Calcium rises locally trigger focal adhesion disassembly and enhance residency of focal adhesion kinase at focal adhesions.
Focal adhesion kinase (FAK) activity and Ca(2+) signaling led to a turnover of focal adhesions (FAs) required for cell spreading and migration. We used yellow Cameleon-2 (Ycam), a fluorescent protein-based Ca(2+) sensor fused to FAK or to a FAK-related non-kinase domain, to measure simultaneously local Ca(2+) variations at FA sites and FA dynamics. Discrete subcellular Ca(2+) oscillators initiate both propagating and abortive Ca(2+) waves in migrating U87 astrocytoma cells. Ca(2+)-dependent FA disassembly occurs when the Ca(2+) wave reaches individual FAs, indicating that local but not global Ca(2+) increases trigger FA disassembly. An unexpectedly rapid flux of FAK between cytosolic and FA compartments was revealed by fluorescence recovery after photobleaching studies. The FAK-Ycam recovery half-time (17 s) at FAs was slowed (to 29 s) by Ca(2+) elevation. FAK-related non-kinase domain-Ycam had a faster, Ca(2+)-insensitive recovery half-time (11 s), which is consistent with the effect of Ca(2+) on FAK-Ycam dynamics not being due to a general modification of the dynamics of FA components. Because FAK association at FAs was prolonged by Ca(2+) and FAK autophosphorylation was correlated to intracellular Ca(2+) levels, we propose that local Ca(2+) elevations increase the residency of FAK at FAs, possibly by means of tyrosine phosphorylation of FAK, thereby leading to increased activation of its effectors involved in FA disassembly.
Giannone
Grégory
G
Laboratoire de Pharmacologie et Physicochimie des Interactions Cellulaires et Moléculaires, Unité Mixte de Recherche CNRS 7034, Université Louis Pasteur de Strasbourg, 67401 Illkirch, France.
Rondé
Philippe
P
Gaire
Mireille
M
Beaudouin
Joël
J
Haiech
Jacques
J
Ellenberg
Jan
J
Takeda
Kenneth
K
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement; D002614:Chelating Agents / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D008027:Light; D008164:Luminescent Proteins / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D002614:Chelating Agents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D015759:Ionomycin; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D002118:Calcium