The mammalian protein Eps15 is phosphorylated by EGF receptor tyrosine kinase and has been shown to interact with several components of the endocytic machinery. A hypomorphic Eps15 mutant was identified in Drosophila that shows reversible paralysis and an altered physiology at restrictive temperatures. In addition, the temperature-sensitive paralytic defect of shibire mutant is enhanced by this mutant. Eps15 is enriched in the larval neuromuscular junction in endocytic 'hot spots' in a pattern similar to Dynamin. Eps15 mutants show a decrease in the α-Adaptin levels at the larval neuromuscular junction synapse. Genetic and biochemical studies of interactions with components of the endocytic machinery suggest that Eps15 has an important role in synaptic vesicle recycling and regulates recruitment of α-Adaptin (Majumdar, 2006).
Different lines of evidence presented in this study suggest that Eps15 is crucial for synaptic vesicle recycling. Homozygous EP2513 animals show lethality and reversible temperature-sensitive paralysis. Overexpression of wild type Eps15 in this mutant using Nirvana Gal4 showed partial rescue of these phenotypes. Since Nirvana Gal4 shows strong nervous system specific expression, the Eps15 overexpression for rescue of the mutant phenotype is occurs exclusively in the nervous system. This implicates a major neuronal requirement for Eps15 in Drosophila. Comparative Western blot and quantitative immunostaining showed homozygous Eps15 mutant animals as hypomorph for Eps15 protein levels. At restrictive temperature, the Eps15 mutant shows impaired synaptic transmission across laminar neurons, as evidenced by loss of on and off transients in the electroretinogram. This is a characteristic feature of defective synaptic vesicle recycling and has been observed in temperature-sensitive alleles of shibire and comatose (Majumdar, 2006).
The shibire locus encodes the fly homologue of mammalian Dynamin, and it has been unequivocally implicated in endocytosis. Eps15 reduction in EP2513 mutants shows specific enhancement of the shibire temperature-sensitive paralysis and loss of the on and off transients at temperatures normally permissive for shibire mutant. Eps15 mutant also exhibits synthetic lethality with another shibire allele shits1. The C. elegans homologue of Eps15, EHS1, also shows enhancement of the phenotype of Dynamin mutant dyn1 (Majumdar, 2006).
A remarkable phenotype of the Eps15 mutant is its synthetic lethality with stoned. The stoned locus in Drosophila encodes two transcripts stnA and stnB. Stoned B has homology to μ-Adaptin and binds to Stoned A, and through its binding with Synaptotagmin, it is essential for recycling of vesicles. Previous observations suggest that stoned mutant alleles show mislocalization of Synaptotagmin and enhance the shibire phenotype of paralysis and synaptic depression. All these observations substantiate an important role of stoned in synaptic vesicle recycling. Studies on a mammalian Stoned B homologue called Stonin2 indicate that it interacts with several molecules of the endocytic pathway including Eps15 and is probably involved in the early steps of the process. In addition, like EP2513 mutants, stoned mutations are lethal in combination with the shits1 but not with other alleles of shibire. In contrast, Eps15 mutant does not show genetic interaction with comatose mutant (comttp7). comatose encodes the Drosophila NSF that has a role in vesicle exocytosis (Majumdar, 2006).
The genetic interaction data are supported by the presence of Dynamin, Stoned B, and α-Adaptin in Eps15 pulled down immunoprecipitate. An earlier study has shown that Stoned B pulls down Synaptotagmin and vice versa, under similar immunoprecipitation conditions as used in this work. The presence of Stoned B and absence of Synaptotagmin in Eps15 immunoprecipitate suggest that Stoned B binding to Eps15 is mutually exclusive from its binding with Synaptotagmin. The genetic and biochemical interaction between Stoned B, Dynamin, and Eps15 possibly signifies a role for each of these proteins in a common pathway of vesicle recycling (Majumdar, 2006).
If Eps15 is acting in endocytosis then it is expected to be localized in endocytic hot spots. Under stimulation at restrictive temperature in shibire mutants, Dynamin immunoreactivity is found in discrete regions on the presynaptic membrane. These have been suggested to be ‘hot spots’ of endocytic retrieval. In addition, Dynamin immunoreactivity is found to exclude active zone markers such as dPAK suggesting that Dynamin is not freely diffusible. Recently endocytically active zones have been found to be present in mammalian neurons. Many proteins have been found to be present in these endocytic regions. These include α-Adaptin, lap/AP180, Stoned, Dap160, and Endophilin. This work shows that Eps15 is enriched in the neuromuscular junction. Its subcellular localization is remarkably identical to that of Dynamin and complementary to that shown by dPAK which is a marker for active zones. The localization of Eps15 is also identical to dynamin in shits1 mutant synaptic terminals when stimulated at restrictive temperatures. This implies that Eps15 is present in restricted zones in the presynaptic membrane, and like Dynamin, it probably has a significant role in synaptic vesicle retrieval (Majumdar, 2006).
The localization of other endocytic proteins was also examined in the Eps15 mutant. A decrease in α-Adaptin level in Eps15 mutant was observed. In vitro studies have shown the C terminus of Eps15 binds to the ear domain of α-Adaptin. A reduction of 70% levels of Eps15 and around 42% levels of α-Adaptin in the Eps15 mutant in context of the immunoprecipitation data has led to the hypothesis that Eps15 is probably acting as an adaptor to the adaptor complex AP-2 by interacting with α-Adaptin and is important for localizing the adaptor complex to sites of endocytosis (Majumdar, 2006).
