Post-translational activation of Mmp2 correlates with patterns of active collagen degradation during the development of the zebrafish tail.

Matrix metalloproteinase-2 (a.k.a. Gelatinase A, or Mmp2 in zebrafish) is known to have roles in pathologies such as arthritis, in which its function is protective, as well as in cancer metastasis, in which it is activated as part of the migration and invasion of metastatic cells. It is also required during development and the regeneration of tissue architecture after wound healing, but its roles in tissue remodelling are not well understood. Gelatinase A is activated post-translationally by proteolytic cleavage, making information about its transcription and even patterns of protein accumulation difficult to relate to biologically relevant activity. Using a transgenic reporter of endogenous Mmp2 activation in zebrafish, we describe its accumulation and post-translational proteolytic activation during the embryonic development of the tail. Though Mmp2 is expressed relatively ubiquitously, it seems to be active only at specific locations and times. Mmp2 is activated robustly in the neural tube and in maturing myotome boundaries. It is also activated in the notochord during body axis straightening, in patches scattered throughout the epidermal epithelium, in the gut, and on cellular protrusions extending from mesenchymal cells in the fin folds. The activation of Mmp2 in the notochord, somite boundaries and fin folds associates with collagen remodelling in the notochord sheath, myotome boundary ECM and actinotrichia respectively. Mmp2 is likely an important effector of ECM remodelling during the morphogenesis of the notochord, a driving structure in vertebrate development. It also appears to function in remodelling the ECM associated with growing epithelia and the maturation of actinotrichia in the fin folds, mediated by mesenchymal cell podosomes.

Early, nonlethal ploidy and genome size quantification using confocal microscopy in zebrafish embryos.

Ploidy transitions through whole genome duplication have shaped evolution by allowing the sub- and neo-functionalization of redundant copies of highly conserved genes to express novel traits. The nuclear:cytoplasmic (n:c) ratio is maintained in polyploid vertebrates resulting in larger cells, but body size is maintained by a concomitant reduction in cell number. Ploidy can be manipulated easily in most teleosts, and the zebrafish, already well established as a model system for biomedical research, is therefore an excellent system in which to study the effects of increased cell size and reduced cell numbers in polyploids on development and physiology. Here we describe a novel technique using confocal microscopy to measure genome size and determine ploidy non-lethally at 48 h post-fertilization (hpf) in transgenic zebrafish expressing fluorescent histones. Volumetric analysis of myofiber nuclei using open-source software can reliably distinguish diploids and triploids from a mixed-ploidy pool of embryos for subsequent experimentation. We present an example of this by comparing heart rate between confirmed diploid and triploid embryos at 54 hpf.

The epitope-mediated MMP activation assay: detection and quantification of the activation of Mmp2 in vivo in the zebrafish embryo.

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.

Experimental Dissection of Metalloproteinase Inhibition-Mediated and Toxic Effects of Phenanthroline on Zebrafish Development.

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.

NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy.

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.

Mmp25ß facilitates elongation of sensory neurons during zebrafish development.

Matrix metalloproteinases (MMPs) are a large and complex family of zinc-dependent endoproteinases widely recognized for their roles in remodeling the extracellular matrix (ECM) during embryonic development, wound healing, and tissue homeostasis. Their misregulation is central to many pathologies, and they have therefore been the focus of biomedical research for decades. These proteases have also recently emerged as mediators of neural development and synaptic plasticity in vertebrates, however, understanding of the mechanistic basis of these roles and the molecular identities of the MMPs involved remains far from complete. We have identified a zebrafish orthologue of mmp25 (a.k.a. leukolysin; MT6-MMP), a membrane-type, furin-activated MMP associated with leukocytes and invasive carcinomas, but which we find is expressed by a subset of the sensory neurons during normal embryonic development. We detect high levels of Mmp25ß expression in the trigeminal, craniofacial, and posterior lateral line ganglia in the hindbrain, and in Rohon-Beard cells in the dorsal neural tube during the first 48 h of embryonic development. Knockdown of Mmp25ß expression with morpholino oligonucleotides results in larvae that are uncoordinated and insensitive to touch, and which exhibit defects in the development of sensory neural structures. Using in vivo zymography, we observe that Mmp25ß morphant embryos show reduced Type IV collagen degradation in regions of the head traversed by elongating axons emanating from the trigeminal ganglion, suggesting that Mmp25ß may play a pivotal role in mediating ECM remodeling in the vicinity of these elongating axons.

