DDR-collagen interactions at the molecular and cellular level
A major focus area of the Agarwal laboratory has been to understand collagen remodeling and its modulation by Discoidin Domain Receptors (DDR1 and DDR2). Collagen type 1 is the most abundant extracellular matrix (ECM) protein in mammalian tissues. Interactions between collagen and cells require specific receptors to modulate cell function and ECM remodeling. In 1997, DDRs were discovered to be a major class of collagen receptors (next to integrins). DDRs are receptor tyrosine kinases (RTKs) that undergo tyrosine phosphorylation upon ligand (collagen) binding. DDR expression and signaling is important for development and its dysregulation is involved in several malignancies and in diseases associated with collagen remodeling such as fibrosis, atherosclerosis and osteoarthritis.
(i) Collagen fibrillogenesis: by analyzing the binding pattern of DDRs onto single collagen molecules, we discovered that DDRs regulate a fundamental aspect of the collagen I molecule: its ability to undergo fibril formation, a process known as fibrillogenesis. This process is central to the macromolecular organization of collagen so that it can fulfil its tissue-supportive function. DDRs not only delay the fibril formation but also modulate the structure of the resulting collagen fibrils.
(ii) Receptor clustering: Another major contribution from our laboratory has been to elucidate the supramolecular organization of DDRs on the cell surface, i.e. the role of DDR oligomerization and clustering and its importance in collagen binding, fibrillogenesis and receptor phosphorylation. Importantly we have elucidated how DDRs can distinguish between the state of its ligand, i.e. monomers vs. fibrils of collagen and identified key differences between the clustering properties of DDR1 and DDR2.
In our ongoing research we aim to examine interactions of DDR1 with Collagen type IV, the major component of the basement membrane.
Functional consequences of structurally altered collagen fibrils
Our studies on collagen fibrillogenesis has enabled us to employ DDRs as a natural means to modulate the collagen fibril structure both in-vitro. In addition we have identified how structural alterations in collagen fibrils exist in-vivo in the aorta and bone tissue of DDR1 knockout mice as well as in matrix remodeling in vascular diseases such as abdominal aortic aneurysms. Our ongoing research employs and/or develops novel technical approaches to understand functional consequences of structurally altered fibrils via the following objectives.
(i) Mechanical properties: by using a passive TEM approach, we determined that collagen fibrils formed in the presence of recombinant DDR2 had reduced persistence length. We also evaluated how DDR2 modulates the mechanical properties of collagen networks. Towards this aim in a collaborative effort (with Lafyatis, Anderson and Powell at OSU), we developed and employed a novel optical tweezer based two-particle active micro-rheology technique and elucidated how DDRs (and decoron) differentially impact macro and micro-mechanics of collagen gels.
(ii) Cell-matrix interaction: structurally altered collagen fibrils can expose/obscure specific binding sites on the collagen triple helix and thus modulate cell-adhesion. Towards this goal we are investigating the thrombogenicity of vessel walls. Using human platelets on murine aortic sections we have found that platelet adhesion is enhanced in the vessel wall of DDR1 knockout mice. We are extending these studies to examine the role of collagen fibril structure and DDR expression in thrombosis and in bleeding disorders.
(iii) Matrix mineralization: deposition of hydroxyapatite (i.e. calcification or mineralization) of collagen fibrils is also dependent on the fibril structure. We reported how matrix mineralization is enhanced by the DDR ectodomains in both cell-based assays as well as biomimetic mineralization protocols. Our ongoing studies aim to employ AFM to map the surface charge of collagen fibrils to further understand the role of collagen fibril structure in modulating matrix mineralization.
NOVEL APPLICATIONS OF AFM
Another vector of our lab is to develop new tools and techniques for biological applications. We have developed and applied many aspects of magnetic force microscopy (MFM) for analysis of superparamagnetic nanoparticles. We have elucidated how MFM can quantitatively characterize magnetic nanoparticles and distinguish between iron-bound vs. unbound ferritin proteins. In collaboration with the. McTigue lab at OSU, we have elucidated how MFM can serve as a label-free tool to characterize nanoscale iron deposits in tissue sections. We have also developed a novel indirect MFM (ID-MFM) technique for detecting magnetic domains in a multimodal, high-throughput manner. Through these studies we have also learnt that iron deposits in biological tissues are characterized by clusters of ferritin(iron) core which may serve as a precursor for biogenic magnetite.