A role for myosin VI in retinal pigment epithelium phagocytosis

https://doi.org/10.1016/j.bbrc.2018.09.006Get rights and content

Highlights

  • Quashing myosin VI activity reduces rates of phagosome trafficking in RPE cells.

  • Trafficked phagosomes in RPE cells undergo two modes of motion.

  • The more randomly directed mode of phagosome trafficking is myosin VI dependent.

  • The more directed mode of phagosome trafficking is myosin VI independent.

Abstract

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells adjacent to the choroid coat, the vascular layer of the eye. Among other functions, these cells are responsible for the phagocytosis of rod and cone cell waste shed from the photoreceptor outer segments. We describe here studies to understand the involvement of the motor protein myosin VI in the trafficking of internalized microspheres by a human retinal pigment epithelium primary cell line (ARPE-19). We perturbed the myosin VI-actin interaction by overexpressing a dominant negative myosin VI construct in these cells. We used single particle tracking to characterize the trajectories of internalized fluorescent microspheres. Analysis of the speed of the microspheres' motions revealed that the magnitude of the average speed over short time scales is reduced in the presence of the dominant negative motor. Analysis of the mean-squared displacement of these trajectories demonstrated that trafficking of phagosomes involves two modes of motion, a rapid, more randomly directed motion that is myosin VI dependent and a slower, more directed motion that is independent of the motor. From these data, we posit that this trafficking process involves an interplay between myosin VI motors, which are involved in motion through the actin periphery of the cell, and microtubule motors.

Introduction

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells adjacent to the choroid coat, the vascular layer of the eye, which plays a critical role in the maintenance of normal vision. One of the roles of RPE cells is to protect the retina from photoreceptor outer segment (POS) waste that is shed daily by rod and cone cells. POS waste contains damaged pigments and free radicals that are harmful to the retina [1]. Through a daily cycle of phagocytosis, the RPE engulfs and degrades this waste [2]. RPE cells are able to phagocytose more material over their lifetime than any other cell in the body given their supervision over numerous photoreceptors as well as their postmitotic state [3]. RPE cells thus help protect the retina from the toxic effects of accumulated photo-oxidative products, and a breakdown in this phagocytic process leads to retinal degeneration in rats [4] and is thought to be associated with degenerative retinal diseases such as age-related macular degeneration [5].

One particular cytoskeletal motor, myosin VI, is a likely candidate for involvement in trafficking of internalized phagosomes into RPE cells. Myosins comprise a superfamily of motor proteins that move along the filamentous protein actin using energy from ATP hydrolysis. They are composed of three domains. The N-terminal motor domain contains the actin-binding and nucleotide-binding sites. Following the motor are one or more light-chain binding domains in the neck region, followed by the C-terminal tail domain. The tail consists of domains that serve a variety of functions such as allowing for interactions with other components of the cell and facilitating self-association of the myosin to form dimers or higher-order oligomers [6]. Within the myosin superfamily, different classes are involved in a variety of processes including intracellular trafficking, cell motility, cytokinesis, and muscle contraction [7].

Using a cultured human RPE cell line (ARPE-19) we examined the role of myosin VI in non-specific (non-receptor mediated) phagocytosis. We hypothesized that myosin VI is involved in trafficking of internalized phagosomes due to the orientation of actin in the periphery of many cells; the plus ends of actin filaments typically point towards the plasma membrane while the minus end points toward the cell interior [8]. Because myosin VI is the only myosin known to move toward the minus end of actin [9], it is well suited for trafficking phagosomes into the cell.

It has been previously demonstrated that myosin VI plays a role in translocation of transferrin-mediated endocytic vesicles through the periphery of ARPE-19 cells, a process that takes on the order of tens of minutes due to the large actin network [10]. We thus hypothesized that this is an ideal cell line for focusing on actomyosin interactions in RPE phagocytosis. Trafficking of internalized fluorescent microspheres was observed in control cells and in cells in which we perturbed the actomyosin interaction by quashing myosin VI motor activity. Our results support a model in which phagosomes are trafficked into the cell via an interplay of myosin VI-dependent and myosin VI-independent processes.

Section snippets

Expression plasmids

The expression plasmid containing our myosin VI construct is described in detail in Refs. [11,12]. The plasmid contains the sequence for a C-terminal fragment of human myosin VI that is capable of binding to cargo, but which lacks the motor domain and is thus unable to generate force or motion (M6-Tail). This construct has been inserted into the pEGFP-C3 expression plasmid (Clontech, Mountain View, CA), resulting in a myosin VI-construct with an N-terminal green fluorescent protein tag

Results and discussion

To elucidate the role of myosin VI in trafficking of phagosomes in ARPE-19 cells, we allowed ARPE-19 cells to internalize micron-diameter fluorescently-tagged polystyrene beads. A phase-contrast image of a cell with an internalized bead in its periphery was captured, followed by the collection of a ten- or 30-min movie consisting of fluorescent images of the bead collected at 2 Hz. Finally, a second phase-contrast image of the cell was captured. A false-color composite of the two phase-contrast

Acknowledgments

We would like to thank Rebekah Daniel and Bianca Nagata for their work developing cell culture protocols and Ellen Rumley and Dylan Tooley for valuable discussions and insights. This work was supported by the M.J. Murdock Charitable Trust and Willamette University's Summer Collaborative Research Program (SCRP) program.

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