Skip to main content Accessibility help
×
Home
Hostname: page-component-59b7f5684b-qn7h5 Total loading time: 0.341 Render date: 2022-09-26T14:16:38.894Z Has data issue: true Feature Flags: { "shouldUseShareProductTool": true, "shouldUseHypothesis": true, "isUnsiloEnabled": true, "useRatesEcommerce": false, "displayNetworkTab": true, "displayNetworkMapGraph": false, "useSa": true } hasContentIssue true

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*
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

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.

Type
Research Articles
Copyright
Copyright © Cambridge University Press 2014 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Abercrombie, M., Heaysman, J.E. & Pegrum, S.M. (1970). The locomotion of fibroblasts in culture III. Movements of particles on the dorsal surface of the leading lamella. Experimental Cell Research 62, 389398.CrossRefGoogle Scholar
Arey, L.B. (1915). The occurrence and the significance of photomechanical changes in the vertebrate retina—an historical survey. The Journal of Comparative Neurology 25, 535554.CrossRefGoogle Scholar
Back, I., Donner, K.O. & Reuter, T. (1965). The screening effect of the pigment epithelium on the retinal rods in the frog. Vision Research 5, 101111.CrossRefGoogle ScholarPubMed
Barsoum, I.B. & King-Smith, C. (2007). Myosin II and Rho kinase activity are required for melanosome aggregation in fish retinal pigment epithelial cells. Cell Motility and the Cytoskeleton 64, 868879.CrossRefGoogle ScholarPubMed
Basciano, P.A. & King-Smith, C. (2002). Actin-dependent, retrograde motility of surface-attached beads and aggregating pigment granules in dissociated teleost retinal pigment epithelial cells. Pigment Cell Research 15, 184191.CrossRefGoogle ScholarPubMed
Boldogh, I., Vojtov, N., Karmon, S. & Pon, L.A. (1998). Interaction between mitochondria and the actin cytoskeleton in budding yeast requires two integral mitochondrial outer membrane proteins, Mmm1p and Mdm10p. The Journal of Cell Biology 141, 13711381.CrossRefGoogle Scholar
Boldogh, I.R., Nowakowski, D.W., Yang, H.C., Chung, H., Karmon, S., Royes, P. & Pon, L.A. (2003). A protein complex containing Mdm10p, Mdm12p, and Mmm1p links mitochondrial membranes and DNA to the cytoskeleton-based segregation machinery. Molecular Biology of the Cell 14, 46184627.CrossRefGoogle ScholarPubMed
Breckler, J., Au, K., Cheng, J., Hasson, T. & Burnside, B. (2000). Novel myosin VI isoform is abundantly expressed in retina. Experimental Eye Research 70, 121134.CrossRefGoogle ScholarPubMed
Bruenner, U. & Burnside, B. (1986). Pigment granule migration in isolated cells of the teleost retinal pigment epithelium. Investigative Ophthalmology and Visual Science 27, 16341643.Google Scholar
Burnside, B., Adler, R. & O’Connor, P. (1983). Retinomotor pigment migration in the teleost retinal pigment epithelium. I. Roles for actin and microtubules in pigment granule transport and cone movement. Investigative Ophthalmology and Visual Science 24, 115.Google Scholar
Burnside, B. & Bost-Usinger, L. (1998). The RPE cytoskeleton. In The Retinal Pigment Epithelium: Current Aspects of Function and Disease, ed. Marmor, M.F. & Wolfensberger, T.J., pp. 4167. Oxford: Oxford University Press.Google Scholar
Burnside, B. & King-Smith, C. (2010). Comparative Eye: Fish Retinomotor Movements. In The Encyclopedia of the Eye, ed. Dartt, D., Besharse, B., Dana, R., Batelle, B., Amsterdam, The Netherlands: Elsevier.Google Scholar
Burnside, B. & Laties, A. (1979). Pigment movement and cellular contractility in the retinal pigment epithelium. In The Retinal Pigment Epithelium, ed. Zinn, K.M. & Marmor, M.F., pp. 175191. Cambridge, MA: Harvard University Press.Google ScholarPubMed
Burnside, B. & Laties, A.M. (1976). Actin filaments in apical projections of the primate pigmented epithelial cell. Investigative ophthalmology 15, 570575.Google ScholarPubMed
Burnside, B. & Nagle, B. (1983). Retinomotor movements of the photoreceptors and retinal pigment epithelium: Mechanisms and regulation. In Progress in Retinal Research, ed. Osborne, N. & Chader, G., pp. 67109. New York: Pergamon Press.Google Scholar
