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