Fig 1.
pH sensor construct and live-imaging setup.
(A) The tpHusion construct consists of a fusion protein of superecliptic pHluorin (SEpHluorin) and FusionRed cloned under control of a tubulin promoter (alpha-Tub84B), which induces ubiquitous expression. An HRas sequence is included to tether the protein to the plasma membrane [20]. (B) Intracellular configuration of tpHusion. SEpHluorin fluoresces more brightly at high pH than at low pH. FusionRed is relatively insensitive to pH. (C) Imaging setup: a CO2 chamber consists of a glass-bottom Petri dish with a CO2 inlet and outlet. CO2 flow rate was ~3.5 l/m (see Methods). The ventral abdomen of the living fly is glued to the glass-bottom coverslip with UV glue and slightly flattened using a small piece of cut coverslip secured with a dab of vacuum grease at each corner. (D) Schematic showing orientation of ovaries within a fly. (D’) A pair of ovaries and an ovariole pulled out from an ovary. Egg chambers develop in assembly-line fashion from anterior to posterior along each ovariole. (D”) Enlarged view of the egg chamber indicated by the rectangle in D’. Fluorescence intensity was measured within a subset of main body follicle cells, excluding dorsal appendage primordia (indicated by the two curved lines on the dorsal side of the egg chamber). The dorsal appendage primordia develop into tubes that form the dorsal appendages. (D‴) Laid egg with dorsal appendages indicated (not to scale).
Fig 2.
pHluorin/FusionRed CO2 response and recovery.
(A, B) Examples of still images extracted from videos (S1 and S2 Videos) of control (y w;; tpHusion) and Idgf-null (w1118 Idgf4Δ; Idgf(1Δ dsRed, 2–3Δ, 6Δ, 5Δ); tpHusion) egg chambers in live, intact, adult Drosophila. Dotted lines indicate ROIs measured within main-body follicle cells. The brightness of the still images was increased for better visualization. Identical adjustments were applied to all images within a series. The videos were not adjusted. (A’, B’) 10-minute time lapse of pHluorin and FusionRed intensities from the same samples shown in panels A and B. (A”, B”) pHluorin/FusionRed intensity ratios normalized to 1.0 just prior to the 1-minute CO2 pulse and showing the decrease and return to 1.0. Note: The images in panel B were scanned as 512x512-pixel images with an acquisition time of less than five seconds. The images in panel A were scanned as 1024x1024-pixel so that at the same scan speed, the images took double the acquisition time of those in panel B. The longer acquisition captured a small decrease in the pH within the 1-minute frame immediately after the CO2 was turned on. This decrease is evident in the second image in panel A and in the graphs in A’ and A”, showing a slight drop in the ratio in the frame starting at 1-minute. All of the wild-type videos (S1 and S3–S12 Videos) and Idgf-null videos S13–S20 Videos were scanned as 1024x1024 images. Idgf-null videos S2, S21 and S22 Videos were scanned as 512x512 images. (C) Normalized 10-minute time-lapse of pHluorin/FusionRed ratios for 11 control and 11 Idgf-null flies. Each line represents the profile for one fly. (C’) Averaged normalized pHluorin/FusionRed ratios—averages of the profiles of the 11 control and 11 Idgf-null flies shown in panel C. Error bars represent 95% confidence limits. (D) Recovery time for pHluorin/FusionRed ratios. Each dot represents the amount of time between when the CO2 was turned off to when the pHluorin/FusionRed ratio returned to 1.0. Error bars represent 95% confidence limits.