EvoprintHD of numb

BIOLOGICAL OVERVIEW

How do two cells, the progeny from a single cell division, develop different fates? This is the fundamental question of developmental biology. Both Prospero and Numb proteins are asymmetrically distributed to progeny cells. For a more detailed discussion of the mechanics of how this asymmetric distribution of both Prospero and Numb takes place, see the prospero site. The current essay is concerned with the functional result and significance of such an uneven distribution.

Numb protein is asymmetrically distributed to the progeny of the MP2 precursors in the central nervous system, and to the progeny of Sensory organ precursor (SOP) cells in the peripheral nervous system. In the case of MP2 progeny, one of the two develops into an interneuron with an anterior axon projection; the other (the recipient of Numb) develops into an interneuron with a posterior axon projection. In SOP cells, one of the two progeny becomes the precursor for both bristle cells and socket cells; the other (the recipient of Numb) becomes the precursor of both neuron and glial (sheath) cells. Mutation of numb results in a transformation of cell fate: the fate of the cell normally receiving Numb is transformed into that of the Numb deficient cell (Spana, 1995 and Knoblich, 1995).

How does Numb determine cell fate? In addition to the intrinsic Numb signal, extrinsic signals are also required to produce a normal SOP lineage. Loss of either Delta, Notch or Suppressor of Hairless function results in neuron and glial fate, the opposite of the numb loss-of-function phenotype. This suggests that Numb might confer resistence to Notch-mediated signals in the neuron and glial fates (Spana, 1996 and references).

Does Notch signaling similarly alter the fate of MP2 progeny? Odd-skipped protein and a ß-galactosidase enhancer-trap marker were used to identify the two progeny of the MP2 lineage (dMP2 and vMP2, respectively). Mutations of either Delta or Notch transform vMP2 into dMP2. Numb protein is segregated into the dMP2 neuron. Loss of Numb transforms cells from dMP2 to vMP2. This is the opposite of the tranformation found in either Delta or Notch mutants. If the function of Numb were to specify the dMP2 fate, and the function of Delta and Notch were to keep Numb out of vMP2, double mutants (numb and Notch or numb and Delta) ought to show the numb phenotype (two vMP2s). Alternatively, if Delta-Notch signaling induces vMP2 fate, and localization of Numb into dMP2 cells inhibits this signal, then double mutants would show the Delta or Notch phenotype (two dMP2s). In both double mutants the dMP2 phenotype predominates. This indicates that the function of Numb is to antagonize the Delta-Notch signal specifying the vMP2 fate (Spana, 1996).

Do the physical distributions of Delta and Notch make sense in terms of their presumed function? Delta is not detected in either dMP2 or vMP2, but rather in adjacent mesoderm (in contact with MP2 and its progeny), while Notch is uniformly distributed throughout all cell types, including the dMP2 and vMP2 neurons. There is no sign of asymmetic Notch localization. If Numb functions to block Notch signaling, as is suspected, then the ubiquitous Notch distribution found is consonant with its proposed function. In this case the Notch ligand (Delta) does not have to be present in either dMP2 or vMP2, but can provide its function from adjacent non-neuronal cells (Spana, 1996).

Numb acts by polarizing the distribution of a-Adaptin, a protein involved in endocytosis

Numb influences cell fate by downregulating Notch through polarized receptor-mediated endocytosis. Numb functions as a linker between α-Adaptin and Notch. α-Adaptin facilitates the endycytosis of Notch. α-Adaptin acts downstream of Numb in the determination of alternative cell fates in asymmetric cell division. During asymmetric cell division in sensory organ precursor cells, Numb protein localizes asymmetrically and segregates into one daughter cell, where it influences cell fate by repressing signal transduction via the Notch receptor. Numb acts by polarizing the distribution of α-Adaptin, a protein involved in receptor-mediated endocytosis. α-Adaptin binds to Numb and localizes asymmetrically in a Numb-dependent fashion. Mutant forms of α-Adaptin that no longer bind to Numb fail to localize asymmetrically and cause numb-like defects in asymmetric cell division. These results suggest a model in which Numb influences cell fate by downregulating Notch through polarized receptor-mediated endocytosis, since Numb also binds to the intracellular domain of Notch (Berdnik, 2002b).

