Control of actin polymerization in live and permeabilized fibroblasts

doi:10.1083/jcb.114.3.503

This Article

Full Text (PDF, 3566K)

Alert me when this article is cited

Citation Map

Services

Email this article

Similar articles in this journal

Similar articles in PubMed

Alert me to new content in the JCB

Download to citation manager

Citing Articles

Citing Articles via HighWire

Citing Articles via CrossRef

Citing Articles via Google Scholar

Google Scholar

Articles by Symons, M. H.

Articles by Mitchison, T. J.

Search for Related Content

PubMed

PubMed Citation

Articles by Symons, M. H.

Articles by Mitchison, T. J.

Pubmed/NCBI databases

Substance via MeSH

Social Bookmarking

What's this?

ARTICLES

Control of actin polymerization in live and permeabilized fibroblasts

MH Symons and TJ Mitchison
Department of Pharmacology, University of California, San Francisco 94143.

We have investigated the spatial control of actin polymerization in fibroblasts using rhodamine-labeled muscle actin in; (a) microinjection experiments to follow actin dynamics in intact cells, and (b) incubation with permeabilized cells to study incorporation sites. Rhodamine-actin was microinjected into NIH-3T3 cells which were then fixed and stained with fluorescein-phalloidin to visualize total actin filaments. The incorporation of newly polymerized actin was assayed using rhodamine/fluorescein ratio-imaging. The results indicated initial incorporation of the injected actin near the tip and subsequent transport towards the base of lamellipodia at rates greater than 4.5 microns/min. Furthermore, both fluorescein- and rhodamine-intensity profiles across lamellipodia revealed a decreasing density of actin filaments from tip to base. From this observation and the presence of centripetal flux of polymerized actin we infer that the actin cytoskeleton partially disassembles before it reaches the base of the lamellipodium. In permeabilized cells we found that, in agreement with the injection studies, rhodamine-actin incorporated predominantly in a narrow strip of less than 1-microns wide, located at the tip of lamellipodia. The critical concentration for the rhodamine-actin incorporation (0.15 microM) and its inhibition by CapZ, a barbed-end capping protein, indicated that the nucleation sites for actin polymerization most likely consist of free barbed ends of actin filaments. Because any potential monomer-sequestering system is bypassed by addition of exogenous rhodamine-actin to the permeabilized cells, these observations indicate that the localization of actin incorporation in intact cells is determined, at least in part, by the presence of specific elongation and/or nucleation sites at the tips of lamellipodia and not solely by localized desequestration of subunits. We propose that the availability of the incorporation sites at the tips of lamellipodia is because of capping activities which preferentially inhibit barbed-end incorporation elsewhere in the cell, but leave barbed ends at the tips of lamellipodia free to add subunits.
Add to CiteULike CiteULike Add to Complore Complore Add to Connotea Connotea Add to Del.icio.us Del.icio.us Add to Digg Digg Facebook Add to Reddit Reddit Add to Technorati Technorati Twitter What's this?

This article has been cited by other articles:

