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).
Protein trafficking is highly regulated in polarized cells. During development, how the trafficking of cell junctional proteins is regulated for cell specialization is largely unknown. In the maturation of Drosophila larval salivary glands (SGs), the Dlg protein is essential for septate junction formation. It was shown that Dlg is enriched in the apical membrane domain of proximal cells and localized basolaterally in distal mature cells. The transition of Dlg distribution is disrupted in Numb-associated kinase (nak) mutants. Nak associates with the AP-2 subunit alpha-Adaptin and the AP-1 subunit AP-1gamma. In SG cells disrupting AP-1 and AP-2 activities, Dlg is enriched in the apical membrane. Therefore, Nak regulates the transition of Dlg distribution likely through endocytosis of Dlg from the apical membrane domain and transcytosis of Dlg to the basolateral membrane domain during the maturation of SGs development (Peng, 2009).
This study describes the re-localization of the Dlg protein during Drosophila SG specialization. From this analyses, Dlg underwent a transition from the apical to the basal membrane domain. In addition, Nak and components of AP-1 or AP-2 complexes were found to be required for the Dlg transition. Two possible models could explain the transition of Dlg re-localization in SG cells. First, in the proximal SG cells, newly synthesized Dlg is transported to and maintained in the apical membrane. In the distal cells, however, the newly synthesized Dlg is directed toward the basal membrane. Apically deposited Dlg has to be depleted in the transition zone cells, which could be mediated through the endocytic and lysosomal degradative pathway. An alternatively model is that newly synthesized Dlg is targeted to the apical membrane in all SG cells. However, the apical membrane-associated Dlg is re-routed to the basolateral membrane domains, starting in the transition zone cells and continuing in distal cells, via endocytosis and transcytosis. This model could explicitly explain the Dlg mislocalization in the mutants that were analyzed. Due to defects in endocytosis in the AP-2 complex mutants, the Dlg protein is retained in the apical membrane. In AP-1 mutants such as the AP47SHE11/EP1112 transheterozygotes, large Dlg-enriched tubular structures were observed. Similar structures have been reported as specific stages, types, or structures of endosome. Therefore, in the absence of AP-1 activity, failure in vesicle budding from TGN or endosomes may lead to the disruption of targeting Dlg to the basal membrane and Dlg retaining in these organelles. Enrichment of Dlg in apical membrane might be an result inadvertently caused by abnormal intracellular transportation or a pool of Dlg being recycled back to the apical membrane through an AP-1-independent route (Peng, 2009).
In nak mutant SG cells, only the transition of Dlg is defective, but not other polarized proteins, such as aPKC, Syx1A, and Arm. In the severe AP47 and α-ada mutants when Dlg was retained at the apical membrane, the ruffled apical membrane might result from the enriched Dlg protein, leading to excess membrane addition. Such phenotype has been described when Dlg was overexpressed during embryonic cellularization. Therefore, the Dlg transition potentially accounts for the establishment of new septate junctions or basolateral membrane addition during SG cell maturation (Peng, 2009).
Clathrin adaptor protein complex-1 (AP-1) and its accessory proteins play a role in the sorting of integral membrane proteins at the trans-Golgi network and endosomes. Their physiological functions in complex organisms, however, are not fully understood. This study found that CG8538p, an uncharacterized Drosophila protein, shares significant structural and functional characteristics with Aftiphilin, a mammalian AP-1 accessory protein. The Drosophila Aftiphilin was shown to interact directly with the ear domain of γ-adaptin of Drosophila AP-1, but not with the GAE domain of Drosophila GGA. In S2 cells, Drosophila Aftiphilin and AP-1 formed a complex and colocalized at the Golgi compartment. Moreover, tissue-specific depletion of AP-1 or Aftiphilin in the developing eyes resulted in a disordered alignment of photoreceptor neurons in larval stage and roughened eyes with aberrant ommatidia in adult flies. Furthermore, AP-1-depleted photoreceptor neurons showed an intracellular accumulation of a Notch regulator, Scabrous, and downregulation of Notch by promoting its degradation in the lysosomes. These results suggest that AP-1 and Aftiphilin are cooperatively involved in the intracellular trafficking of Notch during eye development in Drosophila (Kametaka, 2012).
