Identification and Ultrastructural Characterization of a Novel Nuclear Degradation Complex in Differentiating Lens Fiber Cells

Abstract

An unresolved issue in structural biology is how the encapsulated lens removes membranous organelles to carry out its role as a transparent optical element. In this ultrastructural study, we establish a mechanism for nuclear elimination in the developing chick lens during the formation of the organelle-free zone. Day 12-15 chick embryo lenses were examined by high-resolution confocal light microscopy and thin section transmission electron microscopy (TEM) following fixation in 10% formalin and 4% paraformaldehyde, and then processing for confocal or TEM as described previously. Examination of developing fiber cells revealed normal nuclei with dispersed chromatin and clear nucleoli typical of cells in active ribosome production to support protein synthesis. Early signs of nuclear degradation were observed about 300 μm from the lens capsule in Day 15 lenses where the nuclei display irregular nuclear stain and prominent indentations that sometimes contained a previously undescribed macromolecular aggregate attached to the nuclear envelope. We have termed this novel structure the nuclear excisosome. This complex by confocal is closely adherent to the nuclear envelope and by TEM appears to degrade the outer leaflet of the nuclear envelope, then the inner leaflet up to 500 μm depth. The images suggest that the nuclear excisosome separates nuclear membrane proteins from lipids, which then form multilamellar assemblies that stain intensely in confocal and in TEM have 5 nm spacing consistent with pure lipid bilayers. The denuded nucleoplasm then degrades by condensation and loss of structure in the range 600 to 700 μm depth producing pyknotic nuclear remnants. None of these stages display any classic autophagic vesicles or lysosomes associated with nuclei. Uniquely, the origin of the nuclear excisosome is from filopodial-like projections of adjacent lens fiber cells that initially contact, and then appear to fuse with the outer nuclear membrane. These filopodial-like projections appear to be initiated with a clathrin-like coat and driven by an internal actin network. In summary, a specialized cellular organelle, the nuclear excisosome, generated in part by adjacent fiber cells degrades nuclei during fiber cell differentiation and maturation.

Fig 1. Schematic diagram of fiber cell cross-sections examined by thin section electron microscopy and longitudinal views by confocal microscopy.

Regions sampled are from a typical Vibratome section of a Day 15 embryonic lens near the equatorial plane. Cell cross-sections are derived from electron micrographs at the depths indicated. The approximate position of the organelle-free zone (OFZ) for nuclei is indicated; for other membranous organelles, the OFZ is about 600 μm from the capsule. The mesa of the 180 μm model Vibratome section is raised for cutting 70 nm thin sections from the capsule through the fetal nucleus (fn) to the embryonic nucleus (en). Nuclei are depicted with an intact nuclear envelope (up to 300 μm depth), with modified nuclear envelopes up to 500 μm, with a disrupted nuclear envelope (500–700 μm depth) and without a nuclear envelope (>700 μm depth), roughly represented in the confocal inset. Several bands represent different regions of nuclear breakdown within the fetal nucleus (fn): the outer band (equivalent to the cortex in older lenses) is the region of classical fiber cell appearance, protein production and initiation of organelle degradation by autophagy; the second band represents the formation of the nuclear excisosome and the beginning of nuclear degradation; the next band up to the OFZ represents the breakdown of the nuclear envelope and formation of pyknotic nuclear fragments. Not drawn to scale.

Fig 2. Overview of the capsule and annular pad.

(A) Electron micrograph. (B) Confocal image. The capsule, annular pad and the capsule-epithelial-interface (CEI; double arrow) are indicated. One nucleus (Fig 2A, blue) is highlighted because it is difficult to visualize among the densely stained epithelial cells of the annular pad. Numerous nuclei are stained in the confocal image of an equivalent region of the annular pad (Fig 2B, N). Other membranous organelles such as lysosomes, mitochondria and endoplasmic reticulum are visible as globular objects but not distinguished. EFI is the epithelial to fiber cell interface.

Fig 3. Laser scanning confocal imaging of D15 Vibratome sections.

