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First published online 20 January 2004
doi: 10.1242/jcs.00922

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The C-terminal domain of Drosophila ßHeavy-spectrin exhibits autonomous membrane association and modulates membrane area

Department of Biology, Department of Biochemistry and Molecular Biology, Eberly College of Science, The Pennsylvania State University, University Park, PA 16802, USA

View larger version (27K): [in a new window]   Fig. 1. Transgenic constructs derived from the ßHeavy-spectrin C-terminal segment 33. (A) An (ßH)2 tetramer. Known functional domains are indicated by their segment number: 1, actin binding domain; 2,3, ß dimer nucleation site; 7, Src homology 3 domain; 32, tetramerization site; 33, nonrepetitive C-terminal domain containing the pleckstrin homology (PH) domain. For further details on these domains see Thomas et al. (Thomas et al., 1997) and references therein. Also illustrated are the approximate locations of the truncations arising from the three karst alleles kst1, kst2 and kst14.1 (Medina et al., 2002). (B) Subdomains within segment 33 (ßH33) and its derivatives described in this paper. Myc-tag, epitope for the 9E10 antibody (Munro and Pelham, 1987). Gray boxes, PH domain (pleckstrin homology domain); OPA, short polyglutamine repeat (Wharton et al., 1985); motif III, lysine-rich repeat found at the C-terminus of most ß-spectrins (Lombardo et al., 1993). Black boxes indicate regions conserved (=10 amino acid stretches exhibiting 50% identity) in comparison with the Anopheles homologue of ßH. ßH33PH, ßHPH+3 and ßHPH+5-3 are described in the text. PM loc. indicates the ability of each construct to exhibit stable plasma membrane localization; apoptosis indicates the ability of each construct to induce apoptosis in some tissues; memb. ext./morph. indicates the ability of each construct to inhibit salivary gland invagination and induce the bi-membrane structures described in this paper. (C) Expression of the proteins illustrated in B. Two immunoblots are shown of extracts derived from the heads of adults expressing spectrin derivatives under the control of the GMR-Gal4 driver in the eye. Ten heads were used for each lane, and both blots were probed with the mAb 9E10 to detect the N-terminal myc-tag. Lanes are labeled for the construct expressed (see B) or wt (wild-type). The left-hand blot shows that each construct is expressed and migrates at their predicted size. Marker migration is indicated by black dots for 97, 68, 43 and 29 kDa (top to bottom). The right-hand blot shows the expression of ßspPH (see text), with ßH33 for reference. Marker migration is indicated by black dots for 220, 98, 66, 46, 30 and 14 kDa (top to bottom).

View larger version (129K): [in a new window]   Fig. 6. The ßH segment 33-induced membrane domains have a distinctive bi-membrane topology. All images are transmission electron micrographs of stage 14-16 embryonic salivary glands expressing ßHPH+5-3 under the control of the fkh-Gal4 driver. (A) Low-magnification cross-sectional view of the apical end of salivary gland cells and lumen. Arrows indicate circular bi-membrane structures. Linked arrows delimit the ends of large bi-membrane sheets lying on the apical surface. Bar, 2 µm. (B) Higher-magnification view of a flat bi-membrane structure where one bi-membrane folds back on itself. In the interpretive drawing below, thin lines represent single membrane bilayers; thick lines represent bi-membranes. A single membrane bilayer is always seen at the cytoplasmic side of these stacks and is presumed to be the apical surface. PIT, mid-stage clathrin coated pit. Bar, 250 nm. (C) Circular bi-membrane within the cytoplasm, but closely opposed to the apical plasma membrane. This structure appears to have cytoplasm trapped in its center. An interpretive drawing is to the right. Note how the pattern of single bilayers (thin lines numbered 1) and bi-membranes (thick lines numbered 2) is 2,2,2,1 on the lumenal side and 1,2,2,1 on the cytoplasmic side, indicating that the outermost membrane is adhering to form a bi-membrane with the inner leaflet of the apical membrane. Bar, 300 nm. (D) Circular bi-membrane within the cytoplasm that is not against the plasma membrane in this section. This structure has lumenal material in its center. An interpretive drawing is to the right. In contrast to the structures filled with cytoplasm, the enclosed lumenal material is delimited by a bi-membrane (lines numbered as in C). This, along with the lumenal content, indicates that the center of this structure is contiguous with the lumenal surface of the gland. Bar, 300 nm. (E) A more complex stack of bi-membrane folds lying on the apical surface. Separation of the component bilayers is often seen at the end of the surface bi-membranes (arrow) and occasionally along their length (arrowhead). Typically, such separations contain material resembling cytoplasm. Bar, 500 nm. (F) High-magnification view of part of the circular structure shown in D. Here, the bilayer (1)/ bi-membrane (2) structure is clearly visible. Apart from occasional sites of cytoplasmic inclusion, the individual bilayers in each bi-membrane closely parallel one another. Bar, 50 nm. (G) The plot shows an average density profile across imaged bi-membranes with the positions of the cytoplasmic inner leaflet (IL) and extracellular outer leaflet (OL) indicated (see Materials and Methods). The inner leaflets have an average separation of 8.7 nm.

