Fig. 2

Effects of RFP-zRICH(H334A) expression on PC12 differentiation. A: Differentiation assays with PC12 stable transfectants expressing RFP or RFP-zRICH(H334A). PC12-RFP and PC12-RFP-zRICH(H334A) stable transfectant cells were treated with differentiation medium containing 100 ng/ml of NGF. At day 0, cells are spherical under phase contrast microscopy (panels a and e). After 7 days in the presence of NGF, neuronal differentiation is very apparent under phase contrast microscopy by the presence of long neurites several times the length of the cell body (panels b and f). The expression of RFP and RFP-zRICH(H334A) can be detected in the living cells by fluorescence microscopy (panels c, d, g and h). Scale bars represent 50 μm. B: Summary of the NeuronJ tracing computer-assisted morphometric analysis procedure. The diagram summarizes the procedure to analyze the neurite arbors in differentiated PC12 cells expressing fluorescent proteins. Neurons that are well isolated and express the fluorescent proteins (detected by fluorescence microscopy) are photographed by both fluorescence and phase contrast microscopy. Neurites are traced in the phase contrast images with the help of NeuronJ to facilitate quantitative analysis. C: Example of NeuronJ tracing of a differentiated PC12-RFP-zRICH(H334A) cell. The top panel shows the phase contrast microscopy image and the bottom panel shows the NeuronJ traces. Primary neurite segments arising from the cell body are highlighted in blue and traces of secondary neurite branch segments are highlighted in green. Only neurite segments longer than 1 cell body length were traced. Scale bar represents 50 μm. D: Results of morphometric analysis of differentiation assays with the stable transfectants. From left to right, the graphs represent the number of primary neurite branches, the number of secondary neurite segments, and the total neurite arbor length per cell, respectively. The most pronounced effect of the expression of zRICH(H334A) in differentiated PC12 cells was an approximately 2.4 fold increase in secondary neurite segments when compared with cells expressing RFP. A smaller 1.5 fold increase in the total neurite arbor length was observed, probably due mainly to the larger number of secondary neurites. A modest 1.2 fold increase in primary neurites was observed, but it is not statistically significant. The bars show the average ± SEM; n = 55 PC12-RFP and 43 PC12-zRICH(H334A) cells. Statistics: ** t-test, p < 0.01, *** t-test, p < 0.001. The experiment presented is representative of 4 independent assays