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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.
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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.
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.
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.
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.
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