In this project, a collaboration with Gregory Goldberg at Washington University St. Louis, we employed single-molecule TIRF to study the motion of single matrix metalloproteinases (MMPs) during the digestion of collagen. MMPs play an important role in physiological collagen processing pathways including tissue remodeling, wound healing and cell migration. However, the mechanistic details of MMP interactions with collagen have been refractory to study due to the complex nature of the collagen substrate and the motion of the MMPs. By tracking individual MMPs on isolated native collagen fibers with high spatial and temporal resolution we could characterize the motion of the MMP on the substrate, and how this motion is coupled to proteolytic activity. This approach has provided detailed mechanistic information for this important class of enzymes. We have, for the first time, observed the complex motion of individual MMPs on collagen fibers and have developed a comprehensive quantitative model describing how this motion is coupled to proteolysis of the collagen fiber. We found that MMPs diffuse and frequently pause on collagen and that a small fraction (5%) of long-lived pauses result in the initiation of collagen degradation, which is followed by the rapid and processive degradation of 15 collagen monomers in the fiber. These results were unanticipated and provide unprecedented insight into the interaction of MMPs with collagen while highlighting the unique capabilities of single-molecule methods to measure complex biomolecular processes. In follow-up work we have obtained super-resolution (50 nm) scale dynamic (10 ms) images of MMP binding to fibrillar collagen that reveal a highly periodic (1 micron periodicity) array of high affinity binding sites that slowly migrate over the fibril over time. We can explain these data with an internal strain model that has broad implications for collagen processing and possibly other load bearing protein assemblies. One immediate conclusion is that fibrillar collagen encodes sites of proteolytic attack but that external strain on the fibril eliminates these sites, which provides a mechanism for strain-dependent stabilization of fibrillar collagen against MMP degradation. Immediate goals include the direct observation of the motion and binding of MMPs on fibrillar collagen under varying degrees of mechanical tension. This will require a technique to apply large 10s of nanoNewton loads to individual collagen fibrils while simultaneously tracking individual MMP enzymes with high spatial and temporal resolution. These results were unanticipated and provide unprecedented insight into the interaction of MMPs with collagen while highlighting the unique capabilities of single-molecule methods to measure complex biomolecular processes. The initial measurements and comprehensive modeling are complete and we have submitted the first manuscript. Furthermore, we developed new methodologies to analyze diffusion in single-molecule traces, which are applicable to any single-molecule analysis of diffusion trajectories. Future work on MMP tracking will be focused on improving the temporal and spatial resolution of the tracking in addition to extending the duration of individual trajectories through the use of nitrogen vacancy nano-diamond labels.
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| Hu, Longhua; Vecchiarelli, Anthony G; Mizuuchi, Kiyoshi et al. (2017) Brownian Ratchet Mechanism for Faithful Segregation of Low-Copy-Number Plasmids. Biophys J 112:1489-1502 |
| Yi, Jason; Manna, Asit; Barr, Valarie A et al. (2016) madSTORM: a superresolution technique for large-scale multiplexing at single-molecule accuracy. Mol Biol Cell 27:3591-3600 |
| Vecchiarelli, Anthony G; Li, Min; Mizuuchi, Michiyo et al. (2016) Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD. Proc Natl Acad Sci U S A 113:E1479-88 |
| Dittmore, Andrew; Silver, Jonathan; Sarkar, Susanta K et al. (2016) Internal strain drives spontaneous periodic buckling in collagen and regulates remodeling. Proc Natl Acad Sci U S A 113:8436-41 |
| Sarkar, Susanta K; Marmer, Barry; Goldberg, Gregory et al. (2012) Single-molecule tracking of collagenase on native type I collagen fibrils reveals degradation mechanism. Curr Biol 22:1047-56 |
