A Dynamic Actin Cytoskeleton Functions at Multiple Stages of Clathrin-mediated Endocytosis
10.1091/mbc.e04-09-0774
Crossref journal-article
American Society for Cell Biology (ASCB)
Molecular Biology of the Cell (1076)
Abstract

Clathrin-mediated endocytosis in mammalian cells is critical for a variety of cellular processes including nutrient uptake and cell surface receptor down-regulation. Despite the findings that numerous endocytic accessory proteins directly or indirectly regulate actin dynamics and that actin assembly is spatially and temporally coordinated with endocytosis, direct functional evidence for a role of actin during clathrin-coated vesicle formation is lacking. Here, we take parallel biochemical and microscopic approaches to address the contribution of actin polymerization/depolymerization dynamics to clathrin-mediated endocytosis. When measured using live-cell fluorescence microscopy, disruption of the F-actin assembly and disassembly cycle with latrunculin A or jasplakinolide results in near complete cessation of all aspects of clathrin-coated structure (CCS) dynamics. Stage-specific biochemical assays and quantitative fluorescence and electron microscopic analyses establish that F-actin dynamics are required for multiple distinct stages of clathrin-coated vesicle formation, including coated pit formation, constriction, and internalization. In addition, F-actin dynamics are required for observed diverse CCS behaviors, including splitting of CCSs from larger CCSs, merging of CCSs, and lateral mobility on the cell surface. Our results demonstrate a key role for actin during clathrin-mediated endocytosis in mammalian cells.

Bibliography

Yarar, D., Waterman-Storer, C. M., & Schmid, S. L. (2005). A Dynamic Actin Cytoskeleton Functions at Multiple Stages of Clathrin-mediated Endocytosis. Molecular Biology of the Cell, 16(2), 964–975.

