Browsing by Author "Crawford, Bryan D."

Now showing 1 - 8 of 8
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    Activity-Based Labeling of Matrix Metalloproteinases in Living Vertebrate Embryos
    (PLOS, 2012) Keow, Johnathan Y.; Pond, Eric D.; Cisar, Justin S.; Cravatt, Benjamin F.; Crawford, Bryan D.
    Extracellular matrix (ECM) remodeling is a physiologically and developmentally essential process mediated by a family of zinc-dependent extracellular proteases called matrix metalloproteinases (MMPs). In addition to complex transcriptional control, MMPs are subject to extensive post-translational regulation. Because of this, classical biochemical, molecular and histological techniques that detect the expression of specific gene products provide useful but limited data regarding the biologically relevant activity of MMPs. Using benzophenone-bearing hydroxamate-based probes that interact with the catalytic zinc ion in MMPs, active proteases can be covalently ‘tagged’ by UV cross-linking. This approach has been successfully used to tag MMP-2 in vitro in tissue culture supernatants, and we show here that this probe tags proteins with mobilities consistent with known MMPs and detectable gelatinolytic activity in homogenates of zebrafish embryos. Furthermore, because of the transparency of the zebrafish embryo, UV-photocroslinking can be accomplished in vivo, and rhodamated benzophenone probe is detected in striking spatial patterns consistent with known distributions of active matrix remodeling in embryos. Finally, in metamorphosing Xenopus tadpoles, this probe can be used to biotinylate active MMP-2 by injecting it and cross-linking it in vivo, allowing the protein to be subsequently extracted and biochemically identified.
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    Characterizing the effects of poorly studied cannabinoids on larval zebrafish behaviour
    (University of New Brunswick, 2023-08) Moore, Emily Raeanne; Crawford, Bryan D.
    Cannabis sativa has been used medicinally for thousands of years. However, research on its health effects has been hindered due to prohibition and social stigma. Legalization provides Canadian researchers with the opportunity to apply modern research methods to better understand the benefits and dangers of pharmaceutical cannabinoids. The two main phytocannabinoids are tetrahydrocannabinol (THC) and cannabidiol (CBD). In addition, there are many lesser-known phytocannabinoids such as cannabinol (CBN), and cannabigerol (CBG) each with isomeric variants, which are poorly studied. In this thesis I document the behavioural effects of THC isomers as well as unexpected deleterious effects of CBD and CBN. I also bring attention to the importance of cannabinoid solubility in the context of aquatic research models. This research provides refinements to the use of zebrafish as model organisms for cannabinoid research, improves our fundamental understanding of how cannabinoids affect vertebrate behaviour, and highlights some of their potential as therapeutics.
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    Experimental Dissection of Metalloproteinase Inhibition-Mediated and Toxic Effects of Phenanthroline on Zebrafish Development
    (MDPI AG, 2016) Crawford, Bryan D.; Ellis, Tonya R.
    Metalloproteinases are zinc-dependent endopeptidases that function as primary effectors of tissue remodeling, cell-signaling, and many other roles. Their regulation is ferociously complex, and is exquisitely sensitive to their molecular milieu, making in vivo studies challenging. Phenanthroline (PhN) is an inexpensive, broad-spectrum inhibitor of metalloproteinases that functions by chelating the catalytic zinc ion, however its use in vivo has been limited due to suspected off-target effects. PhN is very similar in structure to phenanthrene (PhE), a well-studied poly aromatic hydrocarbon (PAH) known to cause toxicity in aquatic animals by activating the aryl hydrocarbon receptor (AhR). We show that zebrafish are more sensitive to PhN than PhE, and that PhN causes a superset of the effects caused by PhE. Morpholino knock-down of the AhR rescues the effects of PhN that are shared with PhE, suggesting these are due to PAH toxicity. The effects of PhN that are not shared with PhE (specifically disruption of neural crest development and angiogenesis) involve processes known to depend on metalloproteinase activity. Furthermore these PhN-specific effects are not rescued by AhR knock-down, suggesting that these are bona fide effects of metalloproteinase inhibition, and that PhN can be used as a broad spectrum metalloproteinase inhibitor for studies with zebrafish in vivo.