View larger version (119K): [in a new window]   Fig. 2. Expression of ßH segment 33 induces apoptosis in imaginal tissues. (A) Wild-type wing. (B) Notches (arrows) resulting from the expression of ßH33 under the control of the vg-Gal4 driver along the wing margin. (C) ßH33-induced notching is rescued by co-expression of the baculovirus p35 caspase inhibitor from a UAS-p35 transgene. Curiously, vein L5 (arrow) is incomplete in such wings. (D) Scanning electron micrograph (SEM) of a wild-type eye. (E) SEM of an eye resulting from the expression of ßH33 under the control of the GMR-Gal4 driver in the developing eye disc at 20°C, which produces an intermediate phenotype. (F) The rough eye phenotype shown in E is rescued by co-expression of the baculovirus p35 caspase inhibitor from a GMR-p35 transgene. (G) Live wild-type 3rd instar wing disc stained with acridine orange to reveal cells undergoing apoptosis. (H) Live 3rd instar wing disc expressing ßH33 under the control of the vg-Gal4 driver stained with acridine orange. Numerous dying cells are detected in the region of greatest ßH33 expression along the wing margin (arrows). (I) SEM of a typical eye resulting from the expression of the ß-spectrin C-terminal derivative ßspPH under the control of the GMR-Gal4 driver at 25°C. Only a mild phenotype composed of a few mispositioned bristles is seen despite abundant protein expression (Fig. 1C).

View larger version (150K): [in a new window]   Fig. 3. ßH segment 33 exhibits autonomous membrane localization. Sagittal confocal sections of salivary glands are shown in A-I. A parasagittal section in the plane of the lumenal surface is shown in J-L. All embryos are between stages 13 and 16. (A) A wild-type salivary gland stained for -spectrin. (B) A wild-type salivary gland stained for ß-spectrin. (C) A wild-type salivary gland stained for ßH-spectrin. Inset, parasagittal image at the lumenal surface. (D-L) Salivary glands expressing ßH33 under the control of the fkh-Gal4 driver. Glands are costained for ßH33 with mAb 9E10 and anti-ß-spectrin (D-F), anti-ßH (G-I) or anti-DMoesin (J-L) antibodies. In each case staining for ßH33 is shown on the left (red in the merged image) with the second protein on the right (green in the merged image). Bars, 20 µm.

View larger version (140K): [in a new window]   Fig. 4. Deletion analysis reveals multiple contributions to the membrane binding of ßH segment 33. Sagittal confocal sections of salivary glands expressing ßH33 and its derivatives (Fig. 1B) under the control of the 185Y-Gal4 driver. All images show costaining for the ßH derivative using the mAb 9E10 (left panel, red in merged image) and -spectrin (right panel, green in merged image). Embryos are at stage 14-16. (A-C) Costaining for ßH33. (D-F) Costaining for ßH33PH. (G-I) Costaining for ßHPH+3. (J-L) Costaining for ßHPH+5-3. Bars, 10 µm.

View larger version (112K): [in a new window]   Fig. 5. Overexpression of ßH segment 33 generates a distinct membrane domain associated with dynamin. (A-R) Costaining for ßH derivative ßHPH+5-3 using mAb 9E10 (left panel, red in merged image) and various membrane markers (right panel, green in merged image). All embryos are at stage 15. (A-F) Costaining for the apical transmembrane protein Stranded at second (Sas). (A-C) A parasagittal section at the level of the lumenal surface. (D-F) Sagittal section of the same gland. (G-I) Costaining for the apical cortical protein DMoesin. (J-L) Costaining for the endocytic protein clathrin. Clathrin puncta (see arrowheads) are seen in arrangements that suggest some association with the membrane protrusions. (M-O) Costaining for the endocytic protein amphiphysin. (P-R) Costaining for the protein dynamin (Shibire). (C',F',I',L',O',R') Comparable stainings of wild-type glands for the same antigen as in C, F, I, L, O and R, respectively. Bars, 20 µm.

View larger version (83K): [in a new window]   Fig. 7. ßH segment 33 dominantly interferes with salivary gland development. (A) A stage 16 wild-type embryo expressing a nuclear UAS-LacZ marker and stained for ß-galactosidase protein to illustrate the normal position and morphology of the gland. (B) A stage 16 embryo co-expressing a nuclear UAS-LacZ marker and ßHPH+5-3 under the control of the fkh-Gal4 driver and stained for ß-galactosidase protein. Expression before invagination prevents complete internalization, with cells that remain on the surface being swept forwards to the head region during head involution. (C) Higher-magnification photomontage of the gland shown in B. Note how the posterior cells that first internalized resolutely assume their normal position (bracket 1), and that relatively few cells extend to the location of the cells that do not internalize (bracket 2). (D) Projected confocal series of a salivary gland from a stage 13 embryo expressing ßHPH+5-3 under the control of the fkh-Gal4 driver. The embryo was stained for ßHPH+5-3 (left image, red in merged center panel) using the mAb 9E10 and for the lumenal marker DMoesin (right image, green in merged center panel). For clarity in the projection, the 9E10-staining region was used as a guide to select DMoesin staining that is in the gland cells. DMoesin staining is actually very widespread (inset shows the uncropped outermost section from the series; see also Fig. 3L). Bracket indicates the continuity of the lumen revealed by DMoesin staining.

View larger version (18K): [in a new window]   Fig. 8. Models for the action of ßH segment 33. (A) We suggest that polarized membrane cues recruit ßH (pathway 1) in part to use ßH segment 33 to inhibit endocytosis and thus stabilize that domain (pathway 2; see text for discussion). (B) A model for the origin of the bi-membrane structures caused by ßH expression. We suggest that ßH segment 33 (black oval) is in a complex with one or more proteins (including dynamin (dyn)) and that self association between ßH segment 33 (gray arrows) or an associated protein (black arrows) leads to adhesion between the inner leaflets of the plasma membrane.