Figure 5.
Figure 5. ATP is released from individual LBs upon exocytosis. (A–C) Detection of LB fusion–induced ATP release using genetically encoded ATP sensors. (A) Top: Schematic drawing of ATeam3.10-GL-GPI. Original ATeam3.10 is attached to a GPI-anchor (orange) via a 12xGlycin-linker (black) to facilitate selective expression in the outer leaflet of PM. Bottom: Image illustrating expression of ATeam3.10-GL-GPI on the cell surface (arrows) of ATII cells. Bar, 10 µm. (B) Images of the CFP (mseCFP) and YFP (cp173-mVenus) channel recorded from ATII cell expressing ATeam3.10-GL-GPI. Bottom: Representative YFP/CFP ratio images in the presence of 0 (control) and 100 µM extracellular ATP. Bar, 10 µm. (C) Left: ATeam3.10-GL-GPI was expressed in the outer leaflet of the PM in ATII cells (mseCFP, mVenus). FM1-43 selectively labels LBs after their fusion with the PM (arrowheads; empty arrowheads indicate FM1-43–positive LBs that fused to the PM before the start of the experiment; filled arrowhead indicates LB that fused during the experiment; Haller et al., 1998). Red circle (fused LB) denotes the region where the increase of FM1-43 fluorescence was analyzed. The area within the blue border represents the area where changes in the FRET ratio were analyzed. Bar, 10 µm. Right: Time course of FRET ratio (blue) and FM1-43 fluorescence (red) within the respective areas illustrated in the image on the left. Cells were stimulated at t = 0 s with 300 nM PMA. Dotted line indicates time of LB fusion (onset of FM1-43 fluorescence increase). (D–F) Quantitative analysis of ATP release from individual LBs. (D) Left: Image depicting ATII cells labeled with LTG. The blue shaded area indicates the area under the dual sensor electrode (AUE). LB fusion events were analyzed within the AUE. Middle and right: Magnified view of the area within the yellow rectangle in the left image at t = 0 and 600 s after stimulation of ATII cells with 300 nM PMA. Arrowheads indicate LB that fused within the time course of the experiment (loss of LTG fluorescence in image, t = 600 s). Asterisks denote nuclei of individual primary ATII cells. Bars, 20 µm. (E) Time–current graph recorded with an ATP microbiosensor and cumulative LB fusion activity after stimulation of ATII cells with 300 nM PMA at t = 0 s. Fusion activity starts at ≈t = 180 s and is accompanied by a decrease in current. Addition of ATP (10 µM, t = 700 s) results in a decrease in current, whereas addition of glucose (10 mM, t = 850 s) increases the current. (F) Table representing mean ATP release and LB fusion activity under the electrode from seven independent experiments (as illustrated in C). Based on the observed increase in ATP concentration under the electrode after LB fusions, we calculated a mean ATP concentration of 1.9 mM within individual LBs.

ATP is released from individual LBs upon exocytosis. (A–C) Detection of LB fusion–induced ATP release using genetically encoded ATP sensors. (A) Top: Schematic drawing of ATeam3.10-GL-GPI. Original ATeam3.10 is attached to a GPI-anchor (orange) via a 12xGlycin-linker (black) to facilitate selective expression in the outer leaflet of PM. Bottom: Image illustrating expression of ATeam3.10-GL-GPI on the cell surface (arrows) of ATII cells. Bar, 10 µm. (B) Images of the CFP (mseCFP) and YFP (cp173-mVenus) channel recorded from ATII cell expressing ATeam3.10-GL-GPI. Bottom: Representative YFP/CFP ratio images in the presence of 0 (control) and 100 µM extracellular ATP. Bar, 10 µm. (C) Left: ATeam3.10-GL-GPI was expressed in the outer leaflet of the PM in ATII cells (mseCFP, mVenus). FM1-43 selectively labels LBs after their fusion with the PM (arrowheads; empty arrowheads indicate FM1-43–positive LBs that fused to the PM before the start of the experiment; filled arrowhead indicates LB that fused during the experiment; Haller et al., 1998). Red circle (fused LB) denotes the region where the increase of FM1-43 fluorescence was analyzed. The area within the blue border represents the area where changes in the FRET ratio were analyzed. Bar, 10 µm. Right: Time course of FRET ratio (blue) and FM1-43 fluorescence (red) within the respective areas illustrated in the image on the left. Cells were stimulated at t = 0 s with 300 nM PMA. Dotted line indicates time of LB fusion (onset of FM1-43 fluorescence increase). (D–F) Quantitative analysis of ATP release from individual LBs. (D) Left: Image depicting ATII cells labeled with LTG. The blue shaded area indicates the area under the dual sensor electrode (AUE). LB fusion events were analyzed within the AUE. Middle and right: Magnified view of the area within the yellow rectangle in the left image at t = 0 and 600 s after stimulation of ATII cells with 300 nM PMA. Arrowheads indicate LB that fused within the time course of the experiment (loss of LTG fluorescence in image, t = 600 s). Asterisks denote nuclei of individual primary ATII cells. Bars, 20 µm. (E) Time–current graph recorded with an ATP microbiosensor and cumulative LB fusion activity after stimulation of ATII cells with 300 nM PMA at t = 0 s. Fusion activity starts at ≈t = 180 s and is accompanied by a decrease in current. Addition of ATP (10 µM, t = 700 s) results in a decrease in current, whereas addition of glucose (10 mM, t = 850 s) increases the current. (F) Table representing mean ATP release and LB fusion activity under the electrode from seven independent experiments (as illustrated in C). Based on the observed increase in ATP concentration under the electrode after LB fusions, we calculated a mean ATP concentration of 1.9 mM within individual LBs.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal