Yaffe D., Saxel O. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature. 1977 Dec 22;270(5639):725–727. doi: 10.1038/270725a0. [DOI] [PubMed] [Google Scholar]
- Yisraeli J. K., Sokol S., Melton D. A. A two-step model for the localization of maternal mRNA in Xenopus oocytes: involvement of microtubules and microfilaments in the translocation and anchoring of Vg1 mRNA. Development. 1990 Feb;108(2):289–298. doi: 10.1242/dev.108.2.289. [DOI] [PubMed] [Google Scholar]
- Zuker M. On finding all suboptimal foldings of an RNA molecule. Science. 1989 Apr 7;244(4900):48–52. doi: 10.1126/science.2468181. [DOI] [PubMed] [Google Scholar]