stranded at second: Biological Overview | References
Gene name - stranded at second
Cytological map position - 84C8-84D1
Function - secreted ligand
Keywords - Sas and the receptor-type tyrosine phosphatase PTP10D function as the cell-surface ligand-receptor system that drives tumour-suppressive cell competition - trans-activation of Sas-PTP10D signalling in loser cells restrains EGFR signalling cell elimination and thereby enables elevated JNK signalling in loser cells, triggering cell elimination - expression of Sas in neurons and glia and Ptp10D expressed on longitudinal axons are required to prevent axons from abnormally crossing the midline
Symbol - sas
FlyBase ID: FBgn0002306
Genetic map position - chr3R:7,162,890-7,183,702
NCBI classification - VWC: von Willebrand factor (vWF) type C domain - Fibronectin type III domain
Cellular location - surface transmembrane
Normal epithelial cells often exert anti-tumour effects against nearby oncogenic cells. In the Drosophila imaginal epithelium, clones of oncogenic cells with loss-of-function mutations in the apico-basal polarity genes scribble or discs large are actively eliminated by cell competition when surrounded by wild-type cells. Although c-Jun N-terminal kinase (JNK) signalling plays a crucial role in this cell elimination, the initial event, which occurs at the interface between normal cells and polarity-deficient cells, has not previously been identified. Through a genetic screen in Drosophila, this study identifies the ligand Sas and the receptor-type tyrosine phosphatase PTP10D as the cell-surface ligand-receptor system that drives tumour-suppressive cell competition. At the interface between the wild-type 'winner' and the polarity-deficient 'loser' clones, winner cells relocalize Sas to the lateral cell surface, whereas loser cells relocalize PTP10D there. This leads to the trans-activation of Sas-PTP10D signalling in loser cells, which restrains EGFR signalling and thereby enables elevated JNK signalling in loser cells, triggering cell elimination. In the absence of Sas-PTP10D, elevated EGFR signalling in loser cells switches the role of JNK from pro-apoptotic to pro-proliferative by inactivating the Hippo pathway, thereby driving the overgrowth of polarity-deficient cells. These findings uncover the mechanism by which normal epithelial cells recognize oncogenic polarity-deficient neighbours to drive cell competition (Yamamoto, 2017).
Normal epithelial cells possess an intrinsic tumour-suppression mechanism against oncogenic neighbours. For instance, in canine kidney cell cultures and zebrafish embryos, oncogenic cells that activate Ras or Src are eliminated from an epithelial monolayer when surrounded by normal cells. Similarly, in the Drosophila imaginal epithelium, oncogenic polarity-deficient cells mutant for scribble (scrib) or discs large (dlg1; hereafter dlg) are eliminated from the tissue when surrounded by wild-type cells. The removal of these surrounding wild-type cells abolishes cell elimination and allows scrib- loss-of-function mutant cells to overproliferate; this context-dependent cell elimination is therefore considered to be cell competition. Genetic studies in Drosophila have revealed that this tumour-suppressive cell competition is driven by JNK-dependent cell death, triggered by the Drosophila tumour necrosis factor (TNF) Eiger. However, the initial mechanism by which normal epithelial cells recognize nearby polarity-deficient cells to drive cell competition have remained unknown (Yamamoto, 2017).
To explore the initial event, which occurs at the interface between normal cells and oncogenic polarity-deficient cells, an ethyl methanesulfonate (EMS)-based genetic screen was conducted in Drosophila for genes required for wild-type 'winners' to eliminate neighbouring polarity-deficient 'losers'. In the eye imaginal epithelium, clones of homozygous mutant scrib-/- are eliminated when surrounded by wild-type tissue. The elimination of scrib-/- clones is also evident in adult eyes. Using the FLP/FRT-mediated genetic mosaic technique, EMS-induced homozygous mutations were induced only in wild-type winners and screened for mutations that caused an elimination-defective (eld) phenotype in neighbouring scrib- losers. Among 7,490 mutant strains generated, four elimination-defective mutants (eld-4, eld-6, eld-7, and eld-8) that fell into the same lethal complementation group were generated. Clones of scrib- cells surrounded by eld-4 clones were no longer eliminated but instead grew robustly in the eye disc and survived into adult tissue, causing a characteristic melanization phenotype. Notably, clones of eld-4, eld-6, eld-7, or eld-8 cells showed neither a growth disadvantage of their own nor a suppressive effect on the growth of neighbouring wild-type tissue. Thus, the complementation group eld-4/6/7/8 possesses mutations in a gene required for elimination of neighbouring scrib- clones (Yamamoto, 2017).