The most direct visualization of the role of Drosophila Eps15 in synaptic vesicle recycling came from electron microscopic analysis of the photoreceptor terminals in Drosophila eye and FM1-43 dye uptake in the neuromuscular junction boutons. In the Eps15 mutant, formation of tubular intermediate structures in the photoreceptor synapses was seen at non-permissive temperature. Such structures, seen in shibire mutants, are distinctive feature of defective synaptic vesicle recycling. A probable reason for the origin of these structures is that followed by normal exocytosis, due to a failure in endocytosis in synaptic vesicle recycling mutants, the membrane added by vesicle fusion at the active zones cannot be recovered, and this excess membrane can fold back to form tubular invaginations. This suggests that at the non-permissive temperature in the Eps15 mutant synaptic vesicle recycling is impaired. These data are supported with the earlier observation of absence of synaptic transmission in the Eps15 mutant eyes at non-permissive temperature. Also, at non-permissive temperature, the uptake of the fluorescent endocytic tracer dye FM1-43 was found to be blocked in stimulated Eps15 mutant synapses. This result confirms impaired synaptic vesicle recycling in Eps15 mutant (Majumdar, 2006).
Based on the data presented in this study, it is suggested that Drosophila Eps15 is a critical component of the endocytic 'Hot spots' and is essential for Dynamin-dependent synaptic vesicle recycling (Majumdar, 2006).
Numb has been shown to bind to the intracellular domain of Notch in vitro. Epistasis experiments place alpha-Adaptin between numb and Notch in the pathway for cell fate specification in the bristle lineage. To test whether Numb functions as a linker between alpha-Adaptin and Notch, a physical association between alpha-Adaptin and Numb was tested. In vitro-translated full-length alpha-Adaptin protein binds to a GST-Numb fusion protein. When the C-terminally-truncated Adaear4 protein or the mutant form Adaear26 is used in the binding assay, however, the interaction with Numb is strongly reduced, suggesting that the ear domain is crucial for binding. Conversely, binding of alpha-Adaptin is strongly reduced when the C-terminal amino acids 426–546 are deleted. Interestingly, the C-terminus of Numb contains a consensus alpha-Adaptin binding DPF motif and has been shown to be essential for Notch repression by Numb in cell culture (Berdnik, 2002).
To test whether Numb and alpha-Adaptin also interact in vivo, Numb was immunoprecipitated from embryo extracts using a C-terminal peptide antibody. alpha-Adaptin can be detected in the immunoprecipitate, but the protein is not found in a control experiment where the Numb antibody is preincubated with the peptide that had been used as an antigen. Thus, a fraction of alpha-Adaptin is bound to Numb in vivo, presumably due to a direct physical association between the two proteins that requires the alpha-Adaptin ear domain (Berdnik, 2002).
To test whether the ear domain is also required for Numb binding in vivo, a transgene was generated that expresses the C-terminally truncated, adaear4 mutant form of alpha-Adaptin. Overexpression of this form—like overexpression of full-length alpha-Adaptin—has no effect on asymmetric cell division. In embryos expressing Adaear4, both the truncated form and the endogenous, full-length version can be detected in immunoblots. When Numb is immunoprecipitated from these embryos, however, only the endogenous full-length form of alpha-Adaptin can be detected in the immunoprecipitate. Furthermore, Numb and alpha-Adaptin can be coimmunoprecipitated from wild-type, but not from adaear4/adaear5 transheterozygous mutant larvae. Thus, the in vivo interaction between Numb and alpha-Adaptin is disrupted in adaear mutants. The numb-like cell fate transformations observed in these mutants indicate that binding to alpha-Adaptin is crucial for Numb to carry out its function during asymmetric cell division (Berdnik, 2002).
Numb localizes asymmetrically and segregates into one of the two daughter cells during SOP cell division. To test whether alpha-Adaptin shows a similar subcellular localization, wild-type pupae were stained for Numb, alpha-Adaptin, and the SOP marker Asense. During interphase Numb is uniformly cortical, and the alpha-Adaptin protein is detected in dots that are distributed in the cytoplasm and accumulate at the cell cortex. During metaphase, however, Numb localizes asymmetrically, and the alpha-Adaptin protein is concentrated in the same area of the cell cortex as Numb; asymmetric localization of alpha-Adaptin was seen in 18 of 18 metaphase SOP cells). The asymmetric localization of both proteins is maintained during anaphase and telophase, and, after division, alpha-Adaptin is preferentially found in the pIIb cell that also inherits Numb. Unequal distribution of alpha-Adaptin is only found in a subset of two-cell pairs that show Numb in only one daughter cell, probably indicating a higher turnover rate of alpha-Adaptin protein (Berdnik, 2002).
alpha-Adaptin localization requires Numb. When large mitotic clones homozygous for the strong allele numb15 are induced by the eyeless-Flp/FRT system, no Numb protein can be detected in mutant SOP cells by antibody staining. alpha-Adaptin no longer localizes asymmetrically in these cells but instead is uniformly distributed around the cell cortex throughout mitosis. To test whether the alpha-Adaptin ear domain is required for asymmetric localization of the protein, the distribution of alpha-Adaptin was analyzed in adaear mutant clones. In adaear4 and adaear5 mutant cells, the cortical localization of alpha-Adaptin is very weak, and it is difficult to assess whether the residual cortical protein is asymmetrically localized. In adaear26 mutant SOP cells, in contrast, the protein is concentrated at the cortex but fails to localize asymmetrically during mitosis, even though Numb localization is unaffected. Thus, binding to Numb seems to be required for the asymmetric localization of alpha-Adaptin. Furthermore, the cell fate transformations observed in alpha-Adaptin mutants that disrupt Numb binding suggest that Numb functions by recruiting alpha-Adaptin to one side of the cell cortex during asymmetric cell division (Berdnik, 2002).
A class of retinal degeneration mutants have been identified in Drosophila in which the normally transient interaction between Arrestin2 (Arr2) and rhodopsin is stabilized and the complexes are rapidly internalized into the cell body by receptor-mediated endocytosis. The accumulation of protein complexes in the cytoplasm eventually results in photoreceptor cell death. The endocytic adapter protein AP-2 is essential for rhodopsin endocytosis through an Arr2-AP-2ß interaction, and mutations in Arr2 that disrupt its interaction with the ß subunit of AP-2 prevent endocytosis-induced retinal degeneration. If the interaction between Arr2 and AP-2 is blocked, this also results in retinal degeneration in an otherwise wild-type background. This indicates that the Arr2-AP-2 interaction is necessary for the pathology observed in a number of Drosophila visual system mutants, and suggests that regular rhodopsin turnover in wild-type photoreceptor cells by Arr2-mediated endocytosis is essential for photoreceptor cell maintenance (Orem, 2006).
