Altered stress fibers and integrin expression in the Malpighian epithelium of Drosophila type IV collagen mutants.
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SUBMITTER: Kiss AA
PROVIDER: S-EPMC4816910 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Congenital muscular dystrophy (CMD), a subgroup of myopathies is a genetically and clinically heterogeneous group of inherited muscle disorders and is characterized by progressive muscle weakness, fiber size variability, fibrosis, clustered necrotic fibers, and central myonuclei present in regenerating muscle. Type IV collagen (COL4A1) mutations have recently been identified in patients with intracerebral, vascular, renal, ophthalmologic pathologies and congenital muscular dystrophy, consistent with diagnoses of Walker-Warburg Syndrome or Muscle-Eye-Brain disease. Morphological characteristics of muscular dystrophy have also been demonstrated Col4a1 mutant mice. Yet, several aspects of the pathomechanism of COL4A1-associated muscle defects remained largely uncharacterized. Based on the results of genetic, histological, molecular, and biochemical analyses in an allelic series of Drosophila col4a1 mutants, we provide evidence that col4a1 mutations arise by transitions in glycine triplets, associate with severely compromised muscle fibers within the single-layer striated muscle of the common oviduct, characterized by loss of sarcomere structure, disintegration and streaming of Z-discs, indicating an essential role for the COL4A1 protein. Features of altered cytoskeletal phenotype include actin bundles traversing over sarcomere units, amorphous actin aggregates, atrophy, and aberrant fiber size. The mutant COL4A1-associated defects appear to recapitulate integrin-mediated adhesion phenotypes observed in RNA-inhibitory Drosophila. Our results provide insight into the mechanistic details of COL4A1-associated muscle disorders and suggest a role for integrin-collagen interaction in the maintenance of sarcomeres.
| S-EPMC6829409 | biostudies-literature
Project description:For decades, the work of cell and developmental biologists has demonstrated the striking ability of the mesenchyme and the stroma to instruct epithelial form and function in the mammary gland, but the role of extracellular matrix (ECM) molecules in mammary pattern specification has not been elucidated. Here, we show that stromal collagen I (Col-I) fibers in the mammary fat pad are axially oriented prior to branching morphogenesis. Upon puberty, the branching epithelium orients along these fibers, thereby adopting a similar axial bias. To establish a causal relationship from Col-I fiber to epithelial orientation, we embedded mammary organoids within axially oriented Col-I fiber gels and observed dramatic epithelial co-orientation. Whereas a constitutively active form of Rac1, a molecule implicated in cell motility, prevented a directional epithelial response to Col-I fiber orientation, inhibition of the RhoA/Rho-associated kinase (ROCK) pathway did not. However, time-lapse studies revealed that, within randomly oriented Col-I matrices, the epithelium axially aligns fibers at branch sites via RhoA/ROCK-mediated contractions. Our data provide an explanation for how the stromal ECM encodes architectural cues for branch orientation as well as how the branching epithelium interprets and reinforces these cues through distinct signaling processes.
| S-EPMC3705902 | biostudies-literature
Project description:The angiopoietin (Ang)/Tie2 signaling pathway is essential for maintaining vascular homeostasis, and its dysregulation is associated with several diseases. Interactions between Tie2 and α5β1 integrin have emerged as part of this control; however, the mechanism is incompletely understood. AXT107, a collagen IV-derived peptide, has strong antipermeability activity and has enabled the elucidation of this previously undetermined mechanism. Previously, AXT107 was shown to inhibit VEGFR2 and other growth factor signaling via receptor tyrosine kinase association with specific integrins. AXT107 disrupts α5β1 and stimulates the relocation of Tie2 and α5 to cell junctions. In the presence of Ang2 and AXT107, junctional Tie2 is activated, downstream survival signals are upregulated, F-actin is rearranged to strengthen junctions, and, as a result, endothelial junctional permeability is reduced. These data suggest that α5β1 sequesters Tie2 in nonjunctional locations in endothelial cell membranes and that AXT107-induced disruption of α5β1 promotes clustering of Tie2 at junctions and converts Ang2 into a strong agonist, similar to responses observed when Ang1 levels greatly exceed those of Ang2. The potentiation of Tie2 activation by Ang2 even extended to mouse models in which AXT107 induced Tie2 phosphorylation in a model of hypoxia and inhibited vascular leakage in an Ang2-overexpression transgenic model and an LPS-induced inflammation model. Because Ang2 levels are very high in ischemic diseases, such as diabetic macular edema, neovascular age-related macular degeneration, uveitis, and cancer, targeting α5β1 with AXT107 provides a potentially more effective approach to treat these diseases.
