The ATPase TER94 regulates Notch signaling during Drosophila wing development [RESEARCH ARTICLE]
DISCUSSION
The data presented here described the TER94 ATPase as a novel regulator of the Notch signaling pathway in Drosophila. Although Drosophila TER94 acts broadly to regulate the activity of Hh, Wg and BMP signaling pathways (Zhang et al., 2013; Reim et al., 2014), our report is the first to establish a functional interaction between TER94 and Notch activity during wing development. More specifically, we found that TER94 might regulate Notch protein activity in the signal receiving cells during wing margin formation. We showed that disruption of TER94 activity resulted in profound changes in Notch protein distribution, but had little effect on the ligand Dl. Using cell-type-specific Gal4 drivers, we further demonstrated that TER94 is required in the signal receiving cells for proper activation of Notch signaling. Intriguingly, we observed elevation of Notch protein level and inhibition of Notch signaling activity at the same time when TER94 function was disrupted. These results suggest that the accumulated Notch proteins in TER94 loss-of-function cells were incompetent for signaling activity. Notch proteins with abnormal intracellular localization or modification always accumulate to very high levels in different cellular compartments, but lead to severe inhibition of signaling activity (Okajima et al., 2005; Tien et al., 2008; Acar et al., 2008; Vaccari et al., 2010; Groth et al., 2013). As similar defects were observed for mutations affecting Notch protein folding, exiting from the ER and endocytosis from the plasma membrane, exactly which aspect is regulated by TER94 needs further investigation.
Given the central role of TER94 in a wide range of cellular activities, it is reasonable to assume that TER94 is involved in various developmental events. However, it was difficult to analyze the TER94 loss-of-function phenotypes because of it is essential for cell growth. Massively inhibiting TER94 function by RNAi and TER94AA resulted in early lethality, while the size of TER94k15502 mutant clones was found to be too small to display prominent phenotypes in adult tissues (Zhang et al., 2013; Reim et al., 2014). Interestingly, nicking of wing margin was readily observed in adults bearing TER94k15502 clones (Fig. 1B). We have noticed that small sized mutant clones were able to cause the characteristic wing notch phenotype (Ren et al., 2018). The wing margin is formed by a narrow row of cells, 2–5 cells wide, along the D/V boundary. Therefore, mutant clones with a diameter of 2–5 cells are sufficient to induce marginal defects when they are located across the D/V boundary. As one of the earliest recorded mutant phenotypes in fly, nicked wing margin might still be the most reliable readout for Notch signaling defects in terms of sensitivity. Indeed, when we mildly inhibited TER94 function to avoid cell growth defects in the developing wing, marginal notches are consistently discovered (Figs 2A,B, 5E,F). Our data, however, does not exclude the roles of TER94 in other Notch regulated developmental processes nor other signaling pathways. The adult phenotypes are determined by the inhibition efficiency, as well as the cell types and developmental stages that TER94 RNAi and TER94AA are applied in. Future studies are needed to examine TER94’s function in a more spatial- and temporal-specific fashion, and the outcomes will help us to better understand the comprehensive roles of TER94 during development.
TER94 is a highly pleiotropic AAA-ATPase associated with many essential cellular functions (Schuberth and Buchberger, 2008). Specifications of the diverse activities of TER94 and the fate of its substrates are mainly exerted by the regulatory co-factors (van den Boom and Meyer, 2018). As shown before, distinct co-factors are involved in regulating the Hh and Wg signaling pathway (Zhang et al., 2013; Reim et al., 2014). We show that one of the TER94 co-factors, p47 is required for Notch signaling activity during fly wing margin formation. The p47 co-factor was first identified as an essential component for the TER94-mediated membrane fusion pathway in yeast (Kondo et al., 1997). Subsequent studies revealed that the TER94-p47 complex plays crucial roles in cellular membrane related events, including nuclear envelop formation, ER biogenesis and Golgi assembly (Uchiyama and Kondo, 2005; Meyer et al., 2012). The molecular function of p47 is conserved from yeast to mammalian cells, but its role in animal development is not fully understood. It has been reported that the p47 homolog is essential for photoreceptor morphogenesis in Drosophila by affecting nuclear envelope assembly (Sang and Ready, 2002). Our finding that the TER94-p47 complex positively regulates Notch signaling in the fly wing thus reveals a novel role of TER94-p47 in development. Very recently, the fly TER94-p47 complex was shown to directly bind to and transfer poly-Ubiquitin chains for further processing (Blount et al., 2018). Indeed we observed defects in ubiquitin status in TER94 loss-of-function cells. It is reasonable to assume that the ubiquitin system is involved in TER94-p47 mediated regulation of Notch signaling.
We believe that TER94 regulates Notch protein in the signal receiving cells, but the underlying molecular mechanism is still incomplete. It has been demonstrated that Notch protein is subject to ubiquitin modification in both fly and vertebrates (Lai, 2002; Le Bras et al., 2011). The HECT-type E3 ubiquitin ligase Suppressor of deltex [Su(dx)] interacts and ubiquitinates Notch protein in Drosophila (Cornell et al., 1999; Yao et al., 2018). The mouse homolog of Su(dx), named Itch, adds ubiquitin chains on Notch protein in the same fashion (Qiu et al., 2000). However, the mechanism of regulation of Notch by Su(dx)/Itch remains unclear, they may function on internalization of membrane bound Notch, generation of NICD, and/or free cytoplasmic NICD (Qiu et al., 2000; McGill and McGlade, 2003). First identified in Caenorhabditis elegans, the Sel-10 gene encodes an F-box containing E3 ligase that targets NICD for ubiquitination and proteasomal degradation (Hubbard et al., 1997; Deng and Greenwald, 2016). The mammalian homolog of Sel-10, FBXW7, binds to NICD to promote its ubiquitination and subsequent rapid degradation (Gupta-Rossi et al., 2001; Öberg et al., 2001; Wu et al., 2001). Despite the apparent importance of Sel-10/FBXW7 in regulation of NICD protein stability, a conclusive link between fly Sel-10/FBXW7 and Notch signaling is still missing. Two E3 ubiquitin ligases, Neuralized (Neur) and Mind bomb (Mib), have been shown to regulate a distinct subset of Notch signaling events in the signal-sending cells (Pavlopoulos et al., 2001; Lai et al., 2001; Yeh et al., 2001;Borgne et al., 2005; Lai et al., 2005). Whether the signal-receiving cells also utilize different E3 ligases to regulate distinct developmental processes is still an open question. It would be necessary to examine whether and how TER94 regulates the ubiquitination states of Notch and the relationship between TER94 and Notch E3 ligases to deepen our understanding of the regulatory roles of TER94 in Notch signaling.