Using a series of chromosomal-deficiency lines and subsequent cDNA sequencing, a nonsense mutation in the coding region of the gene stranded at second (sas) was identified in the eld-4 mutant strain. Encoded by sas is a cell-surface ligand protein that has two extracellular domains-von Willebrand factor type C (VWC) and fibronectin type 3 (FN3) domains-as well as a transmembrane domain. Sas is required for proper axon guidance in the nervous system, but its physiological role in epithelia is unknown. Expression of Sas was indeed lost in eld-4 clones, but ectopic expression of Sas within eld-4 clones surrounding scrib-/- clones reversed the elimination-defective phenotype. Moreover, the knockdown of Sas in cells surrounding scrib-/- clones phenocopied the elimination-defective phenotype; a similar elimination-defective phenotype also occurred upon Sas knockdown in cells surrounding dlg-/- mutant eye-disc clones. These data reveal that the cell-surface ligand Sas is required for normal epithelial cells to eliminate neighbouring polarity-deficient cells (Yamamoto, 2017).
Next, attempts were made to understand the mechanism by which Sas drives the elimination of nearby cells. Sas is normally localized at the apical surface of epithelial cells. Notably, however, this study found that Sas relocalized to the lateral cell surface specifically at the interface between wild-type and scrib-/- or dlg-/- clones. This relocalization of Sas at the clone interface was also observed between wild-type and scrib-/- sas-/- double-mutant clones, indicating that the Sas protein that accumulates at the clone interface is derived from surrounding wild-type cells (Yamamoto, 2017).
The fact that normal epithelial cells relocalize Sas laterally to eliminate neighbouring oncogenic cells suggests that normal cells transmit a signal to these cells through a cell-surface receptor for Sas. Attempts were made to identify the Sas receptor expressed in polarity-deficient cells. It has been reported that PTP10D, a receptor-type tyrosine phosphatase (RPTP), interacts and functions with Sas during longitudinal axon guidance in the Drosophila nervous system and that Sas-PTP10D trans-signalling occurs through glial-neuronal communication. It was therefore assumed that PTP10D and/or other RPTPs were strong candidates for the Sas receptor in the imaginal epithelium. Given that two extracellular domains of Sas, VWC and FN3, can form homophilic interactions with the same domains of other proteins and that FN3 is a domain commonly shared by RPTPs, Thirty-two RNA interference (RNAi) fly strains were screened that target expression of Drosophila transmembrane proteins bearing either VWC or FN3 domains. Only one RNAi line targeting PTP10D phenocopied the severe elimination-defective and melanization phenotypes when expressed within scrib-/- or dlg-/- mutant clones. Like Sas, PTP10D was relocalized to the interface between scrib-/- and wild-type clones, whereas it normally localized at the apical surface of epithelial cells. This lateral accumulation of PTP10D was almost eliminated when PTP10D-RNAi was expressed within scrib-/- clones, indicating that the PTP10D accumulating at the clone interface derives from scrib-/- mutant cells. Furthermore, immunostaining analysis of scrib-/-sas-/- double-mutant clones indicated that Sas and PTP10D are localized adjacent to each other in neighbouring cells. Notably, the lateral relocalization of Sas and PTP10D at the clone interface was also observed for the neoplastic non-functional tumour-suppressor mutants vps25-/-, erupted-/-, or Rab5DN-expressing cells, all of which are eliminated as losers of cell competition when surrounded by wild-type cells; however, such relocalization was not observed for non-neoplastic polarity stardust-/- or crumbs-/- mutants. These data suggest that in response to the emergence of neoplastic polarity-deficient cells, adjacent normal cells relocalize Sas laterally whereas nearby polarity-deficient cells relocalize PTP10D laterally, thereby driving elimination of polarity-deficient cells through trans-activated Sas-PTP10D signalling (Yamamoto, 2017).