Canman, J.C. & Bement, W.M. (1997). Microtubules suppress actomyosin-based cortical flow in Xenopus oocytes. Journal of Cell Science 110 (Pt 16), 19071917.Google ScholarPubMed
Cramer, L.P. (1997). Molecular mechanism of actin-dependent retrograde flow in lamellipodia of motile cells. Frontiers in Bioscience 2, d260270.CrossRefGoogle ScholarPubMed
El-Amraoui, A., Schonn, J.S., Kussel-Andermann, P., Blanchard, S., Desnos, C., Henry, J.P., Wolfrum, U., Darchen, F. & Petit, C. (2002). MyRIP, a novel Rab effector, enables myosin VIIa recruitment to retinal melanosomes. EMBO Reports 3, 463470.CrossRefGoogle ScholarPubMed
Ernest, S., Rauch, G.J., Haffter, P., Geisler, R., Petit, C. & Nicolson, T. (2004). Mariner is defective in myosin VIIA: A zebrafish model for human hereditary deafness. Human Molecular Genetics 9, 21892196.CrossRefGoogle ScholarPubMed
Fehrenbacher, K.L., Yang, H.C., Gay, A.C., Huckaba, T.M. & Pon, L.A. (2004). Live cell imaging of mitochondrial movement along actin cables in budding yeast. Current Biology 14, 19962004.CrossRefGoogle Scholar
Forscher, P. & Smith, S.J. (1990). Cytoplasmic actin filaments move particles on the surface of a neuronal growth cone. In Optical Microscopy for Biology, ed. Herman, B. & Jacobson, K., pp. 459471. New York: Wiley-Liss, Inc. Google Scholar
Futter, C.E., Ramalho, J.S., Jaissle, G.B., Seeliger, M.W. & Seabra, M.C. (2004). The role of Rab27a in the regulation of melanosome distribution within retinal pigment epithelial cells. Molecular Biology of the Cell 15, 22642275.CrossRefGoogle ScholarPubMed
Garcia, D.M. & Burnside, B. (1994). Suppression of cAMP-induced pigment granule aggregation in RPE by organic anion transport inhibitors. Investigative Ophthalmology & Visual Science 35, 178188.Google ScholarPubMed
Gibbs, D., Azarian, S.M., Lillo, C., Kitamoto, J., Klomp, A.E., Steel, K.P., Libby, R.T. & Williams, D.S. (2004). Role of myosin VIIa and Rab27a in the motility and localization of RPE melanosomes. Journal of Cell Science 117, 64736483.CrossRefGoogle ScholarPubMed
Huckaba, T.M., Lipkin, T. & Pon, L.A. (2006). Roles of type II myosin and a tropomyosin isoform in retrograde actin flow in budding yeast. The Journal of Cell Biology 175, 957969.CrossRefGoogle Scholar
King-Smith, C. (2009). Melanosome motility in fish retinal pigment epithelial cells. Methods in Molecular Biology 586, 275282.CrossRefGoogle ScholarPubMed
King-Smith, C., Paz, P., Lee, C.W., Lam, W. & Burnside, B. (1997). Bidirectional pigment granule migration in isolated retinal pigment epithelial cells requires actin but not microtubules. Cell Motility and the Cytoskeleton 38, 229249.3.0.CO;2-0>CrossRefGoogle Scholar
Kovar, D.R. (2007). Intracellular motility: Myosin and tropomyosin in actin cable flow. Current Biology 17, R244247.CrossRefGoogle ScholarPubMed
Lin-Jones, J., Sohlberg, L., Dose, A., Breckler, J., Hillman, D.W. & Burnside, B. (2009). Identification and localization of myosin superfamily members in fish retina and retinal pigmented epithelium. The Journal of Comparative Neurology 513, 209223.CrossRefGoogle ScholarPubMed
Liu, X., Ondek, B. & Williams, D.S. (1998). Mutant myosin VIIa causes defective melanosome distribution in the RPE of shaker-1 mice. Nature Genetics 19, 117118.CrossRefGoogle Scholar
McNeil, E.L., Tacelosky, D., Basciano, P., Biallas, B., Williams, R., Damiani, P., Deacon, S., Fox, C., Stewart, B., Petruzzi, N., Osborn, C., Klinger, K., Sellers, J.R. & Smith, C.K. (2004). Actin-dependent motility of melanosomes from fish retinal pigment epithelial (RPE) cells investigated using in vitro motility assays. Cell Motility and the Cytoskeleton 58, 7182.CrossRefGoogle ScholarPubMed
Medeiros, N.A., Burnette, D.T. & Forscher, P. (2006). Myosin II functions in actin-bundle turnover in neuronal growth cones. Nature Cell Biology 8, 215226.CrossRefGoogle Scholar
Nguyen-Legros, J. (1978). Fine structure of the pigment epithelium in the vertebrate retina. International Review of Cytology. Supplement 287328.Google ScholarPubMed
