Figure 1.
Expression of mSpitz::GFP in third instar salivary glands.
A,B A low magnification view showing that the capes are found in every nucleus. Panel A shows the appearance in a single confocal section. The images are dominated by the intranuclear concentrations of mSpi::GFP so the region around one nucleus has been enhanced to allow the perinuclear distribution to become visible. Panel B is a maximum projection of the same group of nuclei. C–G – A high magnification view of five individual nuclei, exemplifying the range of cape size and morphology seen. All images are maximum projections of confocal sections across each nucleus. Scale bar represents 20 μm in A,B, 10 μm in C–G.
Figure 2.
Cytoplasmic capes contain ubiquitylated proteins and endosomal markers and are found in wild-type glands.
Each set of three images shows one nucleus or cape from a salivary gland showing the distribution of two proteins. The center panel is a merged red/green image with the left panel in green. Due to the varied morphology of each cape multiple examples are shown for most markers. A–P express mSPI::GFP and costaining is for the indicated proteins: A-A” – Nucleoplasmic RFP is excluded from capes (arrowheads). This image is a single plane from a nucleus where the image stack had been deconvolved; B-B” – Wheat germ agglutinin (WGA) stains glycosylated proteins of the nuclear envelope and outlines the capes indicating that they are infoldings of this membrane and that the contents are therefore extranuclear. C–F – Lamin C similarly coats the cape membranes. D,E show individual capes from other nuclei. Panel F shows a series of confocal images taken at 0.5 μm intervals through a cape at the top of a nucleus. Chambers associated with individual capes where mSpitz is very low or absent are often seen with this marker (arrowheads). G–H” – Ubiquitin (Ubi) puncta are found in most capes at their periphery (see also P–Q”). H shows a saggital view. I–J” – Occasional Hrs puncta are found in the central space of the terminal chamber; K–O – EPS15 puncta are found in most capes in the central space. L–O show individual capes from other nuclei. Panels P–U” do not express mSpi::GFP demonstrating that capes are present in wild-type glands. Costainings are as indicated: P–Q” – Ubiquitin (Ubi) puncta surround Hrs puncta; R–S” – BicD::GFP is not obviously punctate and fills the central space containing EPS15 puncta. Note the large central vesicle that excludes both markers in example R. T-T” – Rab5 puncta are found in the central space; U-U” – Rab6 is not obviously punctate and fills the central space. Several other Rab proteins do not enter the capes. Due to the widely varying extent and morphology of each cape, some panels show single confocal planes whereas others are maximum projections of up to 8 planes taken at 1 μm intervals. All scale bars represent 10 μm except for panels A”, T’, T” and U” which are 20 μm.
Figure 3.
Ultrastructural analysis of cytoplasmic capes.
TEM images from three capes in wild-type nuclei. A, B – These images shows two capes where regions of perinuclear vesicles (PNV) and the terminal chamber (TC) are visible. NE – nuclear envelope. NP – nucleoplasm. As illustrated in A the PNV region is often associated with an out folding of the outer nuclear membrane (arrowhead), non-membrane bound granules (asterisk) and small vesicles (see D). Scale bars are 1 μm. C – Higher magnification view of part of the membrane from a terminal chamber in B (arrow) showing two membrane bilayers (arrowheads). Scale bar is 200 nm. D–A terminal chamber containing several vesicular and granular organelles (dashed line, asterisk). Two PNV are also visible at the periphery of this chamber (arrows). E–A second cape showing only the region of PNV. Scale bar is 2 μm. F – Higher magnification view of several PNV in C (dashed line with arrow). PNV are bounded by a single bilayer and are separated from the NP by a single bilayer and often protrude into one another. See also serial sections in Movie S1. G – Chart showing the number of capes per nucleus with a given size (arbitrarily estimated in the Z dimension in serial block face SEM image series as the number of 50 nm sections from first appearance to disappearance in serial sections and normalized to the nuclear diameter in the same direction; see Movie S2). Data are shown for wild-type (black bars) and AnxB9RNAi (grey bars). Many more small capes are present when AnxB9 is knocked down. H - Chart showing the number of fully sectioned nuclei containing capes with a terminal chamber. Data are shown for wild-type (black bars) and AnxB9RNAi (grey bars). Whereas all capes end in a terminal chamber in wild-type, few do when AnxB9 is knocked down.
Figure 4.
dpERK levels are elevated when βH is reduced.
A- A wild-type wing imaginal disc stained for βH. B- The same disc stained for activated MAP kinase (dpERK). Staining is most apparent at the anlagen of veins L1 (wing margin) and L3–5 as indicated. The intensities in the two compartments are roughly equal. C- An engrailed-Gal4>βHRNAi disc stained for βH, which is no longer detectable in the posterior compartment. D- The same disc stained for dpERK. Staining is elevated in the posterior half of L1, while L4 and L5 are conspicuously more intense. Experiments were done at 25OC. Scale bar represents 20 μm.
Figure 5.
Cytoplasmic capes are dependent upon βH and AnxB9, but sSpitz production is not affected by βH knockdown.
A – A wild-type salivary gland expressing mSpitz::GFP (AB1>mSpitz::GFP); B,C – Glands expressing mSpitz::GFP where βH was knocked down (AB1>mSpitz::GFP+kstRNAi). Just the occasional cape is seen at this resolution. D – Immunoblot on dissected salivary glands expressing mSpitz::GFP with or without βH knockdown. Five pairs of glands are loaded for each genotype. In the upper panel the blot was probed with an anti-GFP antibody. mSpitz::GFP runs at about 58 kDa and the cleaved ligand, sSpi at 50kDa. The lowest band is non-specific. The lower panel is the same blot probed for Actin as a loading control. The amount of processed sSpi is not to be affected by the loss of the capes. E – The wing pouch region from a wild-type wing disc (MS1096>mSpitz::GFP). mSpitz::GFP marked nuclear envelopes have a rough, slightly punctate appearance. F – The wing pouch region from a wing disc where βH was knocked down (MS1096>mSpitz::GFP+kstRNAi). mSpitz::GFP now smoothly coats the nuclei. All images are maximum projections of multiple confocal sections. Scale bars represent 10 μm (E–F) or 20 μm (A–C).
Figure 6.
A generalized drawing of a cytoplasmic cape.
All wild-type capes have two regions: (1) A complex area of membrane folding at the nuclear periphery that contains abundant perinuclear vesicles. These vesicles are bound by a single membrane and separated from the nucleoplasm by a single membrane bilayer; (2) A terminal chamber where the two membrane bilayers separate single membrane bound organelles and granules (with no obvious membrane layer) from the nucleoplasm. The universal juxtaposition of these two regions seen by extensive serial sectioning leads us to hypothesize that their formation and perhaps function is linked. Blue line – Outer nuclear membrane; Red line – inner nuclear membrane.