The results demonstrate that Drosophila Arr2 plays a role as a mediator of rhodopsin endocytosis by interacting with the AP-2 adaptor complex. The AP-2 adaptor complex is required for the endocytosis of rhodopsin during certain pathological conditions and the disruption of this complex rescues norpA-mediated retinal degeneration. In addition, flies with a single point mutation in the AP-2 binding domain of Arr2, norpA-induced endocytosis of stable rhodopsin-arrestin complexes and the subsequent retinal pathology is blocked. Internalization of rhodopsin by an Arr2-AP-2 interaction is essential for photoreceptor cell viability (Orem, 2006).
Certain Arr2 variants bind tightly to rhodopsin. This results in the recruitment of the endocytic machinery and cell death via excessive rhodopsin endocytosis. In this study, an Arr2 variant (arr2R393A) was found that binds more tightly to rhodopsin than wild-type Arr2. However, this mutant does not trigger extensive retinal degeneration. This is further evidence for the essential role of the Arr2-AP-2 interaction in receptor internalization. In this Arr2 background complexes are formed between Arr2 and Rh1, but since Arr2 cannot interact with AP-2 they are not internalized and no photoreceptor cell death is induced (Orem, 2006).
These data point to an essential role for the endocytosis of rhodopsin through Arr2 in the maintenance of photoreceptor cells. Previous work has implicated an essential role for Drosophila arrestins in endocytosis; however, these studies used mutations that either blocked all endocytosis in the photoreceptor cell or utilized loss-of-function mutants that could have other effects on the photoreceptor. Photoreceptor cell degeneration can be induced by inhibiting dynamin function with a dominant negative mutation or by blocking AP-2 function, as described in this study. However, it is possible that a global inhibition of endocytosis may halt the internalization of compounds essential for cell viability. Therefore, cell death may be unrelated to defects in the phototransduction cascade or the internalization of rhodopsin. In addition, retinal degeneration in Arr1 mutants may be due to the defect in endocytosis but pleiotropic affects associated with the loss of Arr1 may also contribute to the aberrant retinal morphology. By using Arr2 variants that are unable to internalize rhodopsin, the endocytosis of one protein (rhodopsin) was selectively blocked while leaving general endocytosis intact. Therefore, the retinal degeneration observed in this study is due solely to defects in rhodopsin internalization through its interaction with Arr2 (Orem, 2006).
One interesting question concerns the purpose of the essential role for rhodopsin-Arr2 endocytosis. One possibility is that this may be a mechanism to remove damaged rhodopsin from the cell. If rhodopsin is photochemically damaged in such a way that it becomes constitutively active, it would be deleterious to the cell, and would need to be removed. Presumably constitutively active rhodopsin would form a stable complex with Arr2 and be targeted for endocytosis through the interaction of arrestin with the AP-2 adaptor complex. This would provide a surveillance mechanism for the cell, whereby defective rhodopsin molecules are quickly and efficiently removed. Second, it may be an adaptive mechanism. In high light conditions the amount of activated rhodopsin may exceed the ability of arrestin to quickly decouple the metarhodopsin from the phototransduction pathway. This would lead to a loss of visual temporal resolution and be detrimental to cell viability. However, under high light conditions at any given time a higher percentage of the cellular arrestin will be bound to rhodopsin and increase the likelihood that the Arr2 bound to rhodopsin will interact with AP-2 and drive internalization of rhodopsin. This would serve to lower the concentration of rhodopsin available to activate the phototransduction cascade and thereby reduce sensitivity under conditions of intense illumination (Orem, 2006).
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).
In Drosophila melanogaster, external sensory organs develop from a single sensory organ precursor (SOP). The SOP divides asymmetrically to generate daughter cells, whose fates are governed by differential Notch activation. This study shows that the clathrin adaptor AP-1 complex, localized at the trans Golgi network and in recycling endosomes, acts as a negative regulator of Notch signaling. Inactivation of AP-1 causes ligand-dependent activation of Notch, leading to a fate transformation within sensory organs. Loss of AP-1 affects neither cell polarity nor the unequal segregation of the cell fate determinants Numb and Neuralized. Instead, it causes apical accumulation of the Notch activator Sanpodo and stabilization of both Sanpodo and Notch at the interface between SOP daughter cells, where DE-cadherin is localized. Endocytosis-recycling assays reveal that AP-1 acts in recycling endosomes to prevent internalized Spdo from recycling toward adherens junctions. Because AP-1 does not prevent endocytosis and recycling of the Notch ligand Delta, these data indicate that the DE-cadherin junctional domain may act as a launching pad through which endocytosed Notch ligand is trafficked for signaling (Benhra, 2011).
The dorsal thorax of Drosophila pupae, the notum, is a single-layered neuroepithelium that produces epidermal and sensory organ (SO) cells. Each adult SO is composed of four cell types and is derived from a single cell, the sensory organ precursor (SOP, also called the pI cell). Notch regulates binary cell fate decisions in the SO lineage. Each SOP undergoes asymmetric cell division to generate two distinct daughter cells; Notch is activated in the SOP daughter cell that adopts the pIIa fate and is inhibited in the other cell, which becomes a pIIb cell. The pIIa cell divides to generate the external cells of the SO, the shaft and socket cells. The pIIb cell undergoes two rounds of asymmetric cell division to generate the internal cells of the SO, the neuron, the sheath cell, and a glial cell. Although Notch-mediated binary cell fate decision in the SO lineage is tightly controlled by intracellular trafficking, the exact subcellular location of where Notch ligand and receptor interact to produce a signal is subject to debate (Benhra, 2011).
