Nanoscale investigations of collagen using atomic force microscopy
Creators: name: Roth, Jonathan Andrew
Publisher: name: No Publisher Supplied
Year: 2022

description: Collagen is vital in constructing the various organs and tissues necessary for life to exist and is the main component of the extracellular matrix (ECM). However, collagen does not serve only a structural role, but interacts constantly with the diverse components of the ECM environment to ensure proper biological activity. In this dissertation, collagen’s structure, mechanics, and activity will be investigated with the objective of gaining a deeper insight of its role/function in the human body.Crucial for normal cellular function within the ECM are fibrillar collagen-integrin interactions. Collagen fibrils make up the landscape of the ECM and are in constant interaction with cellular integrins. Recently, great efforts have been made to understand the effects of perturbations to the ECM environment. Specifically, glycosaminoglycan (GAGs), anionic polysaccharides that occupy the ECM, have been shown to decrease with age and become mis-regulated in certain diseases such as cancer. The effect of GAGs on the properties of collagen however is still poorly understood. To address this knowledge gap, native and GAG depleted collagen fibrils derived from rat tail tendons were compared in terms of activity, structure, and mechanics using adhesion assays and atomic force microscopy (AFM). Our results indicate that although GAGs did not influence the structure of the collagen fibrils, GAG depleted fibrils had a lower indentation modulus and higher activity to α2I integrin which has implications for how diseases are able to originate and manifest within the ECM. Next, the complex dynamics associated with collagen formation were investigated. Although collagen fibril formation has been extensively studied, early-stage developmental events have never been visualized leading to some still unanswered questions concerning how collagen grows. To achieve this, video rate scanning AFM was used to visualize the collagen fibrillogenesis process in real time (1 frame/second). Collagen monomers were visualized nucleating to a mica surface and over time develop into fully D-banded fibrils. Several unique growth aspects of collagen were also identified such as having uni-directional growth and rope-like characteristics. Together the data serves a benchmark for further growth experiments and provides deeper insight into how collagen structures are initially created. Lastly, as part of a collaboration with Tamr Atieh, and separate from the collagen projects, the effect of DJ-1 on the aggregation of native and glycated alpha synuclein was studied. Alpha synuclein is an intrinsically disordered protein known for aggregating and forming oligomers which are linked to the progression of Parkinson’s disease and other neurodegenerative disorders. Further, glycation of alpha synuclein, the nonenzymatic addition of sugar aldehydes to a protein, alters the dynamics of alpha synuclein and increases oligomer formation. To combat the toxic aggregation, DJ-1, an antioxidative protein which has shown to inhibit fibril formation is used. In this work, through the use of NMR and AFM, it is demonstrated that DJ-1 targets and binds to aggregated forms of alpha synuclein, sequestering them and helping restore monomeric character with similar results occurring for both the native and glycated species. Thus, therapeutics that mimic the action of DJ-1 or upregulate DJ-1 expression may be a desirable method for combatting alpha synuclein aggregation.