The depth from the capsule-epithelium-interface is indicated in the upper right corner of each image here and in subsequent images. (A) At 50 μm depth, the nuclei are large, oval and active indicated by the deep 4’,6-Diamidino-2-Phenylindole (DAPI) staining. The cells have uniform parallel borders except where nuclei distort the cellular shape. The membranes stained with 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine Perchlorate (DiI) are uniform in staining and smooth in topology. Numerous vesicular structures are visible that represent membranous organelles including mitochondria, lysosomes, endoplasmic reticulum and Golgi, as well as large circular objects that may represent autophagic vesicles (arrows). (B) At 100 μm depth, the intensely staining vesicular structures (arrows) predominate the background around nuclei that appear to be smaller in average diameter and lighter staining. (C) At 200 μm depth, a few large vesicular structures are visible (arrows), although the overall number of labeled structures is greatly reduced at the outer edge of the organelle-free zone. The nuclei are thin overall and sometimes irregular in width. (D) At 300 μm depth, the nuclei are thin, irregular in width and decorated with oblong brightly staining objects that appear to be attached to the DiI-stained nuclear envelope (magenta arrows). These appear prominent in part because they are not circular and there are very few vesicular structures visible. These structures are the first clear indication that there is a distinct complex that appears to be modifying the nuclei at the edge of the organelle-free zone. Note that the fiber cells are large in diameter and irregular in shape consistent with their depth from the capsule.

Fig 4. Laser scanning confocal imaging near the organelle-free zone.

(A) At 350 μm depth, the cell shape is irregular and very few vesicular organelles are present within cells. Several examples of the brightly labeled structures located at the nuclear envelope are indicated (magenta arrows), as are potential links to the plasma membrane (black arrowheads). Both these structures can be better appreciated in z-series optical sections (see S1 Fig). (B) At 500 μm depth, the nuclei are irregular in shape and are beginning to degrade. Some nuclei have bright staining close to the nuclear envelope (magenta arrows) while others do not. (C) At 650 μm depth, the cells are larger and more irregular in shape. The nuclei are disrupted with two showing objects associated with the nuclear envelope (magenta arrows, upper left). The two examples in the center have circular shapes, faint staining of associated structures (magenta arrows) and light staining of the nuclear envelope (white arrowheads). These appear to be in the final stages of breakdown of the nuclear envelope as the four examples on the lower right are small globular remnants of nuclei with no visible nuclear envelope. Two small circular dots (white arrows) may be remnants of nuclear envelope breakdown but are not consistent with the vesicular structures in previous images. (D) At 800 μm depth, the cells are very large and irregular with several globular remnants of nuclear breakdown stained brightly with 4,6-diamidino-2-phenylindole (DAPI). These are most likely the pyknotic nuclei described in the early literature.

Fig 5. Fiber cell ultrastructure near the bow region up to a depth of 300 μm.

(A) Nuclei of the bow region are present among well-defined radial cell columns. Many autophagic vesicles (arrows) are visible as well as numerous other smaller organelles. Two nuclei (N) have clear nucleoli (Nu). Day 15 lens imaged 30 μm from the capsule-fiber cell interface. (B) Just beyond the nuclei of the bow region, classical fiber cells with hexagonal 2 μm x 10 μm cross-sections in radial cell columns are observed. Autophagic vesicles (arrows) are present as are other organelles where protein production is still occurring. The dark stained globular clusters in the cytoplasm are probably polysomes. Extensive gap junctions that appear as thin dark lines on broad and narrow faces cover about 50% of the cell surface as indicated for one cell (arrowheads). Day 15 lens about 60 μm from the capsule-fiber cell interface. (C) Fiber cells just outside the organelle-free zone (OFZ) appear normal. Note the nucleus is slightly irregular in shape but otherwise normal in appearance with portions of a nucleolus visible. Autophagosomes with double membranes are indicated (yellow arrows), although they decrease in number corresponding to a decrease in membranous organelles in the cytoplasm through this region. The fiber cells have hexagonal shapes, which are somewhat irregular in size and slightly more rounded. Day 15 lens about 110 μm from the capsule-fiber cell interface. (D) Nuclei at the edge of the OFZ display irregular shapes. Note the pronounced indentation and thin region, which if projected along the nuclear length would suggest a significant reduction in nuclear volume. Irregular nuclear shapes including indentations have been reported previously [29]. Autophagic vesicles (arrows) and other organelles including mitochondria and endoplasmic reticulum are also present although quite rare. An important feature of this region is the irregular shape and rounded edges of the fiber cells. Day 15 lens about 300 μm from the capsule-fiber cell interface.

Fig 6. Indentations often reveal a large macromolecular complex adherent to the nuclear envelope.