To identify new regulators of Notch signaling involved in intracellular trafficking, a double-stranded RNA (dsRNA) screen was carried out for genes affecting SO development and the clathrin adaptor AP-1 complex was identified. AP-1 is an evolutionarily conserved heterotetrameric complex. Drosophila AP-1 complex is composed of AP-1γ (CG9113), β-adaptin (CG12532), AP-1μ1 (encoded by AP-47, CG9388), and AP-1σ (CG5864) subunits. Although mammalian AP-1 is involved in lysosome-related organelle (LRO) biogenesis and in polarized sorting of membrane proteins to the basolateral plasma membrane, the function of Drosophila AP-1 remains largely unknown. Each wild-type SO contains only one socket cell. In contrast, tissue-specific gene silencing of any of the three AP-1 specific subunits, AP-47, AP-1γ, or AP-1σ, gives rise to a Notch gain-of-function phenotype that results in a pIIb-to-pIIa cell fate and/or a shaft-to-socket cell transformation, leading to an excess of socket cells. Following knockdown of AP-1 subunits, 4% to 17% of SO show more than one socket cell. To confirm and extend these dsRNA-induced results, classical mutants were analyzed. Two mutations in AP-47, AP-47SHE11, and AP-47SAE10 were previously recovered as genetic modifiers of presenilin hypomorphic mutations. This stud characterized the AP-47SHE11 allele as a genetic null, whereas the second allele, AP-47SAE10, is hypomorphic. AP-47SHE11/Df(3R)Excel 6264 transheterozygotes die at early first-instar larvae stage, indicating that, as in worms, zebrafish, and mice, AP-47 is essential for viability. To assess the AP-47 loss-of-function phenotype in SO, AP-47 mutant mitotic clones were generated and analyzed in the notum. The same two categories of transformed mutant organs were observed as in the dsRNA experiments. Cell fate transformation was seen in 11% of the mutant organs and in 17% following AP-47dsRNA. The difference could be due to protein perdurance in the mutant clones induced during development. The incomplete penetrance suggests that a compensatory mechanism could bypass the requirement for AP-1. In any case, the results suggest a requirement for the AP-1 complex in Notch-dependent binary cell fate acquisition (Benhra, 2011).
Excess Notch signaling can arise from either disruption of epithelial cell polarity or defects in partitioning of cell fate determinants at mitosis. Because cell polarity relies on the proper apicobasal sorting of membrane proteins, a process requiring both clathrin activity in mammals, this study has analyzed the localization of various polarity markers in AP-47− mutant clones. The Notch gain-of-function phenotype observed in the absence of AP-1 activity cannot be explained by a disruption of epithelial cell polarity, nor by a defect in the partitioning of the cell fate determinants Numb and Neuralized (Neur) at mitosis. Thus, AP-1 activity may be required after unequal segregation of cell fate determinants, possibly at the pIIa/pIIb cell stage to control Notch signaling (Benhra, 2011).
Defects in the endolysosomal degradation, such as in vps25 and erupted mutant cells, result in a Notch gain-of-function phenotype that is caused by ligand-independent mechanisms. Because AP-1 is involved in the biogenesis of LROs in mammals, genetic interaction tests were devised to determine whether excess signaling caused by loss of AP-47 requires the activity of the Notch ligands Delta and Serrate (Ser). Loss of Delta and Ser signaling causes Notch loss-of-function phenotypes, a lateral inhibition defect and a pIIa-to-pIIb cell fate transformation that results in generation of extra neurons, the opposite phenotype to what was observed in AP-47− mutant clones. Loss of external sensory cells accompanied by an excess of neurons is observed in AP-47− Delta− Ser− triple mutant clones, a phenotype indistinguishable from that of Delta− Ser− double mutant clones. The reversal of pIIb-to-pIIa transformation phenotype of AP-47− in AP-47− Delta− Ser− triple mutant clones demonstrates that Delta and Ser are epistatic to AP-47. This finding indicates that the AP-47− mutant phenotype is ligand dependent (Benhra, 2011).
The activity of Delta in the SO lineage is controlled by Neur-dependent endocytosis. Following endocytosis, Delta is recycled, and its trafficking toward apical microvilli requires Arp2/3 and WASp. Mutations in WASp prevent Notch signaling, resulting in a pIIa-to-pIIb cell fate transformation. Excess Notch signaling is observed in AP-47− WASp− clones, as in AP-47− clones. These data demonstrate that AP-47 is required for SO formation even in the absence of WASp. These findings suggest that AP-1 is unlikely to act by preventing Delta recycling and raise the possibility that AP-1 acts on Notch receptor signaling (Benhra, 2011).
Sanpodo (Spdo) is a four-pass transmembrane protein required for Notch signaling in asymmetrically dividing cells. Because mutations in spdo result in reduced Notch signaling, the opposite phenotype to what was observed in AP-47− mutant clones, it could be that AP-1 normally represses Spdo activity. To test this hypothesis, AP-47− spdo− double mutant clones were generated and a phenotype was observed that is indistinguishable from that of spdo− mutant clones. The reversal of the pIIb-to-pIIa transformation phenotype of AP-47− in AP-47− spdo− double mutant clones indicates that AP-1 requires the activity of Spdo to control Notch signaling and suggests that AP-1 might control Spdo trafficking and/or localization (Benhra, 2011).
To test for a role of AP-1 in Spdo localization, the subcellular distribution of Spdo was compared in wild-type and AP-47− SO lineages. In the wild-type SOP, Spdo is found in intracellular compartments. After division, Spdo-positive vesicles remain localized in the pIIb cell as a consequence of the unequal inheritance of Numb during SOP mitosis, whereas Spdo localizes preferentially at the plasma membrane of the posterior pIIa cell. Spdo is also detected at the apical cortex of SOP and pIIa/pIIb cells, albeit at a low level. In contrast, in AP-47− mutant SO cells, Spdo accumulates apically, as well as at the interface between the AP-47− SOP daughter cells, where DE-Cad is present. It is concluded that loss of AP-1 results in the specific accumulation of Spdo at the apical plasma membrane in SO cells, as well as at the level of adherens junction in SOP daughters. It is suggested that this defect in Spdo trafficking could explain the excess Notch signaling (Benhra, 2011).
