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Protein kinase A regulation of pigment granule motility in retinal pigment epithelial cells from fish, Lepomis spp.

Published online by Cambridge University Press:  15 September 2021

Nicole E. Leitner  and
Christina King-Smith Open the ORCID record for Christina King-Smith [Opens in a new window]
Nicole E. Leitner
Affiliation:
Department of Biology, Saint Joseph’s University, Philadelphia, Pennsylvania, USA
Christina King-Smith*
Affiliation:
Department of Biology, Saint Joseph’s University, Philadelphia, Pennsylvania, USA
*
*Corresponding author: Christina King-Smith, email: csmith@sju.edu
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Abstract

Retinomotor movements include elongation and contraction of rod and cone photoreceptors, and mass migration of melanin-containing pigment granules (melanosomes) of the retinal pigment epithelium (RPE) within the eyes of fish, frogs, and other lower vertebrates. Eyes of these animals do not contain dilatable pupils; therefore the repositioning of the rods and cones and a moveable curtain of pigment granules serve to modulate light intensity within the eye. RPE from sunfish (Lepomis spp.) can be isolated from the eye and dissociated into single cells, allowing in vitro studies of the cytoskeletal and regulatory mechanisms of organelle movement. Pigment granule aggregation from distal tips of apical projections into the cell body can be triggered by the application of underivatized cAMP, and dispersion is effected by cAMP washout in the presence of dopamine. While the phenomenon of cAMP-dependent pigment granule aggregation in isolated RPE was described many years ago, whether cAMP acts through the canonical cAMP-PKA pathway to stimulate motility has never been demonstrated. Here, we show that pharmacological inhibition of PKA blocks pigment granule aggregation, and microinjection of protein kinase A catalytic subunit triggers pigment granule aggregation. Treatment with a cAMP agonist that activates the Rap GEF, Epac (Effector protein activated by cAMP), had no effect on pigment granule position. Taken together, these results confirm that cAMP activates RPE pigment granule motility by the canonical cAMP-PKA pathway. Isolated RPE cells labeled with antibodies against PKA RIIα and against PKA-phosphorylated serine/threonine amino acids show diffuse, punctate labeling throughout the RPE cell body and apical projections. Immunoblotting of RPE lysates using the anti-PKA substrate antibody demonstrated seven prominent bands; two bands in particular at 27 and 64 kD showed increased levels of phosphorylation in the presence of cAMP, indicating their phosphorylation could contribute to the pigment granule aggregation mechanism.

Keywords

RPEPKAmelanosomesretinomotor movementsactin-dependent organelle motility
Type
Research Article
Information
Visual Neuroscience , Volume 38 , 2021 , E013
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Copyright
© The Author(s), 2021. Published by Cambridge University Press

