Diffusion of MMPs on the Surface of Collagen Fibrils: The Mobile Cell Surface – Collagen Substratum Interface

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 represents a Mobile Cell Surface – Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions

Fluorescence photobleaching recovery from gelatin-adsorbed fluorescently labeled MMP-2 (A), MMP-9 (B) and their complexes with inhibitors TIMP-2 and TIMP1 (C) and (D).

<p>Fluorescence photobleaching recovery (FPR) curves were obtained from fluorescently labeled enzymes adsorbed to gelatin coated glass coverslips. Fluorescence was excited by an attenuated Argon-ion laser (488 nm) beam (0.25±0.04 µWatt) focused on the decorated gelatin layer. It was monitored through a 40× microscope objective with a photomultiplier tube (PMT) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#pone.0024029-Petersen2" target="_blank">[92]</a> for 36 s before and 72 s after the delivery of a 50 ms bleaching pulse (187±19 µWatt). The fluorescence is normalized to the mean value of the fluorescence signal (PMT response) just prior to the delivery of the bleaching pulse. The solid lines are a non-linear least squares fit of the predicted diffusion dependent recovery (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#pone.0024029-Collier1" target="_blank">[12]</a>, Eq 3.2) to the fluorescence data. The local diffusion coefficients obtained from fitting are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#pone-0024029-t001" target="_blank">Table 1</a>.</p

Biased Diffusion exhibited by MT1-MMP on The Surface of Collagen Fibrils is absent in a catalytically inactive mutant E240->A.

<p>(<b>A</b>). SDS-PAGE analysis of Gel filtration Chromatography of the MT1-MMP expressed in E.Coli: MW standards (lane 1); the leading portion of the major protein peak (lane 2); trailing portion of the major protein peak (lane 3); activation of the MT1-MMP (lane 3) with recombinant Furin for 30 min at 300C (lane 4). (B). Normalized experimental correlation functions obtained from collagen fibrils decorated with either activated MT1-MMP Wild Type (open circles) or MT1-MMP inactive mutant (E240->A, closed circles) were calculated from the 400 µs binned data records as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#s4" target="_blank">Methods</a>. The background was suppressed using the spatial background filter described earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#pone.0024029-Saffarian1" target="_blank">[13]</a>, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#s4" target="_blank">methods</a>). Three experimental correlation functions for each enzyme were normalized and then averaged to obtain the data shown. The experimental correlation function for wild-type enzyme was fitted (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#s4" target="_blank">Methods</a>, equation 3) to a 1D diffusion (D = 6.0±0.05×10⁻⁹ cm² s⁻¹) plus flow (V = 5.8±0.2 µm s⁻¹) model. The correlation function of the inactive mutant exhibits a long tail characteristic of an unbiased 1-D diffusion, accordingly, a fit of the same equation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#s4" target="_blank">Methods</a>, equation 3) yielded a local diffusion coefficient (D = 1.1±0.04×10⁻⁸ cm² s⁻¹) and no significant flow.</p

Effects of inhibition of pericellular collagenolysis on active cell forces (Fc) in stress-strain profiles of 3D tissue constructs.

<p>Embryonic fibroblasts from BALBC (MEF), MT1-MMP wild type (MT1-WT) or MT1-MMP knockout (MT1-KO) mice were cultured in either wild type (WT, black lines) or collagenase resistant (RR, red lines) mouse tail collagens. Each plot is the average of 6–8 tissue samples and the presented differences are highly significant (P<0.001). The results show a consistent reduction in force upon inhibition of collagenolysis either with MMP inhibitor Galardin (GM), use of collagenase resistant collagen or elimination of membrane tethered collagenase MT1-MMP (MT1-KO). Importantly, Galardin has no effect on the force when used to inhibit MMPs in MEF cells on the background of RR collagen (p = 0.78). <b>Fc = Ft–Fm</b>.</p

Zymogram analysis of MMP-9 Cysteine Replacement Mutants C⁴⁶⁸->A, C⁵¹⁶->A, C⁶⁷⁴->A, C⁷⁰⁴->A.

