Autophagy and mitophagy participate in ocular lens organelle degradation - PubMed (original) (raw)

Comparative Study

Autophagy and mitophagy participate in ocular lens organelle degradation

M Joseph Costello et al. Exp Eye Res. 2013 Nov.

Abstract

The eye lens consists of a layer of epithelial cells that overlay a series of differentiating fiber cells that upon maturation lose their mitochondria, nuclei and other organelles. Lens transparency relies on the metabolic function of mitochondria contained in the lens epithelial cells and in the immature fiber cells and the programmed degradation of mitochondria and other organelles occurring upon lens fiber cell maturation. Loss of lens mitochondrial function in the epithelium or failure to degrade mitochondria and other organelles in lens fiber cells results in lens cataract formation. To date, the mechanisms that govern the maintenance of mitochondria in the lens and the degradation of mitochondria during programmed lens fiber cell maturation have not been fully elucidated. Here, we demonstrate using electron microscopy and dual-label confocal imaging the presence of autophagic vesicles containing mitochondria in lens epithelial cells, immature lens fiber cells and during early stages of lens fiber cell differentiation. We also show that mitophagy is induced in primary lens epithelial cells upon serum starvation. These data provide evidence that autophagy occurs throughout the lens and that mitophagy functions in the lens to remove damaged mitochondria from the lens epithelium and to degrade mitochondria in the differentiating lens fiber cells for lens development. The results provide a novel mechanism for how mitochondria are maintained to preserve lens metabolic function and how mitochondria are degraded upon lens fiber cell maturation.

Keywords: ATG5; FYCO1; FYVE and coiled coil domain containing 1; LC3B; OFZ; Pik3c3; TOM20; autophagy; autophagy-related 5; cataract; differentiation; electron microscopy; lens; microtubule-associated protein 1 light chain 3B; mitophagy; organelle free zone; phosphatidylinositol 3-kinase, catalytic subunit type 3; translocase of the outer mitochondrial membranes 20 kDa.

PubMed Disclaimer

Figures

Figure 1. Autophagy and mitophagy in adult human lens epithelial cells

(A) Overview of lens surface from the 22 yo donor lens from India showing the capsule (c), epithelium (e) and fiber cells (f). (B) Two cells of the epithelium, separated by paired plasma membranes, display nuclei (n) and numerous autophagic vesicles within the epithelial cytoplasm (arrowheads). At the epithelial-fiber cell interface fiber cells (f) are stained darkly. The highlighted region is enlarged in (C). (C) An intact mitochondrion (m) is adjacent to an autophagic vesicle containing mitochondrial fragments (arrows). This is a clear example of mitophagy. (D) An autophagic vesical containing a partially degraded mitochondrion (m; based on the cylindrical profile about 0.2 μm × 0.7 μm). (E) An autophagic vesicle probably containing a mitochondrion (m) and a multilamellar membrane aggregate found in many autophagic vesicles (5 nm average membrane thickness). Scale bars: A = 5 μm; B = 0.5 μm; C = 200 nm; D, E = 100 nm.

Figure 2. Autophagy and mitophagy in adult human lens cortical fiber cells

(A) Overview of the outer cortical fiber cells from the 22 yo donor lens from India. Two nuclei of the epithelium and one from a fiber cell are labeled (n). Two autophagic vesicles in the epithelium are marked and five from the fiber cells (arrows). Note the dark staining of the initial layers of elongating fiber cells and the relatively light staining of most of the fiber cells. The density of autophagic vesicles in this region, confirmed at high magnification, is about 16 per a unit area (of about seven fiber cells in cross-section) and drops to less than 5 by 60 μm from the epithelium. (B) An autophagic vesicle between two adjacent fiber cells indicated by the paired membranes at the top and bottom of the image. The limiting membrane of the autophagic vesicle is indicated (arrowhead) and surrounds distinct components including multilamellar membranes (dark staining), vesicles and cell fragments composed of membranes and dispersed proteins. (C) The autophagic vesicle contains multilamellar membranes (dark staining) and a fragment of a mitochondrion (cristae are labeled, arrows). This is an example of mitophagy. (D) A large autophagic vesicle with properties similar to those in (B) and (C) containing a band of multilamellar membranes, dispersed protein in the center, an elongated mitochondrion (arrows) and a smaller mitochondrial fragment in contact with the multilamellar aggregate (cristae are labeled, arrowhead). (E) An autophagic vesicle with an oval core containing protein packed more densely than the adjacent cytoplasm and partially surrounded by tightly packed multiple membranes. (F) An autophagic vesicle near the interface between two fiber cells (Cells 1 and 2) containing a dense uniform core (more dense than the adjacent cytoplasm) and loosely packed multiple membranes. From a 92 yo donor lens from India at a location in the equatorial plane about 120 μm from the capsule. Scale bars: A = 2 μm; B-E = 100 nm; F = 200 nm.