| S-EPMC6478425 | biostudies-literature
Project description:Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382-393 and α1(IV)531-543 sequences, which are binding sites for the α2β1 and α3β1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater. The galactosylation of Hyl(393) in α1(IV)382-393 and Hyl(540) and Hyl(543) in α1(IV)531-543 had a dose-dependent influence on melanoma cell adhesion that was much more pronounced in the case of α3β1 integrin binding. Molecular modeling indicated that galactosylation occurred on the periphery of α2β1 integrin interaction with α1(IV)382-393 but right in the middle of α3β1 integrin interaction with α1(IV)531-543. The possibility of extracellular deglycosylation of type IV collagen was investigated, but no β-galactosidase-like activity capable of collagen modification was found. Thus, glycosylation of collagen can modulate integrin binding, and levels of glycosylation could be altered by reduction in expression of glycosylation enzymes but most likely not by extracellular deglycosylation activity.
| S-EPMC4118119 | biostudies-literature
Project description:BackgroundSec8 is highly expressed in mammalian nervous systems and has been proposed to play a role in several aspects of neural development and function, including neurite outgrowth, calcium-dependent neurotransmitter secretion, trafficking of ionotropic glutamate receptors and regulation of neuronal microtubule assembly. However, these models have never been tested in vivo. Nervous system development and function have not been described after mutation of sec8 in any organism.ResultsWe identified lethal sec8 mutants in an unbiased forward genetic screen for mutations causing defects in development of glutamatergic Drosophila neuromuscular junctions (NMJs). The Drosophila NMJ is genetically malleable and accessible throughout development to electrophysiology and immunocytochemistry, making it ideal for examination of the sec8 mutant synaptic phenotype. We developed antibodies to Drosophila Sec8 and showed that Sec8 is abundant at the NMJ. In our sec8 null mutants, in which the sec8 gene is specifically deleted, Sec8 immunoreactivity at the NMJ is eliminated but immunoblots reveal substantial maternal contribution in the rest of the animal. Contrary to the hypothesis that Sec8 is required for neurite outgrowth or synaptic terminal growth, immunocytochemical examination revealed that sec8 mutant NMJs developed more branches and presynaptic terminals during larval development, compared to controls. Synaptic electrophysiology showed no evidence that Sec8 is required for basal neurotransmission, though glutamate receptor trafficking was mildly disrupted in sec8 mutants. The most dramatic NMJ phenotype in sec8 mutants was an increase in synaptic microtubule density, which was approximately doubled compared to controls.ConclusionSec8 is abundant in the Drosophila NMJ. Sec8 is required in vivo for regulation of synaptic microtubule formation, and (probably secondarily) regulation of synaptic growth and glutamate receptor trafficking. We did not find any evidence that Sec8 is required for basal neurotransmission.
| S-EPMC1326216 | biostudies-literature
Project description:This study aimed to investigate collagen (Coll-I, III, IV) and elastin in canine normal prostate and prostate cancer (PC) using Picrosirius red (PSR) and Immunohistochemical (IHC) analysis. Eight normal prostates and 10 PC from formalin-fixed, paraffin-embedded samples were used. Collagen fibers area was analyzed with ImageJ software. The distribution of Coll-I and Coll-III was approximately 80% around prostatic ducts and acini, 15% among smooth muscle, and 5% surrounding blood vessels, in both normal prostate and PC. There was a higher median area of Coll-III in PC when compared to normal prostatic tissue (p = 0.001 for PSR and p = 0.05 for IHC). Immunostaining for Coll-IV was observed in the basal membrane of prostate acini, smooth muscle, blood vessels, and nerve fibers of normal and PC samples. Although there was no difference in Coll-IV area between normal tissue and PC, tumors with Gleason score 10 showed absence of Coll-IV, when compared to scores 6 and 8 (p = 0.0095). Elastic fibers were found in the septa dividing the lobules and around the prostatic acini of normal samples and were statistically higher in PC compared to normal tissue (p = 0.00229). Investigation of ECM components brings new information and should be correlated with prognosis in future studies.
