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First published online 14 March 2006
doi: 10.1242/jcs.02839

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Brush border spectrin is required for early endosome recycling in Drosophila

Matthew D. Phillips^* and Graham H. Thomas

Departments of Biology, and of Biochemistry and Molecular Biology, The Pennsylvania State University, 208 Erwin W. Mueller Laboratory, University Park, PA 16802, USA

View larger version (73K): [in a new window]   Fig. 1. ßH-spectrin distribution in middle midgut cells. (A) Top: schematic diagram of the larval middle midgut. fg, foregut; pv, proventriculus; gc, gastric caece; am, anterior midgut; cc, cuprophilic cell region; mm, middle midgut; fe, iron cell region; pm, posterior midgut; bb, brush border (illustrates how the long microvilli of the brush border almost fill the apical invagination). Bottom: diagrams illustrating sagittal section (left) and cross-section (right) through the cuprophilic cell (CC) region. For orientation, a dashed box surrounds a cell colored to resemble the staining in C. (B-D) Wild-type third-instar CCs co-stained for -spectrin (B, -Sp, red) and ßH-spectrin (D, ßH-Sp, green) and merged (C). ßH and -spectrin co-label the terminal web subtending the apical surface. is also present on the basolateral membrane and is concentrated at the septate junctions (B, arrowheads). Insets show a top-down view of the septate junction region of another cell. *, CC nucleus. (E,F) karst mutant third-instar cells stained for -spectrin (-Sp). -spectrin is not present at the terminal web in the absence of ßH. The CC in (E) has a properly positioned nucleus (*), whereas the cells in (F) have mispositioned nuclei. Other examples of nuclear mislocalization are seen in Fig. 3J-L and Fig. 6C. (G-I) Parasaggital section of a wild-type third-instar CC co-labeled with -spectrin (G, -Sp, green) and septate junction marker Coracle (I, Cor, red). The merged image (H) shows co-localization at the highly folded junction (bracket). Insets are a top-down view of another septate junction. (J-L) Saggital section of a wild-type third-instar CC co-labeled with ßH-spectrin (J, green) and Coracle (L, red). The apical and basolateral domain abut at the apical margin of the junctions (arrowheads in K). Insets show a top-down view of another septate junction. Bars, 20 µm, except the inset in B, which is 10 µm.

View larger version (74K): [in a new window]   Fig. 2. Acridine Orange staining of third-instar intestines. (A) A segment of wild-type gut stained with AO. The CC apical invaginations (arrows) and gut lumen (L) are filled with a strongly red-shifted AO signal indicating robust acid production. The interstitial cells forming the pores also contain a ring of green fluorescence of unknown origin (arrowheads). (B-D) Wild-type third-instar CC; (B), brightfield view; (C), AO fluorescence. Parasagittal section showing red fluorescence in the apical invagination and pore, whereas the section intersects a green fluorescent ring in the interstitial cells. (D) Illustration of the efficacy of the AO probe by showing the relative red and green channel fluorescence along a transect in the cell in D (see inset). (E) Low-magnification views of representative wild-type guts stained with AO. All show a strongly red-shifted signal in the CC apical invaginations and gut lumen. The first panel is parasagittal, the rest are sagittal. L, lumen. (F) Low-magnification views of representative karst guts stained with AO. There is no AO signal from the CC apical invaginations, whereas the lumens are green to pale orange. Strong accumulation of red-shifted AO is seen in vesicular structures in mutant CCs. The first panel is parasagittal and the rest are sagittal. L, lumen. (G) Higher-magnification view of the segment of karst gut stained with AO in the rightmost panel in F, showing how the CC apical invaginations (arrows) are devoid of AO staining. The gut lumen is filled with mostly unshifted signal, indicating weak acidification. No green rings appear near the pores. Prominent red-shifted vesicles indicating low pH are visible in the CC cytoplasms (e.g. arrowheads). L, lumen. Bars, 20 µm.

View larger version (107K): [in a new window]   Fig. 3. V-ATPase staining of third-instar intestines. All samples were co-stained for -spectrin (A,D,G,J) and V-ATPase (C,F,I,L). Panels B, E, H and K are merged images (-spectrin in green, V-ATPase in red). (A-C) Wild-type gut. Cell shapes are revealed by -spectrin, which outlines both the apical and basolateral domains and exhibits high cytoplasmic concentrations in the interstitial cell cytoplasm. CCs in the center present in cross-section as rounded shapes with the nucleus in the center; by contrast, cells on the top and bottom edges of the gut are parasagittal with a cup-shaped apical invagination around the nucleus. The V-ATPase signal is visible at the apical domain and in clustered vesicular structures. (D-F) karst mutant gut. -spectrin stains only the basolateral membranes in the absence of ßH. V-ATPase staining is diffusely cytoplasmic, lacking both apical and vesicular concentrations. (G-I) Vertical cross-section of an individual wild-type CC. -spectrin stains both basolateral membrane and the terminal web (G, arrows) on either side of the apical invagination (G, asterisk). The terminal web in the center of the cell runs parallel to the image plane and dips into focus near the nucleus where again elevated V-ATPase can be seen (chevron in H). V-ATPase co-localizes with -spectrin in the terminal web at this resolution (H, arrow in I), and is also seen in numerous cytoplasmic vesicles near the basal membrane (H, arrowheads in I). (J-L) Vertical cross-section of an individual karst mutant CC. -spectrin stains the basolateral membrane only in this cell. The V-ATPase signal is diffuse and cytoplasmic, demonstrating neither an apical accumulation nor a vesicular concentration. Note also the misplaced nucleus ( in K). This view is in a similar plane to that illustrated in Fig. S1D (supplementary material). Bars, 20 µm.

