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Two-tiered coupling between flowing actin and immobilized N-cadherin/catenin complexes in neuronal growth cones
- Garcia, Mikael, Leduc, Cécile, Lagardéère, Matthieu, Argento, Améèélie, Sibarita, Jean-Baptiste, Thoumine, Olivier
- Proceedings of the National Academy of Sciences of the United States of America 2015 v.112 no.22 pp. 6997-7002
- actin, adhesion, cell movement, fluorescent proteins, image analysis, locomotion, microfilaments
- Neuronal growth cones move forward by dynamically connecting actin-based motility to substrate adhesion, but the mechanisms at the individual molecular level remain unclear. We cultured primary neurons on N -cadherin–coated micropatterned substrates, and imaged adhesion and cytoskeletal proteins at the ventral surface of growth cones using single particle tracking combined to photoactivated localization microscopy (sptPALM). We demonstrate transient interactions in the second time scale between flowing actin filaments and immobilized N -cadherin/catenin complexes, translating into a local reduction of the actin retrograde flow. Normal actin flow on micropatterns was rescued by expression of a dominant negative N -cadherin construct competing for the coupling between actin and endogenous N -cadherin. Fluorescence recovery after photobleaching (FRAP) experiments confirmed the differential kinetics of actin and N -cadherin, and further revealed a 20% actin population confined at N -cadherin micropatterns, contributing to local actin accumulation. Computer simulations with relevant kinetic parameters modeled N -cadherin and actin turnover well, validating this mechanism. Such a combination of short- and long-lived interactions between the motile actin network and spatially restricted adhesive complexes represents a two-tiered clutch mechanism likely to sustain dynamic environment sensing and provide the force necessary for growth cone migration.