Authors 3
  1. Defne Yarar (first)
  2. Clare M. Waterman-Storer (additional)
  3. Sandra L. Schmid (additional)
References 51 Referenced 377
  1. Ayscough, K. R. (2000). Endocytosis and the development of cell polarity in yeast require a dynamic F-actin cytoskeleton.Curr. Biol.10, 1587-1590. (10.1016/S0960-9822(00)00859-9)
  2. Axelrod, D. (2001). Total internal reflection fluorescence microscopy in cell biology.Traffic2, 764-774. (10.1034/j.1600-0854.2001.21104.x)
  3. Bennett, E. M., Chen, C. Y., Engqvist-Goldstein, A. E., Drubin, D. G., and Brodsky, F. M. (2001). Clathrin hub expression dissociates the actin-binding protein Hip1R from coated pits and disrupts their alignment with the actin cytoskeleton.Traffic2, 851-858. (10.1034/j.1600-0854.2001.21114.x)
  4. Bubb, M. R., Senderowicz, A. M., Sausville, E. A., Duncan, K. L., and Korn, E. D. (1994). Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin.J. Biol. Chem.269, 14869-14871. (10.1016/S0021-9258(17)36545-6)
  5. Carter, L. L., Redelmeier, T. E., Woollenweber, L. A., and Schmid, S. L. (1993). Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis.J. Cell Biol.120, 37-45. (10.1083/jcb.120.1.37)
  6. Conner, S. D., and Schmid, S. L. (2003). Regulated portals of entry into the cell.Nature422, 37-44. (10.1038/nature01451)
  7. Coue, M., Brenner, S. L., Spector, I., and Korn, E. D. (1987). Inhibition of actin polymerization by latrunculin A.FEBS Lett.213, 316-318. (10.1016/0014-5793(87)81513-2)
  8. Cramer, L. P., Briggs, L. J., and Dawe, H. R. (2002). Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells.Cell Motil. Cytoskelet.51, 27-38. (10.1002/cm.10013)
  9. Damke, H., Baba, T., Warnock, D. E., and Schmid, S. L. (1994). Induction of mutant dynamin specifically blocks endocytic coated vesicle formation.J. Cell Biol.127, 915-934. (10.1083/jcb.127.4.915)
  10. Durrbach, A., Louvard, D., and Coudrier, E. (1996). Actin filaments facilitate two steps of endocytosis.J. Cell Sci.109(Pt 2), 457-465. (10.1242/jcs.109.2.457)
  11. 10.1091/mbc.e03-09-0639
  12. Engqvist-Goldstein, A. E., and Drubin, D. G. (2003). Actin assembly and endocytosis: from yeast to mammals.Annu. Rev. Cell Dev. Biol.19, 287-332. (10.1146/annurev.cellbio.19.111401.093127)
  13. Engqvist-Goldstein, A. E., Warren, R. A., Kessels, M. M., Keen, J. H., Heuser, J., and Drubin, D. G. (2001). The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro.J. Cell Biol.154, 1209-1223. (10.1083/jcb.200106089)
  14. Ehrlich, M., Boll, W., Van Oijen, A., Hariharan, R., Chandran, K., Nibert, M. L., and Kirchhausen, T. (2004). Endocytosis by random initiation and stabilization of clathrin-coated pits.Cell118, 591-605. (10.1016/j.cell.2004.08.017)
  15. Frischknecht, F., and Way, M. (2001). Surfing pathogens and the lessons learned for actin polymerization.Trends Cell Biol.11, 30-38. (10.1016/S0962-8924(00)01871-7)
  16. Fujimoto, L. M., Roth, R., Heuser, J. E., and Schmid, S. L. (2000). Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis in mammalian cells.Traffic1, 161-171. (10.1034/j.1600-0854.2000.010208.x)
  17. Gaidarov, I., Santini, F., Warren, R. A., and Keen, J. H. (1999). Spatial control of coated-pit dynamics in living cells.Nat. Cell Biol.1, 1-7. (10.1038/8971)
  18. Giardini, P. A., Fletcher, D. A., and Theriot, J. A. (2003). Compression forces generated by actin comet tails on lipid vesicles.Proc. Natl. Acad. Sci. USA100, 6493-6498. (10.1073/pnas.1031670100)
  19. Gupton, S. L., and Waterman-Storer, C. M. (2005). Live-cell fluorescent speckle microscopy (FSM) of actin cytoskeletal dynamics and their perturbation by drug perfusion. In:Cell Biology: A Laboratory Handbook, 3rd ed., ed J. Celis and J. V. Small, New York: Elsevier (in press).
  20. Heuser, J. E., and Anderson, R. G. (1989). Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation.J. Cell Biol.108, 389-400. (10.1083/jcb.108.2.389)
  21. Kaksonen, M., Sun, Y., Drubin, D. G. (2003). A pathway for association of receptors, adaptors, and actin during endocytic internalization.Cell115,475-487. (10.1016/S0092-8674(03)00883-3)
  22. Keyel, P. A., Watkins, S. C., and Traub, L. M. (2004). Endocytic adaptor molecules reveal an endosomal population of clathrin by total internal reflection fluorescence microscopy.J. Biol. Chem.279, 13190-13204. (10.1074/jbc.M312717200)
  23. Lamaze, C., Fujimoto, L. M., Yin, H. L., and Schmid, S. L. (1997). The actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells.J. Biol. Chem.272, 20332-20335. (10.1074/jbc.272.33.20332)
  24. Loisel, T. P., Boujemaa, R., Pantaloni, D., and Carlier, M. F. (1999). Reconstitution of actin-based motility ofListeriaandShigellausing pure proteins.Nature401, 613-616. (10.1038/44183)
  25. Lunn, J. A., Wong, H., Rozengurt, E., and Walsh, J. H. (2000). Requirement of cortical actin organization for bombesin, endothelin, and epidermal growth factor receptor internalization.Am. J. Physiol. Cell Physiol.279, C2019-C2027.
  26. Ma, L., Cantley, L. C., Janmey, P. A., and Kirschner, M. W. (1998). Corequirement of specific phosphoinositides and small GTP-binding protein Cdc42 in inducing actin assembly inXenopusegg extracts.J. Cell Biol.140, 1125-1136. (10.1083/jcb.140.5.1125)
  27. Maples, C. J., Ruiz, W. G., and Apodaca, G. (1997). Both microtubules and actin filaments are required for efficient postendocytotic traffic of the polymeric immunoglobulin receptor in polarized Madin-Darby canine kidney cells.J. Biol. Chem.272, 6741-6751. (10.1074/jbc.272.10.6741)