View larger version (81K): [in a new window]   Fig. 4. V-ATPase is located in a subset of Rab5-positive endosomes in CCs. Cells shown are all from third-instar guts except where noted; `red' and `green' refer to colors in the central merged image. (A-C) Wild-type cell stained for V-ATPase (A, red) and the Golgi marker Lava Lamp (C, Lva, green). No co-localization is evident. (D-F) Wild-type cell stained for V-ATPase (D, red) and the recycling endosome marker Rab11 (F, green). No co-localization is evident. (G-I) Wild-type cell stained for V-ATPase (G, red) and the early endosome marker Rab5 (I, green), showing that all V-ATPase-positive structures co-label for Rab5. However, not all Rab5-positive endosomes co-label for V-ATPase. (J-M) A myc-2xFYVE construct was expressed in first-instar larval CCs to label phosphatidylinositol 3-phosphate [PtdIns(3)P]-positive endosomes. These were stained for Myc (K/2xFYVE, red), Rab5 (L, green) and V-ATPase (M, blue). Three classes of early endosomes are revealed: those labeled with Rab5 alone (e.g. arrows in L), those co-labeled with Rab5 and myc-2xFYVE (e.g. arrowhead in K and L), or those labeled with Rab5 and V-ATPase (bracket in L and M). Also, myc-2xFYVE stains the basolateral but not the apical membrane. (N-P) karst mutant cell stained for V-ATPase (N, red) and Rab5 (P, green). Not only is the V-ATPase dispersed but none of the Rab5-positive compartments are evident. (Q-S) karst mutant cell stained for Rab11 (Q, red) and Lava Lamp (S, Lva, green), showing that recycling endosomes and Golgi are intact and indistinguishable from wild type in the absence of ßH-spectrin. Bars, 20 µm.

View larger version (81K): [in a new window]   Fig. 5. Lack of ßH prevents dominant-negative Rab5 disruption of the early endosome. All cells are from first-instar guts except (J-L), which are from second-instar guts. Staining is for Rab5 (A,D,G,J,M, green in merge) and V-ATPase (C,F,I,L,O, red in merge). (A-C) Wild-type CC co-stained for Rab5 and V-ATPase, showing co-localization (arrowhead). (D-F) karst mutant CC co-stained for Rab5 and V-ATPase. At this stage, Rab5 endosomes appear intact and V-ATPase co-labels as in wild type (arrowhead). (G-I) Expression of Rab5S43N in wild-type CCs virtually eliminates the Rab5 signal at V-ATPase-positive endosomes (e.g. arrowhead) and eliminates the typical particulate Rab5 pattern. (J-L) By the second instar, wild-type CCs expressing Rab5S43N show non-overlapping distributions of Rab5 and 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 (L, asterisks). (M-O) Expression of Rab5S43N in karst mutant CCs fails to reduce the Rab5 signal at V-ATPase-positive endosomes (e.g. arrowhead) and does not eliminate the typical particulate Rab5 staining pattern. Identical results are seen in second-instar guts that still contain V-ATPase-positive endosomes (complete data set is shown in Fig. S4, supplementary material), except as noted in (J-L) above. Bars, 20 µm.

View larger version (147K): [in a new window]   Fig. 6. Apical delivery of V-ATPase continues in karst mutant cells. All CCs shown are from third-instar guts; `red' and `green' refer to colors in the central merged image. (A) Staining for V-ATPase in a Shibire2Ts (Shi2Ts) mutant cell at the permissive temperature reveals a normal endosomal and apical localization (see Fig. 4G). (B) Staining for Rab5 in a Shi2Ts cell at the permissive temperature reveals a normal distribution (see Fig. 4I). (C) Staining for V-ATPase in a Shi2Ts/karst double-mutant cell at the permissive temperature reveals only the diffuse cytoplasmic staining characteristic for karst mutants. (D) Staining for Rab5 in a Shi2Ts/karst double-mutant cell at the permissive temperature reveals only the diffuse cytoplasmic staining characteristic for karst mutants. (E-G) Staining for V-ATPase (E, red) and Rab5 (G, green) in a Shi2Ts mutant cell at the restrictive temperature reveals a strong apical accumulation for both proteins (arrowheads). The inset shows a co-labeled endosomal compartment from another cell. Here, Rab5 is still present, but the signal appears weaker and somewhat patchy compared with the permissive temperature. (H-M) Staining for V-ATPase (H,K: V, red) and Rab5 (J,M: R5, green) in Shi2Ts/karst double-mutant cells at the restrictive temperature. Both proteins co-accumulate at the apical membrane of CCs (H-J), although V-ATPase is weaker and is variable from larvae to larvae. No V-ATPase- or Rab5-positive endosomes are evident. Rab5 particles also accumulate near the apical membrane in interstitial cells (i), with no evident V-ATPase staining (K-M). Asterisk indicates a CC with apical Rab5 accumulation, but very weak V-ATPase accumulation. Bars, 20 µm.

View larger version (30K): [in a new window]   Fig. 7. A model for the role of brush-border spectrin in the recycling pathway. Newly formed endocytic vesicles must navigate through the F-actin rootlets (gray vertical bars), spectrin (thin black filaments) and myosin II (thick black strands) in the terminal web before proceeding to the early endosome (EE), where a decision is made either to return the protein to the membrane (recycle, 1) or to follow the degradation pathway (lysosome, 2). Our data suggest that brush border spectrin `primes' proteins on vesicles in the terminal web (P) in a way that influences the decision to recycle or degrade them at the early endosome. In ßH-spectrin mutants, we suggest that diversion of membrane flow along pathway 2 results in the run down of the early endosome system at the start of the third instar and the concomitant increase in lysosomal compartments that we observe.

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