Figure 3. Autophagy and mitophagy in day 12 chick embryo lens

(A) Overview of capsule (c) and epithelium (e) of the embryonic chick lens. Two nuclei (n) are indicated from the epithelium, which has multiple layers and many vacuoles (v) typical of the complex avian developing epithelium. Numerous mitochondria and mitophagic vesicles (arrows) are present in the epithelium and in the early fiber cells (f). Large empty vesicles (arrowhead) are also common but may not be autophagic as their perimeter and contents cannot easily be identified. (B) Just beneath the epithelium are developing fiber cells with nuclei (n), large vesicles (arrowhead) and numerous autophagic particles (arrows), eight of which are indicated out of more than twenty visible at higher magnification. This region contains about 25 autophagic vesicles per unit area. (C) About 160 μm deep in the cortex, the fiber cells are clearly defined from their hexagonal shape and nuclei (n). Very few autophagic vesicles (arrow) are present here indicating a pronounced gradient of autophagy through the cortex reducing to less than 5 per unit area by 250 μm from the epithelium. (D) An intact mitochondrion (arrow) is adjacent to a portion of ER with a few ribosomes (arrowheads) from the epithelium (as in A). (E) An autophagic vesicle from the epithelium containing a mitochondrion (arrows) adjacent to multilayered membranes (5 nm average membrane thickness). (F) An autophagic vesicle containing a degrading mitochondrion having one clearly visible cristae (arrows). Images F-I are from young cortical fiber cells as in B where autophagy and mitophagy are very active. (G) An autophagic vesicle most likely containing a cluster of partially degraded mitochondrial cristae (arrow). (H) An autophagic vesicle similar to F except several smaller segments of cristae are visible (arrows). (I) An autophagic vesicle containing a degrading mitochondrion in the upper region with cristae (arrows) and the lower region contains degrading multilamellar membranes. (J) An autophagic vesicle adjacent to an intact mitochondrion (arrow) in the fiber cell displayed in C. (K) An autophagic vesicle containing a dense core of protein surrounded my multiple membrane layers (arrow) from the same region of fiber cells as in C. Scale bars: A = 2 μm; B, C = 1 μm; D-K = 100 nm.

Figure 4. Co-Localization of the autophagy marker LC3B and the mitochondrial marker TOM20 in the E12 embryonic chick lens

Co-localization of TOM20 (green) and LC3B (red) puncta and overlay of the two images (orange/yellow) in E12 chick lenses using confocal fluorescent microscopy. (A) A 20 μm midsagittal section from an E12 whole embryonic chicken lens stained for TOM20 (green), LC3B (red) and nuclear stain TO-PRO-3 (blue). (B) FP (cortical fiber) region of the chick lens and (C). FC (central fiber core) region of the chick lens. (D) EQ (equatorial region) of the chick lens. Fig. 4D is valuable for demonstrating (a) multiple layers of nuclei in the epithelium consistent with the TEM in Fig. 3, (b) the great amount of autophagic vesicles (LC3B dots) and mitochondria (TOM20 dots) in the equatorial region, (c) correspondence of the dots in the overlay suggesting many of the mitochondria are undergoing autophagy, and (d) there is more autophagy here than in the core (Fig. 4C) suggesting a radial decrease in autophagy from the epithelium to the core consistent with the gradient found in the TEM images (Fig. 3). Arrows indicate TOM20 green and LC3B red puncta and their co-localization (orange/yellow color in overlayed images) in each sub-compartment of the lens examined.

Figure 4. Co-Localization of the autophagy marker LC3B and the mitochondrial marker TOM20 in the E12 embryonic chick lens

Figure 5. Co-Localization of the autophagy marker LC3B and the mitochondrial marker TOM20 in control and serum-starved chick lens epithelial cells treated with chloroquine

Chick lens primary cells stained for TOM20 (green) and LC3B (red) in cells maintained in complete media and cells that were serum-starved for 2 h with the addition of 50 μM chloroquine to both. Images were obtained using fluorescent confocal microscopy. Two areas with large numbers of perinuclear localized LC3B positive mitochondria (yellow puncta) are shown at higher magnification.

References

1. Bassnett S. Mitochondrial dynamics in differentiating fiber cells of the mammalian lens. Curr. Eye Res. 1992;11:1227–32. - PubMed
1. Bassnett S. The fate of the Golgi apparatus and the endoplasmic reticulum during lens fiber cell differentiation. Invest Ophthalmol Vis Sci. 1995;36:1793–803. - PubMed
1. Bassnett S. On the mechanism of organelle degradation in the vertebrate lens. Exp Eye Res. 2009;88:133–39. - PMC - PubMed
1. Bassnett S, Shi Y, Vrensen GF. Biological glass: structural determinants of eye lens transparency. Philos Trans R Soc Lond B Biol Sci. 2011;366:1250–64. - PMC - PubMed
1. Bloemendal H, editor. Molecular and cellular biology of the eye lens. Wiley; New York: 1981.

Autophagy and mitophagy participate in ocular lens organelle degradation - PubMed (original) (raw)