Rodriguez, et al (2006): Figure 5

Supplementary Material From Singer Lab Publications:

Visualization of mRNA translation in living cells

J Cell Biol 175(1):67-76 (2006 October 9)

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Figure 5. Localization of the β-actin mRNA transgene correlates with the distribution of mature protein and translation sites. Fluorescence in situ hybridization experiments were performed on C2C12 cells transfected with the FL or ΔZIP construct with Cy-5–labeled probes against the GFP coding sequence. (A) Overlay image of a C2C12 cell transfected with the FL construct showing localized ß-actin transgene mRNA at the cell periphery as assessed by a probe for GFP (white) and DAPI staining of the nucleus (blue). (B) Overlay image of a C2C12 cell transfected with the ΔZIP construct showing the ß-actin transgene mRNA levels highest near the nucleus and dropping off toward the cell periphery as assessed by a probe for GFP (white) and DAPI staining of the nucleus (blue). (C) The relative intensity as a function of distance from the nucleus of the Cy-5–labeled probe for GFP was determined for a population of C2C12 cells containing the FL or ΔZIP construct (see Materials and methods). The fluorescence intensity as a function of normalized distance (the percentage of distance from the nucleus to the cell periphery where the nucleus is defined as 0 and the distal cell membrane was 100) was graphed for cells with the FL construct (red line) and the ΔZIP construct (blue line). The FL mRNA extended to the cell periphery (red line). In contrast, the ΔZIP mRNA was concentrated in the area immediately around the nucleus and declined to the cell periphery (blue line). n = 18. (D) The sites of translation as a function of relative distance from the nucleus was graphed for a population of C2C12 cells containing the FL or ΔZIP constructs as in C. Consistent with the mRNA and protein localizations, the localization of translation sites for the FL construct showed translation of ß-actin in the cell periphery, whereas the ΔZIP construct was confined to the perinuclear area. n = 8. (E) The total fluorescence intensity of the TC–GFP–ß-actin was determined for a population of C2C12 cells containing the FL or ΔZIP construct as above. For the cells with FL construct, there was very little preference throughout the cytoplasm. In contrast, for cells with the ΔZIP construct, there was an enhanced signal closest to the nucleus with decreasing intensity peripherally. n = 18. Error bars are ± SEM.