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Actin filament uncapping localizes to ruffling lamellae and rocketing vesicles

Nature Cell Biology volume 5, pages 972–979 (2003)Cite this article

Abstract

Regulated actin filament assembly is critical for eukaryotic cell physiology. Actin filaments are polar structures1, and those with free high affinity or barbed ends are crucial for actin dynamics and cell motility2. Actin filament barbed-end-capping proteins inhibit filament elongation after binding3, and their regulated disassociation is proposed to provide a source of free filament ends to drive processes dependent on actin polymerization4. To examine whether dissociation of actin filament capping proteins occurs with the correct spatio-temporal constraints to contribute to regulated actin assembly in live cells, I measured the dissociation of an actin capping protein, gelsolin, from actin in cells using a variation of fluorescence resonance energy transfer (FRET). Uncapping was found to occur in cells at sites of active actin assembly, including protruding lamellae and rocketing vesicles, with the correct spatio-temporal properties to provide sites of actin filament polymerization during protrusion. These observations are consistent with models where uncapping of existing filaments provides sites of actin filament elongation.

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Figure 1: Generation and characterization of QSY–gelsolin.
Figure 2: Uncapping occurs at sites of actin assembly in a growth-factor-dependent manner.
Figure 3: PDGF stimulates dissociation of the gelsolin–actin FqRET sensor.
Figure 4: Uncapping at sites of active membrane protrusion and ruffling.
Figure 5: Uncapping associated with rocketing vesicles.

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References

  1. Wegner, A. The mechanism of ATP hydrolysis by polymer actin. Biophys. Chem. 7, 51–58 (1977).

    Article  CAS  PubMed  Google Scholar 

  2. Toyama, S. A variant form of β-actin in a mutant of KB cells resistant to cytochalasin B. Cell 37, 609–614 (1984).

    Article  CAS  PubMed  Google Scholar 

  3. Cooper, J.A. & Schafer, D.A. Control of actin assembly and disassembly at filament ends. Curr. Opin. Cell Biol. 12, 97–103 (2000).

    Article  CAS  PubMed  Google Scholar 

  4. Stossel, T.P., Hartwig, J.H., Janmey, P.A. & Kwiatkowski, D.J. Cell crawling two decades after Abercrombie. Biochem. Soc. Symp. 65, 267–280 (1999).

    CAS  PubMed  Google Scholar 

  5. Oosawa, F. & Asakura, S. Thermodynamics of the Polymerization of Protein (Acadmic Press, New York and London, 1975).

    Google Scholar 

  6. Symons, M.H. & Mitchison, T.J. Control of actin polymerization in live and permeabilized fibroblasts. J. Cell Biol. 114, 503–513 (1991).

    Article  CAS  PubMed  Google Scholar 

  7. Cassimeris, L., Safer, D., Nachmias, V.T. & Zigmond, S.H. Thymosin β4 sequesters the majority of G-actin in resting human polymorphonuclear leukocytes. J. Cell Biol. 119, 1261–1270 (1992).

    Article  CAS  PubMed  Google Scholar 

  8. Carlier, M.F. & Pantaloni, D. Control of actin dynamics in cell motility. J. Mol. Biol. 269, 459–467 (1997).

    Article  CAS  PubMed  Google Scholar 

  9. Pantaloni, D. & Carlier, M.F. How profilin promotes actin filament assembly in the presence of thymosin β4. Cell 75, 1007–1014 (1993).

    Article  CAS  PubMed  Google Scholar 

  10. Glogauer, M., Hartwig, J. & Stossel, T. Two pathways through Cdc42 couple the N-formyl receptor to actin nucleation in permeabilized human neutrophils. J. Cell Biol. 150, 785–796 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hartwig, J.H. et al. Thrombin receptor ligation and activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets. Cell 82, 643–653 (1995).

    Article  CAS  PubMed  Google Scholar 

  12. Condeelis, J. How is actin polymerization nucleated in vivo? Trends Cell Biol. 11, 288–293 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Yin, H.L. & Stull, J.T. Proteins that regulate dynamic actin remodeling in response to membrane signaling minireview series. J. Biol. Chem. 274, 32529–32530 (1999).

    Article  CAS  PubMed  Google Scholar 

  14. Welch, M.D. The world according to Arp: regulation of actin nucleation by the Arp2/3 complex. Trends Cell Biol. 9, 423–427 (1999).

    Article  CAS  PubMed  Google Scholar 

  15. Sun, H.Q., Yamamoto, M., Mejillano, M. & Yin, H.L. Gelsolin, a multifunctional actin regulatory protein. J. Biol. Chem. 274, 33179–33182 (1999).

    Article  CAS  PubMed  Google Scholar 

  16. Selve, N. & Wegner, A. Rate constants and equilibrium constants for binding of the gelsolin–actin complex to the barbed ends of actin filaments in the presence and absence of calcium. Eur. J. Biochem. 160, 379–387 (1986).

    Article  CAS  PubMed  Google Scholar 

  17. Koepf, E.K. & Burtnick, L.D. Horse plasma gelsolin labelled with fluorescein isothiocyanate responds to calcium and actin. Eur. J. Biochem. 212, 713–718 (1993).

    Article  CAS  PubMed  Google Scholar 

  18. Kenworthy, A.K. Imaging protein–protein interactions using fluorescence resonance energy transfer microscopy. Methods 24, 289–296 (2001).