Next the mechanism by which Sas-PTP10D signalling drives elimination of polarity-deficient cells was investigated. It has previously been shown that the activation of Eiger-JNK signalling in polarity-deficient cells is essential for their elimination. Therefore, a possible mechanism by which PTP10D knockdown in scrib-/- clones results in an elimination-defective phenotype is through inhibition of JNK signalling. However, JNK signalling was still strongly activated in scrib-/- clones expressing PTP10D-RNAi, as assessed by the JNK target MMP1. This indicates that loss of PTP10D drives one or more intracellular signalling events that cause an elimination-defective phenotype in the presence of JNK activation. A strong candidate for this signalling event is activation of Ras signalling, as JNK is converted from pro-apoptotic to pro-growth in the presence of Ras activation. Notably, it has been reported that PTP10D and its mammalian orthologue PTPRJ (also known as DEP1/CD148/SCC1/RPTPeta) negatively regulate epidermal growth factor receptor (EGFR) signalling by directly dephosphorylating the intracellular tyrosine kinase domain of EGFR. This study found that EGFR normally localizes apically in wild-type cells but relocalizes to the lateral surface together with PTP10D at the boundaries between scrib-/- and wild-type clones. More pertinently, EGFR-Ras signalling was strongly elevated in scrib-/- clones expressing PTP10D-RNAi, as assessed by downregulation of the transcription factor Capicua. Moreover, co-knockdown of EGFR and PTP10D in scrib-/- clones completely reversed the elimination-defective phenotype, with EGFR-RNAi alone having only a slight effect on the growth of normal tissue. Furthermore, expression of a constitutively active form of EGFR or Ras caused overgrowth of scrib-/- clones, while expression of dominant-negative form of Ras in scrib-/-PTP10D-RNAi clones strongly suppressed their growth. Thus, scrib clones in the absence of PTP10D signalling activate both JNK and Ras signalling and overgrow in a manner dependent on EGFR signalling. The co-activation of EGFR-Ras and Eiger-JNK signalling causes hyper-accumulation of intracellular F-actin, thereby inactivating the tumour-suppressor Hippo pathway. Inactivation of the Hippo pathway triggers nuclear translocation and activation of the downstream transcriptional co-activator Yorkie (Yki), which induces upregulation of various pro-growth and anti-apoptotic genes. Indeed, scrib-/- clones expressing PTP10D-RNAi strongly accumulated intracellular F-actin and showed strong upregulation of the Yki target gene expanded (ex), as well as an increased nuclear signal of Yki protein; however, scrib mutation alone only slightly upregulated F-actin and ex expression. Furthermore, inhibition of Yki activity by the Yki kinase Warts (Wts) or Yki-RNAi significantly suppressed growth of scrib-/- clones in the absence of PTP10D, while Wts-overexpression or Yki-RNAi alone had little effect on tissue growth. Similar upregulations of EGFR signalling and Yki activity were observed in scrib-/- clones when surrounded by sas-/- eld-4 clones. Finally, he number of dying cells at the boundaries between scrib-/- and wild-type clones was found to be significantly reduced by PTP10D-knockdown, whereas cell proliferation was significantly increased in scrib-/- clones expressing PTP10D-RNAi. Together, these data indicate that when neoplastic polarity-deficient cells emerge in the epithelium, neighbouring non-neoplastic cells restrain EGFR signalling of nearby polarity-deficient cells through a Sas-PTP10D trans-interaction, which enables JNK signalling activated in polarity-deficient cells to drive cell elimination. In the absence of Sas-PTP10D, elevated EGFR-Ras signalling in polarity-deficient cells cooperates with JNK signalling to cause Yki activation, thereby leading to an elimination defect and overgrowth of polarity-deficient cells (Yamamoto, 2017).
