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

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, Nature 347:37–44, 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.

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-Pi, followed by the slower dissociation of Pi 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.

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

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.

(A) Schematic diagram of the action of monomer-sequestering agents such as Tβ4. (B) 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. (C) 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. (D) 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. (E) 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. (F) 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. (G) 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.

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.

(A) Schematic view of Arp2/3 complex nucleated actin filament assembly. (B) Schematic view of formin-mediated actin filament assembly.

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.