Brawley, C. M., Rock, R. S. (2009). Unconventional myosin traffic in cells reveals a selective actin cytoskeleton. Proc. Natl. Acad. Sci. USA 106: 9685-9690 [Abstract] [Full Text]
Hotulainen, P., Llano, O., Smirnov, S., Tanhuanpaa, K., Faix, J., Rivera, C., Lappalainen, P. (2009). Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis. JCB 185: 323-339 [Abstract] [Full Text]
Lee, C. W., Peng, H. B. (2008). The Function of Mitochondria in Presynaptic Development at the Neuromuscular Junction. Mol. Biol. Cell 19: 150-158 [Abstract] [Full Text]
Furman, C., Sieminski, A. L., Kwiatkowski, A. V., Rubinson, D. A., Vasile, E., Bronson, R. T., Fassler, R., Gertler, F. B. (2007). Ena/VASP is required for endothelial barrier function in vivo. JCB 179: 761-775 [Abstract] [Full Text]
Gupton, S. L., Eisenmann, K., Alberts, A. S., Waterman-Storer, C. M. (2007). mDia2 regulates actin and focal adhesion dynamics and organization in the lamella for efficient epithelial cell migration. J. Cell Sci. 120: 3475-3487 [Abstract] [Full Text]
Yam, P. T., Wilson, C. A., Ji, L., Hebert, B., Barnhart, E. L., Dye, N. A., Wiseman, P. W., Danuser, G., Theriot, J. A. (2007). Actin myosin network reorganization breaks symmetry at the cell rear to spontaneously initiate polarized cell motility. JCB 178: 1207-1221 [Abstract] [Full Text]
Yamada, S., Nelson, W. J. (2007). Localized zones of Rho and Rac activities drive initiation and expansion of epithelial cell cell adhesion. JCB 178: 517-527 [Abstract] [Full Text]
Robert, A., Smadja-Lamere, N., Landry, M.-C., Champagne, C., Petrie, R., Lamarche-Vane, N., Hosoya, H., Lavoie, J. N. (2006). Adenovirus E4orf4 Hijacks Rho GTPase-dependent Actin Dynamics to Kill Cells: A Role for Endosome-associated Actin Assembly. Mol. Biol. Cell 17: 3329-3344 [Abstract] [Full Text]
Ivanov, A. I., Hunt, D., Utech, M., Nusrat, A., Parkos, C. A. (2005). Differential Roles for Actin Polymerization and a Myosin II Motor in Assembly of the Epithelial Apical Junctional Complex. Mol. Biol. Cell 16: 2636-2650 [Abstract] [Full Text]
Hirshman, C. A., Zhu, D., Pertel, T., Panettieri, R. A., Emala, C. W. (2005). Isoproterenol induces actin depolymerization in human airway smooth muscle cells via activation of an Src kinase and GS. Am. J. Physiol. Lung Cell. Mol. Physiol. 288: L924-L931 [Abstract] [Full Text]
Gupton, S. L., Anderson, K. L., Kole, T. P., Fischer, R. S., Ponti, A., Hitchcock-DeGregori, S. E., Danuser, G., Fowler, V. M., Wirtz, D., Hanein, D., Waterman-Storer, C. M. (2005). Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin. JCB 168: 619-631 [Abstract] [Full Text]
Yam, P. T., Theriot, J. A. (2004). Repeated Cycles of Rapid Actin Assembly and Disassembly on Epithelial Cell Phagosomes. Mol. Biol. Cell 15: 5647-5658 [Abstract] [Full Text]
DesMarais, V., Macaluso, F., Condeelis, J., Bailly, M. (2004). Synergistic interaction between the Arp2/3 complex and cofilin drives stimulated lamellipod extension. J. Cell Sci. 117: 3499-3510 [Abstract] [Full Text]
Vallotton, P., Gupton, S. L., Waterman-Storer, C. M., Danuser, G. (2004). Simultaneous mapping of filamentous actin flow and turnover in migrating cells by quantitative fluorescent speckle microscopy. Proc. Natl. Acad. Sci. USA 101: 9660-9665 [Abstract] [Full Text]
Ivanov, A. I., McCall, I. C., Parkos, C. A., Nusrat, A. (2004). Role for Actin Filament Turnover and a Myosin II Motor in Cytoskeleton-driven Disassembly of the Epithelial Apical Junctional Complex. Mol. Biol. Cell 15: 2639-2651 [Abstract] [Full Text]
Noria, S., Xu, F., McCue, S., Jones, M., Gotlieb, A. I., Langille, B. L. (2004). Assembly and Reorientation of Stress Fibers Drives Morphological Changes to Endothelial Cells Exposed to Shear Stress. Am. J. Pathol. 164: 1211-1223 [Abstract] [Full Text]
Loomis, P. A., Zheng, L., Sekerkova, G., Changyaleket, B., Mugnaini, E., Bartles, J. R. (2003). Espin cross-links cause the elongation of microvillus-type parallel actin bundles in vivo. JCB 163: 1045-1055 [Abstract] [Full Text]
Fischer, R. S., Fritz-Six, K. L., Fowler, V. M. (2003). Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility. JCB 161: 371-380 [Abstract] [Full Text]
