In the nervous system, attractive and repulsive factors guide neuronal growth, pathfinding and target innervation during development, learning and regeneration after injury. Repulsive and growth-inhibitory factors, such as some ephrins, semaphorins, netrins and myelin-associated growth inhibitors, restrict nerve fiber growth, whereas neurotrophins, and other ephrins, semaphorins and netrins attract fibers and promote neurite growth. Several of these guidance molecules also play crucial roles in vasculogenesis, and regulate cell migration and tissue formation in different organs. Precise and highly specific signal transduction in space and time is required in all these cases, which primarily depends on the presence and function of specific receptors. Interestingly, many of these ligands act through multi-subunit receptor complexes. In this Commentary, we review the current knowledge of how complexes of the receptors for attractive and repulsive neurite growth regulatory factors are reorganized in a spatial and temporal manner, and reveal the implications that such dynamics have on the signaling events that coordinate neurite fiber growth.

To ensure precision and specificity of ligand-receptor-induced signaling, co-receptors and modulatory factors play important roles. The membrane-bound ligand Nogo-A (an isoform encoded by RTN4) induces inhibition of neurite outgrowth, cell spreading, adhesion and migration through multi-subunit receptor complexes. Here, we identified the four-transmembrane-spanning protein tetraspanin-3 (TSPAN3) as a new modulatory co-receptor for the Nogo-A inhibitory domain Nogo-A-Δ20. Single-molecule tracking showed that TSPAN3 molecules in the cell membrane reacted to binding of Nogo-A with elevated mobility, which was followed by association with the signaltransducing Nogo-A receptor sphingosine-1-phosphate receptor 2 (S1PR2). Subsequently, TSPAN3 was co-internalized as part of the Nogo-A-ligand-receptor complex into early endosomes, where it subsequently separated from Nogo-A and S1PR2 to be recycled to the cell surface. The functional importance of the Nogo-A-TSPAN3 interaction is shown by the fact that knockdown of TSPAN3 strongly reduced the Nogo-A-induced S1PR2 clustering, RhoA activation, cell spreading and neurite outgrowth inhibition. In addition to the modulatory functions of TSPAN3 on Nogo-A-S1PR2 signaling, these results illustrate the very dynamic spatiotemporal reorganizations of membrane proteins during ligand-induced receptor complex organization.

As a potent radiosensitizer nitric oxide (NO) may be a putative adjuvant in the treatment of malignant gliomas which are known for their radio-and chemoresistance. The NO donor prodrug JS-K (O2-(2.4-dinitrophenyl) 1- [(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1,2-diolate) allows cell-type specific intracellular NO release via enzymatic activation by glutathione-S-transferases overexpressed in glioblastoma multiforme. The cytotoxic and radiosensitizing efficacy of JS-K was assessed in U87 glioma cells in vitro focusing on cell proliferation, induction of DNA damage, and cell death. In vivo efficacy of JS-K and repetitive irradiation were investigated in an orthotopic U87 xenograft model in mice. For the first time, we could show that JS-K acts as a potent cytotoxic and radiosensitizing agent in U87 cells in vitro. This dose-and time-dependent effect is due to an enhanced induction of DNA double-strand breaks leading to mitotic catastrophe as the dominant form of cell death. However, this potent cytotoxic and radiosensitizing effect could not be confirmed in an intracranial U87 xenograft model, possibly due to insufficient delivery into the brain. Although NO donor treatment was well tolerated, neither a retardation of tumor growth nor an extended survival could be observed after JS-K and/ or radiotherapy.