Abstract
The Rac-GEF P-Rex1 promotes membrane ruffling and cell migration in response to Rac activation, but its role in neuritogenesis is unknown. Rac1 promotes neurite differentiation; Rac3, however, may play an opposing role. Here we report that in nerve growth factor (NGF)-differentiated rat PC12 cells, P-Rex1 localised to the distal tips of developing neurites and to the axonal shaft and growth cone of differentiating hippocampal neurons. P-Rex1 expression inhibited NGF-stimulated PC12 neurite differentiation and this was dependent on the Rac-GEF activity of P-Rex1. P-Rex1 inhibition of neurite outgrowth was rescued by low-dose cytochalasin D treatment, which prevents actin polymerisation. P-Rex1 activated Rac3 GTPase activity when coexpressed in PC12 cells. In the absence of NGF stimulation, targeted depletion of P-Rex1 in PC12 cells by RNA interference induced the spontaneous formation of beta-tubulin-enriched projections. Following NGF stimulation, enhanced neurite differentiation, with neurite hyper-elongation correlating with decreased F-actin at the growth cone, was demonstrated in P-Rex1 knockdown cells. Interestingly, P-Rex1-depleted PC12 cells exhibited reduced Rac3 and Rac1 GTPase activity. This study has identified P-Rex1 as a Rac3-GEF in neuronal cells that localises to, and regulates, actin cytoskeletal dynamics at the axonal growth cone to in turn regulate neurite differentiation.
Highlights
Neurite differentiation is a cellular process responsible for neuronal patterning and the formation of connections crucial for the development of the nervous system
P-Rex1 distribution during neuronal differentiation To determine the functional role of P-Rex1 in regulating neuronal differentiation, we first investigated the localisation of P-Rex1 in hippocampal neurons and PC12 cells
P-Rex1-specific polyclonal antibodies were raised to a unique P-Rex1 peptide sequence (Fig. 1A) and immune serum affinity-purified on a peptide-coupled thiopropyl Sepharose column
Summary
Neurite differentiation is a cellular process responsible for neuronal patterning and the formation of connections crucial for the development of the nervous system. The establishment of neuronal networks is regulated by actin cytoskeletal dynamics. In response to specific stimuli, the growth cone of the axon produces and retracts filopodia and membrane ruffles, events that require the temporal and spatial regulation of the actin cytoskeleton (Luo, 2002). The Rho family of small GTPases, which includes Rho, Rac and Cdc, promote morphological changes during neuronal development, including neurite outgrowth, axonal guidance and dendritic development (Dickson, 2001; Govek et al, 2005; Luo, 2000). The temporal and spatial activation of Rho GTPases during neuronal differentiation in response to various extracellular cues is crucial for neurite outgrowth (Aoki et al, 2004)
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