AP-1 and GGAs are the major clathrin adaptors that function at the post-Golgi compartments in species ranging from yeast to mammals. After a decade of biochemical and cell biological approaches, however, functional specificity of each adaptor at a molecular level still remains to be solved. The present study showed that Drosophila AP-1 and its novel accessory protein Aftiphilin, but not GGA, are required for eye development, suggesting that the Drosophila AP-1-Aftiphilin protein complex is involved in the intracellular trafficking of specific cargo molecule(s) distinct from those regulated by GGA during eye development. It has previously been reported that the GAE domain of Drosophila GGA lacks major conserved amino acid residues potentially required for interaction with the accessory molecules that possess the tetrapeptide PsiG[PDE][PsiLM] motif. Consistent with this, this study showed that Drosophila GGA failed to interact with Aftiphilin, suggesting that the GAE domain of GGA is not structurally conserved. This finding might also reflect the physiological functional diversity between Drosophila AP-1 and GGA. However, the interaction between AP-1 and GGA was detected in the coimmunoprecipitation analysis, thus Drosophila AP-1 might also have a certain functional mode to form a complex with GGA, as implicated in mammalian cells (Kametaka, 2012).
It has been suggested that CG8538, an ORF in the Drosophila genome, encodes a protein with a limited homology with human Aftiphilin. This study concluded that Drosophila Aftiphilin/CG8538p is a functional counterpart of mammalian Aftiphilin, because of their common characteristics such as the possession of multiple γ-ear binding motifs, specific interaction with the γ-ear of AP-1, and the colocalization with AP-1 at the trans-Golgi compartments. Interestingly, the molecular basis of the interaction between Aftiphilin and the γ-adaptin of the AP-1 complex was also well conserved over species, because ectopically expressed Drosophila Aftiphilin in HeLa cells was also colocalized with γ1-adaptin of AP-1. Thus, the results indicate that Drosophila could serve a good model system to dissect the molecular mechanisms of AP-1 and Aftiphilin functions (Kametaka, 2012).
In the deduced amino acid sequence of Drosophila Aftiphilin/CG8538p, two WxxF-type binding motifs for the α-subunit of AP-2 complex were found. In mammals, Aftiphilin was shown to interact with AP-1 and AP-2, and was also proposed to function with AP-2 at the endocytic pathway in neuronal cells. In S2 cells, Drosophila Aftiphilin is predominantly associated with AP-1-positive Golgi compartments and forms a stable complex with AP-1. Moreover, the molecular interaction between Drosophila Aftiphilin and AP-2 was detected. Although the interaction seems to be minor compared with the interaction with AP-1, it is likely that Aftiphilin has other functions that are not related to AP-1, because the Aftiphilin-depleted fly occasionally showed much smaller eyes with decreased number of ommatidia in addition to the roughened eye phenotype. Precise analysis of the physiological functions of Drosophila Aftiphilin is ongoing (Kametaka, 2012).
Eye-specific depletion of Drosophila Aftiphilin or of any of the sigma1- or mu1-subunits of AP-1 caused misalignment of the photoreceptor neurons due to generation of extra R8 neurons during eye development. A genetic screening for Notch modifier genes suggested that AP47, which encodes the mu1 subunit of Drosophila AP-1, is involved in Notch signaling. Another genome-wide RNAi screening showed that the subunits of Drosophila AP-1 and Aftiphilin/CG8538 are involved in Notch signaling. Recently, it has also been reported that Drosophila AP-1 depletion led to mislocalization of Notch and its regulator Sanpodo (Spdo) to the apical plasma membrane and the adherens junction in the sensory organ precursor (SOP) daughter cells in developing nota in the fly. It was suggested that the altered trafficking of Notch is primarily due to increased recycling of the Notch regulator Spdo from the recycling endosomes to the plasma membrane, and that the mislocalization of Notch to the cell surface caused the gain-of-function phenotype in the AP-1 mutants. By contrast, in the current study a clear loss-of-function phenotype of Notch was observed by depletion of AP-1 or Aftiphilin in the developing eyes (Kametaka, 2012).
