BIOLOGICAL OVERVIEW

Cellular diversity is a fundamental characteristic of complex organisms, and the Drosophila CNS has proved an informative paradigm for understanding the mechanisms that create cellular diversity. One such mechanism is the asymmetric localization of Numb to ensure that sibling cells respond differently to the extrinsic Notch signal and, thus, adopt distinct fates (A and B). This study focusses on the only genes known to function specifically to regulate Notch-dependent asymmetric divisions: sanpodo and numb. sanpodo, which specifies the Notch-dependent fate (A), encodes a four-pass transmembrane protein that localizes to the cell membrane in the A cell and physically interacts with the Notch receptor. Numb, which inhibits Notch signaling to specify the default fate (B), physically associates with Sanpodo and inhibits Sanpodo membrane localization in the B cell. These findings suggest a model in which Numb inhibits Notch signaling through the regulation of Sanpodo membrane localization (O'Connor-Giles, 2003).

Spdo was initially identified (Dye, 1998) as the homolog of the actin-associated protein Tropomodulin (Tmod), a protein that regulates actin filament length. This study finds that spdo does not encode tmod, but rather a four-pass transmembrane protein that acts upstream of Notch and downstream of Delta to specify the A cell fate. Spdo colocalizes and physically associates with the Notch receptor in vivo. Spdo also exhibits differential subcellular localization between A and B cells during asymmetric divisions, localizing primarily to the cell membrane of the A cell and to the cytoplasm of the B cell. Numb inhibits the cell membrane localization of Spdo in the B cell and Numb and Spdo physically associate in vivo. These findings support a model in which Numb acts in the B cell to block Notch activity by preventing Spdo from localizing to the cell membrane, likely through its link to the endocytic machinery. In the A cell, the absence of Numb allows Spdo to localize to the cell membrane, where it promotes Notch signaling and the A cell fate, likely through a direct association with Notch (O'Connor-Giles, 2003).

Prior studies suggest that spdo acts in the Notch pathway to mediate asymmetric divisions. However, as these studies did not order spdo function relative to members of the Notch pathway, the placement of spdo within the pathway remains uncertain. To order the action of spdo relative to the intramembranous S3 cleavage event that releases the Notch intracellular domain (NICD) from the membrane, two distinct constitutively active forms of Notch, Notch^Intra and Notch^ECN were used. While both Notch constructs function in a ligand-independent manner, Notch^ECN contains the NICD and the Notch transmembrane domain and requires proper execution of the S3 cleavage to activate transcription of Notch target genes. Notch^Intra, which comprises only the NICD, functions independently of the S3 cleavage (O'Connor-Giles, 2003).

In these experiments focus was placed on the development of eight pairs of sibling neurons that arise from spdo/Notch/numb-dependent asymmetric divisions: RP2/RP2sib, dMP2/vMP2, aCC/pCC, and five pairs of U/Usib neurons. Molecular markers can distinguish unambiguously the fate of each of these sibling neurons from their sisters. RP2/RP2sib develop from the Even-skipped (Eve)-expressing GMC4-2a. After division, RP2 retains, while RP2sib extinguishes, Eve expression. Similarly, the U/Usib neurons develop from five Eve-positive GMCs; each GMC divides to produce two initially Eve-positive neurons. The five U neurons retain Eve expression, while the five Usib neurons extinguish Eve. The dMP2/vMP2 interneurons develop from the Odd-skipped (Odd)-positive MP2 precursor. After MP2 division, dMP2 retains Odd expression and extends an axon posteriorly, while vMP2 extinguishes Odd and extends an axon anteriorly. aCC/pCC develop from the Eve-positive GMC1-1a. Both aCC and pCC retain Eve expression; however, aCC expresses 22C10 and extends a motor axon out the intersegmental nerve, while pCC is an interneuron that extends a 22C10-negative axon anteriorly. The RP2sib, pCC, vMP2, and U neurons (A fates) require spdo/Notch function for their specification, while their siblings (B fates) require numb-mediated inhibition of spdo/Notch activity for their development (O'Connor-Giles, 2003).

