RET PLCγ Phosphotyrosine Binding Domain Regulates Ca2+ Signaling and Neocortical Neuronal Migration

The receptor tyrosine kinase RET plays an essential role during embryogenesis in regulating cell proliferation, differentiation, and migration. Upon glial cell line-derived neurotrophic factor (GDNF) stimulation, RET can trigger multiple intracellular signaling pathways that in concert activate various downstream effectors. Here we report that the RET receptor induces calcium (Ca2+) signaling and regulates neocortical neuronal progenitor migration through the Phospholipase-C gamma (PLCγ) binding domain Tyr1015. This signaling cascade releases Ca2+ from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor and stimulates phosphorylation of ERK1/2 and CaMKII. A point mutation at Tyr1015 on RET or small interfering RNA gene silencing of PLCγ block the GDNF-induced signaling cascade. Delivery of the RET mutation to neuronal progenitors in the embryonic ventricular zone using in utero electroporation reveal that Tyr1015 is necessary for GDNF-stimulated migration of neurons to the cortical plate. These findings demonstrate a novel RET mediated signaling pathway that elevates cytosolic Ca2+ and modulates neuronal migration in the developing neocortex through the PLCγ binding domain Tyr1015

Rewiring ret : PTB-adaptor regulated signaling and cell biology

The Ret receptor belongs to a group of transmembrane tyrosine kinase receptors that serve to transduce environmental signals into cellular responses. Upon extracellular ligand activation the receptors dimerize and their structural conformation is altered into one that allows for interaction with proteins in the cell cytosol. Among the most proximal interactors are PTBadaptor molecules that dock to Ret tyrosine residues. As several PTB-adaptors compete for interaction with the same receptor-sequence the molecule that successfully binds to Ret excludes binding of any other PTB-adaptor at that time. Hence, the resulting signals are dependent on which adaptor that successfully engaged the receptor. In this work, Ret has been rewired to preferentially bind one PTB-adaptor on the expense of others to tyrosine 1062. This makes it possible to experimentally assign biochemical events as well as cell biological outcomes to one specific adaptor and so increasingly reveal how one common receptor may serve a plethora of functions depending on the subcellular milieu in which it operates. In paper I the important residues for Ret interaction with the Shc and Frs2alpha adaptors were investigated. Based on data from the effect on adaptor affinity for Ret by specifically substituting amino acids around tyrosine 1062, mutants could be established with selective recruitment of either Shc or Frs2alpha to this tyrosine. Ret interaction with Shc resulted in capacity for the receptor to mediate ligand dependent survival in the setting of apoptotic stimuli or severe starvation while Frs2alpha mediated signaling was insufficient to do so. In paper II the Frs2alpha adaptor docked to Ret were found to be essential for chemotactic directional migration towards Ret ligands. The molecular basis for Ret promoted migration depended on the membrane associated Src family of kinases that bind to Ret at a different tyrosine than Frs2alpha such that these two residues are cooperatively involved in assembling the molecular framework required to execute a migratory response downstream of Ret. In paper III the Dok adaptors, which were recently found to interact with Ret, were investigated. Dok selective Ret mutants could be created and were expressed in neuronally derived cells. Dok binding resulted in prolonged phosphorylation of MAP kinases and allowed for Cdc42 activation. The cellular response was enhanced microspike formation as determined morphologically. In paper IV local membranous Ret interaction with Shc and Frs2alpha were investigated. While Frs2alpha overexpression recruited Ret to distinct lipid raft like partitions of the plasma membrane Shc expression led to Ret being found outside these fractions. A Shc molecule with an appendage forcing its association to lipid rafts resembled Frs2alpha characteristics in terms of downstream biochemical profile and dependence of ordered cholesterol species in the membrane for signaling, suggesting the importance of adaptors for appropriate relocation of Ret. Moreover, the Frs2alpha dependent migration was indeed disrupted by cholesterol oxidation while the survival response promoted via Shc showed little dependence on disruption of membrane architecture

Differential expression and dynamic changes of murine NEDD9 in progenitor cells of diverse tissues.

International audienceNEDD9 is a scaffolding protein in the integrin signaling pathway that is involved in cell adhesion dynamics. Little is known of the cellular localization of NEDD9 expression during embryonic development. In the present study, we have analyzed NEDD9 mRNA expression in the mouse and identified new relevant expression sites. In addition, we have characterized NEDD9 protein expression pattern for the first time in mammals. At E9.5-E10.5, high levels of Nedd9 and the neurogenic transcription factor neurogenin-2 (Ngn2) were found to largely overlap in two discrete domains of the trunk neural tube along its dorso-ventral axis, with Nedd9 extending to more ventral regions of the ventricular zone and Ngn2 differentially expressed in neuronally committed progenitors of the intermediate zone. At encephalic and trunk levels of the neural tube, NEDD9 was present in Sox2(+) progenitor cell populations mostly generating Ngn2(+) and/or Nurr1(+) cells. A sharp down-regulation of NEDD9 expression was found in cells upon lineage commitment, as observed in Nurr1(+) and Ngn2(+) mesencephalic dopaminergic and brainstem neuronal progenitors. In other tissues/organs, i.e. prospective heart, retina, olfactory epithelium, gonads, cartilage, gut and pituitary gland, NEDD9 was found to be co-expressed with Sox2, RXR alpha and/or Nurr1-like proteins, suggesting that NEDD9 expression is confined to early progenitors involved in diverse organogenesis and that it may depend on the repertoire and levels of retinoic acid co-receptors expressed by those cells

