The research focuses on the role of """"""""unconventional"""""""" myosins in vertebrate cells. Myosins are molecular motors that direct the events of cell motility that involve actin filaments, and are widely expressed in vertebrate cells. Since myosins are involved in actin-based motility, we have been studying the role of various classes of myosin in the fundamental processes of phagocytosis. In the vertebrate eye, cells that perform phagocytosis are known as retinal pigmented epithelial (RPE) cells. On a daily basis, the RPE engulf the shed tips of the rod photoreceptors. At least 3 of the 15 classes of myosins have recently been linked with the loss of vision and/or hearing impairment. Failure of RPE phagocytosis leads to photoreceptor degeneration and blindness. The myosins may play several important roles in the process of phagocytosis in conjunction with the actin cytoskeleton. These include, force generation required for membrane protrusion during the engulfment phase, the """"""""pinching off"""""""" of newly formed phagocytic compartments. and the cytoplasmic transport of phagosomal vesicles along actin filaments. The process of phagocytosis involves the rearrangement of the actin cytoskeleton which requires the increased turnover of actin filaments. The actin-binding protein, actin depolymerizing factor (ADF), has been shown to regulate actin filament dynamics in several important cellular processes which require rapid actin turnover. We are interested to determine if ADF plays a role in the regulation of actin filament dynamics during RPE phagocytosis. Interestingly, RPE cells are highly enriched in ADF. Work in progress and continuing throughout the year includes: 1. Biochemical, immunocytological and ultrastructural characterization of class specific myosins in RPE and retina using various vertebrate species including fish, chicken, rat, monkey and human, and characterization of two new fish myosin IV isoforms. 2. Establishment of primary cultures of newborn rat RPE cells and the rat RPE cell line, RPE-J, for use in transfection and quantitative phagocytosis studies. The phagocytosis assay involves feeding the cells fluorescently labelled rat rod outer segments (ROS) and subsequent quantitation by fluorescence activated cell sorting (FACS). 3. The identification of myosin subclasses associated with specific subcellular membrane compartments in vertebrate RPE tissues using subcellular membrane fractionation techniques. Newly formed phagocytotic vesicles will be purified using fluorescent membrane dyes and subsequent fluorescence activated organelle sorting (FAOS) allowing for the identification and characterization of specific phagosome-associated myosins. 4. Functional studies on the role of ADF in RPE cells using adenoviral transfection of RPE cell cultures, including real-time analysis by green fluorescent protein (GFP) and mutant ADF constructs.
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