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. 1986 Jun 1;102(6):2006–2014. doi: 10.1083/jcb.102.6.2006

Determination of the intracellular state of soluble macromolecules by gel filtration in vivo in the cytoplasm of amphibian oocytes

PMCID: PMC2114267  PMID: 3711142

Abstract

A method to examine the diffusible state and the sizes of major cytoplasmic proteins in a living cell is described. Small (40-300 microns) commercially available gel filtration beads of a broad range of Mr exclusion limits were microsurgically implanted into the cytoplasm of oocytes of the frog, Xenopus laevis, usually after metabolic labeling of oocyte proteins with [35S]methionine. After equilibration in vivo for several hours, the appearance of the implanted cells, notably the bead-cytoplasm boundary, was examined by light and electron microscopy of sections and found to be unaffected. After incubation the beads were isolated, briefly rinsed, and their protein contents examined by one- or two-dimensional gel electrophoresis. We show that diffusible proteins can be identified by their inclusion in the pores of the gel filtration beads used and that their approximate sizes can be estimated from the size exclusion values of the specific materials used. The application of this method to important cell biological questions is demonstrated by showing that several "karyophobic proteins," i.e., proteins of the cytosolic fraction which accumulate in the cytoplasm in vivo, are indeed diffusible in the living oocyte and appear with sizes similar to those determined in vitro. This indicates that the nucleo-cytoplasmic distribution of certain diffusible proteins is governed, in addition to size exclusion at nuclear pore complexes and karyophilic "signals," by other, as yet unknown forces. Some possible applications of this method of gel filtration in vivo are discussed.

