Research Article: Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

Date Published: July 26, 2013

Publisher: Public Library of Science

Author(s): Mirco Müller, Ralph P. Diensthuber, Igor Chizhov, Peter Claus, Sarah M. Heissler, Matthias Preller, Manuel H. Taft, Dietmar J. Manstein, Friedrich Frischknecht.

http://doi.org/10.1371/journal.pone.0070636

Abstract

Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

Partial Text

Mammalian actin isoforms are highly conserved and ubiquitously found in eukaryotic cells. The 42 kDa globular actin monomer is composed of four subdomains and can assemble into thin filaments or microfilaments (F-actin). Six actin isoforms can be distinguished: three α-actin isoforms (α-skeletal muscle, α-cardiac muscle and α-vascular), one β-isoform (β-cytoplasmic) and two γ-isoforms (γ-cytoplasmic and γ-smooth muscle). Only subtle sequence variations distinguish the isoactins. The amino acid sequence of α-actin differs from cytoplasmic actin isoforms in more than 20 residues that are spread over the entire molecule. In contrast, differences between β- and γ-actin are restricted to the N-terminus – Asp2-Asp3-Asp4-Ile5 (β-actin) and Glu2-Glu3-Glu4-Ile5 (γ-actin). The N-terminal sequences of α-skeletal and α-cardiac actin correspond to Asp3-Glu4-Asp5-Glu6 and Asp3-Asp4-Glu5-Glu6[1]. Actin isoforms are essential for a wide range of physiological functions. The four muscle actins are restricted to tissues with high tonic activity such as striated heart muscle, skeletal muscle or smooth muscle of blood vessels, gut wall and the urogenital system [2]. By contrast, cytoplasmic actins are ubiquitous and play a pivotal role in cell motility, intracellular transport, cell shape maintenance or mitosis [3]. They undergo spatial and temporal segregation during the formation of stress fibers and actin-based cell protrusions [4], [5]. The resulting structures are regulated by specific actin-binding proteins [6], [7]. There is strong evidence that the isoactins cannot substitute for each other [8], [9], [10], [11], indicating functional intracellular specialization [12], [13]. The β-isoform preferably localizes in stress fibres, circular bundles and at cell-cell contacts as an unbranched filamentous array. Dependent on cellular activities, γ-actin displays a more variable distribution. It is mainly organized as a branched meshwork with cortical and lamellar localization in moving cells, but can colocalize with β-actin in lamellipodia or be recruited into stress fibres [3], [12]. Knockout studies in mice revealed, that both cytoplasmic actin isoforms are required for stereocilia maintenance in hair cells [14]. Aberrant expression of particular isoactins is a significant feature of adaptive and pathological alterations in e.g. wound healing, cardiovasular diseases, myopathies or tumor metastasis [2], [15]. Mutations of β-actin cause pleiotropic diseases and can be linked to neutrophil dysfunction (mutation E364K) [16], malformations, deafness, and delayed-onset dystonia (R183W) [17]. In addition, β-actin mutations are associated with metastasis [18] and tumours like the diffuse large B-cell lymphoma [19], [20]. Multiple missense mutations of γ-actin have been described (T89I, K118M, K118N, I122V, E241K, P264L, T278I, P332A, V370A), all of them are associated with autosomal dominant non-syndromic sensorineural progressive hearing loss [21], [22], [23], [24], [25], [26], [27].

The cytoplasmic actin isoforms were produced in Sf9 cells and purified to homogeneity (Figure 1A). Immunoblots were used to verify the purity of the particular isoactins (Figure 1B). Although actin isoforms display only subtle N-terminal differences (Figure 1C), the monoclonal antibodies used are highly specific for this region and show no cross-reactivity with other isoactins. IEF was performed to determine the amount of contaminating insect actin. Due to differences in their isoelectric points (IEP), the more basic γ-actin (IEP 5.31) can be separated from the more acidic insect actin (IEP 5.29) as depicted in Figure 1D. Insect and human β-actin have similar IEPs and separate less well. IEF and MS analyses indicate that the level of contamination of human cytoplasmic actins with endogenous actin from insect cells is in the range of 5–15% (N = 3).

Our results show clear increases in actin-activation and functional competence for all nonsarcomeric myosins tested, when they were allowed to interact with cytoplasmic isoactins instead of α-actin. The preferred interaction of NM-2A, -2B and -2C1 with cytoplasmic β-actin and γ-actin fits well to the proteins’ overlapping functions in cell migration, adhesion, cytokinesis, and cytoskeletal maintenance [3], [32], [57]. In regard to their interaction with β- or γ-actin, only NM-2A and NM-2B show no clear preference for either cytoplasmic isoactin. In our in vitro experiments, myosin-7A showed a distinct preference for γ-actin over β-actin. In their proper physiological context, myosin-7A, β- and γ-actin play a key role in the development, function and maintenance of cochlear hair cell stereocilia [14], [39]. Although the presence of both cytoplasmic actin isoforms is required for regular hair cell development, differences in the pattern of progressive hearing loss are observed upon their selective ablation. It was reported that γ-actin may be more abundant in particular hair cell structures and that the severity of progressive hearing loss can be related to the γ-actin concentration [14]. Further evidence for specific intracellular interactions between myosin-7A and γ-actin has been provided by studies of MYO7A gene defects [45], [46] and γ-actin mutations [21], [22], [23], [24], [25], [26], [27]. Our results pointing at a preferred interaction of the proteins provide an explanation why both sets of gene defects lead to similar phenotypes. NM-2C also plays a role in the function and maintenance of stereocilia. Mutations in NM-2C are associated with hereditary deafness (DFNA4) [37]. Genome-wide linkage analysis identified an autosomal-dominant mutation which causes a complex phenotype associated with peripheral neuropathy, myopathy, hoarseness, and hearing loss [58]. The results of our in vitro assays show that different from myosin-7A, NM-2C shows a clear preference for β-actin over γ-actin.

 

Source:

http://doi.org/10.1371/journal.pone.0070636