Fig 2. nLDs in hermaphrodite intestinal cells.

(A) Image of two nuclei in an L4 intestine stained for lipid (green, BODIPY) and for the nuclear envelope (red, NPP-9/RanBP2). The nucleus at right has a candidate nLD (arrow). (B) Single nLD (green, BODIPY) viewed in three, orthogonal optical planes from a z stack through the entire nucleus (red, NPP-9/RanBP2). (C) Three examples of D1 nuclei with nLDs; nuclei are stained for lipid (green, BODIPY) and lamin (red, LMN-1). Each nLD is surrounded by a ring of condensed chromatin, or heterochromatin (blue, DNA), and is adjacent to a region of the envelope that appears relatively deficient in peripheral heterochromatin (arrowheads). No = nucleolus in this and all Figures. (D) L4 intestinal nuclei showing examples of giant nLDs (arrows) associated with apparent ruptures of the nuclear envelope (red, LMN-1/lamin). Other giant nLDs were adjacent to lamin-deficient regions of the envelope (see S2 Fig). Several observations suggest the ruptures are not artifacts of sample preparation. First, the positions of ruptured nuclei were not, in general, near dissection sites. Second, sample compression was minimized by supporting coverslips with precision-sized glass beads only slightly smaller than the diameter of the intestine; ruptures also were present in samples prepared for TEM (see Fig 2E) that were never compressed. Finally, intentional compression of fixed intestines caused widespread fusion, and highly abnormal shapes, of cLDs that did not resemble the images presented here. (E) TEM of a D1 intestinal nucleus showing a giant nLD at a nuclear rupture; the inset show both the INM and ONM are broken (arrow). The nLD is partially surrounded by electron-dense material (black arrowheads) resembling heterochromatin at the nuclear periphery and elsewhere. For clarity, the cytoplasm is tinted yellow here and in other micrographs below. (F) TEM showing nLDs (black arrows) in D1 intestinal nuclei. The black arrow in panel 4 indicates the exposed surface of an nLD at a gap in the electron-dense coat. The white arrows in panels 1 and 2 indicate distinct types of nuclear bodies called kernel vesicles (see below). The kernel vesicles are surrounded by conventional lipid bilayer membranes, which stain more intensely than the lipid monolayer surfaces of lipid droplets. (G) High magnification TEM images comparing the exposed surface of an nLD (panel 1) with the lipid monolayer surface of a cLD (panel 2) and with a typical lipid bilayer membrane (panel 3; taken from the plasma membrane around an intestinal microvillus). (H) TEM images of small nLDs by the nuclear envelope, showing variable electron-dense coatings (black arrowheads); white arrowheads indicate nuclear pores. (I) Plot showing the distances of submicron nLDs from the nuclear envelope; data are from optical z-stacks of stained nuclei as in Fig 2C. The smallest nLDs detectable by this technique were about 0.2 μm from the lamina, and appeared to be separated from the lamina by the thickness of the heterochromatin coating (see panel 1 in Fig 2C). Note that by the D2 and D3 stages most of the smallest nLDs are not adjacent to the envelope. (J) Examples of submicron nLDs (arrows) in D2 and D3 nuclei that are not adjacent to the envelope. The insets show that these nLDs do not appear to have distinct heterochromatin coats. Scale bars in microns as indicated.