<p>Individual cDNAs bearing the mutant substitutions as well as the wild type were expressed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#pone.0024029-Goldberg2" target="_blank">[30]</a>. Samples of serum-free conditioned media (5–100 µl) were dialyzed, lyophilized, and subjected to NaDodS04-PAGE using a 7% acrylamide gel impregnated with gelatin. A zymogram was developed for 2.5 h. Lane 1, WT; lane 2, C⁴⁶⁸->A; lane 3, C⁵¹⁶->A; lane 4, C⁶⁷⁴->A; lane5 C⁷⁰⁴->A. The arrows indicate the location of the MMP-9 monomer (lower arrow) and MMP-9 homodimer (upper arrow).</p

Fluorescence Correlation Spectroscopy of MMP-2, MMP-9 and their respective complexes with inhibitors TIMP-2 and TIMP-1 on collagen fibrils.

<p>Normalized experimental correlation functions obtained from collagen fibrils decorated with MMP-2 (A), MMP-2/TIMP-2 (C), MMP-9 (B) and MMP-9/TIMP-1 (D) were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#pone-0024029-g001" target="_blank">Fig. 1</a>. The experimental correlation functions were fitted to an 1-D unbiased diffusion model (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#s4" target="_blank">Methods</a>, Eq. 2). The local diffusion coefficients obtained from the fit are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#pone-0024029-t001" target="_blank">Table 1</a>.</p

Dimerization of MMP-9 Renders the Enzyme Immobile Both on Gelatin Layers and Collagen Fibril surfaces.

<p><b>A.</b> FPR curve for fluorescently labeled gelatin-adsorbed MMP-9 homodimer was obtained as described above in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#pone-0024029-g003" target="_blank">Figure 3</a>. No detectable increase of fluorescence (open circles) occurs after the photobleaching pulse indicating the immobility of the MMP-9 homodimer. Monitoring beam photobleaching alone, indicated by the solid curves (Eq. 3.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#pone.0024029-Collier1" target="_blank">[12]</a> with mobile fraction = 0), accounts for the decay of fluorescence before and after the photobleaching pulse. <b>B (upper panel)</b>. A primary fluorescence record, rebinned to 40 ms (upper curve), was obtained from an individual fibril decorated with fluorescently labeled MMP-9 homodimer. The background noise in the primary record was suppressed by applying the spatial background filter described earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#pone.0024029-Saffarian1" target="_blank">[13]</a> to reveal a flat baseline (lower curve) indicating the absence of single molecule spikes. <b>B (lower panel)</b>. A primary fluoresence record, rebinned to 40 ms (upper curve), was obtained from an individual fibril decorated with fluorescently labeled MMP-9 monomer. The background noise in the primary record was suppressed by applying the same spatial background filter to reveal significant fluctuations in fluoresence (lower curve) indicating the presence of single molecule spikes.</p

The unequal flux of MT1- MMP molecules around a no-transport block on the collagen fibril.

<p>Photobleaching of MT1-MMP decorated collagen fibrils with a laser beam intensity of 80 to 90 mW prevents recovery after photobleaching indicating damage to the fibril, thus blocking enzyme transport across the bleached area. The average number of single enzyme molecules passing through the laser beam in the left (CL) and right (CR) flanks of the no-transport block are calculated by counting (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#s4" target="_blank">Methods</a>) and then averaging the number of spikes in the 300 s fluorescence records of five independent experiments for each form of the enzyme. The difference in transport at the flanks for each experiment is expressed as the asymmetry ratio, |(CL−CR)/(CL+CR)|, which is then averaged over all experiments. A ratio of 1 indicates perfect flow asymmetry and 0 indicates perfectly symmetric flow. The graphic portion is reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024029#pone.0024029-Saffarian1" target="_blank">[13]</a>. License Number 2257830858271 issued Aug 28, 2009 by The American Association for the Advancement of Science.</p