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.