Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2001 Oct;81(4):2010–2019. doi: 10.1016/s0006-3495(01)75851-1

Reduced protein diffusion rate by cytoskeleton in vegetative and polarized dictyostelium cells.

E O Potma 1, W P de Boeij 1, L Bosgraaf 1, J Roelofs 1, P J van Haastert 1, D A Wiersma 1
PMCID: PMC1301675  PMID: 11566774

Abstract

Fluorescence recovery after photobleaching measurements with high spatial resolution are performed to elucidate the impact of the actin cytoskeleton on translational mobility of green fluorescent protein (GFP) in aqueous domains of Dictyostelium discoideum amoebae. In vegetative Dictyostelium cells, GFP molecules experience a 3.6-fold reduction of their translational mobility relative to dilute aqueous solutions. In disrupting the actin filamentous network using latrunculin-A, the intact actin cytoskeletal network is shown to contribute an effective viscosity of 1.36 cP, which accounts for 53% of the restrained molecular diffusion of GFP. The remaining 47% of hindered protein motions is ascribed to other mechanical barriers and the viscosity of the cell liquid. A direct correlation between the density of the actin network and its limiting action on protein diffusion is furthermore established from measurements under different osmotic conditions. In highly locomotive polarized cells, the obstructing effect of the actin filamentous network is seen to decline to 0.46 cP in the non-cortical regions of the cell. Our results indicate that the meshwork of actin filaments constitutes the primary mechanical barrier for protein diffusion and that any noticeable reorganization of the network is accompanied by altered intracellular protein mobility.