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    Laminin and Matrix metalloproteinase 11 regulate Fibronectin levels in the zebrafish myotendinous junction
    (BioMed Central) Jenkins, Molly H.; Alrowaished, Sarah S.; Goody, Michelle F.; Crawford, Bryan D.; Henry, Clarissa A.
    Background: Remodeling of the extracellular matrix (ECM) regulates cell adhesion as well as signaling between cells and their microenvironment. Despite the importance of tightly regulated ECM remodeling for normal muscle development and function, mechanisms underlying ECM remodeling in vivo remain elusive. One excellent paradigm in which to study ECM remodeling in vivo is morphogenesis of the myotendinous junction (MTJ) during zebrafish skeletal muscle development. During MTJ development, there are dramatic shifts in the primary components comprising the MTJ matrix. One such shift involves the replacement of Fibronectin (Fn)-rich matrix, which is essential for both somite and early muscle development, with laminin-rich matrix essential for normal function of the myotome. Here, we investigate the mechanism underlying this transition. Results: We show that laminin polymerization indirectly promotes Fn downregulation at the MTJ, via a matrix metalloproteinase 11 (Mmp11)-dependent mechanism. Laminin deposition and organization is required for localization of Mmp11 to the MTJ, where Mmp11 is both necessary and sufficient for Fn downregulation in vivo. Furthermore, reduction of residual Mmp11 in laminin mutants promotes a Fn-rich MTJ that partially rescues skeletal muscle architecture. Conclusion: These results identify a mechanism for Fn downregulation at the MTJ, highlight crosstalk between laminin and Fn, and identify a new in vivo function for Mmp11. Taken together, our data demonstrate a novel signaling pathway mediating Fn downregulation. Our data revealing new regulatory mechanisms that guide ECM remodeling during morphogenesis in vivo may inform pathological conditions in which Fn is dysregulated.
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    Matrix metalloproteinases in neural development: a phylogenetically diverse perspective
    (Wolters Kluwer Health, 2016) Small, Christopher D.; Crawford, Bryan D.
    The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases originally characterized as secreted proteases responsible for degrading extracellular matrix proteins. Their canonical role in matrix remodelling is of significant importance in neural development and regeneration, but emerging roles for MMPs, especially in signal transduction pathways, are also of obvious importance in a neural context. Misregulation of MMP activity is a hallmark of many neuropathologies, and members of every branch of the MMP family have been implicated in aspects of neural development and disease. However, while extraordinary research efforts have been made to elucidate the molecular mechanisms involving MMPs, methodological constraints and complexities of the research models have impeded progress. Here we discuss the current state of our understanding of the roles of MMPs in neural development using recent examples and advocate a phylogenetically diverse approach to MMP research as a means to both circumvent the challenges associated with specific model organisms, and to provide a broader evolutionary context from which to synthesize an understanding of the underlying biology.
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    Mouse embryonic fibroblasts accumulate differentially on titanium surfaces treated with nanosecond laser pulses
    (AVS: Science & Technology of Materials, Interfaces, and Processing, 2016) Radmanesh, Mitra; Ektasabi, Amin M.; Wyatt, Rachael A.; Crawford, Bryan D.; Kiani, Amirkianoosh
    Biomaterial engineering, specifically in bone implant and osseointegration, is currently facing a critical challenge regarding the response of cells to foreign objects and general biocompatibility of the materials used in the production of these implants. Using the developing technology of the laser surface treatment, this study investigates the effects of the laser repetition rate (frequency) on cell distribution across the surface of the titanium substrates. The main objective of this research is building a fundamental understanding of how cells interact with treated titanium and how different treatments affect cell accumulation. Cells respond differently to surfaces treated with different frequency lasers. The results of this research identify the influence of frequency on surface topography properties and oxidation of titanium, and their subsequent effects on the pattern of cell accumulation on its surface. Despite increased oxidation in laser-treated regions, the authors observe that fibroblast cells prefer untreated titanium to laser-treated regions, except the regions treated with 25 kHz pulses, which become preferentially colonized after 72 h.
