Actin Molecule

Actin is folded into two domains that are stabilized by an adenine nucleotide lying in between (Fig. 33-4). The polypeptide of 375 residues crosses twice between the two domains, with the N- and C-termini located near each other. The two domains are folded similarly, suggesting that the actin gene arose by duplication of an ancestral gene. Remarkably, the adenine nucleotide binding site, fold, and overall shape of actin closely resemble those of two other proteins with very different functions: the glycolytic enzyme hexokinase (see Fig. 3-12) and the heat shock protein Hsc70. All three proteins might have evolved originally in prokaryotes from the same primordial nucleotide-binding protein.

Figure 33-4 atomic structure of actin. A, Ribbon model showing the polypeptide fold and the location of Mg-ATP, shown as space-filling. Numbers indicate the four subdomains.B, Surface rendering. ATP is almost completely buried in the cleft between the two lobes of the protein, where it makes extensive contacts with the protein. The barbed end of the molecule (Fig. 33-9) is at the bottom in this orientation.

(PDB file: 1ATN. Reference: Kabsch W, Mannherz HG, Suck D, et al: Atomic structure of the actin-DNase I complex. Nature 347:37–44, 1990.)

Actin binds adenosine triphosphate (ATP) or adenosine diphosphate (ADP) and a divalent cation, Mg²⁺ in cells, with nanomolar affinity. The affinity of actin for ATP is higher than that for ADP, so given the higher concentration of ATP in cells, unpolymerized actin is saturated with ATP. The bound nucleotide exchanges relatively slowly with nucleotide in the medium (Fig. 33-11). Actin monomer–binding proteins can inhibit or accelerate nucleotide exchange. Bound nucleotide stabilizes the molecule but is not required for polymerization in vitro. ATP-actin and ADP-actin polymerize at different rates.

Figure 33-11 regulation of actin nucleotide exchange by actinbinding proteins. The rate-limiting step is dissociation of the bound nucleotide, which requires opening the nucleotide cleft. ADF/cofilins and β-thymosins inhibit dissociation of both ATP and ADP. Profilin competes with both inhibitors for binding actin monomers and increases the rates of both nucleotide dissociation and binding. The ability of profilin to promote nucleotide exchange, the higher affinity of actin for ATP than for ADP, and the higher concentration of ATP than ADP in cytoplasm drive the reactions to the right, accounting for why essentially all unpolymerized actin in cells has bound ATP.

Posttranslational modifications of actins include acetylation of the N-terminus and (in most cases) methylation of histidine-68. In some insect flight muscles, the small protein ubiquitin (see Fig. 23-7) is attached covalently to about one in six actin molecules, yielding a 55-kD polypeptide that is incorporated with unmodified actin into filaments. Some invertebrate actins are phosphorylated on tyrosine-211. The functional significance of these modifications is still being investigated.

Actin genes originated in prokaryotes, where they are required for rod-shaped bacteria to maintain their asymmetric shapes. Other bacterial actins help to segregate DNA plasmids to the two daughters during cell division. Eukaryotic actin genes are highly conserved, but through divergent evolution, they encode subtly different proteins, some with novel functions. Most organisms have multiple actin genes, and all known actin isoform diversity arises from multiple genes rather than from alternative splicing of mRNAs. Humans have six actin genes; Dictyostelium has more than ten; but budding yeast has only one. Muscle actin genes diverged from cytoplasmic actins in primitive chordates (see Fig. 2-9). To fulfill special developmental functions, plant actin genes diverged among themselves more than animal actin genes.

The biochemical similarities of actin isoforms are more impressive than their differences (Fig. 33-5). The sequences of pairs of actins are generally more than 90% identical, even between highly divergent species. Humans express β and γ isoforms in nonmuscle cells and four different α and β isoforms in various muscle cells. Many nonmuscle cells express both the β and γ isoforms, but red blood cells use only β-actin.

Figure 33-5 sorting of actin isoforms in cells. Fluorescence micrograph of cultured cells doubly stained with fluorescent antibodies specific for β-actin concentrated at the leading edge (orange) and g-actin concentrated in stress fibers (green). Nuclei are stained blue with DAPI.

