Actin Aggregations Mark the Sites of Neurite Initiation

Reorganization of evenly-distributed cortical actin into large aggregations precedes neurite initiation. A Representative images of cultured rat hippocampal neurons at different time points in culture. Fixed neurons were stained with Alexa-488 phalloidin (green in merge) and anti-Tuj 1 antibody (Alex-568, red in merge). B, C Large actin aggregations imaged using SIM; colors as in A. D Live-imaging time frames showing a neurite (arrow, 7 h) initiating from the site of a large actin aggregate, clearly present in previous frames (arrow, 3 and 6 h). F-actin labelled using Lifeact-RFP (red). Yellow stars indicate positions of neighboring, untransfected neurons. E Quantitation of live-imaging results in D. Three independent experiments, consisting of 75 neurites from 62 neurons, were quantified (paired two-tailed t-test P < 0.05). F Schematic of Stages 1a, 1b, and 2 of neuronal development. G Representative images of cultured rat dentate granule neurons at different developmental stages. Prox 1 is a granule neuron-specific marker (labelled with Alexa-633, blue in merge). Scale bars, 10 μm in all images