The Functions of Auxilin and Rab11 in Drosophila Suggest That the Fundamental Role of Ligand Endocytosis in Notch Signaling Cells Is Not Recycling

Abstract

Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. Here, we use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, we find that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, we find that Drosophila female germline cells, the one cell type known to signal without clathrin, also do not require auxilin to signal. Third, we find that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling.

dEHBP1 regulates Scabrous secretion during Notch mediated lateral inhibition

“…Furthermore, the plethora of different context dependent intracellular trafficking regulators provides evidence that different modes of intercellular signaling rely on distinct intracellular trafficking pathways. For example, although endocytosis of Notch ligands always appears to rely on the activity of dynamin, it is not always dependent clathrin and auxillin (Banks et al, 2011;Windler and Bilder, 2010). Similarly, endosomal recycling of Notch ligands, controlled by rab11 and its effector, sec15, is required for the development of asymmetrically dividing mechanosensory lineages (Benhra et al, 2010;Emery et al, 2005;Jafar-Nejad et al, 2005), but not during photoreceptor development or ovary development (Banks et al, 2011;Mehta et al, 2005;Windler and Bilder, 2010).…”

“…In the absence of arp3, Dl recycling towards this specialized microenvironment of the plasma membrane is impaired (Rajan et al, 2009). However, in other tissues, where Dl recycling does not play a role, as for example during development of ovaries (Windler and Bilder, 2010) or photoreceptors (Banks et al, 2011), arp3 may regulate different aspects of Notch signaling. Similarly, our studies support the conclusion that dEHBP1 regulates Notch signaling in different developmental contexts by controlling the intracellular trafficking of different substrates, as for example Dl and Spdo during asymmetric divisions and Sca during lateral inhibition.…”

Functional Analysis of the NHR2 Domain Indicates that Oligomerization of Neuralized Regulates Ubiquitination and Endocytosis of Delta during Notch Signaling

“…The mechanical force or pulling model suggests that Delta endocytosis exerts a force on the Delta-Notch complex that alters the conformation and promotes the cleavage of the Notch extracellular domain (NECD), which is a critical step in Notch activation (17,41,42,51). The recycling model suggests that the modification of an inactive form of Delta in an endosomal compartment makes Delta a more effective ligand, which will be re-presented to the cell surface (perhaps at a microdomain of the plasma membrane) to activate Notch (2,4,14,20,47,52). In the signal-sending cell, Neuralized (Neur) (12,27,43,56) and Mindbomb1 (3,22,25,28,34,44,53) are two E3 ubiquitin ligases that regulate the endocytosis of the Notch ligands Delta and Serrate by ubiquitination.…”

Endocytosis in Notch Signaling Activation

“…Rab11 function, which was found to be essential for Delta trafficking and activation in the SOP system, is not required for Drosophila eye development [189], nor for germinal cell signaling [190]. In both these Notch-dependent events, endocytosis was required and a specific need for epsin, but not recycling, was evident at least in eye development [189].…”