This discrepancy is probably due to the different mechanisms by which intracellular trafficking of Notch is regulated in different tissues. This study focused on Scabrous as a candidate for a Notch regulator that is affected by AP-1 or Aftiphilin depletion. Scabrous is a glycosylated secretory protein expressed in the R8 neurons, and sca mutation as well as AP-1-depletion causes duplication of R8 and other photoreceptor neurons. In addition, Scabrous was also shown to bind to the extracellular domain of Notch and to stabilize Notch at the cell surface. Drosophila AP-1 has been shown to function together with clathrin in the biogenesis of mucin-containing secretory granules in the salivary gland (Burgess, 2011). Because Scabrous was shown to accumulate in the intracellular compartments in the AP-1-deficient eye discs, the observations in the current study suggest that a defect in the secretion of Scabrous and/or other regulatory proteins causes the instability of Notch at the cell surface, which leads to degradation of Notch in the endosomal and lysosomal compartments. The decrease in the amount of Notch on the cell surface then causes defects in the lateral inhibition mechanism required for the photoreceptor cell specification during eye development (Kametaka, 2012).
In addition to the tissue-specific regulation of Notch trafficking, Notch signaling could also be regulated in several ways in the intracellular trafficking pathways. In the AP-1-depleted eye antennal discs, Notch was accumulated at the late endosomal-lysosomal compartment upon treatment with the lysosomal inhibitor chloroquine, suggesting that Notch is missorted for its lysosomal degradation. It has recently been showm that defects in endocytic trafficking caused by mutations of vps25, a component of the ESCRT-II complex, caused endosomal accumulation of Notch and enhanced Notch signaling. This suggests that the cellular output of Notch signal could be affected drastically in several ways through alterations in the intracellular transport machineries for Notch protein. Finally, the possibility cannot be excluded that Notch is a cargo molecule for Drosophila AP-1, although no direct interaction between AP-1 and the cytoplasmic tail of Notch has been observed so far (Kametaka, 2012).
In conclusion, Drosophila AP-1 plays a crucial role in Notch stability in vivo. It is inferred that Drosophila AP-1 is involved in the intracellular trafficking of tissue-specific regulators of Notch at the TGN or endosomal compartments, as proposed by Benhra (2011). Notch trafficking can be regulated by several mechanisms, and a particular regulatory mode would predominate according to the context of the development. Further analysis on the precise molecular mechanisms by which Drosophila AP-1 and Aftiphilin are involved in the sorting of these signaling molecules will uncover the physiological functions of these adaptor proteins in vivo (Kametaka, 2012).
Maintenance of epithelial polarity depends on the correct localization and levels of polarity determinants. The evolutionarily conserved transmembrane protein Crumbs is crucial for the size and identity of the apical membrane, yet little is known about the molecular mechanisms controlling the amount of Crumbs at the surface. This study shows that Crumbs levels on the apical membrane depend on a well-balanced state of endocytosis and stabilization. The Adaptor Protein 2 (AP-2) complex binds to a motif in the cytoplasmic tail of Crumbs that overlaps with the binding site of Stardust, a protein known to stabilize Crumbs on the surface. Preventing endocytosis by mutations in AP-2 causes expansion of the Crumbs-positive plasma membrane and polarity defects, which can be partially rescued by removing one copy of crumbs. Strikingly, knocking-down both AP-2 and Stardust retains Crumbs on the membrane. This study provides evidence for a molecular mechanism, based on stabilization and endocytosis, to adjust surface levels of Crumbs, which are essential for maintaining epithelial polarity (Lin, 2015).
Data presented in this study, obtained using genetic and biochemical assays, has led to the conclusion that a delicate balance of stabilization and internalization controls proper Crb levels at the surface. This balance is mediated by binding of a conserved amino acid sequence of Crb to either Sdt or AP-2, suggesting a competitive binding between Sdt and AP-2. Such a mechanism, based on stabilization by PDZ-ligand interaction and AP-2-mediated endocytosis, has so far only be described for a few proteins, such as the NR2B subunit of the neuronal NMDA receptor and the glutamate transporter excitatory amino acid carrier 1 (EAAC1) in MDCK cells. In the latter case, AP-2 and the PDZ-domain of PDZK1 antagonistically regulate surface levels of EEAC1 by binding to two closely adjacent binding sites in EEAC1 (Lin, 2015).
