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Author ORCID Identifier
Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Analytical Chemistry | Biochemistry | Biophysics | Molecular Biology
Mechanical forces are an integral part in biology, they regulate several cellular properties, such as morphology, proliferation, migration. These forces are also involved in receptor signaling and the differentiation of different cell types. Different proteins and biomolecules such as cadherin, integrin, notch proteins are essential elements of these processes. Measuring these intercellular forces are challenging considering the minimal intensity (piconewton-level) of these molecular forces. In our lab, we have developed a membrane DNA tension probe (MDTP) that uses a DNA hairpin module to sense tensile forces and has a lipid anchor to modify onto live-cell membranes. The programmability of DNA and the dynamic insertion capability of lipids provides us a simple tool to visualize the forces. We believe these probes will be a widely useful tool for investigating the mechanical forces. In this thesis, we have used MDTP as a tool to visualize and measure the mechanical forces involved in different receptor-ligand interaction at the cell-cell junctions.
In the first part of the thesis, we have designed a fluorescence lifetime-based membrane DNA tension probe named as FLIM-MDTP, which uses fluorescence lifetime as a read-out. We used this probe to quantitatively image the E-cadherin-mediated tensile forces at the cell-cell junction. Furthermore, we demonstrated the multiplexed imaging capability of the probe to monitor the mechanical dynamics of the epithelial-to-mesenchymal transition. In the second part, we have used the DNA-based probe to visualize the tensile force involved in Notch activation. Our initial results showed low unfolding efficiency of the probe indicating a weak mechanical force involved in the activation of Notch. To potentially image these low levels of forces, we have further designed a MDTP that uses a lower force threshold hairpin (2.2 pN vs. 4.4 pN) as a force module. Finally, we used these DNA-based probes to measure CD40-induced pulling forces at B cell-T cell junction. Our results indicated that the extent of forces involved in this interaction is also quite small and transient in nature. In short summary, our data indicated that DNA-based probes can be used to measure tensile forces within a diverse type of mechanotransduction at cell-cell junction. Our results also demonstrated that the extent of pulling forces differ largely depending on the type of ligand-receptor pairs.
Keshri, Puspam, "QUANTITATIVE IMAGING OF TENSILE FORCES AT CELL-CELL JUNCTION WITH DNA-BASED PROBES" (2022). Doctoral Dissertations. 2426.
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