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11 The Actin Cytoskeleton: Regulation of Actin Filament Assembly and Disassembly

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Abstract:

Eukaryotic cells possess three kinds of cytoskeletal elements: 5- to 9-nmdiameter actin filaments, 24-nm-diameter microtubules, and 10-nmdiameter intermediate filaments. All these polymer networks are assembled reversibly from monomers. This chapter concentrates on actin. Reorganization of the actin cytoskeleton is required for leukocytes to migrate to sites of infection, for fibroblasts and endothelial cells to migrate to areas of wound healing, and for platelets to plug leaking vessels. Actin polymerization not only drives the motility of leukocytes, fibroblasts, keratocytes, and amoebae, but also provides the propulsive force for the movement of intracellular bacteria including , , and as well as the poxvirus, vaccinia. The severing of actin filaments reduces the viscosity of the peripheral cytoplasm and allows the actin cytoskeleton to be rapidly remodeled. When cells are stimulated to move and change shape, the number of free barbed filament ends markedly increases. This rapid rise in the number of free barbed ends can be accomplished in two ways. First, the barbed ends of preformed actin filaments can be uncapped and second, nucleating proteins can serve as a template to initiate the elongation of new actin filaments. The precise mechanisms regulating the formation of new actin filaments in the motile cell remain to be determined. In addition, filaments can be severed by gelsolin. The combination of uncapping and severing can greatly increase the number of free barbed-filament ends available for rapid actin assembly.

Citation: Southwick F. 2004. 11 The Actin Cytoskeleton: Regulation of Actin Filament Assembly and Disassembly, p 255-274. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch11

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Figures

Image of Figure 11.1
Figure 11.1

Atomic-level structure of an actin monomer showing the ATP-binding site. Based on the work of W. Kabsch, H. G. Mannherz, D. Suck, E. F. Pai, and K. C. Holmes, 37–44, 1990. The vertical axis of the monomer (as depicted in this figure) runs parallel to the long axis of the filament (see Figure 11.3 ). The right-hand side of the molecule is exposed to the outside of the actin filament, whereas the left-hand side is nearest the long axis of the filament. Residues 262 to 274 are thought to reach across this axis and interact with the adjacent actin monomer of the double-stranded helix. As oriented here, the polarity of the filament would correspond to the pointed end at the top and the barbed end at the bottom. The roman numerals delineate the four domains of the molecule.

Citation: Southwick F. 2004. 11 The Actin Cytoskeleton: Regulation of Actin Filament Assembly and Disassembly, p 255-274. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch11
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Image of Figure 11.2
Figure 11.2

Schematic drawing of an actin filament under steady-state conditions. ATP-actin monomers add to the barbed or fast-growing end and are hydrolyzed to ADP-P, followed by the slower dissociation of P to form ADP-actin. ADP-actin monomers dissociate from the pointed or slow-growing end. Under these conditions an actin monomer added to the barbed end will eventually treadmill through the filament and dissociate from the pointed end.

Citation: Southwick F. 2004. 11 The Actin Cytoskeleton: Regulation of Actin Filament Assembly and Disassembly, p 255-274. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch11
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Image of Figure 11.3
Figure 11.3

Schematic drawing to the tertiary structure of an actin filament. Roman numerals correspond to the domains shown in Figure 11.1 .

Citation: Southwick F. 2004. 11 The Actin Cytoskeleton: Regulation of Actin Filament Assembly and Disassembly, p 255-274. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch11
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Image of Figure 11.4
Figure 11.4

Critical-concentration behavior in actin polymerization. (Left) Plot of the steady-state actin filament concentration as a function of monomer concentration. This macroscopic behavior is often measured by the increase in fluorescence when pyrenyl-actin is incorporated into filaments. (Right) When both filament ends are uncapped, the macroscopic critical concentration lies between the microscopic critical concentrations for actin monomer interactions at the barbed end [or (+) end] and the pointed end [or (−) end]. Because the exchange rates are higher at the barbed end, the macroscopic critical concentration is closer to the microscopic critical concentration of the barbed end. At steady state, monomers will naturally dissociate from the pointed ends and will associate with the more stable barbed ends. This phenomenon is known as treadmilling. Because the exchange rates are higher at the barbed end, the macroscopic critical concentration is closer to that of the barbed end.

Citation: Southwick F. 2004. 11 The Actin Cytoskeleton: Regulation of Actin Filament Assembly and Disassembly, p 255-274. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch11
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Image of Figure 11.5
Figure 11.5

