The actin nucleating Arp2/3 complex contributes to the formation of axonal filopodia and branches through the regulation of actin patch precursors to filopodia

Abstract

The emergence of axonal filopodia is the first step in the formation of axon collateral branches. In vitro, axonal filopodia emerge from precursor cytoskeletal structures termed actin patches. However, nothing is known about the cytoskeletal dynamics of the axon leading to the formation of filopodia in the relevant tissue environment. In this study we investigated the role of the actin nucleating Arp2/3 complex in the formation of sensory axon actin patches, filopodia and branches. By combining in ovo chicken embryo electroporation mediated gene delivery with a novel acute ex vivo spinal cord preparation, we demonstrate that actin patches form along sensory axons and give rise to filopodia in situ. Inhibition of Arp2/3 complex function in vitro and in vivo decreases the number of axonal filopodia. In vitro, Arp2/3 complex subunits and upstream regulators localize to actin patches. Analysis of the organization of actin filaments in actin patches using platinum replica electron microscopy reveals that patches consist of networks of actin filaments, and filaments in axonal filopodia exhibit an organization consistent with the Arp2/3-based convergent elongation mechanism. Nerve growth factor (NGF) promotes formation of axonal filopodia and branches through phosphoinositide 3-kinase (PI3K). Inhibition of the Arp2/3 complex impairs NGF/PI3K-induced formation of axonal actin patches, filopodia, and the formation of collateral branches. Collectively, these data reveal that the Arp2/3 complex contributes to the formation of axon collateral branches through its involvement in the formation of actin patches leading to the emergence of axonal filopodia.

Regulation of ECM degradation and axon guidance by growth cone invadosomes

“…2E-H), and we suspect that these two processes could be linked (Chan et al, 2009;Liu et al, 2010). It is noteworthy that the F-actin foci that we observed in growth cones share many similarities to actin patches that have been found to be responsible for collateral branching of chick DRG axons (Ketschek and Gallo, 2010;Spillane et al, 2011).…”

The Adaptor Protein CD2AP Is a Coordinator of Neurotrophin Signaling-Mediated Axon Arbor Plasticity

“…In our model, NGF signaling may be directed by CD2AP to activate the AKT pathway that may drive axon branch formation during arbor expansion. Accordingly, NGF promotes filopodia formation and axonal branching in a PI3K-dependent manner (Ketschek and Gallo, 2010) by inducing local actin nucleation by the Arp2/3 complex (Spillane et al, 2011). Independently, CD2AP has been shown to directly bind Arp2/3 to positively regulate actin nucleation and concomitant actin dynamics (Table 1).…”

Kinesin-1 sorting in axons controls the differential retraction of arbor terminals

“…It is thought that the balance between stabilization and destabilization of F-actin and microtubules determines the formation and growth of axonal branches. For example, activation of the actin nucleation factors, Arp2/3 and Cordon-bleu (Cobl) is required for filopodia formation from the actin patch, and inhibition of this pathway results in decreased branch number (Strasser et al, 2004;Ahuja et al, 2007;Spillane et al, 2011Spillane et al, , 2012. The microtubule-severing enzymes, spastin and katanin, which provide a branching point at the axonal shaft, also control the number of axonal branches .…”