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Figure
7. Newly synthesized β-actin protein localizes to cell contacts and is zipcode dependent. Cells were pulse labeled with FlAsH and chased with ReAsH to follow newly synthesized protein. (A) Schematic representation of the time course of the procedure used in B–D. (B–D) C2C12 cell containing the FL construct. (B) Epifluorescence image of a FlAsH-stained cell. (C) Epifluorescence image of a ReAsH-stained cell in B shown with the contrast stretched to enhance the low staining intensity. (D) Pseudocolored ratio image of C/B, where red indicates a higher red/green ratio. Notice that the ß-actin synthesized during the chase period is localized to the perinuclear region and the leading edge of the cell. (E) Schematic representation of the time course of the procedure used in panels F–K. (F–G) C2C12 cell containing the FL construct touching an untransfected cell (not visible) at its tip. (F) Epifluorescence image of a FlAsH-stained cell. (G) Epifluorescence image of ReAsH-stained cell in F. (H) Pseudocolored ratio image of G/F. Note that the ß-actin synthesized during the chase period is localized to the perinuclear region and the site of cell–cell contact. See Video 2. (I–K) C2C12 cells containing the ΔZIP construct contacting other cells. (I) FlAsH-stained cells. (J) ReAsH-stained cells. (K) Pseudocolored ratio image of J/I. Notice that the majority of the ß-actin synthesized during the chase period is localized to the perinuclear region with little protein accumulated at the sites of cell–cell contact. The highest ratios are shown in red, and the lowest ratios are shown in blue. |
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