| S-EPMC6466295 | biostudies-literature
Project description:In Drosophila, formation of the cardiac extracellular matrix (ECM) starts during embryogenesis. Assembly and incorporation of structural proteins such as Collagen IV, Pericardin, and Laminin A, B1, and B2 into the cardiac ECM is critical to the maintenance of heart integrity and functionality and, therefore, to longevity of the animal. The cardiac ECM connects the heart tube with the alary muscles; thus, the ECM contributes to a flexible positioning of the heart within the animal's body. Moreover, the cardiac ECM holds the larval pericardial nephrocytes in close proximity to the heart tube and the inflow tract, which is assumed to be critical to efficient haemolymph clearance. Mutations in either structural ECM constituents or ECM receptors cause breakdown of the ECM network upon ageing, with disconnection of the heart tube from alary muscles becoming apparent at larval stages. Finally, the heart becomes non-functional. Here, we characterised existing and new pericardin mutants and investigated biosynthesis, secretion, and assembly of Pericardin in matrices. We identified two new pericardin alleles, which turned out to be a null (pericardin3-548) and a hypomorphic allele (pericardin3-21). Both mutants could be rescued with a genomic duplication of a fosmid coding for the pericardin locus. Biochemical analysis revealed that Pericardin is highly glycosylated and forms redox-dependent multimers. Multimer formation is remarkably reduced in animals deficient for the prolyl-4 hydroxylase cluster at 75D3-4.
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Project description:The MRN complex, composed of MRE11, RAD50 and NBS, plays important roles in responding to DNA double-strand breaks (DSBs). In metazoans, functional studies of genes encoding these proteins have been challenging because complete loss-of-function mutations are lethal at the organismal level and because NBS has multiple functions in DNA damage responses. To study functions of Drosophila NBS in DNA damage responses, we used a separation-of-function mutation that causes loss of the forkhead-associated (FHA) domain. Loss of the FHA domain resulted in hypersensitivity to ionizing radiation and defects in gap repair by homologous recombination, but had only a small effect on the DNA damage checkpoint response and did not impair DSB repair by end joining. We also found that heterozygosity for an nbs null mutation caused reduced gap repair and loss of the checkpoint response to low-dose irradiation. These findings shed light on possible sources of the cancer predisposition found in human carriers of NBN mutations.
| S-EPMC2702778 | biostudies-literature
Project description:Obesity is a complex metabolic disorder that often manifests with a strong genetic component in humans. However, the genetic basis for obesity and the accompanying metabolic syndrome is poorly defined. At a metabolic level, obesity arises from an imbalance between the nutritional intake and energy utilization of an organism. Mechanisms that sense the metabolic state of the individual and convey this information to satiety centers help achieve this balance. Mutations in genes that alter or modify such signaling mechanisms are likely to lead to either obese individuals, who in mammals are at high risk for diabetes and cardiovascular disease, or excessively thin individuals with accompanying health problems. Here we show that Drosophila mutants for an intracellular calcium signaling channel, the inositol 1,4,5-trisphosphate receptor (InsP3R) store excess triglycerides in their fat bodies and become unnaturally obese on a normal diet. Although excess insulin signaling can rescue obesity in InsP3R mutants to some extent, we show that it is not the only cause of the defect. Through mass spectrometric analysis of lipids we find that homeostasis of storage and membrane lipids are altered in InsP3R mutants. Possibly as a compensatory mechanism, InsP3R mutant adults also feed excessively. Thus, reduced InsP3R function alters lipid metabolism and causes hyperphagia in adults. Together, the metabolic and behavioral changes lead to obesity. Our results implicate altered InsP3 signaling as a previously unknown causative factor for metabolic syndrome in humans. Importantly, our studies also suggest preventive dietary interventions.
| S-EPMC3634656 | biostudies-literature
Project description:Keratinized mucosa is of fundamental importance to maintain healthy gingival tissue, and understanding the mechanisms of oral mucosa keratinization is crucial to successfully manage healthy gingiva. Previous studies have shown a strong involvement of the basement membrane in the proliferation and differentiation of epithelial cells. Therefore, first, to identify the keratinized mucosa-specific basement membrane components, immunohistochemical analysis for the six alpha chains of type IV collagen was performed in 8-week-old mice. No difference in the expression pattern of type IV collagen ?1(IV) and ?2(IV) chains was observed in the keratinized and non-keratinized mucosa. Interestingly, however, type IV collagen ?5(IV) and ?6(IV) chains specifically were strongly detected in the keratinized mucosa. To analyze the functional roles of the type IV collagen isoform ?6(IV) in oral mucosa keratinization, we analyzed Col4a6-knockout mice. Epithelial developmental delay and low levels of KRT10 were observed in new-born Col4a6-knockout mice. Additionally, in vitro experiments with loss-of function analysis using human gingival epithelial cells confirmed the important role of ?6(IV) chain in epithelial keratinization. These findings indicate that ?112:?556 (IV) network, which is the only network that includes the ?6(IV) chain, is one regulator of KRT10 expression in keratinization of oral mucosal epithelium.
| S-EPMC5805778 | biostudies-literature