The two constitutively active Notch constructs were expressed throughout the CNS of wild-type and spdo mutant embryos using the Gal4/UAS system and the development of the RP2/RP2sib, dMP2/vMP2, and U/Usib neurons was followed. It was reasoned that, if spdo acts upstream of Notch, the Notch gain-of-function phenotype (A/A) should be observed. Conversely, if spdo acts downstream of Notch, the spdo phenotype (B/B) would be seen. The placement of spdo function upstream of Notch^Intra, but downstream of Notch^ECN, would indicate a requirement for spdo in the S3 cleavage of the Notch receptor. In a wild-type background, misexpression of either Notch construct is found to be sufficient to induce cells that would normally acquire the numb-dependent B fate to adopt the A fate at a moderate to high frequency depending upon the sibling pair examined. Misexpression of each Notch construct in spdo embryos is found to yield identical cell fate transformations at frequencies essentially equal to those observed in wild-type embryos misexpressing each construct. These results indicate that spdo functions genetically upstream of the S3 cleavage of Notch during asymmetric divisions (O'Connor-Giles, 2003).

Next, the placement of spdo function relative to Delta was assayed. To do this, Delta was misexpressed throughout the CNS of spdo embryos and U/Usib and RP2/RP2sib neuron development was assayed. It was reasoned that, if spdo acts downstream of, or in parallel to, Delta, then misexpression of Delta would not rescue the spdo phenotype. However, if spdo acts upstream of Delta, rescue of the spdo CNS phenotype would be observed. Misexpressing Delta was found not to rescue the spdo phenotype, indicating that spdo acts genetically downstream of, or in parallel to, Delta to promote asymmetric divisions. Together with the placement of spdo function upstream of the S3 cleavage of Notch, this result suggests that spdo functions at or near the membrane to promote Notch signaling during asymmetric divisions (O'Connor-Giles, 2003).

Spdo was identified as the homolog of the actin-associated protein Tmod, a protein that regulates actin filament length. Since no previous role for tmod in regulating cell fate had been identified, it was important to determine whether spdo function during asymmetric divisions is dependent upon, or separable from, its role in actin regulation. Since chemically induced mutations often cluster in functionally critical protein domains, attempts were made to address this question by identifying the molecular lesions in the EMS-induced spdo alleles. However, despite sequencing the entire coding region, including three alternative 5' exons, the 5' and 3' UTRs, and all splice sites in five alleles, as well as the majority of these sequences in four additional alleles, no molecular lesions were identified in tmod. Since the vast majority of EMS-induced mutations associated with observable phenotypes are found in the coding region of the affected gene, these data suggested that spdo encodes a gene other than tmod (O'Connor-Giles, 2003).

To identify spdo, genetic mapping with single nucleotide polymorphisms (SNPs) was used to localize the molecular lesions responsible for the spdo phenotype to a narrow molecular region. Using this approach, the molecular lesion in spdo^AC85 was localized to an 85 kb region and the lesion in spdo^YY233 to an overlapping 80 kb region. As the two alleles likely map close to one another, efforts were focussed on the 30 kb region of overlap (O'Connor-Giles, 2003).

Sequence analysis of the spdo interval identified nine genes. RNA whole-mount in situ hybridization of the nine genes revealed one gene, CG31020, specifically expressed in the CNS, PNS, and mesoderm during the stages when spdo-dependent cell fate decisions occur in these tissues. Furthermore, spdo^ZZ27 embryos are transcript null for CG31020. To determine whether CG31020 encodes spdo, its open reading frame (ORF) was sequenced in nine remaining spdo alleles as well as three independently generated spdo alleles and molecular lesions were identified in all twelve alleles. Ten alleles contain point mutations. The single base pair changes in six of these alleles (spdo^AC81, spdo^G104, spdo^VV86, spdo^Z143, spdo^ZZ213 and spdo^P46) result in the introduction of premature stop codons. Two alleles, spdo^OO3 and spdo^AB153, contain the identical missense mutation that converts an evolutionarily conserved glycine to an arginine, while spdo^C55 and spdo^K433 contain missense mutations that convert a conserved leucine to an arginine and a serine to a phenylalanine, respectively. The two remaining alleles contain deletions: spdo^AC85 contains a 405 bp in-frame internal deletion, and spdo^YY233 contains a larger deletion that extends beyond the 3' terminus of the transcript. spdo^ZZ27 contains a large multigenic deletion that removes both CG31020 and tmod (O'Connor-Giles, 2003).

