Temporary Disruption of the Retinal Basal Lamina and Its Effect on Retinal Histogenesis

AbstractAn experimental paradigm was devised to remove the retinal basal lamina for defined periods of development: the basal lamina was dissolved by injecting collagenase into the vitreous of embryonic chick eyes, and its regeneration was induced by a chase with mouse laminin-1 and α2-macroglobulin. The laminin-1 was essential in reconstituting a new basal lamina and could not be replaced by laminin-2 or collagen IV, whereas the macroglobulin served as a collagenase inhibitor that did not directly contribute to basal lamina regeneration. The regeneration occurred within 6 h after the laminin-1 chase by forming a morphologically complete basal lamina that included all known basal lamina proteins from chick embryos, such as laminin-1, nidogen-1, collagens IV and XVIII, perlecan, and agrin. The temporary absence of the basal lamina had dramatic effects on retinal histogenesis, such as an irreversible retraction of the endfeet of the neuroepithelial cells from the vitreal surface of the retina, the formation of a disorganized ganglion cell layer with an increase in ganglion cells by 30%, and the appearance of multiple retinal ectopias. Finally, basal lamina regeneration was associated with aberrant axons failing to correctly enter the optic nerve. The present data demonstrate that a transient disruption of the basal lamina leads to dramatic and probably irreversible aberrations in the histogenesis in the developing central nervous system

Tip60-mediated acetylation activates transcription independent apoptotic activity of Abl

<p>Abstract</p> <p>Background</p> <p>The proto-oncogene, c-Abl encodes a ubiquitously expressed tyrosine kinase that critically governs the cell death response induced by genotoxic agents such as ionizing radiation and cisplatin. The catalytic function of Abl, which is essential for executing DNA damage response (DDR), is normally tightly regulated but upregulated several folds upon IR exposure due to ATM-mediated phosphorylation on S465. However, the mechanism/s leading to activation of Abl's apoptotic activity is currently unknown.</p> <p>Results</p> <p>We investigated the role of acetyl modification in regulating apoptotic activity of Abl and the results showed that DNA strand break-inducing agents, ionizing radiation and bleomycin induced Abl acetylation. Using mass spectrophotometry and site-specific acetyl antibody, we identified Abl K921, located in the DNA binding domain, and conforming to one of the lysine residue in the consensus acetylation motif (<b>K</b>XXK--X3-5--SGS) is acetylated following DNA damage. We further observed that the S465 phosphorylated Abl is acetyl modified during DNA damage. Signifying the modification, cells expressing the non acetylatable K921R mutant displayed attenuated apoptosis compared to wild-type in response to IR or bleomycin treatment. WT-Abl induced apoptosis irrespective of new protein synthesis. Furthermore, upon γ-irradiation K921R-Abl displayed reduced chromatin binding compared to wild type. Finally, loss of Abl K921 acetylation in Tip60-knocked down cells and co-precipitation of Abl with Tip60 in DNA damaged cells identified Tip60 as an Abl acetylase.</p> <p>Conclusion</p> <p>Collective data showed that DNA damage-induced K921 Abl acetylation, mediated by Tip60, stimulates transcriptional-independent apoptotic activity and chromatin-associative property thereby defining a new regulatory mechanism governing Abl's DDR function.</p

The Bi-Functional Organization of Human Basement Membranes

The current basement membrane (BM) model proposes a single-layered extracellular matrix (ECM) sheet that is predominantly composed of laminins, collagen IVs and proteoglycans. The present data show that BM proteins and their domains are asymmetrically organized providing human BMs with side-specific properties: A) isolated human BMs roll up in a side-specific pattern, with the epithelial side facing outward and the stromal side inward. The rolling is independent of the curvature of the tissue from which the BMs were isolated. B) The epithelial side of BMs is twice as stiff as the stromal side, and C) epithelial cells adhere to the epithelial side of BMs only. Side-selective cell adhesion was also confirmed for BMs from mice and from chick embryos. We propose that the bi-functional organization of BMs is an inherent property of BMs and helps build the basic tissue architecture of metazoans with alternating epithelial and connective tissue layers

