Activation of Ezrin/Radixin/Moesin Mediates Attractive Growth Cone Guidance through Regulation of Growth Cone Actin and Adhesion Receptors

Abstract

The development of a functioning neural network relies on responses of axonal growth cones to molecular guidance cues that are encountered en route to their target tissue. Nerve growth factor (NGF) and neurotrophin-3 serve as attractive cues for chick embryo sensory growth cones in vitro and in vivo, but little is known about the actin-binding proteins necessary to mediate this response. The evolutionarily conserved ezrin/radixin/moesin (ERM) family proteins can tether actin filaments to the cell membrane when phosphorylated at a conserved threonine residue. Here we show that acute neurotrophin stimulation rapidly increases active phospho-ERM levels in chick sensory neuron growth cone filopodia, coincident with an increase in filopodial L1 and β-integrin. Disrupting ERM function with a dominant-negative construct (DN-ERM) results in smaller and less motile growth cones with disorganized actin filaments. Previously, we found that nerve growth factor (NGF) treatment increases ADF/cofilin activity and growth cone F-actin (Marsick et al., 2010). Here, we show this F-actin increase, as well as attractive turning to NGF, is blocked when ERM function is disrupted, despite normal activation of ADF/cofilin. We further show that DN-ERM expression disrupts leading edge localization of active ADF/cofilin and free F-actin barbed ends. Moreover, filopodial phospho-ERM levels are increased by incorporation of active ADF/cofilin, and reduced by knockdown of L1CAM. Taken together, these data suggest that ERM proteins organize actin filaments in sensory neuron growth cones and are crucial for neurotrophin-induced remodeling of F-actin and re-distribution of adhesion receptors.

The Role of Arp2/3 in Growth Cone Actin Dynamics and Guidance Is Substrate Dependent

“…Consistent with reports that actin polymerization is critical for guidance (Lanier et al, 1999;Marsick et al, 2010), growth cones exhibited reduced turning responses to chemoattractants (NGF or Netrin) and defective chemorepulsive responses (substrate-bound ephrin A2) on L1 during Arp2/3 inhibition. The reduced leading edge protrusion and the veil collapse during Arp2/3 inhibition would hamper growth cone turning toward an attractant.…”

“…Netrin is a potent stimulator of actin polymerization and a chemoattractant (de la Torre et al, 1997;Marsick et al, 2010) in the guidance of RGC axons from the retina into the optic nerve head (Deiner et al, 1997). We inhibited Arp2/3 in DRG or retina explants with CK666 for 4 h before experiments or by overnight expression of the dominant-negative construct EGFP-CA.…”

“…Actin retrograde flow is controlled by three main factors: (1) myosin II activity pulling F-actin back from the leading edge (Lin et al, 1996;Medeiros et al, 2006); (2) elongating actin filaments that push on the membrane at the leading edge, resulting in increased membrane tension, which pushes back on the growing filaments, adding to the retrograde flow of the actin network; (Henson et al, 1999;Medeiros et al, 2006;Craig et al, 2012); and finally (3) linkage of actin filaments to substrate adhesions that restrict their backward flow relative to the substrate (Suter et al, 1998;Shimada et al, 2008;Marsick et al, 2012). Since Arp2/3 inhibition substantially reduced barbed-end numbers and total F-actin, we investigated what effect it would have on the retrograde flow of actin in growth cones on L1.…”

PAK–PIX interactions regulate adhesion dynamics and membrane protrusion to control neurite outgrowth

“…Phosphorylation of these sites in response to growth factors and guidance cues serves to regulate adhesion dynamics and coordinate additional downstream signals. Recent evidence suggests that the assembly, distribution and dynamic turnover of growth cone PCs is a fundamental regulator of axon outgrowth and guidance (Carlstrom et al, 2011;Hines et al, 2010;Marsick et al, 2012;.…”

Roles of BMP6/7 in Actin Dynamics in Amyloid β-Induced Neurotoxicity

“…As we known, ADF and cofilin are both actin-binding pro-teins with similar amino acid sequences and functions (Marsick et al, 2012). The binding of ADF/cofilin to G-actin leads to sequestering actin monomers, while the binding of ADF/cofilin to F-actin leads to severing actin polymers (Jang et al, 2005).…”