(A) Low magnification shows a complex (arrow) that has an unusual asymmetric shape and irregular densities associated with one surface. (B) High magnification is necessary to identify the components of the complex. The core (asterisk) has a uniform texture similar to the adjacent cytoplasm (distinct from nucleoplasm, lysosomes or any type of autophagosome) and is surrounded mainly by two membranes. The outer membrane is closely adhered to the outer nuclear envelope and distorting that membrane (five central blue arrows). The other segments of the outer nuclear membrane (remaining blue arrows) have a normal association with the inner nuclear membrane. The tip is narrowed and covered by a protein layer (yellow arrow) that is 25 nm thick with repeating protein densities that are similar to clathrin and its adapter proteins. The interior of the tip contains fibers (green lines) that are similar actin microfilaments often seen in lens epithelium, fiber cell cytoplasm and elongating ball-and-socket devices. The dense bodies (arrowheads) are multilayered bilayers with 5 nm spacing typical of pure lipid (without integral proteins). Day 15 lens 300 μm from the capsule-fiber cell interface.

Fig 7. A distinct form of the degradation complex appears to attack the inner nuclear membrane.

(A) Low magnification shows a large oblong complex adjacent to the nucleus (arrow). (B) High magnification shows that the complex is nearly completely contained under the outer nuclear membrane (blue arrows). The core (asterisk) is uniform in texture and similar to the adjacent cytoplasm. The core is associated with the inner nuclear membrane and two nuclear pores (NP); however, the position of the multilamellar material suggests that the inner nuclear membrane is being modified. There is no conical tip or clathrin-like coat. A typical gap junction (GJ) is indicated, as is a cluster of thin multilamellar membranes (white arrowhead) associated with the plasma membrane. Day 15 lens about 500 μm from the capsule-fiber cell interface.

Fig 8. Complexes have been observed associated with a small number of thin multilamellar membranes within an indentation.

(A) Low magnification shows a complex (arrow) in an indentation with variable densities. (B) High magnification shows that the complex is beneath the outer nuclear membrane (blue arrows) and the presumed core (asterisk) stains darkly with similar texture to the adjacent cytoplasm. A lighter staining circular region of unknown origin bridges the nuclear envelope with a small accumulation of multilamellar membranes. The molecular events within the complex are unknown, although the proximity of the domains is consistent with breakdown of the inner nuclear membrane. An intact nuclear pore (NP) appears not to be disturbed by the presence of the complex. Day 15 lens 400 μm from the capsule-fiber cell interface.

Fig 9. Many complexes contain large aggregates of thin multilamellar membranes.

(A) Low magnification shows a large complex in a nuclear indentation with an intensely stained aggregate within the complex (white arrow). In addition, there are two extended objects adjacent to the nucleus (black arrows) that are early stages of complex formation as discussed below. (B) High magnification shows that the large dark object is an extensive collection of thin bilayer membranes (arrowheads) that most likely represent pure lipid derived from the breakdown of the nuclear envelope. Bilayer average spacing was 5.2 nm (n = 59). The complex is contained beneath the outer nuclear membrane (blue arrows). The presumed core (asterisk) is small but similar in texture to the adjacent cytoplasm and not to autophagosomes or lysosomes. An intact nuclear pore (NP) is indicated. Day 15 lens 460 μm from the capsule-fiber cell interface.

Fig 10. Extended structures appear to be early stages of complex formation.

One extended structure is adherent to the outer nuclear membrane (blue arrows) with some internal microfilaments labeled (green lines) and potential core noted (asterisk). A second extended structure is capped by a single membrane organelle that is proposed to be smooth endoplasmic reticulum (SER). The needle-like projection and the apparent cap on the core are unique. An intact gap junction (GJ) is noted, as is a nuclear pore (NP). Day 15 lens about 460 μm from the capsule-fiber cell interface.

Fig 11. Degradation complexes originate as filopodial-like projections from adjacent cells.

(A) Low magnification shows one cell sending a projection that bifurcates (white arrow) whereas three other profiles (black arrows) represent projections in different orientations and stages of development. (B) High magnification reveals that the projection from Cell 2 has two arms that have dark staining cores (asterisks) and well-defined clathrin-like caps (yellow arrows). One arm is closely associated with a single membrane flattened or tubular vesicle that appears to be smooth endoplasmic reticulum (SER). All of the cores are covered by double membranes confirming that they are derived from adjacent cells, as are many interlocking devices between fiber cells. Day 15 lens about 500 μm from the capsule-fiber cell interface.

Fig 12. Different morphologies of filopodial-like projections support the hypothesis that they generate the degradation complexes.