Because AP-1 is required for proper localization of Spdo, an anti-AP-1γ antibody was generated to investigate the subcellular distribution of AP-1 relative to Spdo. AP-1γ is closely juxtaposed to the trans Golgi network (TGN) marker GalT::RFP and colocalizes partially with Liquid facet related (LqfR; CG42250), the Drosophila ortholog of Epsin related (Epsin-R), recently reported to localize at the TGN. AP-1γ also partially colocalizes with Rab11-positive recycling endosomes (RE). Thus, in epithelial cells of the notum, AP-1 is found on two membrane-bound compartments, the TGN and RE, as previously reported in tissue culture cells. In SOPs, Spdo was previously shown to partially colocalize with Notch, Hrs, and Rab5. This study reports that Spdo also colocalizes with AP-1γ and Rab11-positive endosomes, suggesting that Spdo traffics within the TGN and RE (Benhra, 2011).
Together with the above genetic data, colocalization of AP-1 with Spdo raises the interesting possibility that AP-1 could control the sorting and transport of Spdo. Furthermore, Spdo contains a conserved N-terminal YTNPAF motif that falls into the Y/FxNPxY/F-consensus sorting signal of the LDL receptor whose localization is regulated by clathrin adaptors. If Spdo is an AP-1 cargo, deletion of the sorting motif of Spdo should prevent its interaction with AP-1. To test this prediction, the localization of AP-47-VenusFP (VFP) was analyzed relative to that of Spdo-mChFP versus Spdo-mChFP deleted of its 18 first amino acids containing the YTNPAF motif (SpdoΔ18-mChFP) in the SOP lineage. On average at the two-cell stage, 69% of the AP-47-VFP-positive vesicles are also positive for Spdo-mChFP, whereas only 14% of AP-47-VFP vesicles are positive for SpdoΔ18-mChFP. Thus, the first 18 amino acids of Spdo may be required for its AP-1-mediated sorting. Nonetheless, SpdoΔ18-mChFP does not accumulate at the apical cortex, suggesting that additional sorting motifs or interacting proteins such as Numb, also interacting with Spdo via the YTNPAF motif, contribute to Spdo apical localization. These data reveal that in addition to AP-2, a second clathrin adaptor complex, AP-1, controls the localization of Spdo and regulates Notch signaling. AP-2 and Numb prevent Spdo accumulation at the plasma membrane, whereas AP-1 prevents Spdo accumulation at the apical plasma membrane. Whether AP-1 binds directly to the YTNPAF motif or indirectly via a yet-to-be-discovered clathrin-associated sorting protein (CLASP) like Numb remains unknown. By analogy to Numb and AP-2, the hypothetical CLASP would function together with AP-1 to sort Spdo at the TGN and/or RE (Benhra, 2011).
Based on its localization at the TGN and the RE, AP-1 may ensure sorting of Spdo from the TGN and/or RE. To test whether AP-1 has a role at RE, a functional Spdo construct was generated in which mChFP is inserted in the second extracellular loop of Spdo (SpdoL2::mChFP) and used in a pulse-chase internalization assay with an anti-RFP that recognizes the extracellularly accessible mChFP tag in epithelial cells of the notum. In the control, following a 45 min chase, the anti-RFP has been efficiently internalized and resides primarily in apically localized endosomes. A small pool of anti-RFP is also detected at the level of adherens junctions labeled with DE-cadherin, suggesting that Spdo can be recycled back to adherens junctions, albeit with low efficiency. In cells depleted of AP-1, anti-RFP internalized from the basolateral membrane is efficiently recycled to the adherens junctions, suggesting that AP-1 acts in RE to limit recycling of Spdo toward adherens junctions. In contrast, when AP-2-dependent endocytosis is prevented, anti-RFP remains mostly localized at the basolateral plasma membrane, even after a chase of 45 min, as predicted for a requirement of AP-2 in the internalization of Spdo. Therefore, the data indicate that AP-1 does not regulate endocytosis of Spdo from the basolateral membrane. To test whether AP-1 could regulate apical endocytosis of SpdoL2::mChFP, a pulse-chase internalization assay was conducted in epithelial cells of the wing imaginal discs, a tissue that, in contrast to the pupal notum, allows for access of anti-RFP at the apical plasma membrane. In cells depleted of AP-47, anti-RFP resides predominantly at the apical side at the level of adherens junction at t = 0 and is internalized with similar kinetics as in the control situation. It is concluded that AP-1 does not regulate SpdoL2::mChFP apical internalization. Altogether, these results indicate that AP-1 acts at the RE to prevent or limit apical recycling of Spdo, giving a rationale for why endogenous Spdo accumulates apically in SO mutant for AP-47 (Benhra, 2011).
Does apical accumulation of Spdo cause the Notch gain-of-function phenotype seen in AP-1 mutant SO? Spdo was previously reported to partially colocalize with Notch in large intracellular structures and at the plasma membrane. In wild-type, Notch localizes at the apical membrane of epidermal cells, SOP cells, and SOP daughter cells. Shortly after SOP division, Notch extracellular domain (NECD) is detected apically together with Spdo at the DE-Cad interface between pIIa and pIIb. This specific localization is transient, because NECD and Spdo are detected at the interface of daughter cells in one-third of the cases and are no longer detectable at the pIIa/pIIb interface when the remodeling of the apical cortex of pIIa/pIIb cells takes place. In AP-47− mutant cells, NECD is stabilized with Spdo at the interface of SOP daughter cells, even at a time when control organs have undergone apical cortex remodeling. Similarly, Notch intracellular domain (NICD) is accumulated at the level of adherens junctions in AP-47− mutant cells, whereas it is detected at the interface of wild-type SOP daughters in only half of the cases. To determine whether the stabilization of Notch at the SOP daughter cell interface is caused specifically by AP-47 loss of function, NECD localization was compared in AP-47− versus spdo− or AP-47− spdo− double mutant clones. Although NECD is enriched at the apical surface in these three mutant situations compared to control cells, stabilization of NECD at the interface of SOP daughter cells occurs in AP-47− single and AP-47− spdo− double clones, but not in spdo− single clones. These data indicate that, upon loss of AP-47, Spdo is not required for NECD to accumulate at the junction between SOP daughter cells, which raises the interesting possibility that Notch itself may be an AP-1 cargo. Because Spdo and Notch are transiently detected at the interface of wild-type SOP daughter cells, it is proposed that sustained elevated levels of Spdo and Notch at the interface cause the excess signaling observed in AP-47− mutants. These effects of AP-1 appear to be specific to Spdo and Notch, because Delta is transiently detected in punctuated structures at the level of junctions together with Spdo in a similar manner in both control and AP-47− SOP daughter cells. Furthermore, endocytosis of Delta is unaffected by the loss of AP-1. It is thus concluded that AP-1 regulates the amount of Notch and Spdo at this junctional domain, which could serve as a launching pad from which endocytosed Notch ligand is trafficked for signaling (Benhra, 2011).
