May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
The Ternary Rab27a:Myrip:MyovIIa Complex Regulates Melanosome Tethering to the Actin Network
Author Affiliations & Notes
  • J. S. Ramalho
    Ophthalmology Dept, IBILI-Faculty of Medicine, University of Coimbra, Coimbra, Portugal
  • V. S. Lopes
    Molecular and Cellular Medicine, Faculty of Natural Sciences, Imperial College London, London, United Kingdom
  • D. M. Owen
    Molecular and Cellular Medicine, Faculty of Natural Sciences, Imperial College London, London, United Kingdom
  • M. O. Karl
    Bereich Experimentelle Ophthalmologie, Klinik und Poliklinik fuer Augenheilkunde, Universitaetsklinikum Hamburg-Eppendorf, Hamburg, Germany
  • O. Strauss
    Bereich Experimentelle Ophthalmologie, Klinik und Poliklinik fuer Augenheilkunde, Universitaetsklinikum Hamburg-Eppendorf, Hamburg, Germany
  • C. Futter
    Division of Cell Biology, Institute of Ophthalmology, University College London, London, United Kingdom
  • M. C. Seabra
    Molecular and Cellular Medicine, Faculty of Natural Sciences, Imperial College London, London, United Kingdom
  • Footnotes
    Commercial Relationships J.S. Ramalho, None; V.S. Lopes, None; D.M. Owen, None; M.O. Karl, None; O. Strauss, None; C. Futter, None; M.C. Seabra, None.
  • Footnotes
    Support None.
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 2534. doi:
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      J. S. Ramalho, V. S. Lopes, D. M. Owen, M. O. Karl, O. Strauss, C. Futter, M. C. Seabra; The Ternary Rab27a:Myrip:MyovIIa Complex Regulates Melanosome Tethering to the Actin Network. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2534.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: Rab27 proteins are small GTPases that interact with cytoskeletal elements and influence the movement of intracellular vesicles. It has been proposed that in retinal pigment epithelium (RPE), Rab27a recruits Myrip, which then recruits MyosinVIIa for the movement of melanosomes into the apical processes. The role of this complex might have a crucial function in the physiology of the retina as supported by the fact that MyosinVIIa is mutated in Usher syndrome type 1B. In this work we establish the formation of the hypothetical tripartite complex and its role in melanosome movement in the RPE.

Methods:: RPE primary cultures were isolated from ashen, shaker-1 and control littermate three week old mice. Immunofluorescence and immuno-electron microscopy for Rab27a and Myrip were performed. Melanosome dynamics was studied using live cell imaging of mouse RPE primary cultures. Adenoviral shRNA technology was used to knock-down Myrip, and nocodazole and cytochalasin D treatment allowed the study of microtubule and actin involvement, respectively.

Results:: In heterologous cultured cells following overexpression, all three proteins co-immunoprecipitated. Rab27a and Myrip localise to the peripheral membrane of RPE melanosomes, forming concentric rings. Melanosome dynamics studies showed that wild-type melanosomes present a peripheral directionality trend in their bursts of fast movement. Significant and similar alterations in melanosome dynamics were observed when anyone of the three components of the putative complex was missing, giving raise to an increase in the number of motile melanosomes, more prolonged and frequent bursts of movement and inversion in directionality. Cytochalasin D treatment resulted in similar alterations to the ones described above, while nocodazole-tretated cells present melanosome paralysis.

Conclusions:: Our results suggest that the tripartite complex Rab27a: Myrip: MyoVIIa regulates tethering of RPE melanosomes onto actin filaments, a process that ensures melanosome movement towards the apical processes.

Keywords: retinal pigment epithelium • melanocytes • cytoskeleton 
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