Drosophila α-Adaptin binds to Numb and the ear domain of α-Adaptin is critical for this interaction. Like Numb, α-Adaptin localizes asymmetrically in dividing SOP cells and preferentially segregates into the pIIb cell. α-Adaptin mutations that affect binding to Numb and abolish asymmetric localization cause cell fate transformations similar to those observed in numb. Epistasis experiments place α-Adaptin downstream of numb and upstream of Notch, suggesting that α-Adaptin is involved in the suppression of Notch signaling by Numb. These results suggest that Numb regulates cell fate by polarizing the distribution of the endocytic protein α-Adaptin which in turn is involved in the endocytosis and consequent inactivation of Notch (Berdnik, 2002b).

To test the epistatic relationship between numb and α-Adaptin, numb was overexpressed in α-Adaptin mutant clones. numb overexpression induces transformations of externally visible outer cells (socket and hair) into inner cells (neuron and sheath), presumably because the protein segregates into both daughter cells and represses Notch. Inner cells do not produce any structures that are visible from the outside, and, therefore, these transformations cause an apparent loss of bristles. If numb acts downstream of α-Adaptin, numb overexpression in α-Adaptin mutant clones should revert the outer cell fate transformations observed in these clones. Conversely, if numb is upstream, outer cell fate transformations should still be observed. Epistasis experiments were carried out in postorbital bristles, which are located at the posterior edge of the eye and can easily be scored in high numbers. When ada^ear4 mutant head clones are generated using eyeless-Flp, about 50% of these bristles show the characteristic transformation of hairs into additional sockets. The other bristles are unaffected, presumably because they are not included in the mutant clones or due to perdurance of α-Adaptin protein. Overexpression of numb in SOP cells, on the other hand, causes a 70% reduction of postorbital bristles. When numb is overexpressed in ada^ear4 mutant head clones, the number of bristles bearing outer cell fate transformations is unchanged. These data show that the ada^ear4 mutant phenotype cannot be reverted upon numb overexpression and indicate that α-Adaptin acts genetically downstream of numb (Berdnik, 2002b).

Numb inhibits membrane localization of Sanpodo, a four-pass transmembrane protein, to promote asymmetric divisions in Drosophila

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; full text of article).

Spdo was initially identified 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).

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).

Numb and alpha-Adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs

During asymmetric cell division in Drosophila sensory organ precursors (SOPs), the Numb protein segregates into one of the two daughter cells, in which it inhibits Notch signalling to specify pIIb cell fate. Numb acts in SOP cells by inducing the endocytosis of Sanpodo, a four-pass transmembrane protein that has been shown to regulate Notch signalling in the central nervous system. In sanpodo mutants, SOP cells divide symmetrically into two pIIb cells. Sanpodo is cortical in pIIa, but colocalizes with Notch and Delta in Rab5- and Rab7-positive endocytic vesicles in pIIb. Sanpodo endocytosis requires alpha-Adaptin, a Numb-binding partner involved in clathrin-mediated endocytosis. In numb or alpha-adaptin mutants, Sanpodo is not endocytosed. Surprisingly, this defect is observed already before and during mitosis, which suggests that Numb not only acts in pIIb, but also regulates endocytosis throughout the cell cycle. Numb binds to Sanpodo by means of its phosphotyrosine-binding domain, a region that is essential for Numb function. These results establish numb- and alpha-adaptin-dependent endocytosis of Sanpodo as the mechanism by which Notch is regulated during external sensory organ development (Hutterer, 2005; full text of article).

This analysis shows that Sanpodo regulates Notch signalling during Drosophila ES organ development. In the pIIa cell, Sanpodo is localized at the plasma membrane and is required for Notch activation. In the pIIb cell, Sanpodo is removed from the plasma membrane by Numb- and alpha-Adaptin-dependent endocytosis. This correlates with the inability of this daughter cell to activate Notch signalling, suggesting that it is the plasma-membrane-localized Sanpodo protein that activates the Notch receptor. Previous epistasis experiments have suggested that Sanpodo acts during the intramembranous (S3) cleavage of the Notch receptor. Assuming that this cleavage occurs at the plasma membrane, it is possible that Notch needs to bind to Sanpodo to become a substrate for the protease Presenilin, which carries out the S3 cleavage (Hutterer, 2005).

