Endocytic Internalization Routes Required for Delta/Notch Signaling

Abstract

Summary The internalization of transmembrane receptors from the cell surface plays a central role in signal regulation. A large body of literature has shown that receptor internalization can occur through different routes[1]; however, because of the difficulty in selectively blocking these routes in vivo, their roles in signaling are poorly understood. Here we investigate this question using null mutations in Drosophila Dynamin, Clathrin, and AP-2 adaptor subunits to analyze internalization requirements for the Delta ligand and its receptor Notch. Bulk Notch is internalized via AP-2-dependent endocytosis, but signaling by Notch requires AP-2-independent Clathrin-dependent endocytosis, highlighting a distinction between Notch endocytic routes required for degradation versus signaling activation. Signaling by the Notch ligand Delta has been shown to require Dynamin, but whether this generates a pulling force of Delta on Notch or allows for Delta entry into a recycling pathway to gain signaling competence is widely debated[2,3]. Surprisingly, we show that signaling by Delta in germline cells can occur by Clathrin-independent endocytosis, when endosomal entry is blocked, and when activity of Rab11 or its effectors is reduced, suggesting that Delta need not pass through a recognized recycling pathway to achieve signaling competence. The absolute requirement for Dynamin-dependent endocytosis but not endosomal entry or Rab11 activity supports ‘pulling force’ rather than ‘recycling’ models for Delta activation.

O-Fucose Monosaccharide of Drosophila Notch Has a Temperature-sensitive Function and Cooperates with O-Glucose Glycan in Notch Transport and Notch Signaling Activation

“…2xUbx-FLP was used to efficiently induce somatic mosaic clones (38). To induce germ line mosaic clones, ovo D G13 (17), ovo D FRT40A, and ovo D FRT80B (39) were used.…”

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].…”

“…However, in invertebrates and, more specifically, in their oogenesis, Delta endocytosis could occur in an AP-2-and clathrinindependent way, as assayed by Notch activation of surrounding follicular cells triggered by germline clones bearing mutations of clathrin and AP-2 adaptor subunits, but not dynamin [190]. In the same system, it was also analyzed the dependence of Notch activation on endosomal trafficking in signal-sending cells: germline clones mutant for small GTPases that critically regulate the endosomal compartment, including Rab5 and Rab11, normally activate Notch in follicular cells.…”

“…Taken together, these data support the absolute requirement for dynamin in DSL uptake. Conversely, neither CME nor endosomal entry of DSLs are universally required for Notch activation [190] (and, see section 3.1.1).…”

Drosophila Ndfip is a novel regulator of Notch signaling

“…[1][2][3] Notch receptor turnover in the absence of ligand is an important mechanism to prevent inappropriate signaling in unstimulated cells, requiring AP-2, AP-3 and HOPSdependent endocytic steps. 4,5 Ubiquitination has a key role in regulating the endocytosis and subsequent downregulation of Notch. 6 In the developing Drosophila wing, members of the Nedd4 family of HECT domain ubiquitin ligases (E3s), dNedd4 and Suppressor of deltex (Su(dx)), ubiquitinate and promote the endocytosis and turnover of Notch in a ligandindependent manner.…”