JCS02839 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Schematic of cuprophilic cell (CC) geometry. All diagrams and cells represent CCs from third-instar larval guts. The CCs are either presented as side views (left, as in Fig. 1A-L) or top-down views, looking down at the CC pore (right, as in Fig. 3G-L), with each in a plane corresponding to the white dashed lines in B. Labeling is as follows: pink, CC cytoplasm; green, CC/interstitial cell basolateral membrane interface; blue, CC terminal web; black, apical microvilli; white, extracellular space; orange, septate junctions between CC and interstitial cells; gray, interstitial cell cytoplasm; red, interstitial cell membranes; n, nuclei; L, gut lumen; IC, interstitial cell; CC, cuprophilic cell. (A,B) Side-view schematics of a CC. A is parasagittal, B sagittal. Note the invaginated apical domain (blue, terminal web; black, microvilli), which remains open to the gut lumen (white, L). In three dimensions, the CC is round, like a flask, although this idealized shape (Filshie, 1971) is quite malleable and we often observe CCs with much wider pores, particularly at the posterior of the CC region. (A’,B’) Duplicate pictures of the CCs shown in A’’ and B’’, but artificially colored to indicate the domains found in the cartoons A and B. These are shown in parasagittal and sagittal planes to match the schematics in A and B. (A”,B”) These are the same images shown in A’ and B’, but uncolored. These cells were stained for a-spectrin, which labels both the apical and basolateral domains plus the septate junctions and structures in the interstitial cell cytoplasm. (C-F) Schematic figures of a CC, from a top-down perspective (looking down through the CC pore). In this orientation, the invaginated apical domain can cause apparent doubling of the apical domain in concentric rings near the base of the cell, as in Fig. 3G-L, Fig. 4G-I and Fig. 6E-G. The four ‘top-down’ cartoons C-F represent visual planes indicated by white dashed lines linked by arrows to the cartoon in B. Note the increasing complexity of the architecture as the planes go deeper into the CC, ending with what appear to be two, mirror-imaged and concentric apical membranes in E and F. (C’-F’) Pictures of CCs at or very close to the planes of the idealized pictures in C-F, respectively. These have been artificially colored to indicate the domains found in the cartoons C, D, E and F. Note in E-E’’ how the peripheral region of the terminal web is roughly perpendicular to the confocal plane, providing a sharp line of staining for proteins resident in this region, whereas in the central region it is in the confocal plane and will thus present an apparently wider distribution for the same protein. (C”-F”) These are the same images shown in C’, D’, E’ and F’, but uncolored. The cells were stained with FITC phalloidin to reveal the F-actin cytoskeleton of the brush border. The bright staining at the edge of the gut is sarcomeric F-actin in muscle cells that lie around the gut circumference.

• Supplemental Figure 2 -

  Fig. S2. Use of Bromophenol Blue feeding to assess loss of acidification in karst mutant guts. Each group of three colored bars represents the results from the single genotype listed below the chart, and every total consisted of at least 70 larvae from at least 3 independent experiments, with the exception of kst^14.1/Df(3L)1226, which represents only 12 larvae owing to the greater lethality of this genotype. The three colors represent the pH transitions for Bromophenol Blue, listed on the right. Every genotype but kst¹/Df(3L)1226 displayed significantly reduced acidification from the mwh red e (mvr) progenitor line, by Chi-square.

• Supplemental Figure 3 -

  Fig. S3. Anti-Manduca antibody C23 recognizes Drosophila V-ATPase subunit B. Immunoblot demonstrating the specificity of the Manduca V-ATPase subunit B antibody C23 (right) in third-instar Drosophila larval extract. A single band of the predicted size (57 kDa) is seen with C23, whereas no bands are visible with secondary antibody alone (left). Black bars indicate size markers as labeled.

• Supplemental Figure 4 -

  Fig. S4. Lack of b_H prevents dominant-negative Rab5 disruption of the early endosome in second-instar larvae. All cells are from second-instar guts. Staining is for Rab5 (A,D,G,J, green in merge) and V-ATPase (C,F,I,L, red in merge). (A-C) Wild-type CCs co-stained for Rab5 and V-ATPase, showing co-localization (e.g. arrowhead). (D-F) karst mutant CCs co-stained for Rab5 and V-ATPase. Rab5 labeling is generally present at the V-ATPase endosomes (arrowhead), but is sporadically missing. V-ATPase co-labels as in wild-type. (G-I) By the second instar, wild-type CCs expressing Rab5S43N show non-overlapping distributions of Rab5 and the V-ATPase. Rab5 is invariably concentrated adjacent to the septate junctions near the pore (arrowhead), whereas the V-ATPase is often seen in multiple compartments, especially in the apical cytoplasm of interstitial cells (I, asterisks). G-I are the same images as Fig. 5J-L and are included here for completeness. (J-L) As in the first instar, expression of Rab5S43N in karst mutant CCs fails to remove Rab5 from V-ATPase-positive endosomes (arrowhead). Bars, 20 mm.