An important unsolved question is how extracellular signals lead to force-generating events needed for cell migration. Cell locomotion requires coordination between localized membrane protrusions and matrix attachments at the cell front, and localized retraction and disassembly of adhesions at the rear. Signaling pathways which control protein movement to the rear of cells are poorly understood, and involve rear-polarized elevation of actomyosin and Ca2+, through mechanisms that have not been fully explained. This proposal addresses a recently discovered intracellular complex, named the ?Wnt5a receptor-actin-myosin-polarity (WRAMP) structure, which integrates endosome-cytoskeleton interactions with mechanisms for cell polarity to achieve a spatially restricted, rear-polarized elevation of actomyosin and cytosolic Ca2+. The goal of this project is to characterize the mechanisms which explain how the WRAMP structure activates rear actomyosin and Ca2+, and how it localizes to the rear to guide directional cell migration.
The Specific Aims will address three key objectives:
Aim 1. Determine the protein interactions in WRAMP structures that induce actomyosin contraction.
This aim will test the hypothesis that MCAM and Wnt5a receptors, contained in a specific pool of endosome vesicles, interact with cytosolic proteins to promote localized activation of RhoA and ROCK, which in turn elevate actomyosin.
Aim 2. Determine the signals responsible for cellular localization of the WRAMP structure.
This aim will test the hypothesis that the polarization of WRAMP structures involves endosome trafficking along cortical microtubules, signaled by interactions between the plasma membrane binding protein, ezrin, and the scaffold, IQGAP1.
Aim 3. Investigate how the WRAMP structure promotes Ca2+ signaling.
This aim will test the hypothesis that the WRAMP structure elevates cytosolic Ca2+ by facilitating ER-PM contacts and STIM-ORAI interactions. The project is innovative, because the WRAMP structure is an unexplored cellular mechanism, which illustrates how intracellular positioning of organelles controls cytoskeletal reorganization and cell polarity. The project also presents multidisciplinary strategies to analyze large heterogeneous complexes, which like the WRAMP structure, involve many components and are temporally and spatially dynamic, and therefore hard to analyze. Our project is supported by a wealth of preliminary data, and integrates the expertise of many collaborators to address a cutting-edge, hypothesis-driven research question. The outcomes will generate a conceptually new knowledge about basic mechanisms for symmetry breaking, front-rear polarity, and cell migration.
The proposed research is relevant to public health because it provides a way to explain how a signaling molecule, named Wnt5a, stimulates front-rear cell polarity, which gives cells the ability to undergo movement. This problem is relevant to cancer, because Wnt5a has been demonstrated to increase malignancy and metastasis in melanoma, lung, gastric, pancreatic and ovarian cancers. However, because Wnt5a is also known to inhibit cancers of colon, thyroid, and blood cells, strategies that simply block it may result in adverse effects. The research in this proposal will build new knowledge about basic mechanisms for Wnt5a, which will be useful for developing more specific therapeutics against cancer.