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Selected References

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  1. Bonner W. M. Protein migration into nuclei. I. Frog oocyte nuclei in vivo accumulate microinjected histones, allow entry to small proteins, and exclude large proteins. J Cell Biol. 1975 Feb;64(2):421–430. doi: 10.1083/jcb.64.2.421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bonner W. M. Protein migration into nuclei. II. Frog oocyte nuclei accumulate a class of microinjected oocyte nuclear proteins and exclude a class of microinjected oocyte cytoplasmic proteins. J Cell Biol. 1975 Feb;64(2):431–437. doi: 10.1083/jcb.64.2.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cleveland D. W., Pittenger M. F., Feramisco J. R. Elevation of tubulin levels by microinjection suppresses new tubulin synthesis. Nature. 1983 Oct 20;305(5936):738–740. doi: 10.1038/305738a0. [DOI] [PubMed] [Google Scholar]
  4. Colman A. Synthesis of RNA in oocytes of Xenopus laevis during culture in vitro. J Embryol Exp Morphol. 1974 Oct;32(2):515–532. [PubMed] [Google Scholar]
  5. Dabauvalle M. C., Franke W. W. Karyophilic proteins: polypeptides synthesized in vitro accumulate in the nucleus on microinjection into the cytoplasm of amphibian oocytes. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5302–5306. doi: 10.1073/pnas.79.17.5302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dabauvalle M. C., Franke W. W. Karyophobic proteins. A category of abundant soluble proteins which accumulate in the cytoplasm. Exp Cell Res. 1984 Aug;153(2):308–326. doi: 10.1016/0014-4827(84)90603-7. [DOI] [PubMed] [Google Scholar]
  7. De Robertis E. M., Longthorne R. F., Gurdon J. B. Intracellular migration of nuclear proteins in Xenopus oocytes. Nature. 1978 Mar 16;272(5650):254–256. doi: 10.1038/272254a0. [DOI] [PubMed] [Google Scholar]
  8. De Robertis E. M. Nucleocytoplasmic segregation of proteins and RNAs. Cell. 1983 Apr;32(4):1021–1025. doi: 10.1016/0092-8674(83)90285-4. [DOI] [PubMed] [Google Scholar]
  9. Dingwall C., Sharnick S. V., Laskey R. A. A polypeptide domain that specifies migration of nucleoplasmin into the nucleus. Cell. 1982 Sep;30(2):449–458. doi: 10.1016/0092-8674(82)90242-2. [DOI] [PubMed] [Google Scholar]
  10. Dumont J. N. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J Morphol. 1972 Feb;136(2):153–179. doi: 10.1002/jmor.1051360203. [DOI] [PubMed] [Google Scholar]
  11. Feldherr C. M., Cohen R. J., Ogburn J. A. Evidence for mediated protein uptake by amphibian oocyte nuclei. J Cell Biol. 1983 May;96(5):1486–1490. doi: 10.1083/jcb.96.5.1486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Feldherr C. M., Kallenbach E., Schultz N. Movement of a karyophilic protein through the nuclear pores of oocytes. J Cell Biol. 1984 Dec;99(6):2216–2222. doi: 10.1083/jcb.99.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Fulton A. B. How crowded is the cytoplasm? Cell. 1982 Sep;30(2):345–347. doi: 10.1016/0092-8674(82)90231-8. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Goldstein L., Ko C. Distribution of proteins between nucleus and cytoplasm of Amoeba proteus. J Cell Biol. 1981 Mar;88(3):516–525. doi: 10.1083/jcb.88.3.516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gurdon J. B. Nuclear transplantation and the control of gene activity in animal development. Proc R Soc Lond B Biol Sci. 1970 Dec 1;176(1044):303–314. doi: 10.1098/rspb.1970.0050. [DOI] [PubMed] [Google Scholar]
  18. Horowitz S. B., Miller D. S. Solvent properties of ground substance studied by cryomicrodissection and intracellular reference-phase techniques. J Cell Biol. 1984 Jul;99(1 Pt 2):172s–179s. doi: 10.1083/jcb.99.1.172s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Horowitz S. B., Paine P. L. Reference phase analysis of free and bound intracellular solutes. II. Isothermal and isotopic studies of cytoplasmic sodium, potassium, and water. Biophys J. 1979 Jan;25(1):45–62. doi: 10.1016/S0006-3495(79)85277-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Horowitz S. B., Paine P. L., Tluczek L., Reynhout J. K. Reference phase analysis of free and bound intracellular solutes. I. Sodium and potassium in amphibian oocytes. Biophys J. 1979 Jan;25(1):33–44. doi: 10.1016/S0006-3495(79)85276-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kalderon D., Richardson W. D., Markham A. F., Smith A. E. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature. 1984 Sep 6;311(5981):33–38. doi: 10.1038/311033a0. [DOI] [PubMed] [Google Scholar]
  22. Kleinschmidt J. A., Scheer U., Dabauvalle M. C., Bustin M., Franke W. W. High mobility group proteins of amphibian oocytes: a large storage pool of a soluble high mobility group-1-like protein and involvement in transcriptional events. J Cell Biol. 1983 Sep;97(3):838–848. doi: 10.1083/jcb.97.3.838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kreis T. E., Birchmeier W. Microinjection of fluorescently labeled proteins into living cells with emphasis on cytoskeletal proteins. Int Rev Cytol. 1982;75:209–214. doi: 10.1016/s0074-7696(08)61005-0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. Lanford R. E., Butel J. S. Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen. Cell. 1984 Jul;37(3):801–813. doi: 10.1016/0092-8674(84)90415-x. [DOI] [PubMed] [Google Scholar]
