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Orientation of actin filaments in teleost retinal pigment epithelial cells, and the effect of the lectin, Concanavalin A, on melanosome motility

Published online by Cambridge University Press:  15 January 2014

CHRISTINA KING-SMITH ,
RONALD J. VAGNOZZI ,
NICOLE E. FISCHER ,
PATRICK GANNON  and
SATYA GUNNAM
CHRISTINA KING-SMITH*
Affiliation:
Department of Biology, Saint Joseph’s University, Philadelphia, Pennsylvania 19131
RONALD J. VAGNOZZI
Affiliation:
Department of Biology, Saint Joseph’s University, Philadelphia, Pennsylvania 19131
NICOLE E. FISCHER
Affiliation:
Department of Biology, Saint Joseph’s University, Philadelphia, Pennsylvania 19131
PATRICK GANNON
Affiliation:
Department of Biology, Saint Joseph’s University, Philadelphia, Pennsylvania 19131
SATYA GUNNAM
Affiliation:
Department of Biology, Saint Joseph’s University, Philadelphia, Pennsylvania 19131
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Abstract

Retinal pigment epithelial cells of teleosts contain numerous melanosomes (pigment granules) that exhibit light-dependent motility. In light, melanosomes disperse out of the retinal pigment epithelium (RPE) cell body (CB) into long apical projections that interdigitate with rod photoreceptors, thus shielding the photoreceptors from bleaching. In darkness, melanosomes aggregate through the apical projections back into the CB. Previous research has demonstrated that melanosome motility in the RPE CB requires microtubules, but in the RPE apical projections, actin filaments are necessary and sufficient for motility. We used myosin S1 labeling and platinum replica shadowing of dissociated RPE cells to determine actin filament polarity in apical projections. Actin filament bundles within RPE apical projections are uniformly oriented with barbed ends toward the distal tips. Treatment of RPE cells with the tetravalent lectin, Concanavalin A, which has been shown to suppress cortical actin flow by crosslinking of cell-surface proteins, inhibited melanosome aggregation and stimulated ectopic filopodia formation but did not block melanosome dispersion. The polarity orientation of F-actin in apical projections suggests that a barbed-end directed myosin motor could effect dispersion of melanosomes from the CB into apical projections. Inhibition of aggregation, but not dispersion, by ConA confirms that different actin-dependent mechanisms control these two processes and suggests that melanosome aggregation is sensitive to treatments previously shown to disrupt actin cortical flow.

Keywords

retinal pigment epitheliumactin filamentsmelanosome transport
Type
Research Articles
Information
Visual Neuroscience , Volume 31 , Issue 1 , January 2014 , pp. 1 - 10
Copyright
Copyright © Cambridge University Press 2014 

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King-Smith Supplementary Material

Phase contrast, time-lapse video of dissociated RPE cell triggered to aggregate melanosomes. Images were collected every 10 s. Perfusion with 1 mM cAMP in HERB began at ca. 290 s, triggering melanosome aggregation.

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Video 129.5 MB

King-Smith Supplementary Material

Time-lapse video of a dissociated RPE cell pretreated with 100 μg/ml ConA in HERB/dopamine, triggered to aggregate using 1 mM cAMP at ca. 200–250 s (cell went out of focus and moved off center briefl y). Images were collected every 10 s. Melanosomes continue to oscillate bidirectionally (shuttling) but fail to aggregate.

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Video 197.1 MB

King-Smith Supplementary Material

Time-lapse video of RPE sheets in situ, placed in cAMP immediately prior to starting the video. Images were collected every 10 s. Melanosomes are visible as phase-bright areas within apical projections extending into the upper part of the fi eld; they migrate toward the basal side of the cell at the bottom of the fi eld. Empty apical projections devoid of melanosomes appear as phase-gray hair-like structures.

Download King-Smith Supplementary Material(Video)
Video 17.3 MB

King-Smith Supplementary Material

Time-lapse video of RPE sheets in situ, after preincubation in 100 μg/ml ConA in HERB/dopamine, then placed in cAMP/ConA immediately prior to starting the video. Basal side is on the left; apical projections extend to the right. Some movement of melanosomes toward the basal side of the cell is visible within projections, but net aggregation is blocked. Images were collected every 10 s.

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Video 17 MB

King-Smith Supplementary Material

Time-lapse video of a dissociated RPE cell with attached latex bead, starting in the lower right. The cell was preincubated in cAMP to aggregate melanosomes, so beads would be easier to visualize. Images were collected every 10 s; the rate of bead motility: 2.06 μm/min

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Video 4.8 MB

King-Smith Supplementary Material

Time-lapse video of a dissociated RPE cell pretreated in 100 μg/ml ConA in cAMP. The bead moves from upper right to lower left at a rate of 0.57 μm/min. (Several beads attached to the substrate remain stationary in the upper right). A few melanosomes from lysed cells that attached to the cell surface are also moving toward the CB. A melanosome that appears to be within a projection (phase gray area that is partly out of focus) at 12:00 also moves in toward the CB. Images were collected every 10 s.

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Video 12.3 MB