Regulation of the autophagy protein LC3 by phosphorylation

PKA puts the brakes on autophagy by inhibiting LC3 recruitment to autophagosomes

Proteomic View of Basement Membranes from Human Retinal Blood Vessels, Inner Limiting Membranes, and Lens Capsules

Basement membranes (BMs) are extracellular matrix sheets comprising the laminins, type-IV collagens, nidogens, and the heparan sulfate proteoglycans, perlecan, collagen XVIII, and agrin. In intact BMs, BM proteins are physiologically insoluble and partially resistant to proteolytic digestion, making BMs a challenge to study. Here three types of BMs from adult human eyes, the inner limiting membrane (ILM), the retinal vascular BMs, and the lens capsule, were isolated for analysis by 1D-SDS-PAGE and LC–MS/MS. Peptide and protein identifications were done using MaxQuant. 1129 proteins were identified with a 1% false discovery rate. Data showed that BMs are composed of multiple laminins, collagen IVs, nidogens, and proteoglycans. The dominant laminin family member in all BMs was laminin α5β2γ1. The dominant collagen IV trimer in lens capsule (LC) and blood vessel (BV) BMs had a chain composition of α1­(IV)₂, α2 (IV), whereas the dominant collagen IV in the ILM had the α3­(IV), α4­(IV), α5­(IV) chain composition. The data also showed that the ratio of laminin and collagen IVs varied among different BM types: the ratio of collagen IV to the other BM proteins is highest in LC, followed by BV and lowest for the ILM. The data have been deposited to the ProteomeXchange with identifier PXD001025

Proteolysis of Rad17 by Cdh1/APC regulates checkpoint termination and recovery from genotoxic stress

Recent studies have shown a critical function for the ubiquitin-proteasome system (UPS) in regulating the signalling network for DNA damage responses and DNA repair. To search for new UPS targets in the DNA damage signalling pathway, we have carried out a non-biased assay to identify fast-turnover proteins induced by various types of genotoxic stress. This endeavour led to the identification of Rad17 as a protein exhibiting a distinctive pattern of upregulation followed by subsequent degradation after exposure to UV radiation in human primary cells. Our characterization showed that UV-induced Rad17 oscillation is mediated by Cdh1/APC, a ubiquitin-protein ligase. Studies using a degradation-resistant Rad17 mutant demonstrated that Rad17 stabilization prevents the termination of checkpoint signalling, which in turn attenuates the cellular re-entry into cell-cycle progression. The findings provide an insight into how the proteolysis of Rad17 by Cdh1/APC regulates the termination of checkpoint signalling and the recovery from genotoxic stress

AFM testing of the two surfaces of the ILM (A–C), the DM (D–F) and LC (G–I).

<p>The AFM imaging mode shows the morphological differences between the retinal (Re)/epithelial (Ep)/endothelial (En) surfaces and the vitreal (Vi)/stromal (St) surfaces of the ILM, DM and the LC. The graphs in (C, F, I) show the quantification of the stiffness measurements obtained by AFM “forced indentation”. The measurements were obtained by probing five ILMs, three DMs and three LCs. The epithelial surfaces of all tested BMs were about twice stiffer than the stromal surfaces. The differences were statistically significant. Scale bar: 10 µm.</p

Isolated human ILM (A), Descemet’s membrane (DM, E) and lens capsule (LC, G) as they appear under a dissecting microscope using dark field.

<p>All BMs are transparent and rolled up, whereby the epithelial surfaces are facing the exterior and the stromal surfaces is facing the interior of the rolled-up BM sheets. SEM micrographs of a folded ILM (B), a DM (F) and a LC (H) show the two surfaces of the BMs. As shown in panel (B), the irregular retinal (R) surface of the ILM can be readily distinguished from the smooth vitreal surface (V), consistent with the morphological differences seen in TEM micrographs of crossections of ILMs in situ (C) and isolated ILMs (D). The endothelial (En) surface of the DM and epidermal (Ep) surface of the LC are indistinguishable from their stromal or vitreal (V) surfaces (F, H). Scale bars: C, D: 1 µm; B, F; H: 10 µm.</p