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    NAD+ Biosynthesis Ameliorates a Zebrafish Model of Muscular Dystrophy
    (PLOS, 2012) Goody, Michelle F.; Kelly, Meghan W.; Reynolds, Christine J.; Khalil, Andre; Crawford, Bryan D.; Henry, Clarissa A.
    Muscular dystrophies are common, currently incurable diseases. A subset of dystrophies result from genetic disruptions in complexes that attach muscle fibers to their surrounding extracellular matrix microenvironment. Cell-matrix adhesions are exquisite sensors of physiological conditions and mediate responses that allow cells to adapt to changing conditions. Thus, one approach towards finding targets for future therapeutic applications is to identify cell adhesion pathways that mediate these dynamic, adaptive responses in vivo. We find that nicotinamide riboside kinase 2b-mediated NAD+ biosynthesis, which functions as a small molecule agonist of muscle fiber-extracellular matrix adhesion, corrects dystrophic phenotypes in zebrafish lacking either a primary component of the dystrophin-glycoprotein complex or integrin alpha7. Exogenous NAD+ or a vitamin precursor to NAD+ reduces muscle fiber degeneration and results in significantly faster escape responses in dystrophic embryos. Overexpression of paxillin, a cell adhesion protein downstream of NAD+ in this novel cell adhesion pathway, reduces muscle degeneration in zebrafish with intact integrin receptors but does not improve motility. Activation of this pathway significantly increases organization of laminin, a major component of the extracellular matrix basement membrane. Our results indicate that the primary protective effects of NAD+ result from changes to the basement membrane, as a wild-type basement membrane is sufficient to increase resilience of dystrophic muscle fibers to damage. The surprising result that NAD+ supplementation ameliorates dystrophy in dystrophin-glycoprotein complex– or integrin alpha7–deficient zebrafish suggests the existence of an additional laminin receptor complex that anchors muscle fibers to the basement membrane. We find that integrin alpha6 participates in this pathway, but either integrin alpha7 or the dystrophin-glycoprotein complex is required in conjunction with integrin alpha6 to reduce muscle degeneration. Taken together, these results define a novel cell adhesion pathway that may have future therapeutic relevance for a broad spectrum of muscular dystrophies.
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    YIGSR domain of laminin binds surface receptors of mesenchyme and stimulates migration during gastrulation in sea urchins
    (The Company of Biologists, 1994) Crawford, Bryan D.; Burke, Robert D.
    During gastrulation in sea urchins, cells at the tip of the archenteron extend filopodia that attach to the blastocoel wall and are thought to assist in the elongation of the archenteron. Upon completion of gastrulation, these cells migrate into the blastocoel. Time-lapse video records were made of preparations from which ectodermal cells have been removed, leaving the archenteron, mesenchyme cells and blastocoelar extracellular matrix (ECM) bounded by the basal lamina. In preparations of late gastrulae, cells at the tip of the archenteron extend filopodia that attach to the basal lamina and pull it inward, collapsing the preparation. This collapse does not occur in preparations made prior to the elongation phase and can be inhibited with cytochalasin B and azide, but not with colchicine. Migratory behavior increased in preparations treated with the laminin-derived peptide Tyr-Ile-Gly-Ser- Arg (YIGSR). Cells extend and retract filopodia, collapse the ECM and migrate out of the preparation. This behavior was not observed in preparations treated with whole laminin, fibronectin or Arg-Gly- Asp-Ser (RGDS) peptides. Cells in preparations treated with YIGSR extend significantly more processes than those incubated in RGDS, laminin, fibronectin or BSA. This effect is titratable between 10–3 and 10–6 M. Whole laminin has a significant inhibitory effect on the number of cell processes observed. Double labelling experiments with biotinylated laminin or biotinylated CDPGYIGSR and a mesenchyme-specific monoclonal antibody (Sp12) reveal that laminin and CDPGYIGSR label mesenchymal and non-mesenchymal cells. A CDPGYIGSR affinity column binds a 125I-labelled cell surface component, which elutes with YIGSR and has an Mr of about 80x103 on SDS-PAGE. We propose that cells at the tip of the archenteron attach to the basal lamina during archenteron elongation, and that domains of laminin containing YIGSR in the basal lamina of the target region stimulate migratory behavior in these cells.