Delineating neurotrophin-3 dependent signaling pathways underlying sympathetic axon growth along intermediate targets.

Postganglionic sympathetic neurons detect vascular derived neurotrophin 3 (NT3) via the axonally expressed receptor tyrosine kinase, TrkA, to promote chemo-attraction along intermediate targets. Once axons arrive to their final target, a structurally related neurotrophic factor, nerve growth factor (NGF), also acts through TrkA to promote final target innervation. Does TrkA signal differently at these different locales? We previously found that Coronin-1 is upregulated in sympathetic neurons upon exposure to NGF, thereby endowing the NGF-TrkA complex with new signaling capabilities (i.e. calcium signaling), which dampens axon growth and branching. Based on the notion that axons do not express functional levels of Coronin-1 prior to final target innervation, we developed an in vitro model for axon growth and branching along intermediate targets using Coro1a-/- neurons grown in NT3. We found that, similar to NGF-TrkA, NT3-TrkA is capable of inducing MAPK and PI3K in the presence or absence of Coronin-1. However, unlike NGF, NT3 does not induce calcium release from intracellular stores. Using a combination of pharmacology, knockout neurons and in vitro functional assays, we suggest that the NT3-TrkA complex uses Ras/MAPK and/or PI3K-AKT signaling to induce axon growth and inhibit axon branching along intermediate targets. However, in the presence of Coronin-1, these signaling pathways lose their ability to impact NT3 dependent axon growth or branching. This is consistent with a role for Coronin-1 as a molecular switch for axon behavior and suggests that Coronin-1 suppresses NT3 dependent axon behavior.

Coronin-1 and calcium signaling governs sympathetic final target innervation.

Development of a functional peripheral nervous system requires axons to rapidly innervate and arborize into final target organs and then slow but not halt their growth to establish stable connections while keeping pace with organ growth. Here we examine the role of the NGF-TrkA effector protein, Coronin-1, on postganglionic sympathetic neuron final target innervation. In the absence of Coronin-1 we find that NGF-TrkA-PI3K signaling drives robust axon growth and branching in part by suppressing GSK3ß. In contrast, the presence of Coronin-1 (wild-type neurons) suppresses but does not halt NGF-TrkA-dependent growth and branching. This relative suppression in axon growth behaviors is due to Coronin-1-dependent calcium release via PLC-γ1 signaling, which releases PI3K-dependent suppression of GSK3ß. Finally, we demonstrate that Coro1a(-/-) mice display sympathetic axon overgrowth and overbranching phenotypes in the developing heart. Together with previous work demonstrating the Coronin-1 expression is NGF dependent, this work suggests that periods before and after NGF-TrkA-induced Coronin-1 expression (and likely other factors) defines two distinct axon growth states, which are critical for proper circuit formation in the sympathetic nervous system.

Coronin-1 is a neurotrophin endosomal effector that is required for developmental competition for survival.

Retrograde communication from axonal targets to neuronal cell bodies is critical for both the development and function of the nervous system. Much progress has been made in recent years linking long-distance, retrograde signaling to a signaling endosome, yet the mechanisms governing the trafficking and signaling of these endosomes remain mostly uncharacterized. Here we report that in mouse sympathetic neurons, the target-derived nerve growth factor (NGF)-tropomyosin-related kinase type 1 (TrkA, also called Ntrk1) signaling endosome, on arrival at the cell body, induces the expression and recruitment of a new effector protein known as Coronin-1 (also called Coro1a). In the absence of Coronin-1, the NGF-TrkA signaling endosome fuses to lysosomes sixfold to tenfold faster than when Coronin-1 is intact. We also define a new Coronin-1-dependent trafficking event in which signaling endosomes recycle and re-internalize on arrival at the cell body. Beyond influencing endosomal trafficking, Coronin-1 is also required for several NGF-TrkA-dependent signaling events, including calcium release, calcineurin activation and phosphorylation of cAMP responsive element binding protein (CREB). These results establish Coronin-1 as an essential component of a feedback loop that mediates NGF-TrkA endosome stability, recycling and signaling as a critical mechanism governing developmental competition for survival.

Trk activation of the ERK1/2 kinase pathway stimulates intermediate chain phosphorylation and recruits cytoplasmic dynein to signaling endosomes for retrograde axonal transport.