Schematic diagram of the action of monomer-sequestering agents such as Tβ4. Schematic diagram of how profilin may facilitate the exchange of ATP for ADP on an actin monomer. When profilin binds to an actin monomer, the central cleft of actin opens, making the nucleotide-binding site more accessible for release and exchange. Because the ATP concentration far exceeds the ADP concentration in living cells, ATP will readily replace ADP from the actin monomer. Simplified kinetic diagram shows how free profilin can take actin monomers from the Tβ4 storage pool and usher them onto the barbed end of an actin filament. For simplicity the reverse arrows profilin-ATP to free profilin and to Tβ4-ATP actin are not shown. Once profilin-ATP binds to the barbed end, profilin rapidly dissociates. Mechanism of a barbed-end capping protein binding to the barbed (or plus) end of an actin filament. Bound capping proteins prevent both association and dissociation of monomers; under such conditions, only the pointed end can interact with the actin monomer pool. Model for the action filament-severing proteins. The severing protein first binds along the side of the actin filament, next interposes itself between neighboring actin subunits within the filament, and then remains tightly bound to the barbed end of one of the severed filaments. Schematic diagram showing the bundling protein α-actinin, which cross-links actin filaments into parallel arrays. α-Actinin molecules form an antiparallel dimer, and each subunit contains an actin filament-binding site. Action of a depolymerizing factor in enhancing disassembly from the pointed end of the actin filament. The dark square with a D represents an ADF or cofilin molecule binding alongside an actin filament at a site near the pointed end. Upon binding, this protein enhances the rate of the pointed end, thereby accelerating the treadmilling rate of uncapped filaments.

Citation: Southwick F. 2004. 11 The Actin Cytoskeleton: Regulation of Actin Filament Assembly and Disassembly, p 255-274. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch11
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Image of Figure 11.6
Figure 11.6

Effects of a barbed-end-capping protein on the critical concentration and rate of depolymerization of actin. (Left) Graph of steady-state actin filament concentration as a function of actin monomer concentration. As shown in Figure 11.5D , capping blocks all exchange at the barbed end. The free pointed end has a lower affinity for actin monomers, and this lower affinity is reflected as an increase in the critical concentration (see Figure 11.4 for comparison). (Right) Plot of the decrease in filamentous actin versus time after diluting actin filaments to below their critical concentration in the absence and presence of a barbed-end-capping protein. Capping of the barbed end retards the depolymerization, because dissociation of actin monomers occurs only at the pointed end.

Citation: Southwick F. 2004. 11 The Actin Cytoskeleton: Regulation of Actin Filament Assembly and Disassembly, p 255-274. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch11
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Image of Figure 11.7
Figure 11.7

Schematic view of Arp2/3 complex nucleated actin filament assembly. Schematic view of formin-mediated actin filament assembly.

Citation: Southwick F. 2004. 11 The Actin Cytoskeleton: Regulation of Actin Filament Assembly and Disassembly, p 255-274. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch11
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Image of Figure 11.8
Figure 11.8

Assembly of an ABM complex. Changes in the structure of vinculin or zyxin act as a molecular switch that exposes ABM-1-binding sequences for attracting and tethering VASP in the polymerization zone. Bound VASP then binds numerous profilin molecules, which increase the local concentrations of actin-ATP within the polymerization zone to stimulate filament assembly.

Citation: Southwick F. 2004. 11 The Actin Cytoskeleton: Regulation of Actin Filament Assembly and Disassembly, p 255-274. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch11
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References

