Mapping the 3D Orientation and Nanoscale Distribution of Piconewton Receptor Forces
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Brockman, Joshua Mark
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Abstract
Molecular forces are vital to many biological systems including hemostasis, immune function, development, and cell-cell communication. The tools currently available to measure cellular forces are incapable of quantifying the 3D orientation and nanoscale distribution of piconewton receptor forces. To address this technological gap, this thesis describes the development of two mechanoimaging techniques, Molecular Force Microscopy (MFM) and tension-Point Accumulation for Imaging in Nanoscale Topography (tPAINT). The first technique, MFM, combines DNA-based molecular tension probes with fluorescence polarization microscopy to measure the 3D orientation of pN molecular forces. MFM revealed that platelets contract anisotropically, a feature missed by previous traction force microscopy measurements, and suggested that integrin forces within focal adhesions are aligned with force-transducing proteins such as talin, linking focal adhesion structure to integrin forces. The second technique, tPAINT, enables single molecule localization microscopy of receptor force events by converting DNA-based molecular tension probes into force-triggered switches that recruit fluorophores only when experiencing pN receptor force. tPAINT enabled measurement of dynamic receptor forces with ~25nm resolution. tPAINT imaging of platelet lamellipodial forces linked these forces with a peripheral ring of actin, demonstrating the capability to link receptor forces to the protein structures that produce them. MFM and tPAINT provide new capabilities to interrogate mechanobiology, and may offer a route to link structural biology and receptor mechanics.
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2020-07-06
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Dissertation