Currently, it is only possible to speculate about the mechanisms that may determine which of the binding partners AP-2 or Sdt - binds to Crb. One possibility is that different binding affinities between Crb and its partners modulate the binding preference. Several arguments suggest that binding of AP-2 to Crb seems to be weaker than the Sdt-Crb interaction. First, AP-2 - Crb interactions could only be shown by liposome recruitment assays, but not by standard pull-down experiments, suggesting that the presence of membranes strengthen the interaction between the adaptor and its cargo. In fact, interaction of AP-2 with PtdIns4,5P2-containing membranes induces a conformational change of the AP-2 complex, thus facilitating binding to its cargo (Lin, 2015).
Second, binding of Sdt only depends on the C-terminal leucine and isoleucine residues of Crb. This is in agreement with recently published structural data using crystallography and fluorescence polarization, which show that the four C-terminal amino acids of human Crb1 interact with the PDZ domain of Pals1 by van der Waals contacts and charged interactions (Ivanova, 2015). In contrast, AP-2 binding to Crb requires the glutamine and arginine residues of Crb besides the leucine and isoleucine residues, at least under the in vitro conditions used in this study. The importance of more than one motif for strong binding of AP-2 is not unprecedented. Binding of the human immunodeficiency (HIV)-1 protein Nef to the α-σ2 -hemicomplex of AP-2 requires both a di-leucine- and a di-acidic-motif. Posttranslational modification of the ligand Crb itself could also contribute to a preferred binding to either Sdt/Pals1 or AP-2. It was recently documented that aPKC-mediated phosphorylation of threonine residues near the FBM of Crb abolishes the Crb- Moesin interaction, but not the Crb/Pals1 interaction (Wei, 2015; Lin, 2015 and references therein).
In the case of Drosophila Gliotactin, a transmembrane protein at the tricellular junction, phosphorylation of tyrosine residues is necessary for its endocytosis and ultimately lysosomal degradation, thus preventing overexpression, which would result in delamination, migration and finally apoptosis of cells (Lin, 2015).
Alternatively, the accessibility of the PDZ domain of Sdt/Pals1 to Crb could be modulated. In fact, a ~100-fold stronger binding of the PDZ domain of Pals1 to the Crb tail is achieved upon intra- or intermolecular interactions between the Src homology 3 (SH3)- and the guanylate kinase (GUK)-domain of Pals1. Similarly, the affinity of the PDZ domain of the postsynaptic density protein PSD-95 to its ligand is reduced upon phosphorylation of a tyrosine residue in a linker region between the third PDZ-domain and the subsequent SH3-domain, thereby weakening the intramolecular interaction between the PDZ- and the SH3 domain (Lin, 2015).
A striking observation was the high degree of phenotypic variability upon knock-down/knock -out of AP-2 in different epithelial tissues, and sometimes even in the same epithelium. This prevented study of the different aspects of the AP-2/Crb relation in just one epithelium. In the follicle epithelium, the phenotypes ranged from minor expansion of the apical surface to complete loss of polarity and overgrowth. Complete loss of AP-2α in wing discs leads to cell lethality, whereas mutant clones survived in eye discs. This variability might be due to the time point of induction of mitotic recombination, which could occur before or after establishment of epithelial polarity, or at time points of high or low Crb expression. Alternatively, additional AP-2-independent polarity regulators could act redundantly and in a tissue- and/or time-specific manner, thus modulating the severity of the phenotype, e.g. in the developing eye (Lin, 2015).
