Abstract
Matrix remodeling is a consequence of tightly regulated matrix metalloproteinase (MMP) activity. MMPs are synthesized as inactive precursors with auto-inhibitory N-terminal propeptides, the proteolytic removal of which exposes the catalytic zinc ion, rendering the protease active. The regulation of MMP activation has been investigated primarily in tissue culture and biochemical assays that lack important biological context. Here we present the epitope-mediated MMP activation (EMMA) assay and use it to observe the activation of Mmp2 (gelatinase A) by endogenous mechanisms in the intact zebrafish embryo. The hemagglutinin (HA) and GFP-tagged reporter construct becomes activated on the surface of specific cells and this activation is abolished by broad-spectrum inhibition of metalloproteinase activity, consistent with existing models of gelatinase A activation. The mechanism(s) acting on the construct are spatially restricted, metalloproteinase-dependent and replacing the HA tag with mCherry abolishes activation, showing that the mechanism(s) are sensitive to the structure of the N-terminal domain. The construct is activated strongly in maturing myotome boundaries, but also intracellularly within myofibrils, consistent with reports implicating this protease in muscle development and function. In addition to general-purpose tools for the production of “EMMAed” MMPs and other proteins, we have established a transgenic line of zebrafish expressing EMMAedMmp2 under control of an inducible promoter to facilitate further investigation into the regulation of this ubiquitous ECM-remodeling protease in vivo.
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Abbreviations
- ECM:
-
Extracellular matrix
- EMMA:
-
Epitope-mediated MMP activation
- HA:
-
Hemagglutinin
- GFP:
-
Green fluorescent protein
- hpf:
-
Hours post-fertilization
- hpHS:
-
Hours post-heat shock
- MMP:
-
Matrix metalloproteinase
- TIMP:
-
Tissue inhibitor of metalloproteinases
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Acknowledgements
This work was supported by a Discovery Grant (341540) to BDC from the Natural Sciences and Engineering Research Council (NSERC) of Canada and the New Brunswick Innovation Foundation (NBIF) STEM Scholarship to EJJ. We thank Rachael Wyatt for her assistance in screening the stable transgenic line, image processing, and for providing feedback on this manuscript. We also thank two anonymous reviewers for valuable constructive criticisms of the manuscript. Finally, we gratefully acknowledge Robyn O’Keefe and Robyn Shortt for their dedication to and maintenance of the University of New Brunswick Zebrafish Aquatic Facility.
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Jeffrey, E.J., Crawford, B.D. The epitope-mediated MMP activation assay: detection and quantification of the activation of Mmp2 in vivo in the zebrafish embryo. Histochem Cell Biol 149, 277–286 (2018). https://doi.org/10.1007/s00418-018-1634-4
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DOI: https://doi.org/10.1007/s00418-018-1634-4