Reticulon 3-dependent ER-PM contact sites control EGFR nonclathrin endocytosis.

The integration of endocytic routes is critical to regulate receptor signaling. A nonclathrin endocytic (NCE) pathway of the epidermal growth factor receptor (EGFR) is activated at high ligand concentrations and targets receptors to degradation, attenuating signaling. Here we performed an unbiased molecular characterization of EGFR-NCE. We identified NCE-specific regulators, including the endoplasmic reticulum (ER)-resident protein reticulon 3 (RTN3) and a specific cargo, CD147. RTN3 was critical for EGFR/CD147-NCE, promoting the creation of plasma membrane (PM)-ER contact sites that were required for the formation and/or maturation of NCE invaginations. Ca2+ release at these sites, triggered by inositol 1,4,5-trisphosphate (IP3)-dependent activation of ER Ca2+ channels, was needed for the completion of EGFR internalization. Thus, we identified a mechanism of EGFR endocytosis that relies on ER-PM contact sites and local Ca2+ signaling.

Threshold-controlled ubiquitination of the EGFR directs receptor fate.

How the cell converts graded signals into threshold-activated responses is a question of great biological relevance. Here, we uncover a nonlinear modality of epidermal growth factor receptor (EGFR)-activated signal transduction, by demonstrating that the ubiquitination of the EGFR at the PM is threshold controlled. The ubiquitination threshold is mechanistically determined by the cooperative recruitment of the E3 ligase Cbl, in complex with Grb2, to the EGFR. This, in turn, is dependent on the simultaneous presence of two phosphotyrosines, pY1045 and either one of pY1068 or pY1086, on the same EGFR moiety. The dose-response curve of EGFR ubiquitination correlate precisely with the non-clathrin endocytosis (NCE) mode of EGFR internalization. Finally, EGFR-NCE mechanistically depends on EGFR ubiquitination, as the two events can be simultaneously re-engineered on a phosphorylation/ubiquitination-incompetent EGFR backbone. Since NCE controls the degradation of the EGFR, our findings have implications for how the cell responds to increasing levels of EGFR signalling, by varying the balance of receptor signalling and degradation/attenuation.

How neuronal migration contributes to the morphogenesis of the CNS: insights from the zebrafish.

We used transgenic zebrafish expressing GFP or YFP in subpopulations of neurons to study the migration, homing process and axon extension of groups of CNS neurons in different regions of the zebrafish brain. We found that extensive migration takes place at all levels of the CNS and gives rise to nuclei or cell populations with specific identities. Here, we describe 4 previously unknown or only partially characterized migratory events taking place in the zebrafish telencephalon and rhombic lip, using 3 different transgenic lines, and identify the phenotypes of the cells undertaking these migrations. The migration of a subgroup of mitral cell precursors from the dorsocaudal telencephalon to the olfactory bulb, visualized in the tg(tbr1:YFP) transgenic line, is coupled with morphogenetic transformation of the dorsal telencephalon. The tg(1.4dlx5a-6a:GFP) transgenic line provides a means to analyze the migration of GABAergic interneurons from the ventral to the dorsal telencephalon, thus extending the occurrence of this migration to another vertebrate. The tg(Xeom:GFP) transgenic line provides the first demonstration of the dorsoventral migration of glutamatergic septal neurons, present in mammals and now described in fish, thus reconciling the contrasting evidence of dorsal patterning genes (tbr1, eomes) expressed in a ventral cell population. Furthermore, migration studies in the tg(1.4dlx5a-6a:GFP) and tg(Xeom:GFP) lines help determine the origin of 2 important cell populations in the fish cerebellum: projection neurons and Purkinje cells. These examples reinforce the concept that migratory events contribute to the distribution of cell types with diverse identities through the CNS and that zebrafish transgenic lines represent excellent tools to study these events.