The results described in this study are compatible with the assumption that several phenotypes obtained by loss/reduction of AP-2 are a consequence of increased Crb levels on the plasma membrane, since similar phenotypes can be obtained upon overexpression of Crb. (1) The Crb-positive plasma membrane in AP-2α mutants cells is expanded both in the imaginal discs and the follicle epithelium, often at the expense of the lateral membrane. (2) Without functional AP-2α, the monolayered epithelium is often disrupted and becomes multilayered. (3) Surviving AP-2α3 clones in eye imaginal discs show strong Crb enrichment, similar as HEK293 cells, in which AP-2α is knocked-down by RNAi. (4) Assuming that a similar increase in Crb also occurs in AP-2α3 mutant cells induced in wing discs, their elimination could be a consequence of cell competition when next to wild-type cells (Lin, 2015).
Finally, the multi-layering phenotype of follicle epithelia lacking AP-2α can be partially suppressed by removing one copy of crb. A more direct insight into the relationship between Crb and AP-2 comes from an analysis of the garland cells, the functional equivalent of vertebrate podocytes. Podocytes are highly specialized epithelial cells in the kidney of vertebrates, which form long 'foot-processes' connected by slit diaphragms to form a filtration barrier in the renal glomerula. Interestingly, Crb2 is expressed in the kidney of both rats and zebrafish, where it localizes at the slit diaphragm of podocytes. crb2b mutant zebrafish show defects in the formation of the slit diaphragm as well as in arborization of the foot-processes. Furthermore, mutations in human Crb2 are linked to Steroid-resistant nephrotic syndrome (Ebarasi, 2015), a disease causing kidney failure due to defects in differentiation and function of podocytes. This study shows that AP-2α3 mutant garland cells are clearly impaired in Crb endocytosis. Whether loss of crb in garland cells affects their excretory function has to be determined (Lin, 2015).
A complex machinery is required to ensure proper Crb surface levels, which is key for the maintenance of apico-basal epithelial cell polarity. Crb levels can be regulated at multiple levels, including stabilization at the membrane via homophilic interactions of the extracellular domains in cis or trans, interactions of the cytoplasmic tail with scaffolding proteins, endocytosis, degradation and recycling by the retromer. The trafficking pathway offers multiple steps for regulation, and results presented here provide further mechanistic insight how binding of two counteracting PDZ motif-binding proteins, Sdt and AP-2, regulate proper Crb levels (Lin, 2015).
Given the finding that the balance between Crb stabilization and internalization/degradation is crucial for surface expression of Crb and hence polarity, future work will aim to identify the mechanisms
that control this balance by regulating the rate of endocytosis, degradation and recycling by the
retromer. Finding these regulators is challenging, but will give important insights into the
mechanisms coordinating endocytosis and polarity, which is important to prevent tumorigenesis
not only in Drosophila, but also in vertebrates (Lin, 2015).
The functional requirement of AP2 complex (see AP-2α) in synaptic membrane retrieval by clathrin mediated endocytosis (CME) is not fully understood. This study isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction synapse, this mutation was named angur (a Hindi dialect meaning grapes). Loss-of-function alleles of angur show more than two-fold overgrowth in bouton numbers and dramatic decrease in bouton size. angur mutation was mapped to σ2-adaptin, the smallest subunit of the adapter complex 2. Reducing neuronal level of any of the subunits of AP2 complex or disrupting AP2 complex assembly in neurons phenocopied σ2-adaptin mutation. Genetic perturbation of σ2-adaptin in neurons leads to a reversible temperature sensitive paralysis at 38°C. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic level of subunits of AP2 complex and clathrin but not other endocytic proteins were reduced in the mutants. Moreover, the BMP/TGFβ signalling was altered in these mutants and was restored by normalizing σ2-adaptin in neurons. Thus, these data suggest that - 1) while σ2-adaptin facilitates SV recycling for basal synaptic transmission, its activity is also required for regenerating SV during high frequency nerve stimulation; and 2) σ2-adaptin regulates NMJ morphology by attenuating TGFβ signalling (Choudhury, 2016).
Synaptic transmission requires fusion of synaptic vesicles (SVs) at the active zones followed by their efficient retrieval and recycling through endocytic mechanisms. Retrieval and sorting of membrane lipids and vesicular proteins at the synapse are mediated by a well-orchestrated and coordinated action of several adapter and endocytic proteins. Clathrin-mediated endocytosis (CME) is the primary pathway operative at the synapses for membrane retrieval. Genetic analysis of the components of the CME pathway in Caenorhabditis elegans and Drosophila has revealed that this pathway is required for SV re-formation, and in many cases, blocking CME at synapses results in temperature-sensitive paralysis. Additionally, CME plays a crucial role in regulating synaptic morphology. At Drosophila NMJs, blocking CME results in enhanced bone morphogenetic protein (BMP) signaling and affects synaptic growth (Choudhury, 2016).