(A) and (B) Moderate magnification shows two projections with cores (asterisks) derived as projections from Cell 2 into Cell 1. Both are double membrane structures in which the outer membrane comes into contact with the outer nuclear membrane. Some internal microfilaments (green lines; also see S2 Fig) are visible and intact nuclear pores (NP) and gap junctions (GJ) are indicated. (C) and (D) Low and high magnification of an extended projection (also see S2 and S4 Figs) with internal microfilaments (green lines) and a clathrin-like cap showing that the projections are not simply ball-and-socket interdigitations, which are usually shorter, have a rounded bulging end and lose their clathrin coat at an early stage of formation [31]. (E) and (F) Circular double membrane profiles that are cross-sections of projections. Both examples have uniform cores (asterisks) and contact the outer nuclear membrane, with the projection making indentation in E and distorting the membrane into a triangular protrusion in F. Day 15 lenses about 600 μm from the capsule-fiber cell interface.

Fig 13. Nuclear envelope breakdown in a localized region reveals multiple staining patterns of the nucleoplasm.

(A) Low magnification shows two projections near the nucleus (arrows) that exhibits several light staining regions as if material has been removed. (B) High magnification of a light-staining region shows that the nuclear envelope has been lost (double arrows) and that the nucleoplasm aggregates into small and large particles (arrowheads), some of which may have departed the nucleoplasm to create the lighter staining zones. This structure may represent a nucleus just prior to becoming pyknotic near the organelle-free zone. Day 15 lens about 600 μm from the capsule-fiber cell interface.

Fig 14. Nuclear envelope breakdown is accompanied by variable aggregations of remaining components of the nucleoplasm.

(A) Low magnification shows multiple projections close to the nucleus (white arrows) even in the final stages of breakdown of the nuclear envelope. (B) High magnification near the center shows three projections (white arrows) adjacent to the nucleoplasm where there is no remaining nuclear envelope. The nucleoplasm displays different sized aggregates, some near 10–30 nm and others that are larger (arrowheads). Day 15 lens about 700 μm from the capsule-fiber cell interface.

Fig 15. Nuclear remnants have many configurations without the nuclear envelope.

A. and B. Two examples of globular remnants where no trace of the nuclear envelope is visible and yet the nucleoplasm forms micron sized aggregates composed of fine particles that have similar textures to the adjacent cytoplasm. Definitive structural data is not available to characterize the final stages of breakdown except to note that the larger aggregates appear to be shedding small dense particles around the surface (arrows). Note the absence of vesicles, intercellular processes and organelles, as well as the prominent irregular cell shapes in this region within the organelle-free zone. Day 15 lenses about 700 μm from the capsule-fiber cell interface.

Fig 16. Diagram illustrating the steps in the formation of the nuclear excisosome and the breakdown of nuclei based on thin section electron micrographs.

(A) Projections from adjacent cells approach an indented nucleus. Projections from Cell 2 into Cell 1 (red arrows) have a dense core containing an actin network (green lines) and a clathrin-like cap (C, yellow). A segment of smooth endoplasmic reticulum (SER, orange) is often seen adjacent to an initial projection. Mitochondria are usually present (M, tan). Based on the electron micrograph in Fig 11A with one process extended to contact the nuclear envelope. (B) Projections make contact with the outer nuclear envelope forming the initial nuclear degradation complex. The tip has a clathrin-like cap (yellow arrow) and actin microfilaments internally (green lines). The core (red) is similar to adjacent cytoplasm. The outer membrane of this double membrane complex appears to be anchored to the outer nuclear membrane (red lines and arrows) distorting the shape of the perinuclear cisternae (blue). Several multilamellar lipid-rich aggregates (black arrowheads) are located near the base of the complex. Based on the electron micrograph in Fig 6B. (C) Modifications of the complex allow close associations with the inner nuclear membrane to facilitate its degradation. Membrane rearrangements permit the core (red) to contact the nuclear envelope and be contained under the outer nuclear membrane within the perinuclear cisternae (blue). Multilamellar objects are also found within this space (black arrowheads). Nuclear pores (blue arrows) can be found within the complex. Based on electron micrograph in Fig 7B. (D) Remnants of the nucleoplasm, after the nuclear envelope has been degraded. Several processes (red) remain even in the absence of the nuclear envelope. Different types of aggregating components of the nucleoplasm (blue arrows) are observed. This type of remnant may represent a pyknotic nuclear fragment as reported in the early literature. Based on the electron micrograph in Fig 14A.