The actin gene family: Function follows isoform^†

Muscle Isoforms

All knockout mice reported thus far have distinct phenotypes suggesting that each isoactin has unique and specialized functions (Table I). α_cardiac-Actin knockout results in lethality during either embryonic or perinatal development with profound disorganization of cardiac myofibrils [Kumar et al., 1997]. α_skeletal-Actin knockout mice appear normal at birth but have weak muscles and all die by 9 days of age [Crawford et al., 2002]. α_smooth-Actin knockout mice are born at Mendelian ratios and are viable. However, these mice have defects in vascular contractility and blood pressure regulation [Schildmeyer et al., 2000]. γ_smooth-Actin knockout mice have not yet been reported.

In all of these mouse models, there is compensatory upregulation of a subset of the remaining actin isoforms. However, since each muscle actin is highly expressed the total level of actin in knockout muscle cells remains less than in wild type. Therefore, transgenic overexpression of other isoforms may rescue the knockout phenotype and provide a useful measure of overlapping function between the isoforms. Along these lines, γ_smooth-actin was overexpressed in α_cardiac-actin knockouts [Kumar et al., 1997] and α_cardiac-actin was overexpressed in α_skeletal-actin knockouts [Nowak et al., 2009]. Overexpression of γ_smooth-actin only partially rescued mice lacking α_cardiac-actin. Even when these mice survived to adulthood, their hearts were hypodynamic and hypertrophic, demonstrating that γ_smooth-actin and α_cardiac-actin each make distinct contributions to cardiac cell function [Kumar et al., 1997].

α_cardiac-Actin and α_skeletal-actin are 99% identical and have overlapping expression patterns [Vandekerckhove et al., 1986]. These similarities suggest that these isoforms have considerable overlapping functions. Correspondingly, transgenic expression of α_cardiac-actin fully rescued the lethality and muscle performance deficits associated with the loss of α_skeletal-actin suggesting a potential treatment for human diseases associated with reduced α_skeletal-actin [Nowak et al., 2009]. In contrast, overexpression of γ_cyto-actin did not rescue lethality due to α_skeletal-actin deficiency suggesting that muscle and nonmuscle actins are more specialized. Interestingly, transgenic expression of γ_cyto-actin in wild-type mice resulted in the substitution of 40% of thin filament α_skeletal-actin with γ_cyto-actin [Jaeger et al., 2009]. Therefore, muscle cell function depends on muscle-specific actin isoforms, but can tolerate a surprising amount of contamination by γ_cyto-actin.

Cytoplasmic Isoforms

β_cyto-Actin and γ_cyto-actin are nearly identical proteins that differ by only four biochemically similar amino acids, all of which are found in the 10 N-terminal residues (Fig. 1A). Mice homozygous for hypomorphic alleles of Actb die during early development of uncharacterized defects [Shawlot et al., 1998; Shmerling et al., 2005]. A conditional knockout has been generated, but not yet described (our unpublished data). In contrast to β_cyto-actin-deficient mice, whole-body γ_cyto-actin knockout mice are viable and can survive to adulthood and beyond [Belyantseva et al., 2009; Bunnell and Ervasti, 2010]. Interestingly, these mice are smaller than wild-type or heterozygous littermates from an early developmental stage, but are born at normal Mendelian ratios. γ_cyto-Actin confers a clear survival advantage as significant numbers of newborn Actg1^−/− mice die as a result of developmental delays and another fraction die stochastically during adulthood [Belyantseva et al., 2009; Bunnell and Ervasti, 2010]. Corresponding to decreased animal survival, primary γ_cyto-actin-deficient fibroblasts cultured from these mice have decreased viability, impaired growth, and increased apoptosis and necrosis. On the other hand, consistent with the viability of Actg1^−/− mice, γ_cyto-actin null fibroblasts have normal migration in a scratch wound assay [Bunnell and Ervasti, 2010].

Loss of γ_cyto-actin has similar consequences for cells in vivo. Sensory hair cells in the inner ear depend on stereocilia formed of β_cyto-actin and γ_cyto-actin for proper function. γ_cyto-Actin-deficient hair cells develop normally but do not maintain stereocilia during aging, which corresponds to progressive hearing loss and deafness [Belyantseva et al., 2009]. Similarly, conditional knockout of γ_cyto-actin in muscle did not have developmental consequences [Sonnemann et al., 2006] despite in vitro evidence that γ_cyto-actin is required for sarcomere assembly [Lloyd et al., 2004]. Instead, mice developed a novel progressive centronuclear myopathy. In all cell types from γ_cyto-actin knockout mice studied thus far, the total cellular concentration of actin remains constant due to compensatory upregulation of the other actin isoforms [Belyantseva et al., 2009; Bunnell and Ervasti, 2010]. Since the actin concentration remains constant, the observed phenotypes can be ascribed to changes in the actin composition.

Distinct Localization Patterns

Skeletal muscle provides a seemingly clear example of differential localization of muscle and nonmuscle actin isoforms with α_skeletal-actin being confined to the sacromeric thin filaments. In contrast, γ_cyto-actin, which has the best-described localization pattern in muscle, appears to be absent from thin filaments, but is instead found in other muscle cell structures. γ_cyto-Actin was initially detected in filamentous structures surrounding mitochondria and adjacent to the sarcolemma [Craig and Pardo, 1983; Pardo et al., 1983]. Subsequent reports found that γ_cyto-actin is the only actin species detected at costameres [Rybakova et al., 2000], which are structures found between the sarcolemma and the z-disk [reviewed in Ervasti, 2003]. Other recent studies detected γ_cyto-actin in costameres as well as in a novel zone adjacent to the z-disk [Kee et al., 2004]. Finally, different groups have detected γ_cyto-actin only in z-disks and not in costameres [Nakata et al., 2001; Papponen et al., 2009].

