Molecular motors have key roles in virtually all cell biology processes. The precise, dynamic organization of cells and tissues depends crucially on these marvellous molecular machines. It is no wonder then that practically every cell has nearly 100 different molecular motors to carry out specific pivotal tasks. And, given the unique demands on the biochemical and biophysical properties of molecular motors to carry out their functions, they have given us a great deal of information about how enzymes work in general.
One such family of molecular motors is the myosin family. Myosins are ATPases that use the chemical energy derived from ATP hydrolysis to produce mechanical work. In 1969, H.E. Huxley1
proposed the swinging crossbridge hypothesis to explain how this energy transduction might occur. According to current versions of this model, following ATP hydrolysis the myosin head domain (the crossbridge between the thick filament of myosin molecules and the thin actin filaments) undergoes a conformational transition into a pre-stroke state. On rebinding to actin and releasing phosphate, the ADP-bound myosin head undergoes a transition from a weak to a strong actin-binding state, which is accompanied by a reverse conformational change to a post-stroke state, resulting in a sliding motion at the actin–myosin interface. The myosin remains tightly bound to actin until ADP is released, at which point ATP rapidly binds and causes release of the myosin from actin. The fraction of the ATPase cycle time that a myosin spends strongly bound to an actin filament is known as the duty ratio. The strongly bound state time determines the maximum velocity of relative movement of myosin along an actin filament. The head cannot move forwards any faster than it can let go.
Crystal structures of several myosins2–5
reveal that they are all composed of a catalytic head that binds to actin and nucleotides, with a converter domain distal to the actin-binding site (). The first myosin crystal structure identified a light chain-binding domain, which extends out from the myosin catalytic domain like a lever arm6
. It has been assumed that for all myosins this light chain-binding region serves as a lever arm to amplify movements of the converter domain, which transitions between pre-stroke and post-stroke configurations (; light chains not shown). This transition is proposed to provide a mechanical stroke in a particular direction along the actin filament, which is a polar structure with barbed (also known as plus) and pointed (also known as minus) ends. Thus, the swinging crossbridge hypothesis was renamed the swinging lever arm hypothesis. What follows the light chain-binding domain varies between different myosins. Some myosins have a coiled-coil domain in this region, which is responsible for dimerization. Many have a cargo-binding domain at their carboxy terminus, which associates with specific cargo-binding proteins.
The swinging lever arm hypothesis
There are ~ 40 different myosin genes in higher eukaryotes7
, and each myosin carries out its own special functions in vivo
. Muscle myosin (of the myosin II class), for example, is highly specialized to provide appropriate forces and velocities during muscle contraction8
. A primary function of non-muscle myosin II is to drive cytokinesis9
. Myosin I, myosin VI and myosin VII are involved in the function of the inner ear, and mutations in them result in deafness10
. Myosin V and myosin VI are involved in membrane trafficking and other aspects of cell organization. How all the different myosins are coordinated to carry out their various specialized tasks is an emerging field of research.
A particularly interesting member of the myosin family is myosin VI. Hasson et al
identified mammalian myosin VI in a kidney proximal tubule cell line, in which it is localized to an apical actin-rich structure in epithelial cells called the brush border. The Drosophila melanogaster
equivalent of myosin VI, 95F
, had been implicated in the transport of cytosplasmic organelles12,13
. Myosin VI is involved in various functions involving multiple cellular organelles (). For example, myosin VI localizes at the base of stereociliary bundles of hair cells (specialized filipodia) of the inner ear and is essential for their structural integrity14
. Myosin VI at the cell periphery also has a role in border cell migration during development15,16
and in cancer metastasis17
. It is also involved in the transfer of endocytic vesicles from clathrin-coated pits to endosomes, along the cortical filamentous actin network. This includes the trafficking of rhodamine-labelled transferrin18,19
, cystic fibrosis transmembrane conductance regulator (CFTR
, lemur tyrosine kinase 2 (LMTK2
; also known as BREK)21,22
and α5β1 integrin23
. Furthermore, myosin VI is recruited to the Golgi complex by optineurin24
, where it is essential for the maintenance of normal Golgi morphology25
. How myosin VI carries out these diverse functions is unclear, although we do know some of the interacting proteins. These include disabled homologue 2 (DAB2
), synapse-associated protein 97 (SAP97
; also known as DLG1)29
, which have been found to target myosin VI to different cellular compartments.
Schematic of myosin VI structure and some of its cellular functions
Similar to other myosins, myosin VI comprises a catalytic head with a converter domain, followed by light chain-binding domains (). The catalytic head is similar to that of myosin II and myosin V, with the exception of two inserts. One insert is near the nucleotide-binding pocket, which changes myosin VI’s nucleotide kinetics relative to other myosins4
, and the second is an insert of 53 amino acids between the converter domain of the catalytic head and a canonical calmodulin-binding IQ motif11,30
. This sequence is known as the unique insert (), and its C-terminal segment binds a single calmodulin31
. The tail sequence beyond the calmodulin-binding region consists of the proximal tail, the medial tail, which is an unusual single α-helix (see below), and the cargo-binding domain4,11,32
Schematic of the myosin VI lever arm and tail domains
When the primary sequence of myosin VI was first analysed, a major portion of its tail region seemed to be a coiled coil, similar to the tails of myosin II and myosin V. Because this motif is found in proteins that dimerize in vivo
, it was assumed that this motor was a dimer. Nearly all in vitro
studies have used a myosin VI construct truncated at Arg992 in the tail domain (near the end of the predicted coiled-coil region), followed by a strong coiled coil — the leucine zipper GCN4 motif — to ensure that the dimer does not dissociate at the low concentrations used for single molecule assays33
. The ~ 300 residues at the C terminus that constitute the cargo-binding domain are absent in this myosin VI construct. We refer to this construct as an artificial dimer, as it was meant to be a substitute for a presumed native dimeric molecule involving interactions of the cargo-binding domain with its appropriate cargo. This artificial dimer has been useful to probe details of myosin VI behaviour; however, it is still unclear whether myosin VI functions as a dimer, a monomer or both in vivo
Myosin VI moves along an actin filament in the opposite direction to all other myosins that have been characterized30
. Myosin I34
, myosin II35
, myosin V36
, myosin X37
and myosin XI38
are directed towards the barbed ends of actin filaments. A truncated form of myosin IX was reported to move towards the pointed ends of actin filaments39
, but the native myosin IX molecule was subsequently shown to be a barbed end-directed motor40
. Myosin VI moves towards the pointed ends of actin filaments, and this ability enables it to carry out specific functions in vivo
. For example, the brush border of epithelial cells is polarized with the pointed ends of the actin filaments directed away from the plasma membrane41
. This is consistent with roles for myosin VI in endocytosis42
and myosin V in exocytosis43
. Similarly, actin bundles in stereocilia are polarized such that myosin VI moves towards their base, thereby increasing membrane tension, which aids in stabilizing and maintaining appropriate tension in the stereocilia.
In addition to having reverse directionality, the artificial dimer of myosin VI has a larger step length on actin filaments than would be predicted from the early interpretations of the structure of the native protein, on the basis of current models of myosin movement44,45
. These observations presented a serious challenge to the swinging lever arm hypothesis. In this Review, we discuss the results from in vitro
motility assays, single molecule analysis, X-ray crystallography and other biophysical approaches, which proved pivotal in elucidating the unexpected, unique features of myosin VI with respect to its movement on actin filaments.