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. 2017 Dec;85:235–246. doi: 10.1016/j.mcn.2017.07.007

Fig. 9.

Fig. 9

Phospho-mimetic and phospho-null mutations affect the localisation of zDHHC2 in primary rat hippocampal neurons. (A) Amino acid sequence of the 330–366 region of zDHHC2. The serine and threonine residues that might potentially modulate zDHHC2 localisation are underlined. All the phospho-mimetic and phospho-null mutations were combined to give rise to 2 new mutants, named DAEEDDDE and AAAAAAAA. These GFP-tagged mutants were expressed together with the wt mCHERRY-zDHHC2 protein in primary rat hippocampal neurons. (B) Representative confocal images of 13 DIV primary rat hippocampal neurons cells co-expressing wt mCHERRY-zDHHC2 (left panel) and the GFP-tagged mutants 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 wt mCHERRY-zDHHC2 coloured in Magenta and GFP-tagged proteins coloured in Green. Scale bar represents 5 μm. (C) Quantification of the intracellular depletion or enrichment of zDHHC2 mutants and of their neurite enrichment or depletion compared to the wt protein. Left panel. The fluorescence intensity values of the intracellular membrane compartment (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 endosomal fluorescence of the GFP construct relative to wt mCHERRY-zDHHC2. Right panel. The mean fluorescence intensity of the neurites related 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 wt mCHERRY-zDHHC2 (n = 17 wt zDHHC2 cells, 15 DAEEDDDE and 22 AAAAAAAA cells). Statistical analysis (ANOVA) shows a significant difference in the localisation of the mutants compared to wt zDHHC2 (⁎, p ≤ 0.05; ⁎⁎⁎, 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.)