To form a properly connected and hence functioning nervous system neurons must make appropriate and specific connections with each other during embryogenesis.
A major mechanism for achieving this is growth cone pathfinding. Growth cones navigate precise routes through developing embryos to locate their appropriate synaptic partners. To do this they respond to a variety of guidance molecules that they encounter along the route. These molecules activate receptors in the growth cone membrane and lead, via intracellular signalling pathways, to changes in the motile behaviour of the growth cone. Necessarily this involves regulating the cytoskeleton of the growth cone and it is this interaction, between guidance molecules and the growth cone cytoskeleton, which we seek to understand. We are approaching this using a wide range of cell and molecular biological techniques including neuronal cell culture, fluorescence and video microscopy and DNA and protein biochemistry.
Microtubule-actin filament interactions in growth cone pathfinding
In growth cones, dynamic microtubules are oriented with their growing tips distal and are continually extending, by plus tip assembly, towards the actin-filament-rich peripheral domain. These microtubules extend into selected filopodia where they become aligned alongside the proximal ends of the filamentous (F)-actin bundles that support filopodia.
The interaction between dynamic microtubules and F-actin is essential for neuritogenesis and growth cone pathfinding. It is suspected that at the core of the molecular machinery enabling interactions between microtubules and F-actin are proteins located at the growing tips of microtubules, so-called +TIP proteins, and that these bind to actin-binding proteins bound to F-actin. A major area of research in the lab relates to the protein complex that mediates this interaction between these two filamentous systems and how it is regulated. We have recently shown that the F-actin binding protein drebrin couples filopodial F-actin to dynamic microtubules in growth cones by binding directly to the +TIP protein EB3. We are now trying to understand how the drebrin interactome functions to couple dynamic microtubules to F-actin and enable growth cone pathfinding.
A master regulator of the growth cone cytoskeleton is the serine/threonine kinase glycogen synthase kinase-3β (GSK-3β). There are two spliced isoforms of GSK-3β in neurons, a short form lacking exon 9 (GSK3β1) that is ubiquitously expressed and a long form containing exon 9 (GSK3β2) that is neuron specific. There is indirect evidence that GSK3β1 and GSK3β2 have separate functions in developing neurons but their distinct phosphorylation substrates in growth cones have not been identified. A major substrate of both GSK-3β isoforms in growth cones is the microtubule-associated protein MAP1B. We have shown that MAP1B controls microtubule dynamics in growth cones and that this function is regulated by GSK-3β phosphorylation. How these two isoforms of GSK-3β are regulated and their down-stream effectors in growth cones are central areas of interest in the lab.