These data have uncover a novel function for AP-1 complex during development. The observations suggest that AP-1 participates in the polarized sorting of Spdo and Notch from the TGN and/or RE toward the plasma membrane. The correlation between the Notch gain-of-function phenotype and the stabilization of Notch and Spdo at the junctions suggests that adherens junctions may be particularly important for Notch activation. Because the effect of loss of AP-1 on Spdo and Notch localization is completely penetrant, it is proposed that a threshold of Spdo and Notch localized at the junctional domain has to be reached in order to cause the cell fate transformation, explaining why only 10% to 20% exhibit the Notch gain-of-function phenotype (Benhra, 2011).
Previous reports have suggested that trafficking of endocytosed Delta to the apical membrane in the pIIb cell is required for its ability to activate Notch that localizes at the apical side in the pIIa cell. Recently, it was reported that most endocytosed vesicles containing the ligand Delta traffic to a prominent apical actin-rich structure (ARS) formed in the SOP daughter cells. Based on phalloidin staining, the ARS appears to be unaffected by the loss of AP-47. Notch and Spdo are stabilized at the junctional domain that is included within the ARS and are therefore poised to receive the Delta signal. This would place this domain of the ARS as an essential site for Delta-Notch interaction, leading to productive ligand-dependent Notch signaling (Benhra, 2011).
Could this novel function for AP-1 be conserved in mammals? Spdo is specifically expressed in Dipterans, and no functional ortholog has been described so far, raising the question of the role of AP-1 in Notch signaling in mammals. Nonetheless, Notch is also mislocalized in AP-1 mutant cells even when Spdo activity is missing. Notch also contains evolutionarily conserved tyrosine-based sorting signals, and it cannot be excluded at present that Notch is itself an AP-1 cargo. Finally, the facts that Notch controls several early steps of T cell development and that mice heterozygous for γ-adaptin exhibit impaired T cell development raise the interesting possibility that Notch-dependent decisions in mammals also required AP-1 function (Benhra, 2011).
In situ hybridization of alphaAdaptin cDNA to preparations of staged whole-mounted embryos indicates that the detection of alphaAdaptin expression is restricted to the CNS and garland cells. In the larvae, alphaAdaptin expression is also found in the developing imaginal discs. High resolution confocal microscopy was performed using antibodies directed against Drosophila alpha-Adaptin. alpha-Adaptin is localized to the plasma membrane of the presynapses; no antibody staining of axons or the soma of neurons was observed. In summary, the results obtained with both in situ hybridization and antibody detection consistently argue that alphaAdaptin expression is highly enriched if not restricted to few locations in developing embryos and larvae (González-Gaitán, 1997).
The P element insertion of the l(2)06694 enhancer trap line interrupts the first exon of the alphaAdaptin gene and thereby causes the mutant alphaAdaptin1 allele. Embryos homozygous for this allele develop into slowly moving larvae that die as pupae. Excision of the P element resulted in normal adults, indicating that the insertion had indeed caused the lethal phenotype. In addition, two imprecise excisions of the P element were obtained. The first imprecise excision caused an internal deletion of P element sequences that weakens the phenotype. Embryos homozygous for the corresponding alphaAdaptin2 allele develop into normal-looking adults that are unable to fly or walk (González-Gaitán, 1997).
The second imprecise excision, giving rise to the alphaAdaptin3 allele, is a deletion encompassing nontranscribed upstream sequences, the two first introns, and the N-terminal region of the protein encoded by both alphaAdaptin transcripts. Homozygous alphaAdaptin 3 embryos develop the most severe phenotype, since they die before hatching. Furthermore, embryos that contain a deletion of the alphaAdaptin gene (Df(2L)al) in trans to the alphaAdaptin 3-bearing chromosome show all aspects of the homozygous alphaAdaptin 3 mutant phenotype, and both Df(2L)al/alphaAdaptin1 and alphaAdaptin 3/alphaAdaptin1 die during the same early larval stage. These observations and the molecular lesion indicate that the alphaAdaptin 3 mutation is a lack-of-function allele of the locus (González-Gaitán, 1997).
alpha-Adaptin-deficient embryos develop into normal-looking first instar larvae. As shown by the normal expression patterns of the neural markers fasciclin II and repo, the architecture of the nervous system of these embryos, including the neuromuscular innervation pattern, is not affected by the lack of alpha-Adaptin. Muscle contractions, however, occur only sporadically, and the larvae fail to hatch from the egg shell. The results therefore suggest that alphaAdaptin is not involved in morphological aspects of neurogenesis but concerns physiological aspects of neuron function, such as the proposed role for alpha-Adaptin in synaptic transmission at the nerve terminals. In order to examine whether and at which step mutant alpha-Adaptin interferes with synaptic vesicle recycling, the FM1-43 assay was applied to neuromuscular junctions of larvae and the synaptic ultrastructure of embryos was examined by electron microscopy (González-Gaitán, 1997).