These data indicate that in response to the emergence of oncogenic polarity-deficient cells, Sas and PTP10D relocalize specifically at the clone interface to the respective lateral surfaces of normal or polarity-deficient cells, enabling the ligand and receptor to interact with each other in trans. Thus, Sas-PTP10D acts as a fail-safe system for epithelial tissue, a system that protects against neoplastic development and is normally latent but activates upon oncogenic cell emergence. Notably, the Sas-PTP10D system was not required for other types of cell competition triggered by Minute, Mahjong, Myc or Yki. Although the mechanism by which Sas and PTP10D relocalize to the clone interface is currently unknown, this study found that the apical proteins Bazooka, Patj, and aPKC and the sub-apical protein E-cadherin also relocalize to the lateral surface of the clone boundary. This suggests that the apical cell surface expands to the lateral region at the clone boundary, meaning that Sas and PTP10D meet each other in trans at the clone interface (Yamamoto, 2017).
The genetic data reveal that Sas and PTP10D act together as tumour suppressors during cell competition. Previous studies have reported that PTPRJ, the mammalian homologue of PTP10D, also acts as a tumour suppressor and negatively regulates EGFR signalling. Although no obvious homologues of Sas have been identified in mammals, thrombospondin-1 and syndecan-2 have been reported to act as ligands for PTPRJ. Given that elimination of scrib-deficient cells by cell competition also occurs in mammalian systems, and that the signalling mechanisms identified in Drosophila are evolutionarily conserved, similar cell-cell recognition mechanisms may help to safeguard human tissues against tumorigenesis (Yamamoto, 2017).
This study developed a screening method for orphan receptor ligands, in which cell-surface proteins are expressed in Drosophila embryos from GAL4-dependent insertion lines and ligand candidates identified by the presence of ectopic staining with receptor fusion proteins. Stranded at second (Sas) binds to the receptor tyrosine phosphatase Ptp10D in embryos and in vitro. Sas and Ptp10D can interact in trans when expressed in cultured cells. Interactions between Sas and Ptp10D on longitudinal axons are required to prevent them from abnormally crossing the midline. Sas is expressed on both neurons and glia, whereas Ptp10D is restricted to CNS axons. Epistasis experiments were conducted by overexpressing Sas in glia and examining how the resulting phenotypes are changed by removal of Ptp10D from neurons. Neuronal Ptp10D was found to restrain signaling by overexpressed glial Sas, which would otherwise produce strong glial and axonal phenotypes (Lee, 2013).
A screen was devised for orphan receptor ligands in which CSS proteins are ectopically expressed in embryos and ligand candidates are identified by the presence of ectopic staining with receptor-AP fusion proteins. A screen of GAL4-dependent insertion lines was performed for 311 CSS protein genes with the XC domain of the Ptp10D RPTP, and a gene encoding the cell-surface protein Sas was identified. A modified ELISA assay was used to show that Sas and Ptp10D selectively bind to each other in the absence of other cofactors, and demonstrated that Sas and Ptp10D-expressing cells can aggregate with each other (Lee, 2013).
Ptp10D Ptp69D and sas Ptp69D double mutants both have strong ectopic midline crossing phenotypes, suggesting that Sas is required for Ptp10D signaling in the context of longitudinal axon guidance. Sas appears to be expressed on all cells in the CNS, including glia. Genetic epistasis experiments were conducted by overexpressing Sas in glia and asking whether loss of the neuron-specific Ptp10D modifies the resulting phenotype. The results suggest that overexpressed Sas can produce a signal that alters glia and affects their communication with neurons. Interactions between Ptp10D and Sas suppress the production of this signal (Lee, 2013).
