Continued support is requested for our investigation of integrin-filamin interactions and their roles in adhesion signaling and cell migration. Integrins are transmembrane receptors that support cell adhesion and migration. They play essential roles throughout development, during hemostasis and in the response to injury and infection. Most integrin functions require a connection to intracellular signaling and cytoskeletal networks and these connections are largely mediated through the regulated interactions of integrin 2 subunit cytoplasmic tails with a variety of intracellular proteins. Characterizing the roles of interacting proteins, the functional consequences of their interaction, the molecular basis for their interactions, how they are regulated, and the cross-talk between different integrin 2 tail binding proteins is therefore central to a complete understanding of this important class of adhesion molecules. Filamins are large actin-crosslinking proteins composed of an N- terminal actin-binding domain and an array of immunoglobulin domains which interact with numerous cytosolic signaling proteins and transmembrane receptors, including integrins. Biochemical, cell biological and genetic data point to roles for FLN in cell migration, differentiation, signaling and the actin cytoskeleton. There are 3 filamin genes in humans, and mutations in filamin result in a wide range of developmental abnormalities and defective neuronal migration. During the current period of support we have characterized integrin-filamin interactions at atomic resolution and identified integrin and filamin mutants with up- or down-regulated affinities. We have identified mechanisms potentially regulating integrin-filamin interactions and revealed a role for filamin in regulating integrin activation state, contractility and tubule formation by breast epithelial cells. We hypothesize that filamin controls migration and adhesion signaling and plays an important role in integrin-mediated sensing and transduction of biomechanical force. To test this we aim to: 1) Characterize the role of different filamin isoforms in cell migration and to identify specific filamin interaction partners important in cell migration;2) Assess the roles of filamin in regulating integrin activation and signaling and 3) characterize the role of filamin in sensing and transducing biomechanical force. To do this we will generate filamin deficient cell lines using knockdown or post-translational targeting techniques and test the ability of different filamin isoforms or mutants defective in specific interactions to reverse phenotypes in an array of assays including cell migration, integrin activation, Rho GTPase activation, tubulogenesis, and cytoskeletal tethering. Mutant integrins with up- or down-regulated filamin binding will be tested in similar assays, and the interaction of filamin with integrin or other regulators characterized using binding assays, X-ray crystallography, NMR an single molecule force spectroscopy.
ll surface receptors called integrins mediate cell adhesion, control cell migration and act as mechano- sensors providing information about the physical environment around cells, these processes are essential during development, for hemostasis and in the response to injury and infection and are perturbed in cancer, cardiovascular and inflammatory diseases. Integrin function depends on interaction with intracellular signaling and structural proteins such as filamins and mutations in filamins result in a range of development disorders and defective neuronal migration. We seek to characterize the integrin-filamin interaction and determine how it controls cell behavior;this should provide insight into essential molecules with important roles in health and disease and may identify novel therapeutic targets.
| Iwamoto, Daniel V; Huehn, Andrew; Simon, Bertrand et al. (2018) Structural basis of the filamin A actin-binding domain interaction with F-actin. Nat Struct Mol Biol 25:918-927 |
| Kadry, Yasmin A; Huet-Calderwood, Clotilde; Simon, Bertrand et al. (2018) Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading. J Cell Sci 131: |
| Huet-Calderwood, Clotilde; Rivera-Molina, Felix; Iwamoto, Daniel V et al. (2017) Novel ecto-tagged integrins reveal their trafficking in live cells. Nat Commun 8:570 |
| Morse, Elizabeth M; Sun, Xiaowen; Olberding, Jordan R et al. (2016) PAK6 targets to cell-cell adhesions through its N-terminus in a Cdc42-dependent manner to drive epithelial colony escape. J Cell Sci 129:380-93 |
| Simpson, Mark A; Bradley, William D; Harburger, David et al. (2015) Direct interactions with the integrin ?1 cytoplasmic tail activate the Abl2/Arg kinase. J Biol Chem 290:8360-72 |
| Iwamoto, Daniel V; Calderwood, David A (2015) Regulation of integrin-mediated adhesions. Curr Opin Cell Biol 36:41-7 |
| Uchil, Pradeep D; Pawliczek, Tobias; Reynolds, Tracy D et al. (2014) TRIM15 is a focal adhesion protein that regulates focal adhesion disassembly. J Cell Sci 127:3928-42 |
| Lee, Monica Y; Skoura, Athanasia; Park, Eon Joo et al. (2014) Dynamin 2 regulation of integrin endocytosis, but not VEGF signaling, is crucial for developmental angiogenesis. Development 141:1465-72 |
| Morse, Elizabeth M; Brahme, Nina N; Calderwood, David A (2014) Integrin cytoplasmic tail interactions. Biochemistry 53:810-20 |
| Ellis, Stephanie J; Lostchuck, Emily; Goult, Benjamin T et al. (2014) The talin head domain reinforces integrin-mediated adhesion by promoting adhesion complex stability and clustering. PLoS Genet 10:e1004756 |
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