Two-tiered coupling between flowing actin and immobilized N -cadherin/catenin complexes in neuronal growth cones

Abstract

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.growth cone | actin flow | N-cadherin adhesion | micropatterned substrates | single-molecule tracking G rowth cones are motile structures at the extremity of axons responsible for path finding and neurite extension during nervous system development and repair. Growth cones translate extracellular signals into directional migration through a coordinated regulation of cytoskeleton, adhesion, and membrane processes (1). At the cytoskeletal level, motility is generated by polarized actin treadmilling, which, together with myosin contraction, generates a continuous retrograde actin flow from the periphery to the base of growth cones (2-7). At the membrane level, adhesion proteins form dynamic bonds with immobilized extracellular ligands, allowing step-by-step locomotion (8).The molecular clutch model postulates that the mechanical coupling between ligand-bound transmembrane adhesion receptors and the actin flow allows traction forces to be transmitted to the substrate, resulting in local diminution of the retrograde flow and forward progression (9-11). Optical tweezers and flexible substrate experiments using microspheres coated with adhesion molecules revealed clutch-like mechanisms for integrins (12, 13), Ig cell adhesion molecules (14, 15), and cadherins (16,17). However, the mechanism of clutch engagement at the individual molecular level remains elusive. For integrin-based adhesion, single-molecule tracking experiments suggested that talin and vinculin could switch between a state bound to flowing actin and a state bound to immobilized integrins (18, 19). For cadherin-based adhesion,...

Defective Gpsm2/Gαi3 signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndrome

“…63. In brief, coverslips were mounted in a chamber and placed on a Nikon Ti Eclipse inverted microscope (Nikon France S.A.S., Champigny-sur-Marne, France) equipped with a TIRF arm coupled to a fibre optic linked to a four-colour laser bench (Roper Scientific).…”

“…Image stacks of 320 frames were acquired in time-lapse mode at a frequency of two images per second, with a camera exposure time of 250 ms. This procedure ensures that fast-diffusing actin monomers which contribute to a blur in the images are eliminated from subsequent analysis, and that only slowly moving actin molecules incorporated in filaments are retained6364. Experiments were done on 11 growth cones for control and 12 growth cones for cKO Gpsm2 neurons, from at least three separate experiments.…”

“…63. Single-molecule localization was performed using a wavelet-based algorithm, and trajectories were computed using a simulated annealing algorithm65.…”

Release activity-dependent control of vesicle endocytosis by the synaptic adhesion molecule N-cadherin

“…adjacent to, but largely outside of the active zone)3839, and thus close to the proposed endocytosis zone of mature presynaptic boutons40. Regarding the molecular mechanism, N-cadherin might exert its enhancing effect by activating a catenin-dependent reorganization of actin filaments4142 that might lead to altered myosin-dependent translocation of vesicles43.…”

Local Arp2/3-dependent actin assembly modulates applied traction force during apCAM adhesion site maturation

“…, 2009; Myers and Gomez, 2011; Thievessen et al. , 2013; Gomez and Letourneau, 2014; San Miguel-Ruiz and Letourneau, 2014; Yamashiro and Watanabe, 2014; Garcia et al. , 2015; Nichol et al.…”