Mechanisms of cytosolic targeting of matrix metalloproteinase-2.

Matrix metalloproteinase-2 (MMP-2) is best understood for its biological actions outside the cell. However, MMP-2 also localizes to intracellular compartments and the cytosol where it has several substrates, including troponin I (TnI). Despite a growing list of cytosolic substrates, we currently do not know the mechanism(s) that give rise to the equilibrium between intracellular and secreted MMP-2 moieties. Therefore, we explored how cells achieve the unique distribution of this protease. Our data show that endogenous MMP-2 targets inefficiently to the endoplasmic reticulum (ER) and shows significant amounts in the cytosol. Transfection of canonical MMP-2 essentially reproduces this targeting pattern, suggesting it is the quality of the MMP-2 signal sequence that predominantly determines MMP-2 targeting. However, we also found that human cardiomyocytes express an MMP-2 splice variant which entirely lacks the signal sequence. Like the fraction of ER-excluded, full-length MMP-2, this variant MMP-2 is restricted to the cytosol and specifically enhances TnI cleavage upon hypoxia-reoxygenation injury in cardiomyocytes. Together, our findings describe for the first time a set of mechanisms that cells utilize to equilibrate MMP-2 both in the extracellular milieu and intracellular, cytosolic locations. Our results also suggest approaches to specifically investigate the overlooked intracellular biology of MMP-2.

Matrix Metalloproteinase 13 Activity is Required for Normal and Hypoxia-Induced Precocious Hatching in Zebrafish Embryos.

Hypoxia induces precocious hatching in zebrafish, but we do not have a clear understanding of the molecular mechanisms regulating the activation of the hatching enzyme or how these mechanisms trigger precocious hatching under unfavorable environmental conditions. Using immunohistochemistry, pharmacological inhibition of matrix metalloproteinase 13 (Mmp13), and in vivo zymography, we show that Mmp13a is present in the hatching gland just as embryos become hatching competent and that Mmp13a activity is required for both normal hatching and hypoxia-induced precocious hatching. We conclude that Mmp13a likely functions in activating the hatching enzyme zymogen and that Mmp13a activity is necessary but not sufficient for hatching in zebrafish. This study highlights the broad nature of MMP function in development and provides a non-mammalian example of extra-embryonic processes mediated by MMP activity.

Paralogues of Mmp11 and Timp4 Interact during the Development of the Myotendinous Junction in the Zebrafish Embryo.

The extracellular matrix (ECM) of the myotendinous junction (MTJ) undergoes dramatic physical and biochemical remodeling during the first 48 h of development in zebrafish, transforming from a rectangular fibronectin-dominated somite boundary to a chevron-shaped laminin-dominated MTJ. Matrix metalloproteinase 11 (Mmp11, a.k.a. Stromelysin-3) is both necessary and sufficient for the removal of fibronectin at the MTJ, but whether this protease acts directly on fibronectin and how its activity is regulated remain unknown. Using immunofluorescence, we show that both paralogues of Mmp11 accumulate at the MTJ during this time period, but with Mmp11a present early and later replaced by Mmp11b. Moreover, Mmp11a also accumulates intracellularly, associated with the Z-discs of sarcomeres within skeletal muscle cells. Using the epitope-mediated MMP activation (EMMA) assay, we show that despite having a weaker paired basic amino acid motif in its propeptide than Mmp11b, Mmp11a is activated by furin, but may also be activated by other mechanisms intracellularly. One or both paralogues of tissue inhibitors of metalloproteinase-4 (Timp4) are also present at the MTJ throughout this process, and yeast two-hybrid assays reveal distinct and specific interactions between various domains of these proteins. We propose a model in which Mmp11a activity is modulated (but not inhibited) by Timp4 during early MTJ remodeling, followed by a phase in which Mmp11b activity is both inhibited and spatially constrained by Timp4 in order to maintain the structural integrity of the mature MTJ.

