A new light chain for myosin-7

Abstract

Recent research has revealed that an adhesion complex based on cadherins and the motor protein myosin-7b (MYO7B) links the tips of intestinal microvilli. Choi et al. now report that a largely uncharacterized protein known as calmodulin-like protein 4 (CALML4) is a component of this adhesion complex and functions as a light chain for myosin-7b. Because the intermicrovillar adhesion complex is homologous to the myosin-7a (MYO7A)-based Usher syndrome complex and Choi et al. also report that CALML4 can bind to myosin-7a, this work also has important implications for research on myosin-7a and hereditary deaf-blindness.

Epithelial cells in many organs are covered by cylindrical protrusions of the apical plasma membrane known as microvilli. Microvilli contain a bundle of actin filaments at their core and provide a powerful model system for cytoskeletal research. Recent work has revealed that the actin-based motor protein myosin-7b localizes to the tips of intestinal microvilli, where its tail interacts with two scaffolding proteins, USH1C and ANKS4B, forming a complex that binds to the cytoplasmic domain of the cadherin-related protein CDHR2 (1–3). The extracellular domain of CDHR2 in turn binds to the extracellular domain of another cadherin-related protein, CDHR5, forming a thin fibril that links the tips of adjacent microvilli (Fig. 1A). The intermicrovillar adhesion complex formed by these proteins is required for the uniform length and packing of microvilli and is remarkably homologous to the Usher syndrome complex found in two microvilli-like structures—the stereocilia of the inner ear and the calyceal processes of photoreceptors. In stereocilia, the Usher syndrome complex forms a link connecting the tip of one stereocilium to the side of its taller neighbor and is required for conveying force from the movement of stereocilia to stretch-activated channels at their tips (Fig. 1B). The stereociliary tip link is essential for the mechanotransduction underlying hearing, and mutations in the Usher syndrome complex are the leading cause of hereditary deaf-blindness. To investigate the much less-studied intermicrovillar adhesion complex, Choi et al. (4) developed a clever affinity purification strategy utilizing the extracellular domain of CDHR5 to pull down CDHR2 and other complex components.

Figure 1.

Schematic diagrams of the intermicrovillar adhesion complex, the Usher syndrome complex, and myosin-7 domain structure. A, simplified diagram of intestinal microvilli illustrating the actin filaments in the microvillar core and the complex formed by the motor protein MYO7B, the PDZ-protein USH1C, the ankyrin-repeat protein ANKS4B, and CDHR2. B, simplified diagram of inner ear stereocilia illustrating the complex formed by MYO7A, an isoform of USH1C, the ankyrin-repeat protein USH1G (a paralog of ANKS4), and cadherin 23 (CDH23). The extracellular domains of CDH23 and protocadherin 15 (PCDH15) bind to one another to form the stereociliary tip link required to open stretch-activated channels located at the tip of the shorter stereocilium. For simplicity, only one copy of each protein is shown in these diagrams even though the myosin-7's and the cadherin superfamily proteins are likely to function as homodimers or higher-order oligomers, and the complexes themselves may form protein condensates (11). For a more detailed summary of protein interactions, see Ref. 2. C, schematic of myosin-7 structure. Myosin-7a and -7b share 52% sequence identity and a similar overall structure consisting of a myosin motor domain, 5 IQ motifs that provide binding sites for light chains of the calmodulin superfamily, and a conserved tail domain.

This affinity purification approach succeeded in pulling down known components of the intermicrovillar adhesion complex along with the calmodulin-like protein CALML4. Although calmodulin is a ubiquitous and intensively studied Ca²⁺-binding protein with hundreds of binding partners, little is known about CALML4 except that it is a member of the calmodulin superfamily. Choi et al. discovered that the major CALML4 transcript in human small intestine had not yet been documented and encodes a 153-amino acid isoform sharing ∼45% identity with the 149-amino acid sequence of calmodulin. Like calmodulin, CALML4 consists largely of four EF-hand motifs, but it is not yet clear whether each of CALML4's EF-hands forms a functional binding site for a Ca²⁺ ion. To provide additional evidence that CALML4 is a bona fide component of the intermicrovillar adhesion complex, Choi et al. used immunofluorescence in intestine and cell culture models to show that endogenous CALM4 exhibits a striking localization to the tips of microvilli along with myosin-7b and other components complex.

These results demonstrated CALML4's location, but what is its function? A strong hint comes from the knowledge that in virtually all myosins the motor domain is followed by a light chain–binding domain consisting of one or more IQ motifs, each of which provides a binding site for a member of the calmodulin superfamily (5). The IQ motifs in many myosins preferentially bind to calmodulin, but other IQ motifs bind to different members of the calmodulin superfamily, such as the essential and the regulatory light chains first identified in muscle myosin-2 (5). Binding of the light chain stabilizes the myosin, enabling more efficient movements along its actin tracks. To test whether CALML4 can serve as a light chain for myosin-7b, Choi et al. showed that a construct consisting of the myosin-7b motor and light chain–binding domain was sufficient to pull down CALML4 and that myosin-7b's five IQ motifs were necessary for the interaction. Importantly, pulldown experiments with myosin-7a constructs yielded similar results, suggesting that CALML4 can serve as a light chain for both myosin-7a and myosin-7b.

Myosin-7b is hypothesized to transport itself and its binding partners to the microvillar tip (6). To test this hypothesis, Choi et al. used myosin-7b knockdown in cell culture, along with a myosin-7b knockout mouse, to clearly demonstrate that CALML4 localization to tips requires myosin-7b. Conversely, CALML4 knockdown disrupted the tip localization of myosin-7b. CALM4 knockdown also disrupted the tip localization myosin-7b's binding partners in the intermicrovillar adhesion complex, and it inhibited the usual ability of microvilli to cluster together via their tips. These CALML4 knockdown results phenocopy those of myosin-7b knockdown, providing crucial evidence that myosin-7b's ability to transport and/or localize itself to microvillar tips is largely dependent on CALML4.

In addition to revealing a new component of the intermicrovillar adhesion complex and a new light chain for myosin-7b, Choi et al. strongly suggest that CALML4 can serve as a light chain for myosin-7a, the intensively studied myosin at the core of the Usher syndrome complex. Previous research using baculovirus to co-express myosin-7a constructs consisting of the motor and light chain–binding domain with calmodulin reported only about three calmodulin molecules bound per heavy chain rather than the five light chains expected (7, 8). These myosin-7a constructs also exhibited relatively low ATPase activities of less than ∼1 ATP/s/head, raising the possibility that their motor activity might be higher in the presence of a full complement of endogenous light chains. This makes it crucial to determine whether CALML4 is an endogenous light chain for myosin-7a and if its presence changes the level or regulation of motor activity. These results also highlight the importance of using proteomics approaches to identify the endogenous light chains for all other myosins whose endogenous light chains remain unclear (5). It will also be important to determine which IQ motifs in the two mammalian myosin-7s bind to CALML4, if they preferentially bind to CALML4 as opposed to other members of the calmodulin superfamily, and if Ca²⁺ regulates their affinity (7). Consistent with a role as a putative myosin-7a light chain, CALML4 is expressed in the inner ear and was recently identified in proteomic analyses of myosin-7a pulldowns (9). Most excitingly, as Choi et al. point out, the gene responsible for Usher syndrome type 1H remains unknown, and CALML4 is one of 27 genes identified within the USH1H candidate region (10). These results, together with the discoveries of Choi et al., make CALML4 a putative myosin-7a light chain and a candidate gene to underlie USH1H hereditary deaf-blindness.