Miyagawa, Y., Tanaka, H., Iguchi, N., Kitamura, K., Nakamura, Y., Takahashi, T., Matsumiya, K., Okuyama, A., Nishimune, Y. (2002). Molecular cloning and characterization of the human orthologue of male germ cell-specific actin capping protein {alpha}3 (cp{alpha}3). Mol Hum Reprod 8: 531-539 [Abstract] [Full Text]
van Leeuwen, H., Elliott, G., O'Hare, P. (2002). Evidence of a Role for Nonmuscle Myosin II in Herpes Simplex Virus Type 1 Egress. J. Virol. 76: 3471-3481 [Abstract] [Full Text]
Watanabe, N., Mitchison, T. J. (2002). Single-Molecule Speckle Analysis of Actin Filament Turnover in Lamellipodia. Science 295: 1083-1086 [Abstract] [Full Text]
Hirshman, C. A., Zhu, D., Panettieri, R. A., Emala, C. W. (2001). Actin depolymerization via the beta -adrenoceptor in airway smooth muscle cells: a novel PKA-independent pathway. Am. J. Physiol. Cell Physiol. 281: C1468-C1476 [Abstract] [Full Text]
Lesh, R. E., Emala, C. W., Lee, H. T., Zhu, D., Panettieri, R. A., Hirshman, C. A. (2001). Inhibition of geranylgeranylation blocks agonist-induced actin reorganization in human airway smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 281: L824-L831 [Abstract] [Full Text]
Shih, J.-Y., Yang, S.-C., Hong, T.-M., Yuan, A., Chen, J. J. W., Yu, C.-J., Chang, Y.-L., Lee, Y.-C., Peck, K., Wu, C.-W., Yang, P.-C. (2001). Collapsin Response Mediator Protein-1 and the Invasion and Metastasis of Cancer Cells. JNCI J Natl Cancer Inst 93: 1392-1400 [Abstract] [Full Text]
Gogarten, W., Emala, C. W., Lindeman, K. S., Hirshman, C. A. (2001). Oxytocin and Lysophosphatidic Acid Induce Stress Fiber Formation in Human Myometrial Cells via a Pathway Involving Rho-Kinase. Biol. Reprod. 65: 401-406 [Abstract] [Full Text]
Sells, M. A., Pfaff, A., Chernoff, J. (2000). Temporal and Spatial Distribution of Activated Pak1 in Fibroblasts. JCB 151: 1449-1458 [Abstract] [Full Text]
Fukui, R., Amakawa, M., Hoshiga, M., Shibata, N., Kohbayashi, E., Seto, M., Sasaki, Y., Ueno, T., Negoro, N., Nakakoji, T., Ii, M., Nishiguchi, F., Ishihara, T., Ohsawa, N. (2000). Increased migration in late G1 phase in cultured smooth muscle cells. Am. J. Physiol. Cell Physiol. 279: C999-C1007 [Abstract] [Full Text]
Price, C. J., Brindle, N. P. J. (2000). Vasodilator-Stimulated Phosphoprotein Is Involved in Stress-Fiber and Membrane Ruffle Formation in Endothelial Cells. Arterioscler. Thromb. Vasc. Bio. 20: 2051-2056 [Abstract] [Full Text]
Moldovan, L., Moldovan, N. I., Sohn, R. H., Parikh, S. A., Goldschmidt-Clermont, P. J. (2000). Redox Changes of Cultured Endothelial Cells and Actin Dynamics. Circ. Res. 86: 549-557 [Abstract] [Full Text]
Chan, A. Y., Bailly, M., Zebda, N., Segall, J. E., Condeelis, J. S. (2000). Role of Cofilin in Epidermal Growth Factor-Stimulated Actin Polymerization and Lamellipod Protrusion. JCB 148: 531-542 [Abstract] [Full Text]
Nishizaka, T., Shi, Q., Sheetz, M. P. (2000). Position-dependent linkages of fibronectin- integrin-cytoskeleton. Proc. Natl. Acad. Sci. USA 97: 692-697 [Abstract] [Full Text]
Raucher, D., Sheetz, M. P. (2000). Cell Spreading and Lamellipodial Extension Rate Is Regulated by Membrane Tension. JCB 148: 127-136 [Abstract] [Full Text]
Mehta, D., Gunst, S. J (1999). Actin polymerization stimulated by contractile activation regulates force development in canine tracheal smooth muscle. J. Physiol. 519: 829-840 [Abstract] [Full Text]
Yanai, M., Butler, J. P., Suzuki, T., Kanda, A., Kurachi, M., Tashiro, H., Sasaki, H. (1999). Intracellular elasticity and viscosity in the body, leading, and trailing regions of locomoting neutrophils. Am. J. Physiol. Cell Physiol. 277: C432-C440 [Abstract] [Full Text]
Svitkina, T. M., Borisy, G. G. (1999). Arp2/3 Complex and Actin Depolymerizing Factor/Cofilin in Dendritic Organization and Treadmilling of Actin Filament Array in Lamellipodia. JCB 145: 1009-1026 [Abstract] [Full Text]
Bailly, M., Macaluso, F., Cammer, M., Chan, A., Segall, J. E., Condeelis, J. S. (1999). Relationship between Arp2/3 Complex and the Barbed Ends of Actin Filaments at the Leading Edge of Carcinoma Cells after Epidermal Growth Factor Stimulation. JCB 145: 331-345 [Abstract] [Full Text]
