Nearly all eukaryotic cells exert forces on their surroundings to generate and maintain tension within their cytoskeleton which is critical for normal cell function. In addition, cells exert forces on their surroundings to orient themselves within an organism, thus gaining information that influences cell fate and behavior, a process called mechanotransduction. In order to study mechanotransduction, a tool is needed that can observe the molecular level sensing events that trigger a decision by a cell as well as the ultimate response that occurs on the whole cell level. There are a number of optical techniques that are used to measure forces from adherent cells at the single cell level; some are good for measuring whole cell forces and some for measuring single molecule level forces, but none have the dynamic range necessary to span both regimes, which is critical for understanding mechanotransduction. To address this need, I have developed a Nano-ElectroMechanical Systems (NEMS) based tool, Single-Cell-Pico-Force-Microscopy (SCPFM), that measures forces exerted by adherent cells with macro-molecular level force sensitivity and sufficient dynamic range to monitor whole cell responses to stimuli with macro-molecular resolution. I have used SCPFM to measure force versus time data from a NIH-3T3 fibroblast as it is perturbed with Cytochalasin D (CD) and allowed to recover in growth media. Within the data there are three excellent examples of previously inaccessible molecular-mechanical processes that illustrate the immense potential of SCPFM to significantly enhance resolution of cell biology at the single cell level: 1) an initial contraction upon exposure to CD followed by the expected force drop, 2) small force oscillations, roughly 400pN peak-to-peak, with frequency that is monotonically dependent on the force being exerted by the lamellipodia, and 3) large, stable, quantized force steps of order 1nN are manifested when a cell’s cytoskeleton is perturbed with CD and allowed to recover in growth media. I propose two complimentary experimental efforts to undertake: a systematic effort to build a library of molecular-mechanical force signatures of various common cytoskeleton reactions and an effort to stimulate and observe compliance sensing and response in adherent cells.ud
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