Composite orbital reconstruction using the vascularized segmentalized osteo-fascio-cutaneous fibula flap

Reconstruction of composite orbital defects must address the orbit and an exposed skull base and/or maxillary region. The orbit should not only be covered but also reshaped to accommodate the orbital contents or an epithesis when warranted. This study presents a rationale for a near-anatomical reconstruction of the orbit, together with adjacent dead space obliteration, using the segmentalized osteo-fascia-cutaneous fibula flap. Before the flap transfer, a cutting template for the fibula is made according to the measures and requirements of the facial defect. The segmentalized bone is then osteosynthesized to the facial skeleton and revascularized. Thus, an orbital depth is created by the bony fibula, whereas the fascio-cutaneous part of the flap may be used for lining the orbit and obliteration of the skull base or the maxillary region, or resurface the palate and/or the nasal cavity

Composite orbital reconstruction using the vascularized segmentalized osteo-fascio-cutaneous fibula flap

Reconstruction of composite orbital defects must address the orbit and an exposed skull base and/or maxillary region. The orbit should not only be covered but also reshaped to accommodate the orbital contents or an epithesis when warranted. This study presents a rationale for a near-anatomical reconstruction of the orbit, together with adjacent dead space obliteration, using the segmentalized osteo-fascia-cutaneous fibula flap. Before the flap transfer, a cutting template for the fibula is made according to the measures and requirements of the facial defect. The segmentalized bone is then osteosynthesized to the facial skeleton and revascularized. Thus, an orbital depth is created by the bony fibula, whereas the fascio-cutaneous part of the flap may be used for lining the orbit and obliteration of the skull base or the maxillary region, or resurface the palate and/or the nasal cavity

RET Tyr1015 mediates GDNF-stimulated migration <i>in vivo</i>.

<p>(<b>A</b>) Cartoon illustrating mouse embryo electroporation and GDNF-bead stimulated migration. (<b>B</b>–<b>D</b>) Migration of cortical progenitors in organotypic brain slices from embryos electroporated with RET^WT (<b>C</b>) or RET¹⁰¹⁵ (<b>D</b>) treated without beads (Control) or with beads (indicated with circles) soaked in PBS (Vehicle) or GDNF (500 ng/ml) placed in the cortical plate (CP). GFP positive RET^WT expressing progenitors (green) stimulated with GDNF beads (<b>B</b>, <b>C</b>) show significantly enhanced migration from the ventricular zone (VZ) towards the CP, as compared to Control, Vehicle, or inhibition of PLC with U73122 (5 µM). In RET¹⁰¹⁵ expressing progenitors GDNF beads failed to stimulate migration (<b>B</b>, <b>D</b>). Scale bars, 100 µm.</p

A RET/PLCγ/InsP₃R-cascade stimulates GDNF-induced Ca²⁺ release.

<p>(<b>A–H</b>) Representative single-cell Ca²⁺ recordings of GFP positive RET^WT expressing cells loaded with Fura-2/AM and preincubated with inhibitors as indicated, following treatment with GDNF (100 ng/ml). Inhibiting PLC with U73122 (5 µM) (<b>A</b>) or knocking down PLCγ with siRNA (<b>B</b>) blocked the cytosolic Ca²⁺ response induced by GDNF. Cells transfected with the Mock-siRNA retain the Ca²⁺ response (<b>C</b>). Inhibiting InsP₃R with 2-APB (5 µM) abolished the Ca²⁺ response induced by GDNF (<b>D</b>), while inhibiting RyR with ryanodine (a, 20 µM) or dantrolene (b, 10 µM) had no effect (<b>E</b>). Depleting intracellular Ca²⁺ stores with the SERCA Ca²⁺-ATPase inhibitor Thapsigargin (1 µM) blocked the Ca²⁺ response (<b>F</b>). Zero extracellular Ca²⁺ eliminated the GDNF-induced Ca²⁺ response (<b>G</b>), whereas a low extracellular concentration of Ca²⁺ (1 mM) produced a normal Ca²⁺ response (<b>H</b>).</p

Characteristics of Ca²⁺ responses triggered by GDNF in RET^WT cells treated with various inhibitors.

a<p>Non-responding cells have no Ca²⁺ increase exceeding 1.25 of the baseline.</p>b<p>Transient responding cells have one Ca²⁺ peak exceeding 1.25 of the baseline.</p>c<p>Oscillatory responding cells have at least three Ca²⁺ peaks exceeding 1.25 of the baseline.</p>d<p>[number of cells/number of experiments].</p>e<p>[concentration of extracellular Ca²⁺].</p