May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Distinctive Patterns of Phosphoinositide Distribution During Retinal Ganglion Cell Differentiation and Neurite Extension
Author Affiliations & Notes
  • R. Beaubien
    University of Montreal, Montreal, Quebec, Canada
    Molecular Biology,
  • L. A. Levin
    University of Montreal, Montreal, Quebec, Canada
    Ophthalmology,
    Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin
  • Footnotes
    Commercial Relationships  R. Beaubien, None; L.A. Levin, US 7,303,915, P.
  • Footnotes
    Support  Canada Research Chair, Canadian Foundation for Innovation
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4898. doi:https://doi.org/
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    • Get Citation

      R. Beaubien, L. A. Levin; Distinctive Patterns of Phosphoinositide Distribution During Retinal Ganglion Cell Differentiation and Neurite Extension. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4898. doi: https://doi.org/.

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

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Abstract

Purpose: : Phosphoinositide metabolism is intimately associated with cell transformation and differentiation via reorganization of the cytoskeleton. We used time-lapse fluorescent imaging of green fluorescent protein (GFP) fusion proteins that bind specific phospholipids to study phosphoinositide distribution during retinal neuronal differentiation.

Methods: : Retinal ganglion cell-like RGC-5 cells were transfected with pEGFP::AktPH (which codes for the pleckstrin homology [PH] domain of Akt that binds 3,4-PIP2), pEGFP::PLCΔPH (coding for the PH domain of phospholipase CΔ that binds 4,5-PIP2), or pEGFP, and then differentiated with the broad spectrum kinase inhibitor staurosporine (316 nM) or the histone deacetylase inhibitor trichostatin A (500 nM). Fluorescent time-lapse imaging with a cooled CCD camera was used to follow the distribution of these phospholipids over time.

Results: : There were distinctive patterns of distribution of these phospholipids in the first few hours after differentiation with staurosporine. The GFP-tagged fusion protein that bound 4,5-PIP2 was found along the cytoplasmic membrane of the soma, and also concentrated at growth cones. In contrast, the marker for 3,4-PIP2 concentrated in the soma subjacent to extending neurites. Both phospholipids were seen to undergo transport in small foci along extending or retracting neurites.

Conclusions: : Metabolism of specific phosphoinositides marks morphological changes in differentiating retinal ganglion cell-like cells. Elucidation of the metabolic pathways underlying neurite extension may shed light on generation and regeneration of retinal ganglion cell axons.

Keywords: ganglion cells • differentiation • imaging/image analysis: non-clinical 
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