Although this model is attractive, it does not explain why Sanpodo colocalizes with Notch in endocytic vesicles and why these vesicles are found in both pIIa and pIIb cells. Furthermore, it was found that ectopic expression of Sanpodo during neurogenesis (where Numb is expressed but not asymmetric) causes a neurogenic phenotype. Thus, Sanpodo can both activate and inhibit Notch signalling depending on the absence or presence of Numb. These observations are more consistent with an alternative model in which Sanpodo regulates the endocytosis of Notch. It was recently shown that ubiquitination and subsequent endocytosis can downregulate Notch. Conversely, endocytosis can also positively influence Notch signalling and was shown to be required for Notch activation in vertebrates. It is speculated that Sanpodo might have a general role in Notch endocytosis. In the absence of Numb, endocytosis could be required for Notch signalling, whereas in its presence, the inhibitory endocytic pathway could prevail. Although this model is speculative, it would also explain why expression of Numb in tissues that do not express Sanpodo has little or no influence on Notch signalling (Hutterer, 2005).

GENE STRUCTURE

There are two transcripts: maternal and zygotic. The promoters and first exon of the two transcripts differ (Uemura, 1989).

cDNA clone length - maternal - 3.1 kb; zygotic - 3.5kb.

Bases in 5' UTR - zygotic, 791

Exons - two for maternal, two for zygotic

Bases in 3' UTR - 1051 for each, maternal and zygotic

PROTEIN STRUCTURE

The maternal and zygotic proteins differ only in the first 41 amino acids, which are absent from the zygotic protein (Uemura, 1989).

Amino Acids - maternal 515, zygotic 557

Structural Domains

The zinc finger, common to maternal and zygotic forms, has a CHX4-CX12-CX4-C motif, one commonly found in zinc fingers. The amino terminal region (partially deleted in the maternal transcript) has many charged amino acids. Both maternal and zygotic forms have multiple PEST sequences, correlating with rapid protein turnover. An N-terminal domain consists of residues predictive of a phosphotyrosine binding domain (PTB domain) (Uemura, 1989 and Zhong, 1996).

Numb protein has an N-terminal phosphotyrosine binding domain. Asymmetric localization but not membrane localization of both Prospero and Numb in Drosophila embryos is inhibited by latrunculin A, an inhibitor of actin assembly. Deletion of either the first 41 aa or aa 41-118 of Numb eliminates both localization to the cell membrane and asymmetric localization during mitosis, whereas C-terminal deletions or deletions of central portions of Numb do not affect its subcellular localization. The N-terminus of Numb protein contains a consensus site for N-myrstoylation, but mutation of this site suggests that it is not required for association with the cell membrane or for asymmetric localization. Fusion of the first 76 or the first 119 aa of Numb to beta-galactosidase results in a fusion protein that localizes to the cell membrane, but fails to localize asymmetrically during mitosis. In contrast, a fusion protein containing the first 227 aa of Numb and beta-galactosidase localizes asymmetrically during mitosis and segregates into the same daughter cell as the endogenous Numb protein, demonstrating that the first 227 aa of the Numb protein are sufficient for asymmetric localization (Knoblich. 1997).

The Numb protein is involved in cell fate determination during Drosophila neural development. Numb has a protein domain homologous to the phosphotyrosine-binding domain (PTB) in the adaptor protein Shc. In Shc, this domain interacts with specific phosphotyrosine containing motifs on receptor tyrosine kinases and other signaling molecules. Residues N-terminal to the phosphotyrosine are also crucial for phosphopeptide binding to the Shc PTB domain. Several amino acid residues in Shc have been implicated by site-directed mutagenesis as being critical for Shc binding to receptor tyrosine kinases. Homologous mutations have been generated in Numb to test whether, in vivo, these changes affect Numb function during Drosophila sensory organ development. Two independent amino acid changes that interfere with Shc binding to phosphotyrosine residues do not affect Numb activity in vivo. In contrast, a mutation shown to abrogate the ability of the Shc PTB domain to bind residues upstream of the phosphotyrosine virtually eliminates Numb function. Similar results were observed in vitro by examining the binding of the Numb PTB domain to proteins from Schneider S2 cells. These data confirm the importance of the PTB domain for Numb function but strongly suggest that the Numb PTB domain is not involved in phosphotyrosine-dependent interactions. The identity of the PTB domain partner(s) of Numb is not yet known (Yaich, 1998).

date revised: 10 August 98  

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