To confirm that CG31020 encodes spdo, RNA interference (RNAi) and gene rescue experiments were conducted. Injection of double-stranded CG31020 RNA into wild-type embryos yields a CNS phenotype essentially identical to that of spdo. In reciprocal experiments using the Gal4/UAS system to express CG31020 throughout the CNS of otherwise spdo mutant embryos, complete to near-complete rescue of the spdo CNS phenotype was observed. The identification of molecular lesions in all spdo alleles, analyzed together with the RNAi and gene rescue experiments all demonstrate that CG31020 identifies spdo (O'Connor-Giles, 2003).

Several notable attributes are observed with respect to the subcellular localization of Spdo. (1) Spdo localizes to the cell membrane as well as to small, intermediate, and large puncta that appear to reside interior to the cell membrane. The relative location of these puncta is consistent with their being cytoplasmic vesicles. For simplicity, from here on these are referred to as cytoplasmic puncta or accumulations of Spdo. (2) Cells that localize Spdo primarily to the cell membrane generally exhibit weak cytoplasmic accumulation of Spdo, while cells that localize Spdo primarily to the cytoplasm generally exhibit weak accumulation of Spdo at the membrane. (3) Spdo localizes uniformly around the cell membrane of cells that localize Spdo predominantly to the cell membrane. The apparent dynamic subcellular localization of Spdo raises the possibility that modulation of Spdo localization may regulate the ability of Spdo to promote Notch signaling during asymmetric divisions (O'Connor-Giles, 2003).

To examine the potential relevance of the subcellular localization of Spdo, colocalization studies were performed between Spdo and Notch, Delta, and Numb. Spdo is found to exhibit extensive colocalization with Notch at the cell membrane and in small and large puncta throughout the cytoplasm. Strong Spdo and Notch colocalization is detected in large cytoplasmic puncta in NBs as well as in smaller puncta near and at the cell membrane of GMCs. Although a significant majority of Notch-expressing puncta in the CNS is observed to colocalize with Spdo, this is not an obligate relationship, as some Notch puncta do not colocalize with Spdo, and many Spdo puncta do not colocalize with Notch. However, the significant overlap between Spdo and Notch suggests that Spdo promotes Notch signaling during asymmetric divisions through a close association with Notch (O'Connor-Giles, 2003).

The relative localization of Spdo and Delta is more complex than that observed for Spdo and Notch. In general, Spdo and Delta are expressed in largely complementary patterns in and around the CNS. This is in agreement with a prior report demonstrating that Delta is expressed at high levels in the mesoderm and at lower levels in NBs and the neurectoderm, but not in GMCs or neurons. However, in regions of close contact between GMCs, neurons, and neighboring Delta-expressing cells, tight juxtaposition of Spdo-expressing and Delta-expressing puncta are observed at or near cell membranes. In most instances, Spdo- and Delta-expressing puncta reside immediately adjacent to one another and exhibit partial overlap. As with Notch, the apposition of Spdo and Delta is not obligate. Most Delta-expressing puncta in these regions are associated with Spdo expression; however, many are not, and most Spdo-positive puncta are not associated with Delta expression. However, the significant colocalization of Spdo with Notch and the frequent juxtaposition of Spdo- and Delta-expressing puncta at or near the cell membrane suggest that Spdo functions in close association with Notch and its ligand Delta to promote productive signaling during asymmetric divisions. Interestingly, no gross changes are observed in the expression or localization of Notch or Delta in spdo mutant embryos (O'Connor-Giles, 2003).

Genetic, molecular, and expression data suggest that Spdo promotes productive Notch signaling through a close association with Notch. To determine whether Spdo physically associates with the Notch receptor, Notch was immunoprecipitated and assayed for the coprecipitation of Spdo. Spdo was found to coprecipitate at roughly equivalent efficiencies with antibodies specific to either the intracellular or extracellular domain of Notch, suggesting that Spdo associates with the full-length Notch receptor. These data indicate that Spdo associates with the Notch receptor in vivo and suggest that Spdo promotes Notch signaling during asymmetric divisions through a physical association with the Notch receptor (O'Connor-Giles, 2003).

