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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 ?m 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 ?m 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 ?m 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.

SUBMITTER: Costello MJ

PROVIDER: S-EPMC4990417 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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

Costello M Joseph MJ Brennan Lisa A LA Mohamed Ashik A Gilliland Kurt O KO Johnsen Sönke S Kantorow Marc M

PloS one 20160818 8

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 ...[more]

PMID: 27536868

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Project description:Background: FGF signaling controls numerous processes during cell lineage specification, organogenesis and terminal differentiation. In lens, FGF signaling was implicated as the key pathway that controls lens fiber cell differentiation, but little is known about its full range and spectrum of regulated genes. Results: Herein, we employed rat lens epithelial explant system and performed RNA and microRNA expression profiling in cells induced to differentiate by FGF2. The primary data were collected at explants grown overnight in the presence of 5 ng/ml of FGF2, followed by a treatment with 100 ng/ml of FGF2 and collection of samples at 2, 4, 12 and 24 hours. Global analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on microRNAs (miRNAs). We identified a total number of 131 FGF2-regulated miRNAs. Forty-four of these microRNAs had at least two predicted and inversely regulated target RNA molecules. The genes regulated by the highest number of miRs include Nfib, Nfat5, c-Maf, Ets1 and N-Myc, all encoding DNA-binding transcription factors. Analysis of RNA data revealed that activated FGF signaling influenced other major signaling pathways known to regulate lens differentiation including BMP/TGF-M-NM-2, Notch, and Wnt signaling. In the M-bM-^@M-^\earlyM-bM-^@M-^] response phase (2-4 hours), miRNAs targeted expression of batteries of genes that control transcription, cell death, cell proliferation, cell junction, and protein serine/threonine kinase activity. In M-bM-^@M-^\lateM-bM-^@M-^] stages (12-24 hours), the main miRNA targets included regulators of cell cycle arrest and cellular differentiation. Specific miRNA:mRNA interaction networks were identified for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Dicer1, Fbx33 and Wdr47 (RNA processing); Ash1l, Med1/PBP and Kdm5b (chromatin remodeling); and c-Maf, Ets1 and Stc1 (FGF signaling). MicroRNAs including miR-9, -143, -155, -455 and -543 downregulated expression of c-Maf in the 3M-bM-^@M-^Y-UTR luciferase reporter asssays. The functional requirement for miRNAs in lens was further demonstrated via disrupted lens fiber cell differentiation in lenses with inactivated Dicer1. Conclusions: These studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and identified novel gene regulatory networks (GRNs) connected by multiple miRNAs. Differential gene expression of FGF2-treated lens cell cultures was analyzed at 0, 2, 4, 12 and 24 hours following the treatment. Two biological replicate experiments were performed.

Project description:Background: FGF signaling controls numerous processes during cell lineage specification, organogenesis and terminal differentiation. In lens, FGF signaling was implicated as the key pathway that controls lens fiber cell differentiation, but little is known about its full range and spectrum of regulated genes. Results: Herein, we employed rat lens epithelial explant system and performed RNA and microRNA expression profiling in cells induced to differentiate by FGF2. The primary data were collected at explants grown overnight in the presence of 5 ng/ml of FGF2, followed by a treatment with 100 ng/ml of FGF2 and collection of samples at 2, 4, 12 and 24 hours. Global analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on microRNAs (miRNAs). We identified a total number of 131 FGF2-regulated miRNAs. Forty-four of these microRNAs had at least two predicted and inversely regulated target RNA molecules. The genes regulated by the highest number of miRs include Nfib, Nfat5, c-Maf, Ets1 and N-Myc, all encoding DNA-binding transcription factors. Analysis of RNA data revealed that activated FGF signaling influenced other major signaling pathways known to regulate lens differentiation including BMP/TGF-β, Notch, and Wnt signaling. In the “early” response phase (2-4 hours), miRNAs targeted expression of batteries of genes that control transcription, cell death, cell proliferation, cell junction, and protein serine/threonine kinase activity. In “late” stages (12-24 hours), the main miRNA targets included regulators of cell cycle arrest and cellular differentiation. Specific miRNA:mRNA interaction networks were identified for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Dicer1, Fbx33 and Wdr47 (RNA processing); Ash1l, Med1/PBP and Kdm5b (chromatin remodeling); and c-Maf, Ets1 and Stc1 (FGF signaling). MicroRNAs including miR-9, -143, -155, -455 and -543 downregulated expression of c-Maf in the 3’-UTR luciferase reporter asssays. The functional requirement for miRNAs in lens was further demonstrated via disrupted lens fiber cell differentiation in lenses with inactivated Dicer1. Conclusions: These studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and identified novel gene regulatory networks (GRNs) connected by multiple miRNAs. Differential gene expression of FGF2-treated lens cell cultures was analyzed at 0, 2, 4, 12 and 24 hours following the treatment.

Project description:Elucidation of both the molecular composition and organization of the ocular lens is a prerequisite to understand its development, function, pathology, regenerative capacity, as well as to model lens development and disease using in vitro differentiation of pluripotent stem cells. Lens is comprised of the anterior lens epithelium and posterior lens fibers, which form the bulk of the lens. Lens fibers differentiate from lens epithelial cells through cell cycle exit-coupled differentiation that includes cellular elongation, accumulation of crystallins, cytoskeleton and membrane remodeling, and degradation of organelles within the central region of the lens. Here, we profiled spatiotemporal expression dynamics of both mRNAs and non-coding RNAs from microdissected mouse nascent lens epithelium and lens fibers at four developmental time points (embryonic [E] day 14.5, E16.5, E18.5, and P0.5) by RNA-seq. During this critical time window, multiple complex biosynthetic and catabolic processes generate the molecular and structural foundation for lens transparency. Throughout this developmental window, 3544 and 3518 genes show consistently and significantly greater expression in the nascent lens epithelium and fibers, respectively. Comprehensive data analysis confirmed major roles of FGF-MAPK, Wnt/β-catenin, PI3K/AKT, TGF-β, and BMP signaling pathways and revealed significant novel contributions of mTOR, EIF2, EIF4, and p70S6K signaling in lens formation. Unbiased motif analysis within promoter regions of these genes with consistent expression changes between epithelium and fiber cells revealed an enrichment for both established (e.g. E2Fs, Etv5, Hsf4, c-Maf, MafG, MafK, N-Myc, and Pax6) transcription factors and a number of novel regulators of lens formation, such as Arntl2, Dmrta2, Stat5a, Stat5b, and Tulp3. In conclusion, the present RNA-seq data serves as a comprehensive reference resource for deciphering molecular principles of normal mammalian lens differentiation, mapping a full spectrum of signaling pathways and DNA-binding transcription factors operating in both lens compartments, and predicting novel pathways required to establish lens transparency.

Dataset Information

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

Publications

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

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