The FM1-43 assay is based on the ability of the fluorescent FM1-43 dye to intercalate into presynaptic membranes of larval neuromuscular junctions. After a labeling pulse, the fluorescent dye can be washed off, provided that the FM1-43-loaded presynaptic membranes were not already internalized by endocytosis. Thus, this assay can be taken to monitor whether endocytotic events in the presynaptic boutons of alphaAdaptin mutant occur at normal rates as compared to wild-type motoneurons. At the presynaptic boutons of wild-type motoneurons, the fluorescent dye was retained after short FM1-43 labeling pulses (5 s) and subsequent washes. This indicates that FM1-43 was internalized normally by endocytosis. No corresponding FM1-43 staining was found in the presynaptic boutons of alphaAdaptin 1 mutant neuromuscular junctions, indicating that the dye could not be internalized after the short labeling pulse. Expanded loading pulses (60 s), however, resulted in extensively stained boutons in both wild-type and mutant neuromuscular junctions. This observation indicates that endocytosis in the weak alphaAdaptin 1 mutants is impaired, but not blocked (González-Gaitán, 1997).
Owing to the small size of the embryonic neuromuscular junctions, the FM1-43 assay is not applicable to embryos. Corresponding effects in homozygous alphaAdaptin 3 mutant embryos could not be examined. Instead the synaptic ultrastructure in the late CNS neuropile of wild-type and alpha-Adaptin-deficient embryos was examined. The mutant synapses were found to be depleted of vesicles and to lack vesicular structures, such as coated or collared pits at the plasma membrane. Instead, the membrane surfaces contained deep folds not seen in wild type. These findings indicate that vesicle recycling is blocked at the initial stage of endocytosis, i.e., the formation of clathrin-coated pits. This results in a fusion of the vesicle compartment with the plasma membrane, which causes an expanded surface of the plasma membrane (González-Gaitán, 1997).
It was asked whether alpha-Adaptin localizes to the sites of endocytosis in the presynaptic terminals of motoneurons. For this, the subcellular localization of alpha-Adaptin was monitored by high resolution confocal microscopy using the distribution of known vesicle components, such as dynamin, Synaptotagmin , and CSP, as reference for the alpha-Adaptin patterns in wild-type and shi mutant synapses. The shi mutant affects the GTPase function of dynamin. Thus, in contrast to the alpha-Adaptin mutant, this allows endocytosis to be initiated normally, but vesicle fission does not occur, i.e., the formation of clathrin-coated vesicles is blocked (González-Gaitán, 1997).
In wild type, synaptotagmin and CSP are distributed between the pools of vesicles in the cytoplasm and at the plasmalemma. The homogeneous distribution of CSP at the plasma membrane was confirmed. In contrast, alpha-Adaptin forms a dense network-like structure at the plasma membrane with alpha-Adaptin-free islands in between. It was also noted that although dynamin is distributed throughout the surface of the presynaptic membrane, its distribution is not homogeneous but shows areas of enrichment in a network-like pattern corresponding to the distribution of alpha-Adaptin. Double-staining experiments involving antibodies directed against Drosophila alpha-Adaptin and dynamin indicate that dynamin is in fact distributed throughout the plasmalemma but exerts an underlying pattern of higher intensities corresponding to the alpha-Adaptin pattern (González-Gaitán, 1997).
In the shi mutant synapses, the network-like pattern of alpha-Adaptin is not altered as compared to wild type. Synaptotagmin and CSP are associated with the plasmalemma, where they remain homogeneously distributed. Dynamin is also associated with the plasmalemma, but its distribution differs from synaptotagmin and CSP. Dynamin is depleted from the alpha-Adaptin-free zones and, instead, becomes restricted to a pattern identical to alpha-Adaptin. In summary, these results establish that alpha-Adaptin is confined to centers at the presynaptic membrane, where endocytosis occurs. In addition, the data suggest that alpha-Adaptin functions upstream of dynamin. This proposal is based on two observations: (i) alpha-Adaptin mutations block endocytosis at an earlier stage than dynamin mutations do, and (ii) the distribution of alpha-Adaptin remains unchanged in shi mutant synapses. Furthermore, the restriction of dynamin to the pattern of alpha-Adaptin suggests that alpha-Adaptin is necessary for the recruitment of dynamin. The results are therefore consistent with the argument (Wang, 1995) that the in vitro association of dynamin and alpha-Adaptin is indeed functional (González-Gaitán, 1997).
In order to elucidate the function of the in vitro association of dynamin and alpha-Adaptin, the possible interaction between the two proteins was explored by genetic means, generating alphaAdaptin 3 and shi double-mutants. shits2 is a temperature-sensitive allele of the X-chromosomal shi locus. At 25°C, a permissive temperature, hemizygous shits2 males develop and behave normally. At the restrictive temperature (29°C), however, such males become paralyzed, and uncoordinated movements can be occasionally observed. shits2;alphaAdaptin3/+ males are indistinguishable from wild type when kept at 18°C. At 25°C, however, a temperature permissive for both hemizygous shits2 males and heterozygous alphaAdaptin 3 males, shits2;alphaAdaptin/+ males can neither fly nor walk, and they show sporadic and uncoordinated movements. This observation demonstrates a synergistic effect between alpha-Adaptin and dynamin mutants, indicating that the two wild-type gene activities interact in vivo. This result and the coinciding patterns of alpha-Adaptin and dynamin in shi mutants suggest that alpha-Adaptin-dependent formation of clathrin-coated pits and the dynamin-dependent internalization of vesicles are linked through the dual function of alpha-Adaptin in endocytosis (González-Gaitán, 1997).
To identify genes required for asymmetric segregation of Numb and for its function in cell fate determination, a large-scale genetic screen was carried out for mutations affecting bristle morphology. Random mutations were induced by EMS treatment and analyzed in large homozygous mutant clones of otherwise heterozygous animals. The eyeless-Flp/FRT/cell-lethal system, that induces mitotic recombination in all tissues expressing Flp recombinase from a particular eyeless promoter fragment, was used. Even though this system was designed to analyze eye development, it can be used to study bristle development, since mutant clones include the whole eye imaginal disc, which gives rise to the head capsule and most of the larger macrochaete and smaller microchaete bristles on the head (Berdnik, 2002).