Significant colocalization is also observed between Spdo and Numb at the cell membrane and in the cytoplasm. However, these studies also reveal a general inverse correlation between the presence of Numb and the membrane localization of Spdo. For example, CNS, PNS, and mesodermal cells that express low levels of Numb generally localize Spdo largely to the cell membrane, whereas cells that express high levels of Numb generally localize Spdo largely to the cytoplasm. The correlation is not absolute; however, together with the genetic placement of numb as an upstream negative regulator of spdo, it raises the possibility that numb inhibits Notch signaling during asymmetric divisions by regulating the subcellular localization of Spdo (O'Connor-Giles, 2003).

To investigate whether numb regulates the subcellular distribution of Spdo, Spdo localization was followed in embryos homozygous mutant for numb. Because of maternal numb product, focus was placed on late stage 11 and older embryos, when minimal levels of maternal Numb protein are detected. Relative to wild-type, in numb embryos, a significant increase in Spdo localization to the cell membrane is observed and a corresponding decrease in Spdo-expressing cytoplasmic puncta in NBs, GMCs, neurons, and mesodermal and PNS precursors. Persistent expression of Spdo is also observed in numb embryos, since most CNS neurons in stage 13 numb embryos express Spdo at high levels, whereas, in stage 13 wild-type embryos, most CNS neurons express Spdo at low levels. Thus, numb appears to regulate the cell membrane localization and levels of Spdo in asymmetrically dividing cells (O'Connor-Giles, 2003).

These data together with the exclusive segregation of Numb to the B cell suggest a model in which Numb blocks Notch signaling by inhibiting the cell membrane localization of Spdo in the B cell. To test this model, Spdo localization was followed in the progeny of the CNS precursor MP2, which divides asymmetrically under the control of spdo and numb. In wild-type, MP2 produces two siblings: a larger dorsal cell, dMP2, and a smaller ventral cell, vMP2. During this division, Numb segregates exclusively into dMP2 (the B cell), where it blocks Notch signaling and promotes the dMP2 fate. Notch signaling is active in vMP2 (the A cell) and specifies the vMP2 fate. If Numb inhibits the cell membrane localization of Spdo in the B cell, strong Spdo membrane localization would be expected in vMP2 and weak membrane localization in dMP2. Using Odd-skipped expression to identify newly born d/vMP2 siblings in wild-type embryos, Spdo is found to localize to the cell membrane of vMP2, but not dMP2. Specifically, in 81.1% of d/vMP2 sibling pairs, Spdo localizes predominantly to the membrane and exhibits minimal cytoplasmic accumulation in vMP2, while, in dMP2, Spdo exhibits minimal or no membrane localization and significant cytoplasmic accumulation. Increased Spdo membrane localization is never detected in dMP2 relative to vMP2 or increased cytoplasmic accumulation in vMP2 relative to dMP2. These results indicate that Spdo exhibits differential subcellular localization between sibling vMP2 (A) and dMP2 (B) cells and suggest that Numb promotes this difference by preventing Spdo from localizing to the cell membrane of dMP2 (O'Connor-Giles, 2003).

To determine whether the differential localization of Spdo between vMP2 and dMP2 depends on numb, Spdo localization was followed during MP2 divisions in numb mutant embryos. In numb embryos, MP2 still produces a smaller ventral cell and a larger dorsal cell; however, both cells acquire the vMP2, or A cell, fate. As in wild-type, the ventral cell always exhibits significant localization of Spdo to the cell membrane and no/minimal cytoplasmic accumulation of Spdo. However, in numb embryos, 93% of the time, the larger dorsal cell is found to exhibit no/minimal cytoplasmic accumulation of Spdo; this cell also exhibits increased localization of Spdo to the cell membrane. Thus, the differential subcellular localization of Spdo between vMP2 and dMP2 observed in wild-type embryos appears to depend on the ability of Numb to restrict Spdo from the cell membrane in the B cell. This numb-dependent asymmetry in the subcellular localization of Spdo, a positive mediator of Notch signaling, suggests that Numb blocks Notch signaling in the B cell through its ability to inhibit the localization of Spdo to the cell membrane (O'Connor-Giles, 2003).

