Development of New Tools for Single-Molecule Force Spectroscopy & The Application of these Tools to Mapping the Role of Force Directionality in the Actin Mechanome
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CitationFeldman, Theodore Charles. 2015. Development of New Tools for Single-Molecule Force Spectroscopy & The Application of these Tools to Mapping the Role of Force Directionality in the Actin Mechanome. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractActin filaments serve as a hub of focal adhesion-based mechanotransduction network in a wide variety of cell types. In this role filaments and networks of filaments are exposed to forces in a range of directions and magnitude. Due to the complexity of the adhesome circuit, many questions remain open, including identification of the fundamental mechanosensitive elements and regulation of interconnected elements. One possibility is that actin itself is mechanosensitive and applied forces regulate the interactions between actin and actin regulatory proteins. To address this question we must be able to characterize the force response of individual actin filaments, the mechanical properties of individual filaments and the mechanical properties of individual actin filament crosslinks. This thesis presents new technologies that are capable of addressing such questions. The design and optimization of a dual-beam optical trap, the workhorse technology of single-molecule force spectroscopy is presented first. The capabilities of force spectroscopy are then expanded in the subsequent chapters. The first development is a new instrument that enables us to probe the energy landscapes of weak, short-lived interactions as well as the strongest, longest-lived interactions. Stable, multiplexed femtoNewton force spectroscopy using particle tracking tools for tether diagnostics is demonstrated. We also show that these capabilities can be used to actively characterize the torsional rigidity of actin cross-links. Finally, the development of an elongational flow hydrodynamic trap, simple tape-based microfluidics and a multiplexed microfluidic microscopy assay are developed and applied to study the role of tension in the regulation of actin filament severing by severing proteins cofilin and gelsolin. Our results using hydrodynamic trapping, a surface-free force spectroscopy tool, in concert with the results of our curvature-sensitive experiments suggest that actin is sensitive to the magnitude and direction of an applied force. Ongoing and future studies will provide mechanistic insight to this observation.
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