Developing methodologies for exploring myosin-5 elasticity under strain

Motor proteins drive the movement of organelles and other types of cargo inside every eukaryotic cell type. These motors include myosins, kinesins and dyneins. Myosin-5a, a ubiquitous motor protein, has been intensively studied and its enzymatic properties and cellular functions have been described in great detail. This dimeric molecule walks processively along actin filaments, enabling it to transport a variety of cargos including mRNA, pigment granules, organelles such as the endoplasmic reticulum and endocytic vesicles. Cargos bind to the C-terminal globular tail domain of the molecule, while the N-terminal motor domain hydrolyses ATP to take regular steps on the actin filament. The structure and enzymatic properties of myosin-5 moving processively along actin under unloaded conditions have already been well characterised. However, while pulling a cargo through the viscoelastic cytoplasm, the molecule will experience variable forces. Therefore it is important to study how these forces alter the structure and kinetic behaviour of myosin-5. The purpose of this study was to design myosin-5 constructs that could be tethered, in a controlled manner, to actin via sequences introduced into the tail region of the molecule. The tethered motor protein is expected to ’stall’ while moving along the actin filament. In this way, the effects of strain on the myosin-5 structure and kinetics could then be investigated. This would simulate a case where inside the cell myosin-5 stalls due to strain, for example, whilst trying to move its cargo through spatially restricted areas of the actin cytoskeleton. To find a new way to attach the tail of myosin-5 to actin, small, artificial actin-binding proteins named Adhirons were raised with phage display assay and their properties were explored. They bind to actin with high affinity and they are also useful for staining actin in cells or for attaching actin to coverslips in various motility assays. Multiple myosin-5 constructs were explored, that had either an Avi-tag or an actin-binding Adhiron at their C-terminal ends. A construct that contained the full predicted coiled-coil motif of myosin-5, could not be attached to the same actin filament via its tail as the motors were bound to. Two further constructs that have an artificial long coiled-coil tail, were cloned and expression trials are currently in progress.