The ability of Numb to regulate the subcellular localization of Spdo together with the known dosage-sensitive interactions between these genes suggests that Numb may physically associate with Spdo to regulate its subcellular localization. To address this possibility, whether Numb and Spdo associate in vivo was assayed via coimmunoprecipitation assays. Antibodies directed against Numb were observed to coprecipitate Spdo from wild-type embryonic cell lysates. Thus, Spdo and Numb appear to physically associate in vivo, consistent with the idea that Numb inhibits the localization of Spdo to the cell membrane and, thus, active Notch signaling in the B cell through this association (O'Connor-Giles, 2003).

A recent model for Numb-dependent inhibition of Notch activity during asymmetric divisions suggests that Numb blocks Notch signaling by targeting Notch for endocytosis in the B cell. In support of this model, Numb can physically interact with Notch and α-Adaptin, a component of the endocytic machinery, and hypomorphic mutations in α-adaptin yield a numb-like phenotype in the PNS. Yet caveats to the model exist. (1) If Numb targets Notch for endocytosis, one would expect to observe lower levels or differential localization of Notch in the B cell relative to the A cell. However, the levels and distribution of Notch appear equivalent between these cells during asymmetric divisions. (2) The presence of Numb and α-Adaptin are not sufficient to inhibit Notch pathway activity in other developmental contexts (O'Connor-Giles, 2003).

The results support a revised model in which Numb interferes with Spdo function to inhibit Notch activity during asymmetric divisions. In this model, Numb inhibits Notch activity in the B cell by blocking the ability of Spdo to localize to the cell membrane. In the A cell the absence of Numb permits Spdo to localize to the cell membrane, where it promotes Notch signaling and the A cell fate, likely through a physical association with Notch. The ability of Numb to associate with Spdo and α-Adaptin suggests that Numb removes Spdo from the cell membrane via the endocytic machinery. Since active Notch signaling appears to require Spdo at the cell membrane, the internalization of Spdo in the B cell is incompatible with productive Notch signaling. While this model does not preclude Notch internalization along with Spdo in the B cell, it does not rely upon differential internalization of Notch between the A and B cells -- a phenomenon not seen in the embryonic CNS (O'Connor-Giles, 2003).

This work and that of others indicate that spdo is generally required to promote Notch/numb-dependent asymmetric divisions. For example, spdo promotes the Notch-dependent fate in all Notch/numb-dependent CNS, heart, and mesoderm precursor divisions assayed to date. spdo also appears to play a role in all Notch/numb-dependent asymmetric divisions in the PNS. In the canonical external sensory organ lineage, a single precursor (SOPI) and its progeny (SOPIIa, SOPIIb, and SOPIIIb) divide asymmetrically under Notch/numb control to produce the distinct cell types that make up the sensory organ. In addition, mitotic spdo clones in the eye proper and notum lack bristles, a phenotype indicative of spdo promoting the asymmetric division of SOPI. These studies indicate that spdo likely plays an important role in mediating all Notch/numb-dependent asymmetric divisions in Drosophila (O'Connor-Giles, 2003).

Although spdo and numb appear to regulate all Notch-dependent asymmetric divisions in Drosophila, neither has been shown to regulate Notch pathway activity in any other developmental context. The limited effect of Numb on Notch signaling cannot be explained by a restricted expression pattern, since Numb (and α-Adaptin) exhibits a relatively general expression pattern. The apparent inability of Numb to inhibit Notch signaling in developmental contexts other than asymmetric divisions suggests that it may function through a protein or proteins specifically required for Notch-dependent asymmetric divisions. Critically, Notch signaling requires Spdo only during asymmetric divisions, and, in this context, Spdo appears to act at the cell membrane to promote Notch signaling. The ability of Numb to inhibit the cell membrane localization of Spdo suggests that Spdo may be the key factor that links Numb to the regulation of Notch pathway activity. If this model is correct, then Numb will only be able to inhibit Notch signaling in those developmental contexts in which Notch activity requires Spdo function-asymmetric divisions. This model then provides a rational explanation for why Numb appears to inhibit Notch signaling only during asymmetric divisions (O'Connor-Giles, 2003).

