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. 2012 Dec 4;10(12):e1001439. doi: 10.1371/journal.pbio.1001439

Figure 5. Identification of determinants that allow MKK7 mRNA localization and functional assessment of MKK7 mRNA localization.

Figure 5

(A) Schematic of MKK7-GFP expression constructs flanked with different 3′-UTR sequences. (B) Relative neurite enrichment of exogenously expressed mRNAs determined as performed in Figure S5D. n = two experiments. (C) Subcellular localization of exogenously expressed MKK7-GFP mRNAs. Representative FISH micrographs of exogenously expressed MKK7-GFP/-, MKK7-GFP/3′-UTR1, and MKK7-GFP/3′-UTR2 mRNAs using a GFP antisense probe. F-actin signal (phalloidin staining) is also shown. Images are in ibw contrast. Black arrows point to growth cones, red arrows point to somata. Note different mRNA localizations: MKK7-GFP/-, soma; MKK7-GFP/3′-UTR1, soma and growth cone; MKK7-GFP/3′-UTR2, growth cone. Scale bar: 25 µm. (D) Representative micrographs of differentiated N1E-115 MKK7 KN cells rescued with different exogenously expressed MKK7-GFP mRNAs. Cells were immunostained for α-tubulin and are shown in ibw contrast. Note that with siRNA and plasmid transfection efficiencies of 100% and 80%, respectively (unpublished data), average whole population measurements can be made. Scale bar: 100 µm. (E) Neurite outgrowth measurements from micrographs in (D) are shown. Measurements from 10% cells with longest neurites are shown, n = 1,000 cells. (F) Representative micrographs of pMKK7 signals of differentiated N1E-115 MKK7 KN cells rescued with different exogenously expressed MKK7-GFP constructs. Images are color-coded so that warm and cold colors represent high and low pMKK7 signal. Coverslips were stained, imaged, and fluorescence intensities scaled with identical conditions within one experiment. Images were acquired with a confocal microscope with a maximally open pinhole for adequate signal quantification. Red arrowheads point to the soma, green arrowheads point to the neurite. Scale bar: 25 µm. (G) Quantification of pMKK7 signals in the neurite. Mean neurite fluorescence intensities per neurite are shown. Note robust neurite pMKK7 signal recovery with MKK7-GFP/3-UTR1 and 3′UTR2 constructs. Error bars represent SD, n = 20 cells.