Measuring the effect of mechanical forces on receptor signals through nanoscale AFM measurement of the cytoskeleton

Stanford University, Stanford CA

Investigators

Abstract

PI: Manish J. Butte Proposal ID: 1264833 The research objective of this proposal is to test the hypothesis that mechanical forces upon the cytoskeleton regulate signals from cell-surface receptors. In T cells it has been shown that exogenous mechanical forces in addition to ligation of the T cell receptor seem to be required for the T cell to become activated. In cancer cells, it is known that mechanical forces plus ligation of integrin receptors by the extracellular matrix can lead to an invasive and proliferative phenotype. How does force act as a cue to modify molecular signaling events? Recent discoveries in my lab using biological Atomic Force Microscopy (AFM) showed that naive T cells have a stiff cytoskeletal architecture, which softens immediately upon TCR triggering, and that the facility with which the cytoskeleton softens, mediated by kinases and tethering molecules, actually controls the threshold for activation. This proposal seeks to test generally the capability of cytoskeletal structures to modulate the signals of cell-surface receptors, thus tying together the fields of mechanobiology and signaling. While the cytoskeleton's important roles in cell motility and intracellular trafficking are well characterized, its functional roles during receptor signaling have been largely unexplored. The PI lab's results prompt to investigate the role of mechanical forces at the cellular level, to study how durable cytoskeletal structures control the activation or invasiveness of cells, and at the single receptor level, to show how force upon the receptors modulates its signaling. The Intellectual Merit of this program lies in PI's unique ability to control and study the linkage between nanoscale forces and receptor signaling. The ability to ligate receptors on live cells and deliver forces by AFM, plus image simultaneously by spinning disk confocal microscopy, is a unique and powerful capability of my lab. What will emerge will be an improved understanding of how force signals are sensed, exerted, and "stored" by cells. It is inferred from PI's findings in T cells that, generally, loosening of cytoskeletal tethers controls the threshold of receptor signaling. Cells may interpret mechanical forces to gate receptor signals as a way of preventing inadvertent triggering (i.e., autoimmunity), or as a way of utilizing inter-cellular forces that occur during certain developmental, embryonic checkpoints as a trigger for proliferation and growth. "Storing" these receptor set-points in the form of cytoskeletal structures may be more durable than, for example, tyrosine phosphorylation or gene transcription. The Broader Impact of this work includes an Educational Outreach Plan that proposes to develop tools to teach Nanoscale Biology by direct experience. The PI has utilized the latest 3D printing technology to print palm-sized cell models complete with nanoscale features. These cell models can be held and touched to sample cellular features, and will inspire interest in nanoscale science and biology. In conjunction with the models, the investigators are developing a haptic interface that allows users to experience "touching" the nanoscale features of cells when using a 3D joystick and the AFM. It is proposed to share these cell models and haptic interfaces with local classrooms and science museums, to inspire participants of all ages to interact with the nanoscale features and dynamic movements of cells. Beyond this educational impact, this project will help build a unique research group with expertise in cellular biomechanics, enriching interdisciplinary group of collaborators who are interested in applying the methods developed through this work to their cells and their biological questions. The findings will be disseminated though publications and the website, making the AFM techniques developed accessible to students and researchers. Finally, the PI will collaborate with Molecular Vista, a local AFM startup, and with Agilent Technologies to build the mechanobiological insights into their AFM instruments. This proposal addresses one of the National Academy of Engineering's Grand Challenges: to engineer the tools of biological scientific discovery.