Owaribe, K., Kartenbeck, J., Rungger-Brandle, E. & Franke, W.W. (1988). Cytoskeletons of retinal pigment epithelial cells: Interspecies differences of expression patterns indicate independence of cell function from the specific complement of cytoskeletal proteins. Cell and Tissue Research 254, 301315.CrossRefGoogle ScholarPubMed
Peraza-Reyes, L., Crider, D.G. & Pon, L.A. (2010). Mitochondrial manoeuvres: Latest insights and hypotheses on mitochondrial partitioning during mitosis in Saccharomyces cerevisiae. BioEssays 32, 10401049.CrossRefGoogle ScholarPubMed
Perkins, B.D., Matsui, J.I., Murphy, M.K. & Dowling, J.E. (2004). Histological and physiological abnormalities in myosin VIIA mutant zebrafish. Investigative Ophthalmology and Visual Science 45, 3591 Google Scholar
Ponti, A., Machacek, M., Gupton, S.L., Waterman-Storer, C.M. & Danuser, G. (2004). Two distinct actin networks drive the protrusion of migrating cells. Science 305, 17821786.CrossRefGoogle ScholarPubMed
Rosenblatt, J., Cramer, L.P., Baum, B. & McGee, K.M. (2004). Myosin II-dependent cortical movement is required for centrosome separation and positioning during mitotic spindle assembly. Cell 117, 361372.CrossRefGoogle ScholarPubMed
Steinberg, R.H. & Miller, S.S. (1979). Transport and membrane properties of the retinal pigment epithelium. In The Retinal Pigment Epithelium, ed. Zinn, K.M. & Marmor, M.F., pp. 205225. Cambridge, MA: Harvard University Press.Google ScholarPubMed
Svitkina, T.M. & Borisy, G.G. (1998). Correlative light and electron microscopy of the cytoskeleton of cultured cells. Methods in Enzymology 298, 570592.CrossRefGoogle ScholarPubMed
Theriot, J.A. & Mitchison, T.J. (1992). Comparison of actin and cell surface dynamics in motile fibroblasts. The Journal of Cell Biology 119, 367377.CrossRefGoogle ScholarPubMed
Travis, G.H., Golczak, M., Moise, A.R. & Palczewski, K. (2007). Diseases caused by defects in the visual cycle: Retinoids as potential therapeutic agents. Annual Review of Pharmacology and Toxicology 47, 469512.CrossRefGoogle ScholarPubMed
Troutt, L.L. & Burnside, B. (1989). Role of microtubules in pigment granule migration in teleost retinal pigment epithelial cells. Experimental Eye Research 48, 433443.CrossRefGoogle Scholar
Verkhovsky, A.B., Svitkina, T.M. & Borisy, G.G. (1997). Polarity sorting of actin filaments in cytochalasin-treated fibroblasts. Journal of Cell Science 110 (Pt 15), 16931704.Google ScholarPubMed

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.

Download King-Smith Supplementary Material(Video)
Video 129 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.

Download King-Smith Supplementary Material(Video)
Video 197 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 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.

Download King-Smith Supplementary Material(Video)
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

Download King-Smith Supplementary Material(Video)
Video 5 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.

Download King-Smith Supplementary Material(Video)
Video 12 MB
8
Cited by

Save article to Kindle

To save this article to your Kindle, first ensure coreplatform@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about saving to your Kindle.

Note you can select to save to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be saved to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

Find out more about the Kindle Personal Document Service.

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

Save article to Dropbox

To save this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you used this feature, you will be asked to authorise Cambridge Core to connect with your Dropbox account. Find out more about saving content to Dropbox.

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

Save article to Google Drive

To save this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you used this feature, you will be asked to authorise Cambridge Core to connect with your Google Drive account. Find out more about saving content to Google Drive.

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

Reply to: Submit a response

Please enter your response.

Your details

Please enter a valid email address.

Conflicting interests

Do you have any conflicting interests? *