Clathrin adaptor AP-1 and Stratum act in parallel pathways to control Notch activation inDrosophilaSensory Organ Precursor Cells

Abstract

Drosophila sensory organ precursors divide asymmetrically to generate pIIa/pIIb cells whose identity relies on the differential activation of Notch at cytokinesis. While Notch is present apically and basally relative to the midbody at the pIIa-pIIb interface, only the basal pool of Notch is reported to contribute to Notch activation in the pIIa cell. Correct intra-lineage signalling requires appropriate apico-basal targeting of Notch, its ligand Delta and its trafficking partner Sanpodo. We previously reported that AP-1 and Stratum regulate the intracellular trafficking of Notch and Sanpodo from the trans-Golgi network to the basolateral membrane. Loss of AP-1 or Stratum caused mild Notch gain-of-function phenotypes. Here, we report that the concomitant loss of AP-1 and Stratum results in a much more penetrant Notch gain-of-function phenotype indicating that AP-1 and Strat control two parallel pathways. While unequal partitioning of cell fate determinants and cell polarity were unaffected, Numb-mediated symmetry breaking is impaired. We further observed increased amounts of signaling competent Notch as well as Delta and Sanpodo at the apical pIIa-pIIb interface and the loss of the basal pool of Notch. We propose that AP-1 and Stratum operate in two parallel pathways to ensure the correct apico-basal localization of Notch controlling where receptor activation takes place.

Bazooka/Par3 cooperates with Sanpodo for the assembly of Notch signaling clusters following asymmetric division ofDrosophilasensory organ precursor cells

“…The distribution of Baz is reminiscent of that reported for a GFP tagged version of Notch, NiGFP, that transiently distributes in clusters at the apical and lateral pIIa-pIIb interface prior to proteolytic activation and targeting of the resulting intracellular domain of Notch tagged with GFP into the nucleus of the pIIa cell (Fig. 1D,D”, (Bellec et al, 2018; Bellec et al, 2021; Couturier et al, 2012; Trylinski et al, 2017). As Baz, Notch is not detected in clusters at the lateral interface of epidermal daughter cells (Fig.…”

“…3A, A’). However, the apical NiGFP fluorescence intensity measured as described in (Bellec et al, 2021) is significantly reduced (Fig. 3B) and the numbers and intensity of the lateral clusters are decreased upon loss of Baz (Fig.…”

“…Photobleaching and phototracking experiments during SOP cytokinesis revealed that among the two pools of Notch, the basolateral pool located basal to the midbody is the main contributor (Trylinski et al, 2017). While the apical pool of Notch also contributes to NICD production, it is yet unclear whether NICD is directly produced from the apical pIIa-pIIb interface or if basolateral relocation is a prerequisite (Bellec et al, 2021; Couturier et al, 2012; Trylinski et al, 2017).…”

“…To generate Neur-H2B-iRFP670 and Neur-iRFP670-GAP43 transgenic strains, we first ordered to Genewiz pUC57-Amp plasmids containing H2B-iRFP670 or iRFP670-GAP43 sequences flanked by StuI and SpeI restriction sites respectively on 5’- and 3’-ends. For this, we used the following sequences of H2B from (Bellec et al, 2021), GAP43 from (Mavrakis et al, 2009), iRFP670 (genbank KC991142) from (Shcherbakova and Verkhusha, 2013) and pHStinger-NeurGFP from (Aerts et al, 2010; Barolo et al, 2000). Details of cloning will be provided upon request.…”

“…Numb causes the targeting of Spdo/Notch to late endosomes (Cotton et al, 2013; Couturier et al, 2013), whereas Neur promotes the endocytosis of the Notch ligand Delta (Dl) (Le Borgne and Schweisguth, 2003), so that Notch is inhibited in pIIb and activated in pIIa. During SOP cytokinesis two pools of Notch, located apical and basal to the midbody, are present at the pIIb-pIIa interface and both contribute to Notch signaling (Bellec et al, 2021; Trylinski et al, 2017). Previous studies based on photobleaching and photoconversion experiments revealed that the basal pool of Notch is the main contributor of NICD (Trylinski et al, 2017).…”