Structural analysis of the alpha(2) integrin I domain/procollagenase-1 (matrix metalloproteinase-1) interaction.

Previous studies have established that ligation of keratinocyte alpha(2)beta(1) integrin by type I collagen induces expression of matrix metalloproteinase-1 (MMP-1) and that MMP-1 activity is required for the alpha(2)beta(1) integrin-dependent migration of primary keratinocytes across collagenous matrices. We now present evidence that MMP-1 binds the alpha(2)beta(1) integrin via the I domain of the alpha(2) integrin subunit. Using an enzyme-linked immunosorbent assay with purified human MMP-1 and recombinant alpha(2) integrin I domain, we showed that the alpha(2) integrin I domain specifically bound in a divalent cation-dependent manner to both the pro and active forms of MMP-1, but not to MMP-3 or MMP-13. Although both the I domain and MMP-1 bind divalent cations, MMP-1 bound, in a divalent cation-dependent manner, to alpha(2) integrin I domains containing metal ion-dependent adhesion sites motif mutations that prevent divalent cation binding to the I domain, demonstrating that the metal ion dependence is a function of MMP-1. Using a series of MMP-1-MMP-3 and MMP-1-MMP-13 chimeras, we determined that both the linker domain and the hemopexin-like domain of MMP-1 were required for optimal binding to the I domain. The alpha(2) integrin/MMP-1 interaction described here extends an emerging paradigm in matrix biology involving anchoring of proteinases to the cell surface to regulate their biological activities.

Pro-collagenase-1 (matrix metalloproteinase-1) binds the alpha(2)beta(1) integrin upon release from keratinocytes migrating on type I collagen.

In injured skin, collagenase-1 (matrix metalloproteinase-1 (MMP-1)) is induced in migrating keratinocytes. This site-specific expression is regulated by binding of the alpha(2)beta(1) integrin with dermal type I collagen, and the catalytic activity of MMP-1 is required for keratinocyte migration. Because of this functional association among substrate/ligand, receptor, and proteinase, we assessed whether the integrin also directs the compartmentalization of MMP-1 to its matrix target. Indeed, pro-MMP-1 co-localized to sites of alpha(2)beta(1) contacts in migrating keratinocytes. Furthermore, pro-MMP-1 co-immunoprecipitated with alpha(2)beta(1) from keratinocytes, and alpha(2)beta(1) co-immunoprecipitated with pro-MMP-1. No other MMPs bound alpha(2)beta(1), and no other integrins interacted with MMP-1. Pro-MMP-1 also provided a substrate for alpha(2)beta(1)-dependent adhesion of platelets. Complex formation on keratinocytes was most efficient on native type I collagen and reduced or ablated on denatured or cleaved collagen. Competition studies suggested that the alpha(2) I domain interacts with the linker and hemopexin domains of pro-MMP-1, not with the pro-domain. These data indicate that the interaction of pro-MMP-1 with alpha(2)beta(1) confines this proteinase to points of cell contact with collagen and that the ternary complex of integrin, enzyme, and substrate function together to drive and regulate keratinocyte migration.

Fluorescent detection of Zn(2+)-rich vesicles with Zinquin: mechanism of action in lipid environments.

High concentrations of free Zn2+ ions are found in certain glutamatergic synaptic vesicles in the mammalian brain. These terminals can be visualized histochemically with quinoline sulfonamide compounds that form fluorescent complexes with Zn2+. The present study was undertaken to examine the interaction of the water-soluble quinoline sulfonamide probe, Zinquin (2-methyl-8-(toluene-p-sulfonamido)-6-quinolyloxyacetic acid) with the complex heterogeneous cellular environment. Experiments on rat hippocampal and neocortical slices gave indications that Zinquin in its free acid form was able to diffuse across the plasma and synaptic vesicle membranes. Further experiments were undertaken on unilamellar liposomes to study the interaction of Zinquin and its metal complexes in membranes. These experiments confirmed that Zinquin is able to diffuse across lipid bilayers. Steady-state and time-resolved fluorimetric studies showed that Zinquin in aqueous solution mainly forms a 1:2 (metal:ligand) complex with small amounts of a 1:1 complex. Formation of the 1:1 complex was favored by the presence of lipid, suggesting that it partitions into membranes. Evidence is presented that Zinquin can act as a Zn(2+)-ionophore, exchanging Zn2+ for two protons. The presence of a pH gradient across vesicles traps the Zn(2+)-probe complex within the vesicles. Zinquin is useful as a qualitative probe for detecting the presence of vesicular Zn2+; however, its tendency to partition into membranes and to serve as an ionophore should be borne in mind.

Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.

The fluorescent probe FM1-43 has been used extensively for imaging vesicle recycling; however, high nonspecific adsorption resulting in elevated background levels has precluded its use in certain tissues, notably brain slices. We have found that a sulfobutylated derivative of beta-cyclodextrin (ADVASEP-7) has a higher affinity for FM1-43 than the plasma membrane. ADVASEP-7 was used as a carrier to remove FM1-43 nonspecifically bound to the outer leaflet of the plasma membrane or extracellular molecules, significantly reducing background staining. This has enabled us to visualize synaptic vesicle recycling in the nematode C. elegans, intact lamprey spinal cord, and rat brain slices.