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Warchol, Ewa (2016): Expression, purification and characterization of human myosin 9a. Dissertation, LMU München: Medizinische Fakultät



Myosins form a large family of actin-based motor proteins that are involved in different forms of cellular motility. Those include muscle contraction, intracellular transport, endo- and exocytosis and cell division and locomotion. Certain myosin isoforms are also involved in hearing and vision processes. So far, very little is known about the myosin class 9. Myosin 9 has been proposed to be involved in cell differentiation and morphology. Mammals express two class 9 myosins; myosin 9a is expressed in brain and its loss results in the formation of hydrocephalus, while myosin 9b appears to be involved in the directional movement of dendritic cells. On the structural level, myosin 9 contains a RhoGAP domain in its tail region, indicating it could link its mechanical activity along actin filaments to intracellular protein G signaling. So far, only myosin 9b orthologs have been investigated. Here, I used the baculovirus/insect cell system to express recombinant human myosin 9a constructs, optimized the purification procedure and examined the function of myosin 9a motor in vitro. Designed constructs contained the motor and neck domains of myosin 9a, fused to different N- and C-terminal tags to facilitate purification. I addressed the ATPase activity of myosin 9a (particularly the Minimal Motor Domain construct) in the absence and presence of F-actin by means of an NADH enzyme-linked assay. The mechanical activity of myosin 9a was addressed by means of the gliding filament assay. The velocity of actin gliding over the lawn of surface-bound myosin 9a molecules was largely independent from the motor density on the surface of a flow cell. In summary, this work presents the first successful purification of recombinant human myosin 9a motor constructs and describes basic mechanochemical properties of human myosin 9a motor. The results presented in this work provide framework for further investigation of myosin 9a properties, including investigation of the regulatory role of the tail domain of myosin 9a.