A central process in connective tissue homeostasis is cell migration, which involves dynamic interactions between focal adhesions, the actin cytoskeleton and mitochondria, but the role of focal adhesion proteins in cell migration is not wholly defined. We examined focal adhesion-associated proteins from mouse fibroblasts and identified Flightless I (FliI) as a potential focal adhesion protein. We determined that FliI is distributed in the cytosol and co-localizes with actin monomers and mitochondria, but partially with paxillin. Biochemical assays showed that FliI associates with both actin monomers and short oligomers/filaments. Migration assay determined that cells with reduced FliI expression migrated more quickly and that FliI knockdown inhibited activation of β1 integrins. Consistent with these data, cell adhesion assay demonstrated that FliI knockdown cells were less adherent than wildtype cells. Our findings indicate that FliI may regulate cell migration by interacting with the actin monomers and the mitochondria to affect cell adhesion.

A central process in connective tissue homeostasis is cell migration, which involves dynamic interactions between focal adhesions, the actin cytoskeleton and mitochondria, but the role of focal adhesion proteins in cell migration is not wholly defined. We examined focal adhesion-associated proteins from mouse fibroblasts and identified Flightless I (FliI) as a potential focal adhesion protein. We determined that FliI is distributed in the cytosol and co-localizes with actin monomers and mitochondria, but partially with paxillin. Biochemical assays showed that FliI associates with both actin monomers and short oligomers/filaments. Migration assay determined that cells with reduced FliI expression migrated more quickly and that FliI knockdown inhibited activation of β1 integrins. Consistent with these data, cell adhesion assay demonstrated that FliI knockdown cells were less adherent than wildtype cells. Our findings indicate that FliI may regulate cell migration by interacting with the actin monomers and the mitochondria to affect cell adhesion.

Ca2+/calmodulin-dependent protein kinase type-II (CaMK-II) is a Ser/Thr protein kinase regulated by Ca2+ and Calmodulin. It is a highly conserved and broadly expressed enzyme and has a unique structure and dynamic regulation. It has the ability to remain active in the absence of Ca 2+ as a result of Ca2+ dependent autophosphorylation. CaMK-II phospliorylates proteins involved in neurotransmitter secretion, long term potentiation, cytoskeletal dynamics, gene transcription, and cell motility. To support existing and identify new intracellular roles of CaMK-II, potential binding partners were identified. This was accomplished by transfecting and purifying "FLAG-tagged" CaMK-II's (α, βE, δC, and δE). CaMK-II associated proteins were then identified using tandem mass spectrometry. Known binding partners were identified using this approach, including CaMK-II and calmodulin, verifying the approach's validity. Additionally several unexpected but interesting proteins were identified, including the gelsolin related actin binding protein, Flightless-I. Fli-I is an actin binding and capping protein that also functions as a transcriptional coactivator. The CaMK-II-Fli-I interaction was confirmed with endogenous (un-tagged) proteins. The association and localization of Fli-I are dependent on CaMK-II's activity state, although Fli-I is not a substrate of CaMK-II. When CaMK-II is inhibited, Fli-I translocates to the nucleus. Conversely when CaMK-II is artificially activated using a Ca2+ ionophore, Fli-I returns to the cytosol. The discovery of this reversible interaction epresents a potentially new CaMK-II regulated pathway and likely serves as a link between Ca2+ based signal transduction pathways and regulation of the actin component of the cytoskeleton and transcription.