  28. Merrifield, C. J. (2004). Seeing is believing: imaging actin dynamics at single sites of endocytosis.Trends Cell Biol.14, 352-358. (10.1016/j.tcb.2004.05.008)
  29. Merrifield, C. J., Feldman, M. E., Wan, L., and Almers, W. (2002). Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits.Nat. Cell Biol.4, 691-698. (10.1038/ncb837)
  30. Merrifield, C. J., Moss, S. E., Ballestrem, C., Imhof, B. A., Giese, G., Wunderlich, I., and Almers, W. (1999). Endocytic vesicles move at the tips of actin tails in cultured mast cells.Nat. Cell Biol.1, 72-74. (10.1038/9048)
  31. Merrifield, C. J., Qualmann, B., Kessels, M. M., and Almers, W. (2004). Neural Wiskott Aldrich Syndrome Protein (N-WASP) and the Arp2/3 complex are recruited to sites of clathrin-mediated endocytosis in cultured fibroblasts.Eur. J. Cell Biol.83, 13-18. (10.1078/0171-9335-00356)
  32. 10.1091/mbc.e03-04-0230
  33. Pantaloni, D., Le Clainche, C., and Carlier, M. F. (2001). Mechanism of actin-based motility.Science292, 1502-1506. (10.1126/science.1059975)
  34. Qualmann, B., and Kessels, M. M. (2002). Endocytosis and the cytoskeleton.Int. Rev. Cytol.220, 93-144. (10.1016/S0074-7696(02)20004-2)
  35. Qualmann, B., Kessels, M. M., and Kelly, R. B. (2000). Molecular links between endocytosis and the actin cytoskeleton.J. Cell Biol.150, F111-F116. (10.1083/jcb.150.5.F111)
  36. Rappoport, J., Simon, S., and Benmerah, A. (2004). Understanding living clathrin-coated pits.Traffic5, 327-337. (10.1111/j.1398-9219.2004.00187.x)
  37. Rappoport, J. Z., and Simon, S. M. (2003). Real-time analysis of clathrin-mediated endocytosis during cell migration.J. Cell Sci.116, 847-855. (10.1242/jcs.00289)
  38. Rappoport, J. Z., Taha, B. W., Lemeer, S., Benmerah, A., and Simon, S. M. (2003). The AP-2 complex is excluded from the dynamic population of plasma membrane-associated clathrin.J. Biol. Chem.278, 47357-47360. (10.1074/jbc.C300390200)
  39. Rozelle, A. L., Machesky, L. M., Yamamoto, M., Driessens, M. H., Insall, R. H., Roth, M. G., Luby-Phelps, K., Marriott, G., Hall, A., and Yin, H. L. (2000). Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3.Curr. Biol.10, 311-320. (10.1016/S0960-9822(00)00384-5)
  40. Santini, F., Gaidarov, I., and Keen, J. H. (2002). G protein-coupled receptor/arrestin3 modulation of the endocytic machinery.J. Cell Biol.156, 665-676. (10.1083/jcb.200110132)
  41. Santini, F., and Keen, J. H. (2002). A glimpse of coated vesicle creation? Well almost!Nat. Cell Biol.4, E230-E232.
  42. Schmid, S. L., and Carter, L. (1990). ATP is required for receptor-mediated endocytosis in intact cells.J. Cell Biol.111, 2307-2318. (10.1083/jcb.111.6.2307)
  43. Sever, S., Damke, H., and Schmid, S. L. (2000). Dynamin:GTP control the formation of constricted coated pits, the rate limiting step in clathrin-mediated endocytosis.J. Cell Biol.150, 1137-1147. (10.1083/jcb.150.5.1137)
  44. Slepnev, V. I., and De Camilli, P. (2000). Accessory factors in clathrin-dependent synaptic vesicle endocytosis.Nat. Rev. Neurosci.1, 161-172. (10.1038/35044540)
  45. Smythe, E., Carter, L. L., and Schmid, S. L. (1992). Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors.J. Cell Biol.119, 1163-1171. (10.1083/jcb.119.5.1163)
  46. Steyer, J. A., and Almers, W. (2001). A real-time view of life within 100 nm of the plasma membrane.Nat. Rev. Mol. Cell. Biol.2, 268-275. (10.1038/35067069)
  47. Taunton, J., Rowning, B. A., Coughlin, M. L., Wu, M., Moon, R. T., Mitchison, T. J., and Larabell, C. A. (2000). Actin-dependent propulsion of endosomes and lysosomes by recruitment of N-WASP.J. Cell Biol.148, 519-530. (10.1083/jcb.148.3.519)
  48. Theriot, J. A., Mitchison, T. J., Tilney, L. G., and Portnoy, D. A. (1992). The rate of actin-based motility of intracellularListeria monocytogenesequals the rate of actin polymerization.Nature357, 257-260. (10.1038/357257a0)
  49. Vale, R. D., Reese, T. S., and Sheetz, M. P. (1985). Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility.Cell42, 39-50. (10.1016/S0092-8674(85)80099-4)
  50. Vallee, R. B., Wall, J. S., Paschal, B. M., and Shpetner, H. S. (1988). Microtubule-associated protein 1C from brain is a two-headed cytosolic dynein.Nature332, 561-563. (10.1038/332561a0)
  51. Waterman-Storer, C. M. (2002). Fluorescent speckle microscopy (FSM) of microtubules and actin in living cells. In:Current Protocols in Cell Biology, ed. J. S. Bonifacino, M. Dasso, J. B. Harford, J. Lippincott-Schwartz, and K. M. Yamada, New York: John Wiley, Unit 4.10.
Dates
Type When
Created 20 years, 8 months ago (Dec. 15, 2004, 8:33 p.m.)
Deposited 4 years, 1 month ago (July 1, 2021, 10:32 a.m.)
Indexed 1 month ago (July 24, 2025, 7:43 a.m.)
Issued 20 years, 6 months ago (Feb. 1, 2005)
Published 20 years, 6 months ago (Feb. 1, 2005)
Published Print 20 years, 6 months ago (Feb. 1, 2005)
Funders 0

None

@article{Yarar_2005, title={A Dynamic Actin Cytoskeleton Functions at Multiple Stages of Clathrin-mediated Endocytosis}, volume={16}, ISSN={1939-4586}, url={http://dx.doi.org/10.1091/mbc.e04-09-0774}, DOI={10.1091/mbc.e04-09-0774}, number={2}, journal={Molecular Biology of the Cell}, publisher={American Society for Cell Biology (ASCB)}, author={Yarar, Defne and Waterman-Storer, Clare M. and Schmid, Sandra L.}, year={2005}, month=feb, pages={964–975} }