In other cell types, γ_cyto-actin seems to be uniformly distributed in all actin-containing structures [Otey et al., 1986]. In contrast, β_cyto-actin seems to have a more polarized distribution [Hoock et al., 1991; Bassell et al., 1998]. The differences between β_cyto-actin and γ_cyto-actin localization may be due to a 54-nucleotide ÓzipcodeÓ sequence found in the 3′ UTR of β_cyto-actin but not γ_cyto-actin transcripts. The zipcode sequence binds to zipcode-binding protein (ZBP1, also known as IMP-1), which facilitates targeting of the transcript and regulates translation [reviewed in Condeelis and Singer, 2005]. ZPB1 binds numerous different transcripts [Jonson et al., 2007], perhaps integrating β_cyto-actin into a broader cellular program.

ZBP1-mediated regulation of β_cyto-actin has important functional consequences in different cell types. In neurons cultured from X. laevis, growth cones exposed to an attractive cue require targeted β_cyto-actin transcripts and newly synthesized β_cyto-actin protein for normal turning behavior [Leung et al., 2006; Yao et al., 2006]. β_cyto-Actin is enriched compared to γ_cyto-actin on the side of the growth cone exposed to attractant [Yao et al., 2006] and growth cones fail to turn when neurons are treated with antisense oligos to β_cyto-actin [Leung et al., 2006]. Together, this evidence strongly predicts that β_cyto-actin is essential for normal neuronal development. However, this hypothesis has yet to be tested in an intact mammalian system.

Zbp1-mediated targeting of β_cyto-actin transcripts appears to be important in other cell types, including fibroblasts and adenocarcinoma cells, because interference with the ZBP1 activity alters cell morphology and migration [Kislauskis et al., 1994; Shestakova et al., 2001]. Consistent with targeting of β_cyto-actin transcripts, several groups report that β_cyto-actin is enriched at the leading edge of cultured fibroblasts and myoblasts as compared to stress fibers found in the central region of the cell [Hoock et al., 1991; Hill and Gunning, 1993; Kislauskis et al., 1997; Shestakova et al., 2001]. In contrast, γ_cyto-actin appears to be uniformly distributed in all actin-containing structures in fibroblasts [Otey et al., 1986].

In a conflicting report, recent work using newly generated isoform-specific antibodies and a different fixation technique suggests that β_cyto-actin predominates in stress fibers while γ_cyto-actin is enriched at the leading edge [Dugina et al., 2009]. Discrepancies in β_cyto and γ_cyto-actin localization have also been noted in auditory hair cell stereocilia. Here, immunofluorescent labeling indicated that γ_cyto-actin was localized at the periphery of stereocilia [Belyantseva et al., 2009], while a study using immuno-TEM found that β_cyto-actin was peripheral and γ_cyto-actin was more centrally localized [Furness et al., 2005].

Study-to-study differences in observations of cytoplasmic actin localization in cultured cells, stereocilia, and muscle indicate that actin localization presents a formidable challenge. The slight amino acid sequence differences between isoforms require establishing antibody specificity, ideally using knockout tissue. Importantly, antibody binding interactions vary with experimental conditions; for example, the specificity of the anti-a_cardiac-actin antibody depends on the salt concentration [Franke et al., 1996]. Even with validated antibodies, it is difficult to interpret cases where there is a lack of staining (i.e., costameres, the central regions of stereocilia, stress fibers) because it seems that some actin-based structures may be inherently difficult to immunostain. Tightly bundled actin filaments, such as those found in stereocilia and stress fibers, may resist staining due to epitope masking or limited antibody penetration. Other actin-based structures, such as costameres, are labile and easily lost during fixation.

Actin as an Alloy

Differential localization as a mechanism to explain the distinct functions of actin isoforms is an attractive hypothesis because of its intuitive simplicity. However, it remains unclear how relative enrichment leads to changes in actin function. New biochemical evidence suggests that F-actin properties may vary according to the mix of isoforms in the filament. Bergeron et al. [ 2010] recently characterized purified β_cyto and γ_cyto-actin produced in a recombinant baculovirus system. They observed that under calcium-bound conditions, β_cyto-actin exhibited more dynamic behavior than γ_cyto-actin with faster polymerization and depolymerization rates. Intriguingly, biochemical assays demonstrated that β_cyto and γ_cyto-actin readily copolymerize and that the resulting filaments have polymerization and depolymerization rates that vary according to the ratio of β_cyto-actin to γ_cyto-actin [Bergeron et al., 2010]. In addition to polymerization dynamics, different mixtures of actin isoforms may well have distinct biophysical properties or distinct interactions with stability regulators such as AIP1 or cofilin.

Varying the mixture of actin may be a useful mechanism for adapting the cytoskeleton for a variety of functions. By way of analogy, steel is an alloy composed of numerous different metals. The ratio of each component determines the characteristics of the particular kind of steel, which is engineered to meet the desired compromises between properties such as weight, tensile strength, and cost. It is important that alloys can be manipulated because innumerable different steels are required to meet the varied demands of buildings and machines. Actin is cellular steel. Altering the ratio of actin isoforms within a filament may tune its properties to meet the specific requirements of different cells or subcellular structures.