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References

Bäck, I., Donner, K.O. & Reuter, T. (1965). The screening effect of the pigment epithelium on the retinal rods in the frog. Vision Research 5, 101–111.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, 868–879.CrossRefGoogle ScholarPubMed
Berridge, M.J. (2014). Module 2: Cell signalling pathways. Cell Signalling Biology 6, csb0001002.CrossRefGoogle Scholar
Bruenner, U. & Burnside, B. (1986). Pigment granule migration in isolated cells of the teleost retinal pigment epithelium. Investigative Ophthalmology and Visual Science 27, 1634–1643.Google ScholarPubMed
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, 1–15.Google ScholarPubMed
Burnside, B. & Basinger, S. (1983). Retinomotor pigment migration in the teleost retinal pigment epithelium. II. Cyclic-3′,5′-adenosine monophosphate induction of dark-adaptive movement in vitro. Investigative Ophthalmology and Visual Science 24, 16–23.Google ScholarPubMed
Burnside, B., Evans, M., Fletcher, R.T. & Chader, G.J. (1982). Induction of dark-adaptive retinomotor movement (cell elongation) in teleost retinal cones by cyclic adenosine 3′,′5-monophosphate. Journal of General Physiology 79, 759–774.CrossRefGoogle ScholarPubMed
Burnside, B. & King-Smith, C. (2017). Retinomotor movements. In Reference Module in Neuroscience and Biobehavioral Psychology. Amsterdam, The Netherlands: Elsevier.Google Scholar
Burnside, B. & Laties, A.M. (1979). Pigment movement and cellular contractility in the retinal pigment epithelium. In The Retinal Pigment Epithelium, pp. 175–191. Cambridge, MA: Harvard University Press.Google Scholar
Burnside, B. & Nagle, B. (1983). Retinomotor movements of photoreceptors and retinal pigment epithelium: Mechanisms and regulation. In Progress in Retinal Research, pp. 67–109. Oxford: Pergamon Press.Google Scholar
Dearry, A. & Burnside, B. (1988). Stimulation of distinct D2 dopaminergic and alpha 2-adrenergic receptors induces light-adaptive pigment dispersion in teleost retinal pigment epithelium. Journal of Neurochemistry 51, 1516–1523.CrossRefGoogle ScholarPubMed
DeVries, G.W., Cohen, A.I., Hall, I.A. & Ferrendelli, J.A. (1978). Cyclic nucleotide levels in normal and biologically fractionated mouse retina: Effects of light and dark adaptation. Journal of Neurochemistry 31, 1345–1351.CrossRefGoogle ScholarPubMed
Farber, D.B., Souza, D.W., Chase, D.G. & Lolley, R.N. (1981). Cyclic nucleotides of cone-dominant retinas. Reduction of cyclic AMP levels by light and by cone degeneration. Investigative Ophthalmology and Visual Science 20, 24–31.Google 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, 1996–2004.CrossRefGoogle ScholarPubMed
Fimia, G.M. & Sassone-Corsi, P. (2001). Cyclic AMP signalling. Journal of Cell Science 114, 1971–1972.CrossRefGoogle ScholarPubMed
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, 2264–2275.CrossRefGoogle ScholarPubMed
Garcia, D.M. (1993). Pigment Granule Aggregation in the Retinal Pigment Epithelium of Green Sunfish (Thesis). Berkeley, CA: University of California.Google Scholar
García, D.M. & Burnside, B. (1994). Suppression of cAMP-induced pigment granule aggregation in RPE by organic anion transport inhibitors. Investigative Ophthalmology and Visual Science 35, 178–188.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, 6473–6483.CrossRefGoogle ScholarPubMed
Gilson, C.A., Ackland, N. & Burnside, B. (1986). Regulation of reactivated elongation in lysed cell models of teleost retinal cones by cAMP and calcium - Google search. Journal of Cell Biology 102, 1047–1059.CrossRefGoogle Scholar
González, A., Crittenden, E.L. & García, D.M. (2004). Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill. BioMed Central Neuroscience 5, 23Google ScholarPubMed
Grønborg, M., Kristiansen, T.Z., Stensballe, A., Andersen, J.S., Ohara, O., Mann, M., Jensen, O.N. & Pandey, A. (2002). A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: Identification of a novel protein, Frigg, as a protein kinase A substrate. Molecular & Cellular Proteomics 1, 517–527.CrossRefGoogle ScholarPubMed
Jiang, M., Paniagua, A.E., Volland, S., Wang, H., Balaji, A., Li, D.G., Lopes, V.S., Burgess, B.L. & Williams, D.S. (2020). Microtubule motor transport in the delivery of melanosomes to the actin-rich apical domain of the retinal pigment epithelium. Journal of Cell Science 133, jcs242214. https://doi.org/10.1242/jcs.242214CrossRefGoogle ScholarPubMed