Among the mutations identified were nine new alleles of numb, the strongest of which, numb15, causes an almost complete cell fate transformation of all head bristle cells into four sockets. Another complementation group consists of the two mutations adaear4 and adaear5, that also cause the generation of extra sockets and the loss of shafts, and adaear26, a third allele, which shows these transformations at a lower frequency and also has missing bristles. To test whether these morphological changes are due to cell fate transformations in the bristle lineage, pupae carrying adaear4 and adaear5 mutant head clones were stained for cell-type-specific markers of the bristle lineage. Wild-type external sensory organs consist of two small inner cells, which express Prospero (sheath cell) or Elav (neuron), and two larger outer cells, which express Su(H) (socket cell) or none of the markers analyzed (hair cell). In adaear4 mutant clones, 92% of the macrochaetae on the head show cell fate transformations of inner into outer cells (either pIIb into pIIa or hair to socket). In 66% of the macrochaetae, all cells are transformed into four sockets. Similar defects, even though at a lower frequency, are observed in microchaetae (44%, outer cell fate transformations; 4%, four sockets). It is concluded that adaear4 mutations cause cell fate transformations that are similar to, although somewhat weaker than, those observed in numb mutants (Berdnik, 2002).
adaear mutations were mapped to the tip of chromosome arm 2L by recombination with marked P elements of known cytological position. Based on noncomplementation of the deficiency Df(2L)al, the cytological location was further narrowed down to 21C1–C7. This deficiency deletes the Drosophila homolog of alpha-Adaptin, and, indeed, all three alleles of this complementation group fail to complement a previously identified mutation in alpha-Adaptin (ada3) (Gonzalez-Gaitan, 1997). It is concluded that the adaear mutations are alleles of Drosophila alpha-adaptin (Berdnik, 2002).
ada3, an amorphic mutation in alpha-Adaptin, is late embryonic or early larval lethal and causes defects in synaptic vesicle recycling (Gonzalez-Gaitan, 1997). Animals carrying the weaker allele ada1 develop into larvae that move slowly, due to defects in synaptic vesicle recycling, and die as pupae. No obvious cell fate transformations in the bristle lineage were detected in ada3 or ada1 mutant clones. ada3 mosaic animals die as pupae with severe cuticle defects, presumably due to a requirement of alpha-Adaptin for general endocytosis in all cells. Flies carrying ada1 mutant clones, in contrast, are viable and show only very mild loss of bristles on the head. Furthermore, adaear/ada1 transheterozygous flies are viable and have no obvious phenotype, suggesting that the adaear mutant protein can still function in the endocytosis of synaptic vesicles. Taken together, these results suggest that adaear mutations preferentially affect functions of alpha-Adaptin that are required during bristle development (Berdnik, 2002).
To determine which part of the alpha-Adaptin protein is affected in these alleles, the exact nucleotide exchanges in adaear mutations were analyzed. adaear4 and adaear5 are C to T transitions that change an arginine at position 841 and a glutamine at position 850 into stop codons and are therefore predicted to result in C-terminal truncations of the protein. adaear26 is a six-nucleotide deletion that removes valine 824 and asparagine 825 but leaves the rest of the protein intact. Thus, all of the new alpha-Adaptin alleles affect the alpha-Adaptin ear domain, which extends from amino acids (aa) 713 to 940 of the protein. Based on the available crystal structure for this domain, it is predicted that the identified mutations delete or modify a C-terminal subdomain that makes all of the important protein interactions (Berdnik, 2002).
alpha-Adaptin could act at several levels during cell fate specification in the bristle lineage. It could be required for Numb localization, for Notch repression by Numb, or for signal transduction downstream of Notch. To distinguish between these possibilities, the asymmetric localization of Numb was examined in adaear4 mutants and epistasis experiments were performed between alpha-Adaptin, numb, and Notch (Berdnik, 2002).
Numb localization was analyzed by staining SOP cells of the developing eye in adaear4 mutant clones for DNA and Numb. Like in wild-type SOP cells, Numb localizes into a cortical crescent overlying one of the two spindle poles from prophase to anaphase and segregates into one of the two daughter cells upon cytokinesis. Similar results were obtained for the other adaear alleles. Thus, alpha-Adaptin is not required for the asymmetric localization of Numb (Berdnik, 2002).
To test the epistatic relationship between numb and alpha-Adaptin, numb was overexpressed in alpha-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 alpha-Adaptin, numb overexpression in alpha-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 adaear4 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 alpha-Adaptin protein. Overexpression of numb in SOP cells, on the other hand, causes a 70% reduction of postorbital bristles. When numb is overexpressed in adaear4 mutant head clones, the number of bristles bearing outer cell fate transformations is unchanged. These data show that the adaear4 mutant phenotype cannot be reverted upon numb overexpression and indicate that alpha-Adaptin acts genetically downstream of numb (Berdnik, 2002).
To test whether alpha-Adaptin acts upstream or downstream of Notch, the Notch, alpha-Adaptin double mutant phenotype was examined. Inactivation of Notch during SOP division causes transformations of hair and socket cells into inner cells and leads to an apparent loss of bristles. Notch, alpha-Adaptin double mutants should have the Notch phenotype if alpha-Adaptin is upstream, but the alpha-Adaptin phenotype if it is downstream, of Notch. A temperature-sensitive allele of Notch (Notchts) was used that has no bristle phenotype at 18°C but causes essentially a complete loss of postorbital bristles when shifted to 29°C during the time of asymmetric cell divisions in the bristle lineage. When adaear4 mutant clones are generated in a Notchts background, outer cell fate transformations are observed at the permissive temperature, but not at the restrictive temperature. Thus, Notchts, adaear4 double mutant SOP cells have the Notch mutant phenotype, indicating that alpha-Adaptin acts upstream of, or in parallel to, Notch (Berdnik, 2002).
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date revised: 15 April 2011
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