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Figure
6. Translation sites colocalize with their cognate mRNA near the sites of cell contact. ReAsH staining experiments were performed on live cells that were subsequently fixed and processed for FISH, showing a colocalization between the red translation puncta and mRNA at cell contacts. Shown are contacting C2C12 cells containing either the FL construct (B, D, F, H, J, and L) or the ΔZIP construct (C, E, G, I, K, and M). The transfected cell exhibits positive GFP staining (H and I). (A) Schematic representation of the time course of the procedure. (B and C) DAPI staining and differential interference contrast (DIC) image overlaid. (D and E) A single plane of a deconvolved z series of C2C12 cells stained with ReAsH, showing sites of translation. Note that in the contacting cell (D) with the FL construct, the translation sites are restricted to the contact, whereas in E, translation sites are absent from the contact with the ΔZIP construct. (F and G) A single plane of a deconvolved z series of C2C12 cells hybridized with Cy-5–labeled probes against the GFP coding sequence, showing the distribution of the mRNA. (H and I) Deconvolved plane from a z series of C2C12 cells, showing GFP fluorescence from mature actin. (J and K) Overlay images of the translation site staining (red) and mRNA hybridizations (green). Note the colocalization of the translation sites and mRNA only at the contact with the FL construct (J) and the lack of colocalization with the ΔZIP construct (K). (L and M) Overlay of the translation site staining (red), mRNA hybridization (green), GFP actin fluorescence (blue), and differential interference contrast images. |
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