MultiBac System-Based Purification and Biophysical Characterization of Human Myosin-7a

Myosins are molecular motor proteins that interact with actin to drive numerous cellular processes^1,2,3,4. Humans possess 12 classes and 39 myosin genes⁵, which are involved in a wide range of physiological functions, such as muscle contraction⁶ and sensory processes⁷. Each myosin molecule is a multimeric complex composed of a heavy chain and light chains. The heavy chain is divided into head, neck, and tail regions. The head contains actin- and nucleotide-binding sites that are responsible for ATP hydrolysis and generating force on actin filaments². The neck is formed by several α-helical IQ motifs where a specific set of light chains are bound. They together function as a lever arm to amplify the motor's conformational changes into large movements^8,9,10. The tail contains class-specific subdomains and plays a regulatory role in tuning myosin's motor activity and mediating interactions with cellular binding partners^2,11.

Human myosin-7a, a member of class-7 myosins, is essential for auditory and visual processes^12,13. The IQ motifs of human myosin-7a are associated with a unique combination of light chains, including the regulatory light chain (RLC), calmodulin, and calmodulin-like protein 4 (CALML4)^14,15,16. Besides stabilizing the lever arm, these light chains regulate the mechanical properties of myosin-7a in response to calcium signaling, a feature that appears to be unique to the mammalian isoform¹⁴.

Defects in the gene encoding the myosin-7a heavy chain (MYO7A/USH1B) are responsible for Usher syndrome type 1, the most severe form of combined vision and hearing loss in humans¹⁷. Additionally, the light chain gene CALML4, is among the candidate genes mapped to contain the causative allele for USH1H, another variant of type 1 Usher syndrome^15,18. In the retina, myosin-7a is expressed in the retinal pigment epithelium and photoreceptor cells¹³. It has been implicated in the localization of melanosomes in the retinal pigment epithelium (RPE)¹⁹ and phagocytosis of photoreceptor outer segment disks by the RPE cells²⁰. In the inner ear, myosin-7a is primarily found in the stereocilia, where it plays a critical role in establishing hair bundles and in gating the mechano-electrical transduction process^12,21,22.

While the importance of myosin-7a in sensory cells is well established, its functional mechanisms at the molecular level remain poorly understood. This gap in knowledge is partly due to the challenges in purifying the intact protein, especially the mammalian isoform. Recently, significant progress has been made using the MultiBac system to recombinantly express the complete human myosin-7a holoenzyme¹⁴. This advancement has enabled structural and biophysical characterizations of this motor protein, leading to the discovery of several unique properties of human myosin-7a that are specifically adapted for mammalian auditory functions^14,23.

The MultiBac system is an advanced baculovirus/insect cell platform specifically designed for the expression of eukaryotic multimeric complexes^24,25. A key feature of this system is its ability to host multiple gene expression cassettes, each encoding a subunit of the complex, within a single MultiBac baculovirus. The assembly of the multigene expression cassettes is facilitated through a so-called multiplication module: a homing endonuclease (HE) site and a matching designed BstXI site flanking the multiple cloning sites (MCS). This module enables the iterative assembly of a single expression cassette by restriction/ligation, leveraging the fact that the HE and BstXI restriction sites are eliminated upon their ligation. In this paper, human myosin-7a heavy chain, RLC, calmodulin, and CALML4 are each cloned into the multiplication module within the pACEBac1 vector (Figure 1A), which are then assembled into a multigene expression cassette through the iterative process (Figure 1B). The myosin-7a multigene cassette is integrated into the baculoviral genome (bacmid) through the transposition of the mini-Tn7 element from the pACEBac1 vector to the mini-attTn7 target site in the genome (Figure 1C). Following procedures for bacmid purification, baculovirus production, and amplification (Figure 1D,E), the recombinant myosin-7a MultiBac baculovirus is prepared and can be used for large-scale protein production (Figure 1F). Additionally, the myosin-7a light chains can be produced separately in E. coli and purified using a cleavable His6-SUMO tag^26,27,28. The purified light chains are useful for studying their binding dynamics and regulation of myosin-7a.

The purified myosin-7a protein can be subjected to structural, biochemical, and biophysical studies to gain insights into the structural-functional regulation of this motor protein. Additionally, its interactions with the actin network and other binding proteins²⁹ can be examined using a variety of in vitro reconstitution approaches. Findings from these analyses will inform the biophysical properties of this myosin, leading to a mechanistic understanding of how myosin-7a drives the cytoskeletal changes and ultimately shapes the unique morphology and function of sensory cells. In this paper, we detail a workflow for actin filament gliding assay that has been specifically adapted for mammalian myosin-7a. Actin filament gliding assay is a robust in vitro motility assay that quantitatively studies the movement of fluorescent actin filaments propelled by a large number of myosin motors immobilized on a coverslip surface^30,31,32. The advantages of this assay include its simplicity of setup, minimal equipment requirements (a wide-field fluorescence microscope equipped with a digital camera), and high reproducibility. Additionally, because the motion of actin filaments is driven by a cluster of immobilized myosin motors, this assay is particularly useful for studying the motility of monomeric myosins such as myosin-7a^14,33. The protocols include several modifications, from experimental procedures to imaging analysis, specifically tailored to the unique motile properties of mammalian myosin-7a. With the availability of intact myosin-7a protein and the functional characterization protocol outlined here, this paper lays the groundwork for further investigation into the molecular roles of myosin-7a in both physiological and pathological processes.