RPTP signaling controls the decisions by axonal growth cones to choose longitudinal versus commissural pathways, because in a quadruple Rptp mutant (Ptp10D Lar Ptp69D Ptp99A), all 1D4 (FasII)-positive longitudinal axons are diverted into the commissures and the longitudinal bundles are absent. Ptp10D and Ptp69D are key to these guidance decisions, because triple Rptp mutants in which either Ptp10D or Ptp69D is wild-type have a relatively normal 1D4 pattern, but any mutant combination that includes both Ptp10D and Ptp69D mutations has thick 1D4-positive commissures. This suggests that Ptp10D and Ptp69D share some critical substrate(s) or interacting protein(s) that controls these decisions (Lee, 2013).
sas Ptp69D double mutants also have strong ectopic midline crossing phenotypes that are rescued by selective expression of Sas in FasII neurons. The simplest model to explain these findings is that Ptp10D forms a complex with Sas in FasII-expressing longitudinal tract neurons in order to activate the downstream signaling pathway(s) that it shares with Ptp69D. However, the axons of FasII neurons bundle together, so Sas on one axon could contact Ptp10D on another axon. The sas Ptp69D phenotype can also be rescued by expression of Sas in glia, and Sas protein(s) appear to be deposited in the ECM. Thus, signaling interactions relevant to midline crossing might also be mediated by binding of soluble Sas to Ptp10D on axons (Lee, 2013).
Longitudinal axon guidance and interface glial development are intertwined processes. Perturbation of interface glia can cause longitudinal axons to cross the midline. Conversely, the fates of longitudinal glia, which are a subset of the interface glia, are controlled by signals from neurons (Lee, 2013).
The analysis of glial-neuronal interactions provides an excellent system in which to examine whether signaling through Sas can be regulated by interaction with Ptp10D. Ptp10D is only on axons, whereas Sas is expressed on glia. Driving Sas overexpression in glia with Repo-GAL4 produces only subtle phenotypes. However, genetic removal of Ptp10D from Repo > Sas embryos generates strong ectopic midline crossing phenotypes. These phenotypes are accompanied by disorganization of interface glia. Glial mispositioning might be sufficient to affect axon guidance. However, given the severity of the axonal phenotype, it is thought more likely that the disruption of the glial lattice is reflective of changes in gene expression that cause the glia to send abnormal axon guidance signals to the neurons (Lee, 2013).
The 37 aa Sas cytoplasmic domain interacts with Numb in the yeast two-hybrid assay. Numb is an inhibitor of Notch signaling, and both elevation and loss of Notch signaling affect longitudinal glia. It is suggested that when Sas is overexpressed in glia and is not restrained by Ptp10D binding, it might sequester Numb, thereby increasing Notch signaling (Lee, 2013).
Binding of Sas to Ptp10D on longitudinal axons facilitates Ptp10D's functions in regulation of CNS axon guidance. In glia, overexpressed Sas produces a signal that is suppressed by interactions with neuronal Ptp10D. Other receptors involved in axon guidance exhibit interactions with ligands that produce different signaling outcomes depending on whether the ligands and receptors are expressed on the same or on different cells. In retinal ganglion cells and spinal motor neurons, Eph RTKs interact with Ephrin ligands both on other cells (in trans) and on the same cells (in cis), and cis interactions attenuate the responses of the RTKs to ligand presented in trans (Lee, 2013).
Like Sas, Ephrins and type III neuregulin 1 (a ligand for ErbB RTKs) also generate 'reverse' signals in the cells that express them. These signals are important for axon pathfinding. However, Ephrin and neuregulin signals are produced upon engagement of the ligands with their receptors, not blocked by receptor engagement as in the case of Sas and Ptp10D (Lee, 2013).
Search PubMed for articles about Drosophila Sas
Lee, H. K., Cording, A., Vielmetter, J. and Zinn, K. (2013). Interactions between a receptor tyrosine phosphatase and a cell surface ligand regulate axon guidance and glial-neuronal communication. Neuron 78(5): 813-826. PubMed ID: 23764287
Yamamoto, M., Ohsawa, S., Kunimasa, K. and Igaki, T. (2017). The ligand Sas and its receptor PTP10D drive tumour-suppressive cell competition. Nature 542(7640): 246-250. PubMed ID: 28092921
date revised: 20 September 2018
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