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J Cell Biol. 1989 June 1; 108(6): 2343–2353.
PMCID: PMC2115600

Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization


We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed that when eight gold particles were seen in this iterated array, the signal to noise ratio was greater than 30:1. Furthermore, these gold particles were colinear, often spiral, or circular suggesting detection of a single nucleic acid molecule. Antibodies against actin, vimentin, or tubulin proteins were used after in situ hybridization, allowing simultaneous detection of the protein and its cognate message on the same sample. This revealed that cytoskeletal mRNAs are likely to be extremely close to actin protein (5 nm or less) and unlikely to be within 20 nm of vimentin or tubulin filaments. Actin mRNA was found to be more predominant in lamellipodia of motile cells, confirming previous results. These results indicate that this high resolution in situ hybridization approach is a powerful tool by which to investigate the association of mRNA with the cytoskeleton.

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Selected References

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  • Ben-Ze'ev A, Horowitz M, Skolnik H, Abulafia R, Laub O, Aloni Y. The metabolism of SV40 RNA is associated with the cytoskeletal framework. Virology. 1981 Jun;111(2):475–487. [PubMed]
  • Binder M, Tourmente S, Roth J, Renaud M, Gehring WJ. In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes. J Cell Biol. 1986 May;102(5):1646–1653. [PMC free article] [PubMed]
  • Bird RC, Sells BH. Cytoskeleton involvement in the distribution of mRNP complexes and small cytoplasmic RNAs. Biochim Biophys Acta. 1986 Dec 18;868(4):215–225. [PubMed]
  • Bloemendal H, Benedetti EL, Ramaekers FC, Dunia I, Kibbelaar MA, Vermorken AJ. Is the cytoskeleton-plasma membrane complex involved in lens protein biosynthesis? Mol Biol Rep. 1979 May 31;5(1-2):99–103. [PubMed]
  • Bonneau AM, Darveau A, Sonenberg N. Effect of viral infection on host protein synthesis and mRNA association with the cytoplasmic cytoskeletal structure. J Cell Biol. 1985 Apr;100(4):1209–1218. [PMC free article] [PubMed]
  • Cervera M, Dreyfuss G, Penman S. Messenger RNA is translated when associated with the cytoskeletal framework in normal and VSV-infected HeLa cells. Cell. 1981 Jan;23(1):113–120. [PubMed]
  • Christensen AK, Kahn LE, Bourne CM. Circular polysomes predominate on the rough endoplasmic reticulum of somatotropes and mammotropes in the rat anterior pituitary. Am J Anat. 1987 Jan;178(1):1–10. [PubMed]
  • Chua NH, Blobel G, Siekevitz P, Palade GE. Periodic variations in the ratio of free to thylakoid-bound chloroplast ribosomes during the cell cycle of Chlamydomonas reinhardtii. J Cell Biol. 1976 Nov;71(2):497–514. [PMC free article] [PubMed]
  • Farmer SR, Ben-Ze'av A, Benecke BJ, Penman S. Altered translatability of messenger RNA from suspended anchorage-dependent fibroblasts: reversal upon cell attachment to a surface. Cell. 1978 Oct;15(2):627–637. [PubMed]
  • Fulton AB, Wan KM, Penman S. The spatial distribution of polyribosomes in 3T3 cells and the associated assembly of proteins into the skeletal framework. Cell. 1980 Jul;20(3):849–857. [PubMed]
  • Howe JG, Hershey JW. Translational initiation factor and ribosome association with the cytoskeletal framework fraction from HeLa cells. Cell. 1984 May;37(1):85–93. [PubMed]
  • Hutchison NJ, Langer-Safer PR, Ward DC, Hamkalo BA. In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold. J Cell Biol. 1982 Nov;95(2 Pt 1):609–618. [PMC free article] [PubMed]
  • Isaacs WB, Fulton AB. Cotranslational assembly of myosin heavy chain in developing cultured skeletal muscle. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6174–6178. [PubMed]
  • Kost TA, Theodorakis N, Hughes SH. The nucleotide sequence of the chick cytoplasmic beta-actin gene. Nucleic Acids Res. 1983 Dec 10;11(23):8287–8301. [PMC free article] [PubMed]
  • Langer PR, Waldrop AA, Ward DC. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6633–6637. [PubMed]
  • Lawrence JB, Singer RH. Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Res. 1985 Mar 11;13(5):1777–1799. [PMC free article] [PubMed]
  • Lawrence JB, Singer RH, Villnave CA, Stein JL, Stein GS. Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization. Proc Natl Acad Sci U S A. 1988 Jan;85(2):463–467. [PubMed]
  • Lenk R, Ransom L, Kaufmann Y, Penman S. A cytoskeletal structure with associated polyribosomes obtained from HeLa cells. Cell. 1977 Jan;10(1):67–78. [PubMed]
  • Lenk R, Ransom L, Kaufmann Y, Penman S. A cytoskeletal structure with associated polyribosomes obtained from HeLa cells. Cell. 1977 Jan;10(1):67–78. [PubMed]
  • Ornelles DA, Fey EG, Penman S. Cytochalasin releases mRNA from the cytoskeletal framework and inhibits protein synthesis. Mol Cell Biol. 1986 May;6(5):1650–1662. [PMC free article] [PubMed]
  • Pudney J, Singer RH. Electron microscopic visualization of the filamentous reticulum in whole cultured presumptive chick myoblasts. Am J Anat. 1979 Nov;156(3):321–336. [PubMed]
  • Pudney J, Singer RH. Intracellular filament bundles in whole mounts of chick and human myoblasts extracted with triton X-100. Tissue Cell. 1980;12(4):595–612. [PubMed]
  • Radic MZ, Lundgren K, Hamkalo BA. Curvature of mouse satellite DNA and condensation of heterochromatin. Cell. 1987 Sep 25;50(7):1101–1108. [PubMed]
  • Ramaekers FC, Benedetti EL, Dunia I, Vorstenbosch P, Bloemendal H. Polyribosomes associated with microfilaments in cultured lens cells. Biochim Biophys Acta. 1983 Sep 9;740(4):441–448. [PubMed]
  • Schwartz RJ, Rothblum KN. Gene switching in myogenesis: differential expression of the chicken actin multigene family. Biochemistry. 1981 Jul 7;20(14):4122–4129. [PubMed]
  • Singer RH, Ward DC. Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7331–7335. [PubMed]
  • Singer RH, Pudney JA. Filament-directed intercellular contacts during differentiation of cultured chick myoblasts. Tissue Cell. 1984;16(1):17–29. [PubMed]
  • Toh BH, Lolait SJ, Mathy JP, Baum R. Association of mitochondria with intermediate filaments and of polyribosomes with cytoplasmic actin. Cell Tissue Res. 1980;211(1):163–169. [PubMed]
  • Traub P, Nelson WJ. Polyribosomes are not associated with vimentin-type intermediate filaments in Ehrlich ascites tumor cells. Hoppe Seylers Z Physiol Chem. 1982 Oct;363(10):1177–1185. [PubMed]
  • van Venrooij WJ, Sillekens PT, van Eekelen CA, Reinders RJ. On the association of mRNA with the cytoskeleton in uninfected and adenovirus-infected human KB cells. Exp Cell Res. 1981 Sep;135(1):79–91. [PubMed]
  • Webster HF, Lamperth L, Favilla JT, Lemke G, Tesin D, Manuelidis L. Use of a biotinylated probe and in situ hybridization for light and electron microscopic localization of Po mRNA in myelin-forming Schwann cells. Histochemistry. 1987;86(5):441–444. [PubMed]

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