Abstract
Growth cones of extending axons navigate to correct targets by sensing a guidance cue gradient via membrane protein receptors. Although most signaling mechanisms have been clarified using an in vitro approach, it is still difficult to investigate the growth cone behavior in complicated extracellular environment of living animals due to the lack of tools. We develop a system for the light-dependent activation of a guidance receptor, Deleted in Colorectal Cancer (DCC), using Arabidopsis thaliana Cryptochrome 2, which oligomerizes upon blue-light absorption. Blue-light illumination transiently activates DCC via its oligomerization, which initiates downstream signaling in the illuminated subcellular region. The extending axons are attracted by illumination in cultured chick dorsal root ganglion neurons. Moreover, light-mediated navigation of the growth cones is achieved in living Caenorhabditis elegans. The photo-manipulation system is applicable to investigate the relationship between the growth cone behavior and its surrounding environment in living tissue.
Highlights
We show a photo-activation system for DCC, an axon guidance receptor protein embedded in the plasma membrane
DCC connected with photo-insensitive CRY2 mutant (D387A)[22], represented as photo-activatable DCC (PA-DCC) (D387A), and DCC without CRY2 (DCC) were prepared as a non-light reactive control (Fig. 1a)
These results indicate that photo-conversion of CRY2 triggered PA-DCC oligomerization
Summary
We show a photo-activation system for DCC, an axon guidance receptor protein embedded in the plasma membrane. Cytoplasmic domain of DCC oligomerizes upon its extracellular ligand binding, which is crucial for the activation of intracellular signaling to induce cytoskeletal remodeling[20]. To control the oligomerization by external light, CRY2 is connected genetically with DCC, named photo-activatable DCC (PA-DCC). PA-DCC enables us to induce axon attraction by blue light illumination in chick DRG neurons. We demonstrate its applicability for the in vivo light-induction of growth cone turning in the nematode Caenorhabditis elegans
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