  28. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  29. Mattaj I. W., Lienhard S., Zeller R., DeRobertis E. M. Nuclear exclusion of transcription factor IIIA and the 42s particle transfer RNA-binding protein in Xenopus oocytes: a possible mechanism for gene control? J Cell Biol. 1983 Oct;97(4):1261–1265. doi: 10.1083/jcb.97.4.1261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Miller D. S., Lau Y. T., Horowitz S. B. Artifacts caused by cell microinjection. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1426–1430. doi: 10.1073/pnas.81.5.1426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mills A. D., Laskey R. A., Black P., De Robertis E. M. An acidic protein which assembles nucleosomes in vitro is the most abundant protein in Xenopus oocyte nuclei. J Mol Biol. 1980 May 25;139(3):561–568. doi: 10.1016/0022-2836(80)90148-5. [DOI] [PubMed] [Google Scholar]
  32. O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
  33. Paine P. L., Austerberry C. F., Desjarlais L. J., Horowitz S. B. Protein loss during nuclear isolation. J Cell Biol. 1983 Oct;97(4):1240–1242. doi: 10.1083/jcb.97.4.1240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Paine P. L. Diffusive and nondiffusive proteins in vivo. J Cell Biol. 1984 Jul;99(1 Pt 2):188s–195s. doi: 10.1083/jcb.99.1.188s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Paine P. L., Feldherr C. M. Nucleocytoplasmic exchange of macromolecules. Exp Cell Res. 1972 Sep;74(1):81–98. doi: 10.1016/0014-4827(72)90483-1. [DOI] [PubMed] [Google Scholar]
  36. Paine P. L., Moore L. C., Horowitz S. B. Nuclear envelope permeability. Nature. 1975 Mar 13;254(5496):109–114. doi: 10.1038/254109a0. [DOI] [PubMed] [Google Scholar]
  37. Peters R. Nucleo-cytoplasmic flux and intracellular mobility in single hepatocytes measured by fluorescence microphotolysis. EMBO J. 1984 Aug;3(8):1831–1836. doi: 10.1002/j.1460-2075.1984.tb02055.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rechsteiner M., Kuehl L. Microinjection of the nonhistone chromosomal protein HMG1 into bovine fibroblasts and HeLa cells. Cell. 1979 Apr;16(4):901–908. doi: 10.1016/0092-8674(79)90105-3. [DOI] [PubMed] [Google Scholar]
  39. Scheer U., Trendelenburg M. F., Franke W. W. Regulation of transcription of genes of ribosomal rna during amphibian oogenesis. A biochemical and morphological study. J Cell Biol. 1976 May;69(2):465–489. doi: 10.1083/jcb.69.2.465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stacey D. W., Allfrey V. G. Evidence for the autophagy of microinjected proteins in HeLA cells. J Cell Biol. 1977 Dec;75(3):807–817. doi: 10.1083/jcb.75.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stacey D. W., Allfrey V. G. Microinjection studies of protein transit across the nuclear envelope of human cells. Exp Cell Res. 1984 Sep;154(1):283–292. doi: 10.1016/0014-4827(84)90687-6. [DOI] [PubMed] [Google Scholar]
  42. Sugawa H., Imamoto N., Wataya-Kaneda M., Uchida T. Foreign protein can be carried into the nucleus of mammalian cell by conjugation with nucleoplasmin. Exp Cell Res. 1985 Aug;159(2):419–429. doi: 10.1016/s0014-4827(85)80015-x. [DOI] [PubMed] [Google Scholar]
  43. Taylor D. L., Wang Y. L. Molecular cytochemistry: incorporation of fluorescently labeled actin into living cells. Proc Natl Acad Sci U S A. 1978 Feb;75(2):857–861. doi: 10.1073/pnas.75.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Thomas J. O., Kornberg R. D. An octamer of histones in chromatin and free in solution. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2626–2630. doi: 10.1073/pnas.72.7.2626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wadsworth P., Sloboda R. D. Microinjection of fluorescent tubulin into dividing sea urchin cells. J Cell Biol. 1983 Oct;97(4):1249–1254. doi: 10.1083/jcb.97.4.1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wallace R. A., Hollinger T. G. Turnover of endogenous, microinjected, and sequestered protein in Xenopus oocytes. Exp Cell Res. 1979 Mar 15;119(2):277–287. doi: 10.1016/0014-4827(79)90355-0. [DOI] [PubMed] [Google Scholar]
  47. Wojcieszyn J. W., Schlegel R. A., Wu E. S., Jacobson K. A. Diffusion of injected macromolecules within the cytoplasm of living cells. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4407–4410. doi: 10.1073/pnas.78.7.4407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wu L., Rechsteiner M., Kuehl L. Comparative studies on microinjected high-mobility-group chromosomal proteins, HMG1 and HMG2. J Cell Biol. 1981 Nov;91(2 Pt 1):488–496. doi: 10.1083/jcb.91.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yamaizumi M., Uchida T., Okada Y., Furusawa M., Mitsui H. Rapid transfer of non-histone chromosomal proteins to the nucleus of living cells. Nature. 1978 Jun 29;273(5665):782–784. doi: 10.1038/273782a0. [DOI] [PubMed] [Google Scholar]
  50. Zeller R., Nyffenegger T., De Robertis E. M. Nucleocytoplasmic distribution of snRNPs and stockpiled snRNA-binding proteins during oogenesis and early development in Xenopus laevis. Cell. 1983 Feb;32(2):425–434. doi: 10.1016/0092-8674(83)90462-2. [DOI] [PubMed] [Google Scholar]

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