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R. Beaubien, M. Catrinescu, A. Kanamori, L. A. Levin; Role of (3,4,5) PIP3 in RGC-5 Cell Differentiation and Neurite Extension. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2526.
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© ARVO (1962-2015); The Authors (2016-present)
During development, neurons extend neurites in response to external cues in order to form connections. To do this, the developing neuron need to be able to integrate guidance cues received from the plasma membrane receptor to the cytoskeleton via intracellular signaling. One candidate for signaling is phosphatidylinositol 3,4,5-triphosphate (PtdInsP3 or 3,4,5-PIP3), which has been shown to be an important regulator of cytoskeletal form and cell motility in response to multiple signals. Through protein effectors containing pleckstrin-homology (PH) domains, 3,4,5-PIP3 is known to modulate the activities of a number of signaling molecules. 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 3,4,5-PIP3 and 3,4-PIP2) and then differentiated with the broad spectrum kinase inhibitor staurosporine (316 nM). Fluorescent time-lapse imaging with a cooled CCD camera was used to follow the distribution of these phospholipids over time. Time-lapse imaging was correlated with immunofluorescence of GAP-43, β-III-tubulin, and F-actin.
Stimulation of the PI3K pathways caused redistribution of AktPH::GFP fluorescence at the plasma membrane, as expected. RGC-5 cell differentiation resulted in distinctive patterns of distribution of AktPH::GFP. The extension of a neurite was significantly (p < 0.01) more likely to be preceded by a focus of AktPH::GFP signal at the plasma membrane. AktPH::GFP at the emerging growth cone co-localized with GAP-43, and foci of AktPH::GFP signal at the tip of neurites were associated with F-actin.
Elucidation of the phospholipid-dependent pathways underlying neurite extension may shed light on generation and regeneration of retinal ganglion cell axons.
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