PROJECT SUMMARY
Directional sensing of force plays a critical role in a wide variety of physiologic processes including migration,
angiogenesis, morphogenesis, and mechanical homeostasis. An important aspect of this force sensing is that
many of these forces are dynamic in nature, requiring that cells constantly probe, sense, and respond to force
in both time and space using force sensitive proteins. While these forces exist at the tissue scale, they are
sensed through molecular deformations at the protein scale that drive changes in binding, reinforcement, and
downstream signaling. Despite the critical importance of directional force sensing, the molecular mechanisms
that allow for this behavior are unclear. While many tools now exist for measuring biophysical forces and different
length scales, there are several unresolved questions with respect to the scale at which this directional force
sensing occurs. Recent work has indicated that mechanosensitive molecules in the cytoskeleton display direction
dependent reinforcement of bond lifetimes at the molecular scale. While this behavior has the potential to drive
directional force sensing at the cellular and tissue scales, tools for measuring the orientation of molecular forces
in living cells are limited. One established method for measuring orientation, polarized fluorescence microscopy,
is limited by its lack of intracellular force and dynamics information. The goal of this work is to develop methods
for simultaneous measurement of these dynamic forces and material systems for interrogation of cellular
responses to directional mechanical cues. To accomplish this, this research program will consist of three
research themes. Theme 1: Do molecular level forces and force orientations drive emergent directional
mechanosensing behaviors at the cellular scale? We will develop imaging and analysis methods for
simultaneous measurement of multiple biophysical variables in living cells that allow for cross-correlation of
molecular measurements with emergent behaviors. Theme 2: Can we engineer environments to manipulate and
study directional mechanosensing behaviors at the molecular and cellular scale? By engineering cellular
microenvironments with independently controllable directional cues that drive durotaxis and contact guidance,
we will investigate the molecular basis of these phenomena. Theme 3: Are environmental deformations
directionally sensed through individual proteins with directionally reinforced domains? By developing systems
for high resolution analysis of cellular deformation mechanosensing using engineered laminates with
independently controllable stiffness and Poisson’s ratio, we will assess biophysical changes at the molecular
and cellular scales in response to directional deformation fields. The combined imaging modalities and material
systems developed in this proposal will allow for detailed dissection of directional force sensing at the molecular
level, providing an important new toolset for interrogating and manipulating molecular mechanisms of directional
force sensing.
