Localization of the Unconventional Myosin, Myo3A, to Photoreceptor Calycal Processes Requires an Active Motor Domain and a Highly Conserved Terminal Tail Domain

Abstract

Abstract: : Purpose: The class III myosin Myo3A is preferentially expressed in vertebrate retina and has been localized to the calycal processes in fish photoreceptors. While the functions of neither Myo3A nor calycal processes are understood, the localization of Myo3A to a specific population of actin filaments in photoreceptor calycal processes suggests that it may play an important role in photoreceptor function at this location. Methods: In order to understand how Myo3A is targeted to this specific region of photoreceptors, we have expressed Myo3A or various fragments of Myo3A fused to enhanced green fluorescent protein (EGFP) in both HeLa cells and Xenopus laevis tadpoles. Stable transgenic expression in frogs is driven by the Xenopus rod opsin promoter that restricts expression to rod photoreceptors. Myo3A protein consists of kinase, motor, neck and tail domains. Results: Expression of full-length EGFP:Myo3A fusion causes an accumulation of fluorescence in the distal portion of actin bundles in cell extensions of HeLa cells and photoreceptor inner segments. A point mutation in the motor domain active site produces diffuse cytoplasmic localization of the Myo3A fusion protein in HeLa cells, indicating that motor activity is required for the distal localization. A deletion of the tail domain from the Myo3A fusion transgene produces a similar diffuse cytoplasmic localization indicating that the tail domain is also required for distal localization. Expression of the tail domain alone shows fluorescence associated with actin stress fibers in HeLa cells and with calycal process actin filament bundles in photoreceptors. Deletion analysis of this tail region indicates that the C-terminal 20 amino acid domain, which is highly conserved between fish and human myosin 3A, is required for colocalization with actin bundles. Conclusions: These results indicate that the localization of Myo3A to the distal ends of actin bundles in HeLa cells and in photoreceptor inner segments requires both an active motor domain and the presence of the conserved terminal tail domain.

Keywords: 517 photoreceptors • 383 cytoskeleton • 476 molecular biology