It remains unclear why Spdo is required for Notch signaling only during asymmetric divisions. The context-specific requirement of spdo suggests that spdo does not promote an event generally required for Notch activity (such as Notch presentation at the membrane or Notch proteolysis) but rather an event specifically required for Notch activity during asymmetric divisions. Insight into this question may come from the observation that most spdo-independent Notch-mediated decisions occur in an epithelium, while spdo/Notch-dependent asymmetric divisions occur in nonepithelial cells. Thus, it is possible that, during asymmetric divisions, Notch signaling requires accessory proteins not needed in epithelial cells to stabilize or otherwise to promote Notch-Delta interaction and/or signaling-proteins such as Spdo. The relative expression patterns of Spdo, Notch, and Delta are consistent with this, as is the observation that asymmetric divisions that produce siblings that retain a close association with the epithelium (e.g., the SOPIIa division that produces the socket and bristle) exhibit a weaker requirement for Spdo than those that produce siblings that do not retain close contact with the epithelium (e.g., GMCs, heart precursors, and SOPIIIb) (O'Connor-Giles, 2003).

Notch and Numb localize asymmetrically within CNS precursors in the mammalian brain, and molecular and genetic studies indicate that Notch and Numb regulate the asymmetric division of these precursors. These observations together with the apparent link Spdo provides between Numb and the Notch pathway in Drosophila leads to the speculation that mammalian orthologs of Spdo mediate Notch/Numb-dependent asymmetric divisions in mammals (O'Connor-Giles, 2003).

Standard computational approaches, however, fail to identify mammalian Spdo orthologs. The Anopheles Spdo ortholog shares 32% amino acid identity with Drosophila Spdo. This degree of identity is significantly lower than the average identity of 56% observed between orthologous pairs of Drosophila and Anopheles proteins, identifying spdo as a fast-evolving gene with limited constraints on amino acid substitutions. Thus, it will likely be difficult to identify Spdo orthologs in distantly related species through standard computational approaches. However, additional research on the Notch pathway as well as work on vertebrate and invertebrate odorant receptors suggests that alternate strategies may identify mammalian Spdo orthologs (O'Connor-Giles, 2003).

LAG-3, a C. elegans glutamine/proline-rich protein, forms a ternary complex with the Notch pathway transcription factor LAG-1 [CSL/Su(H)] and the Notch intracellular domain to activate transcription of Notch target genes (Petcherski, 2000a). Database searches do not identify LAG-3 orthologs in other species. Despite this, Petcherski (2000b) used a modified yeast two-hybrid system to search for functional LAG-3 homologs. This work identified Mastermind, a glutamine/proline-rich protein and canonical member of the Notch signaling pathway in Drosophila, and a murine homolog, mMam1, as functional LAG-3 homologs (Petcherski, 2000b). The identical roles LAG-3 and Mastermind play in Notch signaling together with their similar structural composition led to the model that LAG-3 and Mastermind share a common ancestor but that this relationship is occluded by a high rate of amino acid substitution in these proteins (Petcherski, 2000b). As with LAG-3, the identification of Spdo-interacting proteins may provide a tool for identifying functional Spdo homologs, while also elucidating the molecular basis by which Spdo regulates Notch signaling (O'Connor-Giles, 2003).

Endocytosis by Numb breaks Notch symmetry at cytokinesis

Cell-fate diversity can be generated by the unequal segregation of the Notch regulator Numb at mitosis in both vertebrates and invertebrates. Whereas the mechanisms underlying unequal inheritance of Numb are understood, how Numb antagonizes Notch has remained unsolved. Live imaging of Notch in sensory organ precursor cells revealed that nuclear Notch is detected at cytokinesis in the daughter cell that does not inherit Numb. Numb and Sanpodo act together to regulate Notch trafficking and establish directional Notch signalling at cytokinesis. It is proposed that unequal segregation of Numb results in increased endocytosis in one daughter cell, hence asymmetry of Notch at the cytokinetic furrow, directional signalling and binary fate choice (Couturier, 2012).

This analysis supports the following model for the control of Notch by Numb and Spdo in the context of asymmetric cell division. Before mitosis, endocytosis of Notch by Spdo decreases cortical Notch levels. At mitosis, cortical Notch moves along the apical cortex towards the apical pIIa/pIIb interface whereas internalized Notch is delivered to the newly formed plasma membrane along the pIIa/pIIb interface. At cytokinesis, Numb acts in pIIb to regulate the endocytosis of Notch, thereby removing Notch from the pIIb membrane. As a result, Notch is activated only in pIIa. In the absence of Spdo, Notch accumulates at the apical cortex before mitosis, resulting in increased cortical Notch at the apical pIIa/pIIb interface as well as decreased levels of Notch in endosomes, hence reduced levels of Notch delivered to the cytokinetic furrow. In the absence of Numb, similar amounts of Notch localize in pIIb and pIIa at the pIIa/pIIb interface, hence resulting in symmetric activation. In this model, the unequal segregation of Numb at mitosis results in an early asymmetry of Notch localization at the pIIa/pIIb interface, hence leading to asymmetric signalling and binary fate choice (Couturier, 2012).

