High temporal resolution tracking reveals region-specific control of focal adhesion nucleation, dynamics, and translocation

Abstract Focal adhesions (FAs) are dynamic multilayer protein complexes linking actin cytoskeleton to extracellular matrix and responsible for cell attachment, spreading and motility. To explore FA regulation and dynamics at the population level, we have developed an improved algorithm for the automated detection and tracking of FAs in living cells. We confirmed a predominantly exponential decay distribution of FA sizes and lifespans, indicating dominance of small, short-lived FAs. Automated analysis at high temporal resolution (up to 30 frames per minute) revealed several novel FA characteristics: (I) high occurrence frequency of short-living FAs with rapid growth and disassembly; (II) robust correlation between FA area and paxillin/vinculin content; (III) short-term upregulation of FA formation behind rapidly advancing lamellae, and (IV) fast sliding of FAs in retracting edges, accompanied by instances of separation of cytosolic FA layers (paxillin and vinculin). Depolymerization of microtubules resulted in stabilization of FAs (increased lifetime and protein content, decreased assembly/disassembly rates) along with decreased frequency of FA formation in rapidly advancing lamella but did not affect regional differences in FA dynamics. We suggest microtubules play a dual role in FA dynamics, accelerating turnover of nascent FAs in the entire cell and stimulating formation of FAs behind advancing lamellipodia.