Transient Increase in Cyclic AMP Localized to Macrophage Phagosomes
Figure 4
Transient burst of cAMP at the developing phagosome.
RAW cells expressing C4 or the Epac-camps biosensor were fed opsonized targets and component images for phase-contrast and FRET were taken every 30 sec to capture the phagocytic process from initiation to closure of the phagocytic cup. A. and B. Left insert: A phase-contrast (top) and corresponding EA image (bottom) of intact live macrophages transfected with C4 control (A) or Epac-camps (B), from the designated time intervals. Right inserts: One minute time course of a magnified portion of the cell transfected with the specified plasmid (beginning immediately after the sRBCs were added to the culture). The red circle denotes the location of the opsonized sRBC on the EA image. Color bar indicates scale of ratio and scale bar is 10 µm in the left insert and 5 µm.in the right insert C. To normalize for non-specific cAMP-mediated effects during phagocytosis, the data are shown as the phagosome-specific difference in EA between C4 control and Epac-camps (ec) biosensor-expressing cells. (ie., (EA(C4-phago)/EA(C4-cell)) − (EA(ec-phago)/EA(ec-cell)); n = 10 cells) D. To verify that the differences seen between cells transfected with the C4 control construct and the Epac-camps construct were not due to selective bleaching of one of the fluorescent proteins, the RI values are plotted (ie., (RI(C4-phago)/RI(C4-cell)) − (RI(ec-phago)/RI(ec-cell))).