Single-Molecule Tracking of Collagenase on Native Type I Collagen Fibrils Reveals Degradation Mechanism

SummaryBackgroundCollagen, the most abundant human protein, is the principal component of the extracellular matrix and plays important roles in maintaining tissue and organ integrity. Highly resistant to proteolysis, fibrillar collagen is degraded by specific matrix metalloproteases (MMPs). Degradation of fibrillar collagen underlies processes including tissue remodeling, wound healing, and cancer metastasis. However, the mechanism of native collagen fibril degradation remains poorly understood.ResultsHere we present the results of high-resolution tracking of individual MMPs degrading type I collagen fibrils. MMP1 exhibits cleavage-dependent biased and hindered diffusion but spends 90% ± 3% of the time in one of at least two distinct pause states. One class of exponentially distributed pauses (class I pauses) occurs randomly along the fibril, whereas a second class of pauses (class II pauses) exhibits multistep escape kinetics and occurs periodically at intervals of 1.3 ± 0.2 μm and 1.5 ± 0.2 μm along the fibril. After these class II pauses, MMP1 moved faster and farther in one direction along the fibril, indicative of biased motion associated with cleavage. Simulations indicate that 5% ± 2% of the class II pauses result in the initiation of processive collagen degradation, which continues for bursts of 15 ± 4 consecutive cleavage events.ConclusionsThese findings provide a mechanistic paradigm for type I collagen degradation by MMP1 and establish a general approach to investigate MMP-fibrillar collagen interactions. More generally, this work demonstrates the fundamental role of enzyme-substrate interactions including binding and motion in determining the activity of an enzyme on an extended substrate

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

Delayed skin wound repair in proline-rich protein tyrosine kinase 2 knockout mice

Proline-rich protein tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family. We used Pyk2 knockout (Pyk2-KO) mice to study the role of Pyk2 in cutaneous wound repair. We report that the rate of wound closure was delayed in Pyk2-KO compared with control mice. To examine whether impaired wound healing of Pyk2-KO mice was caused by a keratinocyte cell-autonomous defect, the capacities of primary keratinocytes from Pyk2-KO and wild-type (WT) littermates to heal scratch wounds in vitro were compared. The rate of scratch wound repair was decreased in Pyk2-KO keratinocytes compared with WT cells. Moreover, cultured human epidermal keratinocytes overexpressing the dominant-negative mutant of Pyk2 failed to heal scratch wounds. Conversely, stimulation of Pyk2-dependent signaling via WT Pyk2 overexpression induced accelerated scratch wound closure and was associated with increased expression of matrix metalloproteinase (MMP)-1, MMP-9, and MMP-10. The Pyk2-stimulated increase in the rate of scratch wound repair was abolished by coexpression of the dominant-negative mutant of PKCδ and by GM-6001, a broad-spectrum inhibitor of MMP activity. These results suggest that Pyk2 is essential for skin wound reepithelialization in vivo and in vitro and that it regulates epidermal keratinocyte migration via a pathway that requires PKCδ and MMP functions

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

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

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