Fig. 5.

The mutation of the LL and NP sites affects the localisation of zDHHC2 in primary rat hippocampal neurons. (A) Representative confocal images of 13 DIV primary rat hippocampal neurons cells co-expressing wt mCHERRY-zDHHC2 (left panel) and the GFP tagged wt or endocytic mutant of zDHHC2 (middle panel). Both are represented in FIRE pseudocolour. Below each cell is a zoom image of a neurite region (defined by a box in the corresponding full size merged image). The right panel is the merge of the left and middle panels, with the wt mCHERRY-zDHHC2 coloured in Magenta and GFP tagged proteins coloured in Green. Scale bar represents 5 μm. (B) Quantification of the depletion of the mutant of zDHHC2 from intracellular membranes and of its neurite enrichment. Left panel. The fluorescence intensity values of the intracellular compartments (intra) as a fraction of the whole soma fluorescence were calculated as described in material and methods for both GFP and mCHERRY. The graph shows the normalized mean ratio ± SEM of intracellular fluorescence of the GFP construct relative to the wt zDHHC2 mCHERRY-tagged protein. Right panel. The mean fluorescence intensity of the neurites relative to the soma was calculated for both GFP- and mCHERRY-tagged proteins. The graph shows the normalized ratio ± SEM of neurite fluorescence intensity for the GFP construct relative to the wt mCHERRY zDHHC2 protein (n = 13 wt zDHHC2 cells and 14 L334/335A_N357A cells). Statistical analysis (Student's t-test) shows a significant difference in the localisation of the mutant compared to wt zDHHC2 (^⁎⁎⁎, p ≤ 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)