Intracellular Localization in Zebrafish Muscle and Conserved Sequence Features Suggest Roles for Gelatinase A Moonlighting in Sarcomere Maintenance.

Gelatinase A (Mmp2 in zebrafish) is a well-characterized effector of extracellular matrix remodeling, extracellular signaling, and along with other matrix metalloproteinases (MMPs) and extracellular proteases, it plays important roles in the establishment and maintenance of tissue architecture. Gelatinase A is also found moonlighting inside mammalian striated muscle cells, where it has been implicated in the pathology of ischemia-reperfusion injury. Gelatinase A has no known physiological function in muscle cells, and its localization within mammalian cells appears to be due to inefficient recognition of its N-terminal secretory signal. Here we show that Mmp2 is abundant within the skeletal muscle cells of zebrafish, where it localizes to the M-line of sarcomeres and degrades muscle myosin. The N-terminal secretory signal of zebrafish Mmp2 is also challenging to identify, and this is a conserved characteristic of gelatinase A orthologues, suggesting a selective pressure acting to prevent the efficient secretion of this protease. Furthermore, there are several strongly conserved phosphorylation sites within the catalytic domain of gelatinase A orthologues, some of which are phosphorylated in vivo, and which are known to regulate the activity of this protease. We conclude that gelatinase A likely participates in uncharacterized physiological functions within the striated muscle, possibly in the maintenance of sarcomere proteostasis, that are likely regulated by kinases and phosphatases present in the sarcomere.

Regulation of matrix metalloproteinase-2 (MMP-2) activity by phosphorylation.

The regulation of matrix metalloproteinases (MMP) has been studied extensively due to the fundamental roles these zinc-endopeptidases play in diverse physiological and pathological processes. However, phosphorylation has not previously been considered as a potential modulator of MMP activity. The ubiquitously expressed MMP-2 contains 29 potential phosphorylation sites. Mass spectrometry reveals that at least five of these sites are phosphorylated in hrMMP-2 expressed in mammalian cells. Treatment of HT1080 cells with an activator of protein kinase C results in a change in MMP-2 immunoreactivity on 2D immunoblots consistent with phosphorylation, and purified MMP-2 is phosphorylated by protein kinase C in vitro. Furthermore, MMP-2 from HT1080 cell-conditioned medium is immunoreactive with antibodies directed against phosphothreonine and phosphoserine, which suggests that it is phosphorylated. Analysis of MMP-2 activity by zymography, gelatin dequenching assays, and measurement of kinetic parameters shows that the phosphorylation status of MMP-2 significantly affects its enzymatic properties. Consistent with this, dephosphorylation of MMP-2 immunoprecipitated from HT1080 conditioned medium with alkaline phosphatase significantly increases its activity. We conclude that MMP-2 is modulated by phosphorylation on multiple sites and that protein kinase C may be a regulator of this protease in vivo.

In Vitro Model-Systems to Understand the Biology and Clinical Significance of Circulating Tumor Cell Clusters.

The isolation of clusters of circulating tumor cells (CTCs) from cancer patients has recently challenged the accepted view that the initiation of secondary tumors during metastasis involves the dissemination of individual cancer cells. As such clusters appear to be more aggressive than single tumor cells, CTC clusters are now considered a main player in the metastatic process, and many studies are exploring their diagnostic, prognostic, and clinical significance. However, several technical challenges limit advances in this area. Here, we suggest the use of established cancer cell lines that grow as cell clusters in suspension as a complementary approach that can help in understanding the biology of CTC clusters and their clinical significance. We argue that the many similarities between these "surrogate" clusters and the CTC clusters isolated from patients (e.g., in terms of size, morphology, heterogeneous expression of epithelial and mesenchymal markers, and type of cell-cell junctions) make these cell lines ideal systems for the development of strategies aimed at preventing or slowing down the metastatic process by targeting CTC clusters.

Activity and distribution of paxillin, focal adhesion kinase, and cadherin indicate cooperative roles during zebrafish morphogenesis.