Nabi, I. (1999). The polarization of the motile cell. J. Cell Sci. 112: 1803-1811 [Abstract]
Fukui, Y, Kitanishi-Yumura, T, Yumura, S (1999). Myosin II-independent F-actin flow contributes to cell locomotion in dictyostelium. J. Cell Sci. 112: 877-886 [Abstract]
Schafer, D. A., Welch, M. D., Machesky, L. M., Bridgman, P. C., Meyer, S. M., Cooper, J. A. (1998). Visualization and Molecular Analysis of Actin Assembly in Living Cells. JCB 143: 1919-1930 [Abstract] [Full Text]
Le Ma, , Cantley, L. C., Janmey, P. A., Kirschner, M. W. (1998). Corequirement of Specific Phosphoinositides and Small GTP-binding Protein Cdc42 in Inducing Actin Assembly in Xenopus Egg Extracts. JCB 140: 1125-1136 [Abstract] [Full Text]
Ballestrem, C, Wehrle-Haller, B, Imhof, B. (1998). Actin dynamics in living mammalian cells. J. Cell Sci. 111: 1649-1658 [Abstract]
Chan, A., Raft, S, Bailly, M, Wyckoff, J., Segall, J., Condeelis, J. (1998). EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cells. J. Cell Sci. 111: 199-211 [Abstract]
Eddy, R.J., Han, J., Condeelis, J.S. (1997). Capping Protein Terminates but Does Not Initiate Chemoattractant-induced Actin Assembly in Dictyostelium. JCB 139: 1243-1253 [Abstract] [Full Text]
Schindelholz, B., Reber, B. F.�X. (1997). Bradykinin-Induced Collapse of Rat Pheochromocytoma (PC12) Cell Growth Cones: A Role for Tyrosine Kinase Activity. J. Neurosci. 17: 8391-8401 [Abstract] [Full Text]
Svitkina, T. M., Verkhovsky, A. B., McQuade, K. M., Borisy, G. G. (1997). Analysis of the Actin-Myosin II System in Fish Epidermal Keratocytes: Mechanism of Cell Body Translocation. JCB 139: 397-415 [Abstract] [Full Text]
Waterman-Storer, C. M., Salmon, E.D. (1997). Actomyosin-based Retrograde Flow of Microtubules in the Lamella of Migrating Epithelial Cells Influences Microtubule Dynamic Instability and Turnover and Is Associated with Microtubule Breakage and Treadmilling. JCB 139: 417-434 [Abstract] [Full Text]
Machesky, L. M., Hall, A. (1997). Role of Actin Polymerization and Adhesion to Extracellular Matrix in Rac- and Rho-induced Cytoskeletal Reorganization. JCB 138: 913-926 [Abstract] [Full Text]
Shorte, S. L. (1997). N-Methyl-D-Aspartate Evokes Rapid Net Depolymerization of Filamentous Actin in Cultured Rat Cerebellar Granule Cells. J. Neurophysiol. 78: 1135-1143 [Abstract] [Full Text]
Musch, A., Cohen, D., Rodriguez-Boulan, E. (1997). Myosin II Is Involved in the Production of Constitutive Transport Vesicles from the TGN. JCB 138: 291-306 [Abstract] [Full Text]
Welch, M. D., DePace, A. H., Verma, S., Iwamatsu, A., Mitchison, T. J. (1997). The Human Arp2/3 Complex Is Composed of Evolutionarily Conserved Subunits and Is Localized to Cellular Regions of Dynamic Actin Filament Assembly. JCB 138: 375-384 [Abstract] [Full Text]
Cramer, L. P., Siebert, M., Mitchison, T. J. (1997). Identification of Novel Graded Polarity Actin Filament Bundles in Locomoting Heart Fibroblasts: Implications for the Generation of Motile Force. JCB 136: 1287-1305 [Abstract] [Full Text]
Allen, P. G., Laham, L. E., Way, M., Janmey, P. A. (1996). Binding of Phosphate, Aluminum Fluoride, or Beryllium Fluoride to F-actin Inhibits Severing by Gelsolin. J. Biol. Chem. 271: 4665-4670 [Abstract] [Full Text]
Cudmore, S, Reckmann, I, Griffiths, G, Way, M (1996). Vaccinia virus: a model system for actin-membrane interactions. J. Cell Sci. 109: 1739-1747 [Abstract]
Winklbauer, R, Stoltz, C (1995). Fibronectin fibril growth in the extracellular matrix of the Xenopus embryo. J. Cell Sci. 108: 1575-1586 [Abstract]
Appeddu, P., Shur, B. (1994). Control of stable lamellipodia formation by expression of cell surface beta 1,4-galactosyltransferase cytoplasmic domains. J. Cell Sci. 107: 2535-2545 [Abstract]
Stossel, T. (1993). On the crawling of animal cells. Science 260: 1086-1094 [Abstract]
Tilney, L., Cotanche, D., Tilney, M. (1992). Actin filaments, stereocilia and hair cells of the bird cochlea. VI. How the number and arrangement of stereocilia are determined. Development 116: 213-226 [Abstract]