(Courtesy of I. Herman, Tufts Medical School, Boston, Massachusetts.)

In every case that was examined, actin isoforms copolymerize in the test tube, so it is remarkable that cells can sort actin isoforms into different structures. For example, b-actin is concentrated near the plasma membrane of cultured cells, whereas g-actin is concentrated in stress fibers (Fig. 33-6). In muscle, α-actin forms the thin filaments of the contractile apparatus, whereas g-actin localizes around mitochondria. Even the full array of known actin-binding proteins cannot yet explain how cells prevent copolymerization of the isoforms or concentrate isoforms at different locations.

Figure 33-6 comparison of actin and actin-related proteins. Space-filling models are based on the atomic structure of actin and the sequences of the Arps.Yellow residues are identical to actin,green residues are conservative substitutions,blue residues are nonconservative substitutions, and red residues are insertions. All of these proteins have similar internal architectures, including identical contacts with ATP, but their surfaces differ considerably. The phylogenetic tree, based on sequence comparisons, shows that the genes for actins and for all of the Arps had a common ancestor.

(From the work of J. Kelleher, Johns Hopkins Medical School, Baltimore, Maryland; illustration redrawn from Mullins D, Kelleher J, Pollard TD: Actin’ like actin. Trends Cell Biol 6:208–212, 1996.)

Actin-Related Proteins

After believing for 20 years that actins are one of the most evolutionarily conserved protein families, scientists discovered several families of highly divergent actin-related proteins (Arps) in the 1990s (Fig. 33-6). Genes for Arps diverged from actin genes after the earliest branches in the eukaryotic tree and are now found in eukaryotic species ranging from amoebas to humans. Arps share with actin the fold of the polypeptide chain and residues forming the nucleotide-binding site, but fewer than 60% of the residues are identical to actin. Divergent surface residues allow Arps to participate in molecular interactions different from actin. Arp1 forms a short filament as part of the dynactin complex that promotes cargo movement by the microtubule motor dynein (see Fig. 37-2). Arp2 and Arp3 are two of seven subunits in a protein complex that nucleates branched actin filaments in the cell cortex (Fig. 33-13). Eight additional types of Arps are widespread in eukaryotes. Several participate in complexes that regulate chromatin structure.

Figure 33-13 Nucleation of branched actin filaments by Arp2/3 complex.A, Ribbon diagram of the crystal structure of Arp2/3 complex. The seven subunits are color coded and labeled. Numbers label the subdomains of Arp2 and Arp3.B, Model of branch based on a 3D reconstruction from electron micrographs.C, Steps in branch formation (also refer back to panel A): A WASp/Scar nucleation promoting factor binds an actin monomer (1). This binary complex binds Arp2/3 complex, bringing together the actin subunit with Arp2 and Arp3 (2). The ternary complex binds to the side of an actin filament, completing the activation process (3). A new daughter filament grows at its barbed end from the side of the older mother filament (4). B is the barbed end. P is the pointed end.

(A, PDB file: 1K8K. References: Robinson R, Turbedsky K, Kaiser DA, et al: Crystal structure of Arp2/3 complex. Science 294:1679–1684, 2001. B, Based on the work of I. Rouiller, N. Volkmann, and D. Hanein, Burnham Institute, La Jolla, California. C, Marchand J-B, Kaiser DA, Pollard TD, Higgs HN: Interaction of WASp/Scar proteins with actin and vertebrate Arp2/3 complex. Nat Cell Biol 3:76–82, 2001.)

Actin Polymerization

Actin filaments are polarized, owing to the uniform orientation of the asymmetrical subunits along the polymer (Fig. 33-7). One end is called the barbed end, the other is called the pointed end. This nomenclature arises from the asymmetrical arrowhead pattern created when myosin bind along the length of actin filaments (Fig. 33-8). The helical arrangement of subunits in actin filaments was originally revealed in the 1960s by electron microscopy and X-ray fiber diffraction of whole muscle and actin gels. These low-resolution data are used to orient the atomic structure of the actin monomer in current models.