We investigated the focal adhesion proteins paxillin and Fak, and the cell-cell adhesion protein cadherin in developing zebrafish (Danio rerio) embryos. Cadherins are expressed in presomitic mesoderm where they delineate cells. The initiation of somite formation coincides with an increase in the phosphorylation of Fak, and the accumulation of Fak, phosphorylated Fak, paxillin, and fibronectin at nascent somite boundaries. In the notochord, cadherins are expressed on cells during intercalation, and phosphorylated Fak accumulates in circumferential rings where the notochord cells contact laminin in the perichordal sheath. Subsequently, changes in the orientations of collagen fibers in the sheath suggest that Fak-mediated adhesion allows longitudinal expansion of the notochord, but not lateral expansion, resulting in notochord elongation. Novel observations showed that focal adhesion kinase and paxillin concentrate at sites of cell-cell adhesion in the epithelial enveloping layer and may associate with actin cytoskeleton at epithelial junctions containing cadherins. Fak is phosphorylated at these epithelial junctions but is not phosphorylated on Tyr397, implicating a noncanonical mechanism of regulation. These data suggest that Fak and paxillin may function in the integration of cadherin-based and integrin-based cell adhesion during the morphogenesis of the early zebrafish embryo.

Zebrafish Xenograft: An Evolutionary Experiment in Tumour Biology.

Though the cancer research community has used mouse xenografts for decades more than zebrafish xenografts, zebrafish have much to offer: they are cheap, easy to work with, and the embryonic model is relatively easy to use in high-throughput assays. Zebrafish can be imaged live, allowing us to observe cellular and molecular processes in vivo in real time. Opponents dismiss the zebrafish model due to the evolutionary distance between zebrafish and humans, as compared to mice, but proponents argue for the zebrafish xenograft's superiority to cell culture systems and its advantages in imaging. This review places the zebrafish xenograft in the context of current views on cancer and gives an overview of how several aspects of this evolutionary disease can be addressed in the zebrafish model. Zebrafish are missing homologs of some human proteins and (of particular interest) several members of the matrix metalloproteinase (MMP) family of proteases, which are known for their importance in tumour biology. This review draws attention to the implicit evolutionary experiment taking place when the molecular ecology of the xenograft host is significantly different than that of the donor.

Mammalian fibroblast cells avoid residual stress zone caused by nanosecond laser pulses.

This study investigates the effects of laser irradiation on crystalline silicon and its application in biomaterials. We used an analytical model to predict the ablation depth and pit size resulting from laser exposure of silicon samples. The temperatures generated are predicted correlate with laser power, and to result in the formation of a residual stress zone bordering the ablated groove. Different crystal orientations found in the substrate confirm that there was crystal distortion, which consequently induces these residual stress zones. Mouse embryonic fibroblasts avoid the stress areas and accumulate outside of these zones. Higher laser power results in broader residual stress zone and a larger zone of cellular exclusion. We argue that residual stress resulting from high-energy laser ablation of silicon may be a promising avenue to explore as a method for patterning cell growth on these materials.

Mammalian fibroblast cells show strong preference for laser-generated hybrid amorphous silicon-SiO2 textures.

BACKGROUND: In this study, we investigated a method to produce bioactive hybrid amorphous silicon and silicon oxide patterns using nanosecond laser pulses. METHODS: Microscale line patterns were made by laser pulses on silicon wafers at different frequencies (25, 70 and 100 kHz), resulting in ablation patterns with frequency-dependent physical and chemical properties. RESULTS: Incubating the laser-treated silicon substrates with simulated body fluid demonstrated that the physicochemical properties of the laser-treated samples were stable under these conditions, and favored the deposition of bone-like apatite. More importantly, while NIH 3T3 fibroblasts did colonize the untreated regions of the silicon wafers, they showed a strong preference for the laser-treated regions, and further discriminated between substrates treated with different frequencies. CONCLUSIONS: Taken together, these data suggest that laser materials processing of silicon-based devices is a promising avenue to pursue in the production of biosensors and other bionic devices.

Matrix metalloproteinases in neural development: a phylogenetically diverse perspective.

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.

Laminin and Matrix metalloproteinase 11 regulate Fibronectin levels in the zebrafish myotendinous junction.

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. CONCLUSIONS: 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.

Mouse embryonic fibroblasts accumulate differentially on titanium surfaces treated with nanosecond laser pulses.

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.

Activity-based labeling of matrix metalloproteinases in living vertebrate embryos.

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.