Regulation of 3D Axon Guidance By Growth Cone Invadosomes

While extensive studies have investigated the molecular mechanisms that regulate axon guidance over two-dimensional substrata in vitro or along axonal tracks in vivo, little is known about the signals that control axon guidance across three dimensional tissues. Our preliminary and recently published data suggest that along with planar filopodia and lamellipodia, growth cones generate orthogonal protrusions in vitro and in vivo that resemble podosomes or invadopodia (Figure 1). Podosomes and invadopodia, collectively referred to as invadosomes, are actin-based cellular protrusions associated with extracellular matrix (ECM) degradation and tissue invasion. We hypothesize that growth cone invadosomes function to actively detect ligands through receptor interactions that regulate actin polymerization and participate in ligand and receptor degradation to modulate ligand-mediated guidance.

invadosome schematic 1
On the left is a super resolution fluorescence microscope image of a human forebrain neuron growth cone labeled for F-actin (green) and microtubules (purple) with an F-actin foci within its central domain indicated (box). F-actin foci are believed to be precursors to invadosomes, which are basal protrusions that penetrate into the underlying substratum. See image rotation (below) of 3D rendering view of Structured Illumination Microscope (SIM) image of a human cortical neuron growth cone triple-labeled for F-actin (red), ßIII tubulin (blue) and ADAM17 (green). On the right is a simplified schematic representation of key proteins believed to be involved in growth cone invadosome function. See Santiago-Medina et. al., Development 2015 for details.

Current Open Questions:

  1. How do grow factors and axon guidance cues influence invadosome formation?
  2. What roles do scaffolding and signaling proteins such as Tks5, Cortactin and Src play in growth cone invadopodia formation and function?
  3. What matrix metalloproteases and ADAM proteins are targeted to growth cone invadosomes and what roles do they play in basal lamina degradation and ligand activation?
  4. How and where do invadosomes function in 3D axon guidance in vivo?
Regulation of axon guidance by ECM adhesion and degradation. A representative neuronal growth cone (above) labeled for F-actin (red) and paxillin (green), which indicate invadosomes in the central domain (F-actin, arrows) and peripheral point contact adhesions (paxillin, arrowheads), respectively. The schematic drawing below shows common signaling pathways that regulate peripheral point contact adhesions and basally directed invadosomal protrusions. Integrin adhesions and invadosomes are regulated by extracellular signals from ECM proteins, growth factors (GF), guidance cues (GC) and chemokines in the environment. Receptors on growth cones activate signaling pathways that modulate adhesion site dynamics and peripheral actin polymerization to support planar axon extension upon the ECM. However, activation of key lipid modulators and other signaling pathways by specific GFs likely redirects actin polymerization regulators and adaptor proteins toward basal invadosomal protrusions. Mature invadosomes are targeted by microtubules, which support Kinesin motor and Rab-dependent trafficking of MMP containing vesicles. Targeted secretion of MMPs leads to local degradation of ECM, allowing developing axons to penetrate ECM barriers to cross into new tissues.