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1. De La Cruz, E. M.,, E. M. Ostap,, R. A. Brundage,, K. S. Reddy,, H. L. Sweeney,, and D. Safer. 2000. Thymosin-beta(4) changes the conformation and dynamics of actin monomers. Biophys. J. 78:25162527.
2. Dickinson, R. B.,, F. S. Southwick,, and D. L. Purich. 2002. A direct-transfer polymerization model explains how the multiple profilin-binding sites in the actoclampin motor promote rapid actin-based motility. Arch. Biochem. Biophys. 406:296301.
3. Kang, F.,, D. L. Purich,, and F. S. Southwick. 1999. Profilin promotes barbed-end actin filament assembly without lowering the critical concentration. J. Biol. Chem. 274:3696336972.
4. Nyman, T.,, R. Page,, C. E. Schutt,, R. Karlsson,, and U. Lindberg. 2002. A cross-linked profilin-actin heterodimer interferes with elongation at the fast-growing end of F-actin. J. Biol. Chem. 277:1582815833. Experiments with this cross-linked complex emphasize the role of the profilin-actin heterodimer in barbed-end actin filament assembly.
5. Safer, D.,, T. R. Sosnick,, and M. Elzinga. 1997. Thymosin beta 4 binds actin in an extended conformation and contacts both the barbed and pointed ends. Biochemistry 36:58065816. Thymosin beta 4 is predominantly unstructured in solution. Upon binding to actin, the molecule contacts specific actin residues located at the barbed end as well as a residue in domain 2 at the pointed end, allowing thymosin beta 4 to sterically block actin polymerization at both ends of the actin monomer.
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8. Schafer, D. A.,, P. B. Jennings,, and J. A. Cooper. 1996. Dynamics of capping protein and actin assembly in vitro: uncapping barbed ends by polyphosphoinositides. J. Cell Biol. 135:169179. CapZ has a slow dissociation rate, but rapidly dissociates when exposed to PIP2. The relatively slow on-rate of this capping protein may explain the length distribution of actin filaments in living cells.
9. Witke, W.,, W. Li,, D. J. Kwiatkowski,, and F. S. Southwick. 2001. Comparisons of CapG and gelsolin-null macrophages: demonstration of a unique role for CapG in receptor-mediated ruffling, phagocytosis, and vesicle rocketing. J. Cell Biol. 154:775784. CapG is shown to have distinct in vivo functions that differ from its family member gelsolin. This capping protein is required for receptor-mediated ruffling of macrophages and plays an important role in phagocytosis.
10. Lin, K. M.,, M. Mejillano,, and H. L. Yin. 2000. Ca2+ regulation of gelsolin by its C-terminal tail. J. Biol. Chem. 275:2774627752.
11. McGrath, J. L.,, E. A. Osborn,, Y. S. Tardy,, C. F. Dewey, Jr.,, and J. H. Hartwig. 2000. Regulation of the actin cycle in vivo by actin filament severing. Proc. Natl. Acad. Sci. USA 97:65326537. These elegant studies emphasize the importance of gelsolin in actin filament turnover.
12. Flanagan, L. A.,, J. Chou,, H. Falet,, R. Neujahr,, J. H. Hartwig,, and T. P. Stossel. 2001. Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells. J. Cell Biol. 155:511517.
13. Nakamura, F.,, E. Osborn,, P. A. Janmey,, and T. P. Stossel. 2002. Comparison of filamin A-induced cross-linking and Arp2/3 complex-mediated branching on the mechanics of actin filaments. J. Biol. Chem. 277:91489154. The rheological experiments prove convincingly that filamin, but not Arp2/3 complex, can form gels in vitro. Polymer branching in the absence of cross-linking does not lead to polymer gelation.
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17. Evangelista, M.,, D. Pruyne,, D. C. Amberg,, C. Boone,, and A. Bretscher. 2002. Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast. Nat. Cell Biol. 4:260269.
18. Falet, H.,, K. M. Hoffmeister,, R. Neujahr,, J. E. Italiano, Jr.,, T. P. Stossel,, F. S. Southwick,, and J. H. Hartwig. 2002. Importance of free actin filament barbed ends for Arp2/3 complex function in platelets and fibroblasts. Proc. Natl. Acad. Sci. USA 99:1678216787. This third study utilizes gelsolin-null cells and CapG to prove that free barbed ends are required for efficient Arp2/3 complex nucleation and localization.
19. Pantaloni, D.,, R. Boujemaa,, D. Didry,, P. Gounon,, and M. F. Carlier. 2000. The Arp2/3 complex branches-filament barbed ends: functional antagonism with capping proteins. Nat. Cell Biol. 2:385391. Demonstrates that free barbed-filament ends are required for efficient Arp2/3 complex formation.
20. Pruyne, D.,, M. Evangelista,, C. Yang,, E. Bi,, S. Zigmond,, A. Bretscher,, and C. Boone. 2002. Role of formins in actin assembly: nucleation and barbed-end association. Science 297:612615. Formins produce an unbranched filament architecture that is distinctly different than Arp2/3 complex nucleated actin assembly. Formins remain bound to the barbed ends of growing actin filaments.
21. Volkmann, N.,, K. J. Amann,, S. Stoilova-McPhie,, C. Egile,, D. C. Winter,, L. Hazelwood,, J. E. Heuser,, R. Li,, T. D. Pollard,, and D. Hanein. 2001. Structure of Arp2/3 complex in its activated state and in actin filament branch junctions. Science 293:24562459.
22. Bear, J. E.,, T. M. Svitkina,, M. Krause,, D. A. Schafer,, J. J. Loureiro,, G. A. Strasser,, I. V. Maly,, O. Y. Chaga,, J. A. Cooper,, G. G. Borisy,, and F. B. Gertler. 2002. Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell 109:509521. VASP-transfected cells contain longer, less branched actin filaments compared with VASP-deficient cells. At μM concentrations VASP can inhibit barbed-end capping by nM concentrations of CapZ.
23. Castellano, F.,, C. Le Clainche,, D. Patin,, M. F. Carlier,, and P. Chavrier. 2001. A WASp-VASP complex regulates actin polymerization at the plasma membrane. EMBO J. 20:56035614.
24. DeMali, K. A.,, C. A. Barlow,, and K. Burridge. 2002. Recruitment of the Arp2/3 complex to vinculin: coupling membrane protrusion to matrix adhesion. J. Cell Biol. 159:881891.
25. Howe, A. K.,, B. P. Hogan,, and R. L. Juliano. 2002. Regulation of vasodilator-stimulated phosphoprotein phosphorylation and interaction with Abl by protein kinase A and cell adhesion. J. Biol. Chem. 277:3812138126.
26. Rottner, K.,, M. Krause,, M. Gimona,, J. V. Small,, and J. Wehland. 2001. Zyxin is not colocalized with vasodilator-stimulated phosphoprotein (VASP) at lamellipodial tips and exhibits different dynamics to vinculin, paxillin, and VASP in focal adhesions. Mol. Biol. Cell. 12:31033113.

Tables

Generic image for table
Table 11.1

Actin-regulatory proteins

Citation: Southwick F. 2004. 11 The Actin Cytoskeleton: Regulation of Actin Filament Assembly and Disassembly, p 255-274. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch11

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