Sunday, September 15, 2024
9/15/2024
Summary. This proposal seeks to test whether mechanical affinity of the complex between RGD, a tripeptide domain found in proteins of extracellular medium, and integrin, a cell membrane receptor protein, is correlated with cell migrations. By attaching the RGD ligand to a magnetic bead via a DNA handle, integrins on cell surface can be evaluated for their binding affinity to the RGD by mechanically pulling away the binding complex using a pair of magnets in magnetic-tweezers instrument. During the pulling, a camera in the magnetic-tweezers setup is employed to record the distance between the magnetic bead and the magnet pair, which is converted to the binding force at the time of bead detachment. The same camera is also used to simultaneously monitor cell morphology changes that are related to cell migrations. Such a setup allows to correlate the mechanical affinity of the RGD-integrin complex to the cell migration. This new technique has also been successful in measuring the extraction force of lipid molecules such as cholesterol away from the cell membrane. Cholesterol is a critical component to increase the rigidity of the lipid rafts in the cell membrane. Cell migration is shown to be inhibited when lipid rafts are dissolved. Cell membrane receptors such as EGFR, HER2, and CD36 are found within lipid rafts. They may be impacted by compromised lipid rafts after cholesterol depletion. All these three receptors play a role in cell migration through cell signaling pathways such as the Src-Fak axis. We propose that these receptors may induce changes in cell migration when the structure and property of lipid rafts are altered by factors such as cholesterol supplement/depletion or overexpression/knock-down of EGFR/HER2/CD36 receptors. Integrin-RGD mechanical affinity will be used as a marker to follow changes in cell migration as integrin binds to the extracellular matrix via peptide domains such as RGD. When lipid rafts change their properties, integrin-RGD mechanical affinity may alter, which leads to variation in cell migration. Previously, mechanical affinity has been shown to correlate with binding affinity; however, it is unknown if mechanical affinity coordinates with intracellular signaling. By depleting cholesterol, migration will be inhibited. We expect that supplementation of cholesterol after depletion may restore cell migration. To investigate this restoration, we will evaluate changes in cell migration at either extracellular or intracellular level. At the extracellular level, we will again use the mechanical affinity of integrin-RGD as a marker for cell migration. At the intracellular level, western blot analysis will be used to follow changes in cell signaling by monitoring expression levels of phosphorylated proteins associated with cell migration. We aim to test if extracellular mechanical binding force corresponds with intercellular signaling as changes in migration occur. In summary, we will evaluate cell migration using integrin-RGD mechanical affinity when changes in lipid raft composition occur (Aim 1) and test if changes in mechanical affinity correspond with cell signaling intensity (Aim 2).
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