Johnson, A.S. & García, D.M. (2007). Carbachol-mediated pigment granule dispersion in retinal pigment epithelium requires Ca2+ and calcineurin. BioMed Central Cell Biology 8, 53.CrossRefGoogle ScholarPubMed
Kashina, A.S., Semenova, I.V., Ivanov, P.A., Potekhina, E.S., Zaliapin, I. & Rodionov, V.I. (2004). Protein kinase A, which regulates intracellular transport, forms complexes with molecular motors on organelles. Current Biology 14, 1877–1881.CrossRefGoogle ScholarPubMed
King-Smith, C. (2016). Melanosome motility in fish retinal pigment epithelial cells. In Cytoskeleton Methods and Protocols, Methods in Molecular Biology (3rd ed.), pp. 315–322. New York: Springer.CrossRefGoogle Scholar
King-Smith, C., Chen, P., Garcia, D., Rey, H., & Burnside, B. (1996). Calcium-independent regulation of pigment granule aggregation and dispersion in teleost retinal pigment epithelial cells. Journal of Cell Science 109, 33–43.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, 229–249.3.0.CO;2-0>CrossRefGoogle Scholar
King-Smith, C., Vagnozzi, R.J., Fischer, N.E., Gannon, P. & Gunnam, S. (2014). Orientation of actin filaments in teleost retinal pigment epithelial cells, and the effect of the lectin, Concanavalin A, on melanosome motility. Visual Neuroscience 31, 1–10.CrossRefGoogle ScholarPubMed
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.CrossRefGoogle ScholarPubMed
Leemhuis, J., Boutillier, S., Schmidt, G., & Meyer, D. K. (2002). The protein kinase A inhibitor H89 acts on cell morphology by inhibiting Rho kinase. Journal of Pharmacology and Experimental Therapeutics 300, 1000–1007.CrossRefGoogle ScholarPubMed
Liepe, B.A. & Burnside, B. (1993). Cyclic nucleotide regulation of teleost rod photoreceptor inner segment length. Journal of General Physiology 102, 75–98.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, 117–118.CrossRefGoogle ScholarPubMed
Lopes, V. S., Gibbs, D., Libby, R. T., Aleman, T. S., Welch, D. L., Lillo, C.,… Williams, D. S. (2011). The Usher 1B protein, MYO7A, is required for normal localization and function of the visual retinoid cycle enzyme, RPE65. Human Molecular Genetics 20, 2560‑2570.CrossRefGoogle ScholarPubMed
Murray, A.J. (2008). Pharmacological PKA inhibition: All may not be what it seems. Science Signal 1, re4.CrossRefGoogle Scholar
Nascimento, A.A., Roland, J.T. & Gelfand, V.I. (2003). Pigment cells: A model for the study of organelle transport. Annual Review of Cell and Developmental Biology 19, 469–491.CrossRefGoogle Scholar
Pagh-Roehl, K., Han, E. & Burnside, B. (1993). Identification of cyclic nucleotide-regulated phosphoproteins, including phosducin, in motile rod inner-outer segments of teleosts. Experimental Eye Reearch 57, 679–691.CrossRefGoogle ScholarPubMed
Pagh-Roehl, K., Lin, D., Su, L. & Burnside, B. (1995). Phosducin and PP33 are in vivo targets of PKA and type 1 or 2A phosphatases, regulators of cell elongation in teleost rod inner-outer segments. Journal of Neuroscience 15, 6475–6488.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, 1040–1049.CrossRefGoogle ScholarPubMed
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, 1782–1786.CrossRefGoogle ScholarPubMed
Rasmussen, H. (1981). Calciun and cAMP as Synarchic Messengers. New York: Wiley.Google Scholar
Sammak, P.J., Adams, S.R., Harootunian, A.T., Schliwa, M. & Tsien, R.Y. (1992). Intracellular cyclic AMP not calcium, determines the direction of vesicle movement in melanophores: Direct measurement by fluorescence ratio imaging. Journal of Cell Biology 117, 57–72.CrossRefGoogle Scholar
Semenova, I., Ikeda, K., Ivanov, P. & Rodionov, V. (2009). The protein kinase A anchoring protein moesin is bound to pigment granules in melanophores. Traffic 10, 153–160.CrossRefGoogle ScholarPubMed
Skroblin, P., Grossmann, S., Schäfer, G., Rosenthal, W. & Klussmann, E. (2010). Mechanisms of protein kinase A anchoring. Internationat Review of Cell and Molecular Biology 283, 235–330.CrossRefGoogle ScholarPubMed
Sokal, F.J. & Rohlf, R.R. (1981). Biometry (2nd ed.). New York: W.H. Freeman & Company.Google Scholar
Tang, P. H., Kono, M., Koutalos, Y., Ablonczy, Z., & Crouch, R. K. (2013). New insights into retinoid metabolism and cycling within the retina. Progress in Retinal and Eye Research 32, 48–63.CrossRefGoogle ScholarPubMed
Verkhovsky, A.B., Svitkina, T.M. & Borisy, G.G. (1999). Network contraction model for cell translocation and retrograde flow. Biochemical Society Symposia 65, 207–222.Google ScholarPubMed

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