Full Text

The Full Text of this article is available as a PDF (211.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Axelrod D., Koppel D. E., Schlessinger J., Elson E., Webb W. W. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J. 1976 Sep;16(9):1055–1069. doi: 10.1016/S0006-3495(76)85755-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ayscough K. R., Stryker J., Pokala N., Sanders M., Crews P., Drubin D. G. High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A. J Cell Biol. 1997 Apr 21;137(2):399–416. doi: 10.1083/jcb.137.2.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Brown E. B., Wu E. S., Zipfel W., Webb W. W. Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery. Biophys J. 1999 Nov;77(5):2837–2849. doi: 10.1016/S0006-3495(99)77115-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Condeelis J. Life at the leading edge: the formation of cell protrusions. Annu Rev Cell Biol. 1993;9:411–444. doi: 10.1146/annurev.cb.09.110193.002211. [DOI] [PubMed] [Google Scholar]
  6. Cooper J. A. The role of actin polymerization in cell motility. Annu Rev Physiol. 1991;53:585–605. doi: 10.1146/annurev.ph.53.030191.003101. [DOI] [PubMed] [Google Scholar]
  7. Dictyostelium discoideum: molecular approaches to cell biology. Methods Cell Biol. 1987;28:1–516. [PubMed] [Google Scholar]
  8. Fishkind D. J., Wang Y. L. New horizons for cytokinesis. Curr Opin Cell Biol. 1995 Feb;7(1):23–31. doi: 10.1016/0955-0674(95)80041-7. [DOI] [PubMed] [Google Scholar]
  9. Garcia-Parajo M. F., Segers-Nolten G. M., Veerman J. A., Greve J., van Hulst N. F. Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules. Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7237–7242. doi: 10.1073/pnas.97.13.7237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Houtsmuller A. B., Rademakers S., Nigg A. L., Hoogstraten D., Hoeijmakers J. H., Vermeulen W. Action of DNA repair endonuclease ERCC1/XPF in living cells. Science. 1999 May 7;284(5416):958–961. doi: 10.1126/science.284.5416.958. [DOI] [PubMed] [Google Scholar]
  11. Johnson E. M., Berk D. A., Jain R. K., Deen W. M. Hindered diffusion in agarose gels: test of effective medium model. Biophys J. 1996 Feb;70(2):1017–1023. doi: 10.1016/S0006-3495(96)79645-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Konzok A., Weber I., Simmeth E., Hacker U., Maniak M., Müller-Taubenberger A. DAip1, a Dictyostelium homologue of the yeast actin-interacting protein 1, is involved in endocytosis, cytokinesis, and motility. J Cell Biol. 1999 Jul 26;146(2):453–464. doi: 10.1083/jcb.146.2.453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kuwayama H., Ecke M., Gerisch G., Van Haastert P. J. Protection against osmotic stress by cGMP-mediated myosin phosphorylation. Science. 1996 Jan 12;271(5246):207–209. doi: 10.1126/science.271.5246.207. [DOI] [PubMed] [Google Scholar]
  14. Luby-Phelps K., Castle P. E., Taylor D. L., Lanni F. Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4910–4913. doi: 10.1073/pnas.84.14.4910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Luby-Phelps K. Cytoarchitecture and physical properties of cytoplasm: volume, viscosity, diffusion, intracellular surface area. Int Rev Cytol. 2000;192:189–221. doi: 10.1016/s0074-7696(08)60527-6. [DOI] [PubMed] [Google Scholar]
  16. Luby-Phelps K., Taylor D. L., Lanni F. Probing the structure of cytoplasm. J Cell Biol. 1986 Jun;102(6):2015–2022. doi: 10.1083/jcb.102.6.2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nolta K. V., Steck T. L. Isolation and initial characterization of the bipartite contractile vacuole complex from Dictyostelium discoideum. J Biol Chem. 1994 Jan 21;269(3):2225–2233. [PubMed] [Google Scholar]
  18. Novak K. D., Titus M. A. Myosin I overexpression impairs cell migration. J Cell Biol. 1997 Feb 10;136(3):633–647. doi: 10.1083/jcb.136.3.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Olveczky B. P., Verkman A. S. Monte Carlo analysis of obstructed diffusion in three dimensions: application to molecular diffusion in organelles. Biophys J. 1998 May;74(5):2722–2730. doi: 10.1016/S0006-3495(98)77978-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pang K. M., Lee E., Knecht D. A. Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures. Curr Biol. 1998 Mar 26;8(7):405–408. doi: 10.1016/s0960-9822(98)70159-9. [DOI] [PubMed] [Google Scholar]
  21. Parent C. A., Blacklock B. J., Froehlich W. M., Murphy D. B., Devreotes P. N. G protein signaling events are activated at the leading edge of chemotactic cells. Cell. 1998 Oct 2;95(1):81–91. doi: 10.1016/s0092-8674(00)81784-5. [DOI] [PubMed] [Google Scholar]
  22. Periasamy N., Verkman A. S. Analysis of fluorophore diffusion by continuous distributions of diffusion coefficients: application to photobleaching measurements of multicomponent and anomalous diffusion. Biophys J. 1998 Jul;75(1):557–567. doi: 10.1016/S0006-3495(98)77545-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Phair R. D., Misteli T. High mobility of proteins in the mammalian cell nucleus. Nature. 2000 Apr 6;404(6778):604–609. doi: 10.1038/35007077. [DOI] [PubMed] [Google Scholar]
  24. Popov S., Poo M. M. Diffusional transport of macromolecules in developing nerve processes. J Neurosci. 1992 Jan;12(1):77–85. doi: 10.1523/JNEUROSCI.12-01-00077.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schwille P., Kummer S., Heikal A. A., Moerner W. E., Webb W. W. Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins. Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):151–156. doi: 10.1073/pnas.97.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Seksek O., Biwersi J., Verkman A. S. Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus. J Cell Biol. 1997 Jul 14;138(1):131–142. doi: 10.1083/jcb.138.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Swaminathan R., Hoang C. P., Verkman A. S. Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion. Biophys J. 1997 Apr;72(4):1900–1907. doi: 10.1016/S0006-3495(97)78835-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Terry B. R., Matthews E. K., Haseloff J. Molecular characterisation of recombinant green fluorescent protein by fluorescence correlation microscopy. Biochem Biophys Res Commun. 1995 Dec 5;217(1):21–27. doi: 10.1006/bbrc.1995.2740. [DOI] [PubMed] [Google Scholar]
  29. Wessels D., Soll D. R. Myosin II heavy chain null mutant of Dictyostelium exhibits defective intracellular particle movement. J Cell Biol. 1990 Sep;111(3):1137–1148. doi: 10.1083/jcb.111.3.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yumura S., Fukui Y. Spatiotemporal dynamics of actin concentration during cytokinesis and locomotion in Dictyostelium. J Cell Sci. 1998 Aug;111(Pt 15):2097–2108. doi: 10.1242/jcs.111.15.2097. [DOI] [PubMed] [Google Scholar]
  31. Zischka H., Oehme F., Pintsch T., Ott A., Keller H., Kellermann J., Schuster S. C. Rearrangement of cortex proteins constitutes an osmoprotective mechanism in Dictyostelium. EMBO J. 1999 Aug 2;18(15):4241–4249. doi: 10.1093/emboj/18.15.4241. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES