(E-161) Investigating Membrane-cortex Adhesion Using FRET-based Tension Sensor

The mechanical properties of the cellular cortex, which are comprised of the plasma membrane and the underlying actin cortex, play critical roles in cell morphology, polarization, protrusion, and migration. Furthermore, defects in the cellular cortex, particularly the ability of the two sub-structures to maintain adhesion in response to applied forces, have been implicated in the progression of diseases such as cancer metastasis. The most common physiological linker that mediates cellular cortex adhesion is the ERM family protein (ezrin, radixin, moesin), especially ezrin, which is reported to contribute to cell mechanical properties changes during many key developmental or pathological processes of cells, such as stem cell differentiation and tumorigenesis. Moreover, there are a wide variety of well-established conserved domains that bind either membrane composition or the actin filament with different affinities, which provide the basis for engineering exogenous linkers that interfere with cortex mechanics. Therefore, in this work, we seed to probe and manipulate the mechanical properties of the cell cortex, focusing on the role membrane-cortical-actin(MCA) linkers play in mechanotransduction and cortical mechanics.

First, we explored the mechanical functions of the ERM family protein, ezrin. We utilized stable shRNA-mediated knockdown to create a cellular system where the mechanical function of ezrin could be easily probed. Next, we produced and validated an Ezrin tension sensor (EzrTS) by engineering a FRET tension sensor module into Ezrin. Then we use sensitized-emission FRET imaging technics to acquire FRET signals from EzrTS reconstituted Ezrin-KD A431 cells, a type of epithelial cell where ezrin is first found. Secondly, to investigate the possibility of manipulating cell behaviors by controlling membrane-to-cortex adhesion, we created a suite of artificial membrane-actin cross-linkers comprised of various protein domains with different binding affinities to the membrane and cortical actin containing as well as tension sensors and tested if they can successfully report MCA adhesive force while altering cell shape dynamics.

FRET images of EzrTS reconstituted cells revealed that ezrin is subject to mechanical forces that are reduced in response to biochemical perturbation, such as actomyosin inhibitors, and mechanical stimulus such as hypertonic shock. The initial design of artificial MCA linkers with F-tractin that binds actin cortex and kCaax motif targeting membrane by prenylation show that increased membrane adhesion increases molecular tension and reduces membrane protrusion. Together, these results advance our understanding of the molecular mechanisms mediating membrane mechanics and cell migration. In the future, we can promote our understanding of how MCA adhesion directs cell behavior by utilizing other biological or mechanical tools, such as micropatterned ECM substrate to study cell migration, polyacrylamide-based gel to study stiffness-dependent cell shape control,  and the magnetic tweezer for site-specific mechanical perturbation. We look forward to seeing a great variety of applications brought up by our MCA adhesion sensor.