Actin and Actin-Binding Proteins

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CHAPTER 33 Actin and Actin-Binding Proteins

Actin filaments form a cytoskeletal and motility system in all eukaryotes (Fig. 33-1). Cross-linked actin filaments resist deformation, transmit forces, and restrict diffusion of organelles. A network of cortical actin filaments excludes organelles (Fig. 33-2C), reinforces the plasma membrane, and restricts the lateral motion of some integral membrane proteins. The cortex varies in thickness from a monolayer of actin filaments in red blood cells (see Fig. 7-10) to more than 1μm in amoeboid cells (Fig. 33-2C). Like fingers in a glove, bundles of actin filaments support slender protrusions of plasma membrane called microvilli or filopodia (Fig. 33-2B). Microvilli expand the cell surface for transport of nutrients and participate in sensory processes, including hearing. The actin cytoskeleton complements and interacts physically with cytoskeletal structures composed of microtubules (see Chapter 34) and intermediate filaments (see Chapter 35).

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Figure 33-2 electron micrographs of actin filaments. A, Filaments of purified actin prepared by negative staining.B, A thin section of an intestinal epithelial cell illustrating finger-like microvilli with tightly packed bundles of actin filaments linked to the surrounding plasma membrane by myosin-I. The barbed ends of these filaments (see Fig. 33-9) are located at the tips of the microvilli.C, A thin section of Acanthamoeba showing the actin filament meshwork in the cortex beneath the plasma membrane.D–E, Cultured fish scale keratocytes, fixed while actively migrating toward thetop of the figure. D, Electron micrograph of a whole mount of a cell illustrating the meshwork of branched filaments near the leading edge and longer, unbranched filaments deeper in the cytoplasm. Most filaments are oriented with their barbed ends forward.E, Fluorescence micrograph with phalloidin staining actin filaments (blue) and antibodies staining myosin II (red).

(A, Courtesy of U. Aebi, University of Basel, Switzerland. B, Courtesy of L. Tilney, University of Pennsylvania, Philadelphia, and M. Mooseker, Yale University, New Haven, Connecticut. D–E, Courtesy of T. Svitkina and G. Borisy, University of Wisconsin, Madison.)

Actin contributes to cell movements in two ways. First, polymerization and depolymerization of the network of actin filaments just inside the plasma membrane contribute to the extension of pseudopods, cell locomotion (Fig. 33-2D-E), and phagocytosis (see Fig. 22-3). Second, actin filaments are tracks for movements of the myosin family of motor proteins (see Fig. 36-7). Actin filaments and myosin filaments form the highly ordered, stable contractile apparatus of muscles (Fig. 33-3B; also see Fig. 39-3), as well as the transient contractile ring that pinches the two daughter cells apart at the end of mitosis (Fig. 33-3A; also see Fig. 44-23). Myosins also power movements of membranes and other cargo along actin filaments, complementing organelle movements by other motors along microtubules (see Fig. 37-1). Actin, myosin, and accessory proteins form intracellular bundles called stress fibers (Fig. 33-1B) that apply tension between adhesive junctions on the plasma membrane (see Fig. 30-11), where cells attach to each other or to the extracellular matrix. Stress fibers are prominent in tissue culture cells grown on glass or plastic and in endothelial cells lining major arteries.

Actin and myosin are thought to be among the five most abundant eukaryotic proteins on the earth. Actin is often the most abundant protein in a cell, composing up to 15% of total protein, and the many types of actin-binding proteins may account for another 10% of cellular protein. In muscle, actin and myosin constitute more than 60% of the total protein. Given this abundance, it is curious that actin was discovered in muscle only in the 1940s and in nonmuscle cells in the late 1960s. Since the 1970s, scientists have discovered new actin-binding proteins every year, but the inventory is probably still incomplete. Genetic defects in components of the actin cytoskeletal and motility system cause many human diseases, including muscular dystrophy (see Table 39-2), hereditary fragility of red blood cells (i.e., hemolytic anemias, see Fig. 7-10), and hereditary heart diseases called cardiomyopathies (see Table 39-4).

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.

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.

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.

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.

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.

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.

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.

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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