As Numb interacts with Spdo through its phosphotyrosine-binding domain and with the ear domain of the α-adaptin through its NPF motif, Numb probably regulates the endocytosis, that is internalization and/or endosomal sorting, of Spdo-Notch complexes. Numb has also been proposed to interact directly with Notch, suggesting that Numb may also regulate the endocytosis of Notch receptors that are not in a complex with Spdo. Thus, these molecular interactions suggest that Numb may link both Notch and Spdo-Notch complexes to the AP-2 endocytic machinery to promote their internalization. Alternatively, but non-exclusively, Numb could block the recycling of both Notch and Spdo-Notch complexes back to the pIIa/pIIb interface. In both molecular models, the presence of Numb in pIIb would lead to the depletion of Notch from the pIIb membrane (Couturier, 2012).

How Spdo positively regulates Notch is not entirely clear. It is proposed that Spdo may act positively by increasing the pool of endosomal Notch in SOPs before mitosis to ensure that an appropriate number of receptors are targeted towards the cytokinetic furrow to localize along the basal pIIa/pIIb interface at cytokinesis. Alternatively, the data showing that Spdo interacts and co-traffics with Notch in pIIa indicate that Spdo may regulate endocytosis and activation of Notch in pIIa, that is in the absence of Numb. Spdo could, for instance, regulate the trafficking of active membrane-tethered S2-cleaved Notch towards a compartment where it is further processed by the γ-secretase. As extracellular epitopes are separated from activated membrane-tethered Notch on S2 cleavage, this potential role of Spdo was not examined in the antibody uptake assay (Couturier, 2012).

A recent study has suggested that Notch is directionally trafficked into pIIa at cytokinesis. This conclusion was based on the live-imaging analysis of anti-NECD/anti-Mouse Fab complexes that had been internalized in SOPs just before mitosis. However, this study did not provide evidence that endogenous Notch localized into the pool of endosomes that are directionally trafficked at cytokinesis. This study did not observe directional trafficking of NiGFP at cytokinesis. Thus, these data do not support the notion that Notch is directionally trafficked towards pIIa (Couturier, 2012).

In conclusion, this live-imaging study of Notch in Drosophila has revealed that directional Notch signalling is established at the end of mitosis through the regulated internalization and/or endosomal sorting of Notch-Spdo complexes by Numb in one of the two daughter cells. This model provides a simple and possibly general mechanism for the role of Numb in asymmetric division in animal cells (Couturier, 2012).

GENE STRUCTURE

cDNA clone length - 2099

Bases in 5' UTR - 124

Exons - 2

Bases in 3' UTR - 277

PROTEIN STRUCTURE

Amino Acids - 565

Structural Domains

Conceptual translation of CG31020 indicates that spdo encodes a 565-amino acid protein with four predicted transmembrane domains at its extreme C terminus. Protein topology prediction algorithms indicate that Spdo is likely a type IIIa transmembrane protein, with a 431-amino acid N-terminal cytoplasmic domain. Consistent with this, Spdo protein is found to accumulate abnormally in the cytoplasm and exhibits minimal membrane targeting in embryos homozygous for spdo alleles containing nonsense mutations prior to the predicted transmembrane domains. Except for the transmembrane domains and a glutamine-rich N-terminal domain (amino acids 71-94), Spdo contains no characterized protein motifs (O'Connor-Giles, 2003).

Spdo orthologs were identified in Drosophila pseudoobscura and Anopheles gambiae via comparative sequence analysis. The two Drosophila proteins share 80% identity, while D. melanogaster and Anopheles Spdo are 32% identical and 46% similar at the amino acid level. Most of the conservation resides in the transmembrane and intervening loop domains, as well as in a 60-amino acid N-terminal region that maintains 75% identity and 93% similarity (O'Connor-Giles, 2003).

date revised: 10 February 2004

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