Figure 33-7 structure of the actin filament. A, Electron micrograph of a negatively stained actin filament. B, Reconstruction of an actin filament by image processing of electron micrographs (blue) with ribbon models of the subunits(gold) along one strand of the double helix. One subunit is enlarged to the right. The pointed end of the subunits with the nucleotide cleft is at thetop, and the faster-growing barbed end is at thebottom. This orientation of the actin molecule in the filament uniquely accounts for the X-ray fiber diffraction pattern of oriented filaments and agrees with electron microscopy with probes on specific actin residues and with chemical cross-linking between residues of adjacent subunits. C, Surface rendering of the molecular model. Subunits in the two long-pitch helices are shown asyellow-orange andblue-green (see Fig. 5-5 for nomenclature). The short pitch helix, including every subunit, follows a yellow-green-orange-blue pattern.D, Scale drawing used throughout this text.

(A–B, Courtesy of U. Aebi, University of Basel, Switzerland. C, Courtesy of R. Milligan, Scripps Research Institute, La Jolla, California.)

Figure 33-8 actin filament elongation. A, Electron micrograph of growth from an actin filament seed decorated with myosin heads to reveal the polarity. Growth is faster at the barbed end than at the pointed end.B, Rate constants for association (units: μM⁻¹s⁻¹) and dissociation (units: s⁻¹) for Mg-ATP-actin (T) and Mg-ADP-actin (D) were determined by measuring the rate of elongation at the two ends as a function of monomer concentration. Ratios of the rate constants yield critical concentrations (K, units: mM) for the various reactions. The critical concentrations at the two ends are the same for ADP-actin but differ for ATP-actin.

(A, Courtesy of M. Runge, Johns Hopkins Medical School, Baltimore, Maryland. B, Reference: Pollard TD: Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments. J Cell Biol 103:2747–2754, 1986.)

Actin self-assembles into filaments by means of a series of bimolecular reactions (Fig. 33-9; see also Fig. 5-6). Actin is isolated from cells as a monomer at low salt concentrations. Physiological concentrations of monovalent and divalent cations bind to low-affinity sites on actin and promote polymerization. In vitro, actin trimers appear to be the nucleus that initiates polymer growth in the sense that the reactions that are required to form trimers are very unfavorable in comparison with reactions for elongation of polymers larger than trimers. To initiate new filaments, cells use regulatory proteins to overcome these unfavorable nucleation reactions.

Figure 33-9 actin filament nucleation, growth, and nucleotide hydrolysis. A, Nucleation. Formation of dimers and trimers is very unfavorable, owing to rapid dissociation of subunits. Actin trimers are called nuclei because they initiate the highly favorable elongation reactions. This mechanism is based on kinetic modeling of experimental data. Estimated rate constants have units of μM⁻¹s⁻¹ for association reactions and s⁻¹ for dissociation reactions.B, ATP hydrolysis by a polymer of ATP-actin(yellow subunits) is random and irreversible at a rate of 0.3s⁻¹, yielding subunits with bound ADP and inorganic phosphate (orange). Phosphate dissociates slowly at a rate of 0.002s⁻¹, converting half of the newly polymerized subunits to ADP-actin (pink) in 6 minutes. ADP bound to polymerized subunits does not exchange with nucleotide in the medium. Phosphate binding is reversible, but the affinity is low, so most subunits bind only ADP.

(Reference: Pollard TD, Blanchoin L, Mullins RD: Biophysics of actin filament dynamics in nonmuscle cells. Annu Rev Biophys Biomol Struct 29:545–576, 2000.)

Actin filaments grow and shrink by the addition and loss of subunits at the two ends of the polymer. The reactions at the two ends have different rate constants (Fig. 33-8). Association of subunits is rapid at both ends. Subunit association is a diffusion-limited reaction (see Chapter 4) at the rapidly growing barbed end and somewhat slower at the other end. Subunit dissociation is relatively slow at both ends, between 0.3 and 8 subunits per second. The rates of these reactions depend on the nucleotide bound to the monomer associating with or dissociating from a filament.

In the presence of ATP, purified actin assembles almost completely, leaving as monomers the critical concentration of about 0.1μM ATP-actin. The critical concentration is the monomer concentration giving equal rates of association and dissociation, 1.4s⁻¹

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