I took notice of a Nature Neuroscience paper recently that came out back in September, and brings together a few of my interests in cell migration: axon guidance, extracellular matrix (ECM), and focal adhesion kinase (FAK). In their paper, Focal adhesion kinase signaling at sites of integrin-mediated adhesion controls axon pathfinding, Robles and Gomez lay out their rationale and findings as follows:
Neuronal growth cones contain accumulations of PY-containing proteins within adhesion complexes and at the tips of filopodia. We recently demonstrated that filopodial extension is regulated by PY accumulations at their tips. To further clarify the role of tyrosine kinase signaling in axonal growth and guidance, we characterized growth cone PY signaling at sites of ECM adhesion. We found that the formation of PY-containing point contacts is determined by the ECM substratum and requires FAK activity. Signaling through FAK promotes integrin-dependent growth cone motility and is required for proper neurite outgrowth and guidance in vitro and in vivo.
Robles and Gomez used two strategies to inhibit FAK activity in growth cones, and thus identify what FAK’s functions are in these cell structures – expression of the inhibitory splice variant of FAK, the FAK-related non-kinase (FRNK); and RNAi knockdown of endogenous FAK. Plasmids for both of these strategies (and controls) were injected into Xenopus blastomeres, and derived embryonic spinal explants were harvested for culture. From this, they obtained a number of nice fluorescent micrographs, including immunostaining for paxillin, FAK, and phosphotyrosines (PY). Two areas stood out as of interest to me, in studying the role of FAK in cell migration…
First, they describe in the results the live imaging of neurons with impaired FAK activity, growing on laminin, and found a strong reduction in the rate of neurite outgrowth and increased filopodial lifetimes (Figure 3; l,m). They concluded that the stable and protrusive filopodia suggested that FAK activity promotes the turnover of filopodia independently of their ability to elongate – much like FAK is thought to promote turnover of focal adhesions. In contrast, they found that lamellipodial protrusions generated by FAK-impaired growth cones were typically more transient than those observed under control conditions. So while FAK might promote filopodial turnover, it appeared to do the opposite – stabilize – lamellipodial protrusions.
Second, Robles and Gomez examined whether assymetric FAK activation could control the direction of neurite outgrowth through selective lamellipodial stabilization, and found that substratum-dependent growth cone turning requires FAK. They did this by culturing spinal neuron explants with adjacent regions of the ECM proteins laminin and tenascin. Given that both laminin and tenascin supported adhesion and neurite extension, yet differentially stimulated FAK activity, this assay tested whether the ability to form point contacts and stabilize lamellipodia selectively in one direction promotes growth cone turning. And, sure enough, they found an assymetric stabilization of lamellipodia and subsequent redirection of growth cones (Figure 5).
While other mechanisms are surely at work in growth cone guidance, making such biochemical signaling networks clogged and confusing to the researcher, these findings aren’t radical news. And further, based on canonical pathways for polarity and actin dynamics, the obvious conclusion is that FAK is differentially effecting Rac (lamellipodia) and Cdc42 (filopodia).
- Robles E, Gomez TM. Focal adhesion kinase signaling at sites of integrin-mediated adhesion controls axon pathfinding. Nat Neurosci. 2006 Oct;9(10):1274-83. Pubmed.