Expression Studies of Other Unconventional Myosins

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Myosin 7a is an actin based motor protein essential for vision and hearing. Mutations of myosin 7a cause Usher syndrome type 1, the most common and severe form of deaf blindness in humans. The molecular mechanisms that governs its mechanochemistry remain poorly understood, primarily due to the difficulty of purifying stable, intact protein. Here, we recombinantly produce the complete human myosin 7a holoenzyme in insect cells and characterize its biochemical and motile properties. Unlike the Drosophila ortholog which primarily associates with calmodulin, we found that human myosin 7a utilizes a unique combination of light chains including regulatory light chain, calmodulin, and calmodulin like protein 4 (CALML4). Our results further reveal that CALML4 does not function as a Ca2+ sensor but plays a crucial role in maintaining the lever arms structural functional integrity. Using our recombinant protein system, we purified two myosin 7a splicing isoforms which have been shown to be differentially expressed along the cochlear tonotopic axis. We show that they possess distinct mechano enzymatic properties despite differing by only 11 amino acids at their N termini. Using single molecule in vitro motility assays, we demonstrate that human myosin 7a exists as an autoinhibited monomer and can move processively along actin when artificially dimerized or bound to cargo adaptor proteins such as MyRIP. These results suggest that myosin 7a can serve multiple roles in the sensory systems such as a transporter or an anchor/force sensor. Furthermore, our research highlights that human myosin 7a has evolved unique regulatory elements that enable precise tuning of its mechanical properties suitable for mammalian auditory functions. In collaboration with John Hammer we have begun to study myosin 19, a mitochondrial associated myosin to complement ongoing cell biological studies. We have expressed an fragment containing the motor domain and a single IQ motif with binds a light chain. In addition we have created a mutant myosin19 which was designed to eliminate the actin-activated enzymatic activity of the protein as a control for ongoing in vivo studies in the Hammer lab. Enzymatic studies in our lab has indicated that the mutant is indeed inactive. We are using minflux microscopy to study the movement of myosin 6 on actin filaments with very high spatial and temporal resolution. We are purifying both full length and a truncated motor only version of myosin 10. The full length protein will be used to study its regulation and will be used in assays to reconstitute elements of filopodial formation. The truncated version will be used to screen for inhibitory small molecules. In collaboration with Kathleen Trybus of the University of Vermont, we have expressed a myosin 14 paralog, termed PfMyoA, from Plasmodium falciparus, the malaria parasite. This myosin has been shown to be essential for the infection of RBC by the parasite through a mechanism that involved two membrane proteins, GAP40 and GAP45 and another protein called GAP45 that binds the myosin and GAP50. Together, this complex is called the glidiosome which links the membrane to actin filaments. We would like to physically reconstitute the glidiosome, in vitro. To this end we first compare the ability of PfMyoA to move actin filaments in solution when the myosin is either bound to the glass surface or bound to a supported lipid bilayer. There is little difference in the rate of actin sliding between these modes of myosin attachment. Now we have purified the other proteins of the glidiosome and are in the process of measuring their relative affinity.