The retrograde transport of Trk-containing endosomes from the axon to the cell body by cytoplasmic dynein is necessary for axonal and neuronal survival. We investigated the recruitment of dynein to signaling endosomes in rat embryonic neurons and PC12 cells. We identified a novel phosphoserine on the dynein intermediate chains (ICs), and we observed a time-dependent neurotrophin-stimulated increase in intermediate chain phosphorylation on this site in both cell types. Pharmacological studies, overexpression of constitutively active MAP kinase kinase, and an in vitro assay with recombinant proteins demonstrated that the intermediate chains are phosphorylated by the MAP kinase ERK1/2, extracellular signal-regulated kinase, a major downstream effector of Trk. Live cell imaging with fluorescently tagged IC mutants demonstrated that the dephosphomimic mutants had significantly reduced colocalization with Trk and Rab7, but not a mitochondrial marker. The phosphorylated intermediate chains were enriched on immunoaffinity-purified Trk-containing organelles. Inhibition of ERK reduced the amount of phospho-IC and the total amount of dynein that copurified with the signaling endosomes. In addition, inhibition of ERK1/2 reduced the motility of Rab7- and TrkB-containing endosomes and the extent of their colocalization with dynein in axons. NGF-dependent survival of sympathetic neurons was significantly reduced by the overexpression of the dephosphomimic mutant IC-1B-S80A, but not WT IC-1B, further demonstrating the functional significance of phosphorylation on this site. These results demonstrate that neurotrophin binding to Trk initiates the recruitment of cytoplasmic dynein to signaling endosomes through ERK1/2 phosphorylation of intermediate chains for their subsequent retrograde transport in axons.

Nitric oxide is the shared signalling molecule in phosphorus- and iron-deficiency-induced formation of cluster roots in white lupin (Lupinus albus).

BACKGROUND AND AIMS: Formation of cluster roots is one of the most specific root adaptations to nutrient deficiency. In white lupin (Lupinus albus), cluster roots can be induced by phosphorus (P) or iron (Fe) deficiency. The aim of the present work was to investigate the potential shared signalling pathway in P- and Fe-deficiency-induced cluster root formation. METHODS: Measurements were made of the internal concentration of nutrients, levels of nitric oxide (NO), citrate exudation and expression of some specific genes under four P × Fe combinations, namely (1) 50 µm P and 10 µm Fe (+P + Fe); (2) 0 P and 10 µm Fe (-P + Fe); (3) 50 µm P and 0 Fe (+P-Fe); and (4) 0 P and 0 Fe (-P-Fe), and these were examined in relation to the formation of cluster roots. KEY RESULTS: The deficiency of P, Fe or both increased the cluster root number and cluster zones. It also enhanced NO accumulation in pericycle cells and rootlet primordia at various stages of cluster root development. The formation of cluster roots and rootlet primordia, together with the expression of LaSCR1 and LaSCR2 which is crucial in cluster root formation, were induced by the exogenous NO donor S-nitrosoglutathione (GSNO) under the +P + Fe condition, but were inhibited by the NO-specific endogenous scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl- 3-oxide (cPTIO) under -P + Fe, +P-Fe and -P-Fe conditions. However, cluster roots induced by an exogenous supply of the NO donor did not secrete citrate, unlike those formed under -P or -Fe conditions. CONCLUSIONS: NO plays an important role in the shared signalling pathway of the P- and Fe-deficiency-induced formation of cluster roots in white lupin.

[Development of transient pyrometer based on multi-spectral radiation technology].

In modern dynamics system, the radiant temperature of the flame, which caused by the transient plasma stimulated by high-energy-level electromagnetism field, takes an important role in the description of the flying object's status as well as cauterization of the trajectory. Due to its extremely high temperature and transient process, the radiant temperature of the flame can hardly be measured through contracted ways, either static ways such as traditional pyrophotometer or CCD arrays. In the present paper, the authors bring forward a novel pyrophotometer based on classical theory of Planck's law (blackbody radiation law) and multi-channel spectrums radiation method. With this new type pyrophotometer, any spectrum can be selected out from the wavelength of 300 to 860 nm within 2 ns. Also, the application of high-definition diffraction grating and fibers can'ensure the accuracy of selected spectrum. The results through a serial of experiments by using this theory as well as high-speed photodetector indicate that this method is valid and accurate for the measurement of the object's surface's radiant temperature.