Modern microscopy and image analysis, together with fluorescent probe technology, has evolved to quantify signaling in living cells and animals with seconds and microns resolution. More recently, optogenetics and chemogenetics have made it possible to control signaling in vivo, and thereby explore causal relationships among signaling molecules as they are regulated by spatio-temporal dynamics. We propose here to combine protein visualization and control in the same cell, for unprecedented quantitative accuracy in studying how Rho GTPase signals are coordinated by feed-back and feed-forward relationships. To generate proteins controlled by light or small molecules, we will use novel approaches that provide ready access to many different structures and minimize perturbation of living cells. These include dye-based biosensors of endogenous protein conformation, engineered allosteric control for inhibition or activation by light, and the use of photoresponsive protein analogs that can serve as substitutes for endogenous proteins. We will study ?frustrated phagocytosis?, a system where the complex dynamics driving phagocytosis are preserved, but are restricted to two dimensions and occur in precise geometries generated by patterned substrates. We will examine communication between spatially restricted zones of signaling using single molecule microscopy of protein conformational change. Precise control of activation gradients, kinetics and localization will be used to inform mathematical models examining how precisely segregated signaling domains are maintained. In a second project, we will work with our collaborators Eric Betzig and Leong Chew of Janelia Farm to adapt biosensor and optogenetic technologies to lattice light sheet microscopy, for visualization and control of the complex morphological changes megakaryocytes undergo as they produce platelets. There our ultimate goal will be optogenetic modification of signaling to enhance platelet production. We will focus on enabling technologies to generate minimally perturbing biosensors and optogenetic tools that can be applied by other researchers in a wide range of fields.
This proposal describes a broad program to understand basic mechanisms of signaling in three different areas relevant to disease. We will develop new methods to simultaneously visualize and control protein activities in living cells and animals. This will enable us to dissect the flow of information through the cell as it integrates extracellular stimuli and ?decides? how to behave. We will focus on phagocytosis, platelet formation, and metastasis.
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| Tajadura-Ortega, Virginia; Garg, Ritu; Allen, Richard et al. (2018) An RNAi screen of Rho signalling networks identifies RhoH as a regulator of Rac1 in prostate cancer cell migration. BMC Biol 16:29 |
| van Haren, Jeffrey; Charafeddine, Rabab A; Ettinger, Andreas et al. (2018) Local control of intracellular microtubule dynamics by EB1 photodissociation. Nat Cell Biol 20:252-261 |
| Stefanini, Lucia; Lee, Robert H; Paul, David S et al. (2018) Functional redundancy between RAP1 isoforms in murine platelet production and function. Blood 132:1951-1962 |
| Woroniuk, Anna; Porter, Andrew; White, Gavin et al. (2018) STEF/TIAM2-mediated Rac1 activity at the nuclear envelope regulates the perinuclear actin cap. Nat Commun 9:2124 |