    Article  CAS  PubMed  Google Scholar 

  19. Rozelle, A.L. et al. Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3. Curr. Biol. 10, 311–320 (2000).

    Article  CAS  PubMed  Google Scholar 

  20. Suchy, S.F. & Nussbaum, R.L. The deficiency of PIP2 5-phosphatase in Lowe syndrome affects actin polymerization. Am. J. Hum. Genet. 71, 1420–1427 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Cooper, J.A. Effects of cytochalasin and phalloidin on actin. J. Cell Biol. 105, 1473–1478 (1987).

    Article  CAS  PubMed  Google Scholar 

  22. Kraynov, V.S. et al. Localized Rac activation dynamics visualized in living cells. Science 290, 333–337 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Varnai, P., Rother, K.I. & Balla, T. Phosphatidylinositol 3-kinase-dependent membrane association of the Bruton's tyrosine kinase pleckstrin homology domain visualized in single living cells. J. Biol. Chem. 274, 10983–10989 (1999).

    Article  CAS  PubMed  Google Scholar 

  24. Botelho, R.J. et al. Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis. J. Cell Biol. 151, 1353–1368 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Oatey, P.B. et al. Confocal imaging of the subcellular distribution of phosphatidylinositol 3,4,5-trisphosphate in insulin- and PDGF-stimulated 3T3-L1 adipocytes. Biochem. J. 344, 511–518 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Carpenter, C.L., Tolias, K.F., Van Vugt, A. & Hartwig, J. Lipid kinases are novel effectors of the GTPase Rac1. Adv. Enzyme Regul. 39, 299–312 (1999).

    Article  CAS  PubMed  Google Scholar 

  27. Carpenter, C.L. Actin cytoskeleton and cell signaling. Crit. Care Med. 28, N94–N99 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Janmey, P.A., Xian, W. & Flanagan, L.A. Controlling cytoskeleton structure by phosphoinositide–protein interactions: phosphoinositide binding protein domains and effects of lipid packing. Chem. Phys. Lipids 101, 93–107 (1999).

    Article  CAS  PubMed  Google Scholar 

  29. Wang, Q. et al. Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin. J. Cell Biol. 160, 565–575 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Selve, N. & Wegner, A. Rate constants and equilibrium constants for binding of the gelsolin–actin complex to the barbed ends of actin filaments in the presence and absence of calcium. Eur. J. Biochem. 160, 379–387 (1986).

    Article  CAS  PubMed  Google Scholar 

  31. Bearer, E.L., Prakash, J.M., Manchester, R.D. & Allen, P.G. VASP protects actin filaments from gelsolin: an in vitro study with implications for platelet actin reorganizations. Cell Motil. Cytoskeleton 47, 351–364 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Rohatgi, R. et al. The interaction between N-WASP and the Arp2/3 complex links Cdc42- dependent signals to actin assembly. Cell 97, 221–231 (1999).

    Article  CAS  PubMed  Google Scholar 

  33. Blanchoin, L., Pollard, T.D. & Mullins, R.D. Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks. Curr. Biol. 10, 1273–1282 (2000).

    Article  CAS  PubMed  Google Scholar 

  34. Falet, H. et al. Importance of free actin filament barbed ends for Arp2/3 complex function in platelets and fibroblasts. Proc. Natl Acad. Sci. USA 99, 16782–16787 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Pantaloni, D., Boujemaa, R., Didry, D., Gounon, P. & Carlier, M.F. The Arp2/3 complex branches filament barbed ends: functional antagonism with capping proteins. Nature Cell Biol. 2, 385–391 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Schafer, D.A. & Cooper, J.A. Control of actin assembly at filament ends. Annu. Rev. Cell Dev. Biol. 11, 497–518 (1995).

    Article  CAS  PubMed  Google Scholar 

  37. Taunton, J. et al. Actin-dependent propulsion of endosomes and lysosomes by recruitment of N-WASP. J. Cell Biol. 148, 519–530 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ichetovkin, I., Grant, W. & Condeelis, J. Cofilin produces newly polymerized actin filaments that are preferred for dendritic nucleation by the Arp2/3 complex. Curr. Biol. 12, 79–84 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Lamb, J.A., Allen, P.G., Tuan, B.Y. & Janmey, P.A. Modulation of gelsolin function. Activation at low pH overrides Ca2+ requirement. J. Biol. Chem. 268, 8999–9004 (1993).

    CAS  PubMed  Google Scholar 

  40. Allen, P.G. & Janmey, P.A. Gelsolin displaces phalloidin from actin filaments. A new fluorescence method shows that both Ca2+ and Mg2+ affect the rate at which gelsolin severs F-actin. J. Biol. Chem. 269, 32916–32923 (1994).

    CAS  PubMed  Google Scholar 

  41. Palmgren, S., Ojala, P.J., Wear, M.A., Cooper, J.A. & Lappalainen, P. Interactions with PIP2, ADP–actin monomers, and capping protein regulate the activity and localization of yeast twinfilin. J. Cell Biol. 155, 251–260 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Azuma, T., Witke, W., Stossel, T.P., Hartwig, J.H. & Kwiatkowski, D.J. Gelsolin is a downstream effector of rac for fibroblast motility. EMBO J. 17, 1362–1370 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

I would like to thank J. Hartwig, G. Cicchetti, M. Biernacki and J. Farquharson for critical reading of this manuscript. Initial samples of OCRL-deficient fibroblasts were provided by S. Suchy (National Institutes of Health). This work was supported by NIH grant RO1GM57256.

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  1. Hematology Division, Brigham and Women's Hospital and Harvard Medical School, Boston, 02115, MA, USA

    Philip G. Allen

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Allen, P. Actin filament uncapping localizes to ruffling lamellae and rocketing vesicles. Nat Cell Biol 5, 972–979 (2003). https://doi.org/10.1038/ncb1059

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