Mechanisms of axon guidance during development

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications & trials

Abstract

The experimental system we use in our lab is regulation of axon growth and guidance by the receptor Notch, via its regulation of the Abl tyrosine kinase signaling network. In the past year, we published two papers that significantly advance our understanding of this molecular machine. First, as a corollary to dissecting the role of Notch in cell morphogenesis, it became necessary to understand how cell morphogenesis and the cytoskeleton modulate Notch. In Hunter, et al., we found that actomyosin regulates the mechanism of Notch signaling in two ways. First, it is directly involved in generating the pulling force that stretches the Notch extracellular domain upon binding its ligand Delta, thereby exposing an extracellular protease cleavage site and initiating the proteolytic cascade of Notch receptor activation. Second, we found (unexpectedly) that activation of Notch is profoundly sensitive to the rigidity of the associated plasma membrane: without a rigid surface against which to pull, Delta endocytosis evidently cannot produce sufficient force to expose the Notch protease cleavage site and thereby initiate signaling. This is likely to be one of the molecular mechanisms underlying recent results implicating local membrane rigidity of the stem cell niche in the choice of stem cells to self-renew or differentiate. Second, we attacked more directly the molecular nature of the Notch-Abl signaling pathway, and in particular the similarities and differences of its initiating events with those of canonical, nuclear Notch signaling. In Kannan, et al., we found that activation of Notch-Abl signaling requires precisely the same proteolytic cascade as does canonical Notch signaling: the very same Notch cleavage event that releases the Notch intracellular domain from the membrane to transit to the nucleus to build a transcriptional transactivating complex, also induces disassembly of membrane-tethered complexes of Notch with components of the Abl network, thereby locally inactivating Abl signaling by the same mechanism that activates Notch-CSL nuclear signaling. What distinguishes these two machines is that Notch-Abl complexes employ a small, specialized population of Notch protein that is phosphorylated on juxtamembrane tyrosine residues. This provides us with a specific biochemical handle on that subset of Notch protein molecules that signal locally to the cytoskeleton via Abl, in addition to revealing the molecular mechanism by which Notch locally suppresses Abl network signaling. In parallel to these molecular studies of Notch itself, we have also advanced our live-imaging analysis of the mechanism of growth and guidance of a Notch-dependent axon, the TSM1 neuron of the Drosophila wing. In a paper that we have released on bioRxiv (and that is currently under review elsewhere), we have performed a detailed, high resolution analysis of the actin dynamics that underly axon growth, their relationship to growth cone motility, and their regulation by Abl network signaling. What we have shown is that actin organization is the key target of Abl, and that actin, in turn, controls axon growth by locally controlling filopodial dynamics. In particular, in vivo, the distal axon of TSM1 maintains a local accumulation of actin. This mass of actin undergoes intrinsic, stochastic fluctuations in size, but Abl (presumably in response to external guidance signals) introduces a spatial bias into those fluctuations. This causes the actin mass to advance along a defined trajectory, and as it does, regions that gain actin density also gain the ability to promote filopodial extension, while regions that lose their actin lose the ability to maintain filopodia and thus convert to smooth, definitive, axon shaft. In the process, the axon automatically lengthens, and does so along a defined trajectory. We are currently extending these studies by investigating the contributions of other signaling molecules, including other components of the Abl network, performing detailed computational simulations of actin dynamics to dissect the molecular events that underly this cell biology, and investigating directly the effect of Notch activation on the cytoskeletal events we have been imaging in the developing growth cone.