Project Summary Many cellular processes, such as spreading, motility, division, and morphogenesis generate membrane tension gradients. Such gradients drive membrane flows, which relax the initial gradients. In addition, quiescent cells maintain a constant surface area and a relatively stable membrane tension, ๐, by balancing the rates at which membrane is added (via exocytosis) and removed (via endocytosis) to and from the cell surface. Changes in ๐ have been proposed to provide rapid, long-range cellular signaling. Yet, how the plasma membrane flows and how gradients of ๐ relax are very poorly understood, with estimates of membrane tension equilibration times in cells ranging from milliseconds to tens of minutes. One of the major reasons underlying this dearth of knowledge is the lack of suitable methods for measuring membrane tension changes in live cells. In the past, two classes of membrane tension measurements have been developed, but both have severe limitations. The first class is based on changes in optical properties of small molecules. These sensors probe local properties of cell membranes. Due to large heterogeneities in biological membranes, and potential interactions of the probes with various membrane components, correlating ๐ with the local properties probed by these small molecule sensors is not straightforward. The second approach relies on pulling a thin membrane tether from the cell surface and measuring the tether force using optical trapping or atomic force microscopy. The tether force is related to the in-line membrane tension, membrane bending modulus, and the adhesion energy between the plasma membrane and the cytoskeleton. This approach allows a "true" membrane tension to be measured, but requires specialized equipment, is very difficult to implement when cells undergo physiological changes when tension gradients are most likely to arise, and only provides a local measurement. Thus, despite the urgent need, there are no direct and convenient probes to quantify membrane tension gradients during cellular processes. We propose to close this gap by developing a radically new class of membrane tension sensors based on DNA-based self-assembly of an elastic network over cell surfaces, called LEMONADE, for Lego-like membrane tension analyzer based on self-assembled DNA elastic networks. We aim to 1) develop a library of DNA tiles and connector-springs that self-assemble on cell surfaces into a network with tunable properties. A variety of DNA tile and connector-spring designs will be generated and optimized for self-assembly on membranes. The connectivity and elasticity of the network will be tunable by substitution of components with different properties. Expansion or contraction of the network due to changes in membrane area will be detected using FRET dye pairs located on the connector- spring modules. 2) Characterize the response of the DNA-based membrane tension sensor to controlled membrane tension perturbations in various ...