The heterotetrameric adapter protein 2 (AP2) complex is a major effector of the CME pathway. AP2 serves as a major hub for a large number of molecular interactions and links plasma membrane, cargo/signaling molecules, clathrin, and accessory proteins in the CME pathway and hence can directly influence synaptic signaling. The AP2 complex is pseudo-asymmetric and contains four subunits-one each of large α and β2 subunits, one medium μ2 subunit, and a small σ2 subunit. Depletion of clathrin or its major adapter, AP2, in either Drosophila or mammalian central synapses results in accumulation of endosome-like vacuoles and reduction of SVs, suggesting that CME may not be essential for membrane retrieval. Similarly, genetic perturbation of μ2-adaptin or α-adaptin shows only mild defects in vesicle biogenesis at C. elegans synapses, but simultaneous loss of both adaptins leads to severely compromised SV biogenesis and accumulation of large vacuoles at nerve terminals. While Drosophila loss-of-function mutations in α-adaptin are embryonic lethal, hypomorphic mutants exhibit reduced FM1-43 uptake, suggesting a compromised endocytosis in these mutants. Whether reduced endocytosis reflects a defect in membrane retrieval or a defect in SV biogenesis remains unclear. Moreover, the consequences of AP2 reduction on synaptic morphology and physiology remain unknown (Choudhury, 2016).
This study presents an analysis of Drosophila σ2-adaptin in the context of regulating NMJ morphological plasticity and physiology. A mutation was identified that dramatically altered NMJ morphology. Next, this mutation was mapped to σ2-adaptin by deficiency mapping. This study shows that AP2-dependent vesicle endocytosis regulates both synaptic growth and transmitter release. The AP2 complex is a heterotetramer, and these studies in Drosophila show that the four subunits are obligate partners of each other and are required for a functional AP2 complex. This finding is in contrast to the hemicomplex model in C. elegans, in which α/σ2 and β2/μ2 can sustain the function, if any one of the subunits is mutated. Loss of AP2 disrupts stable microtubule loops of the presynaptic cytoskeleton and exacerbates growth signaling through the phosphorylated Mothers Against Decapentaplegic (pMAD) pathway, suggesting that normal AP2 constrains the TGFβ signaling module. Reducing σ2-adaptin level results in synaptic fatigue at the larval NMJ synapses during high-frequency stimulation and causes temperature-sensitive paralysis in adults. Based on these results, it is suggestd that AP2 is essential for attenuating synaptic growth signaling mediated by the TGFβ pathway in addition to its requirement in regenerating SVs under high-frequency nerve firing (Choudhury, 2016).
AP2/CME has been proposed to play an essential role in SV endocytosis. Moreover, mutation in the proteins affecting CME also results in altered NMJ development in Drosophila, suggesting regulation of synaptic signaling by CME. This study analyzed the role of AP2, one of the major adapters for clathrin at synapses, in the context of its physiological relevance and synaptic signaling. The results demonstrate that AP2 facilitates basal synaptic transmission and SV endocytosis but also is essential during high-frequency nerve firing to regenerate SVs. Moreover, evidence is provided that AP2 regulates morphological plasticity at the Drosophila NMJ by stabilizing the microtubule loops and attenuating the BMP signaling pathway (Choudhury, 2016).
The biogenesis of rhabdomeres is regulated by endocytosis and intracellular trafficking. Components of the endocytic machinery, when perturbed, lead to defects in rhabdomere formation. One such example is the disruption of the rhabdomere base when shits flies are grown at restrictive temperature for 4 hr. Rh1-Gal4-driven shits1 flies raised at 19° led to an enlargement of photoreceptor cell bodies with the inter-rhabdomeric space being reduced. Similarly, when α-adaptin was knocked down using GMR-Gal4, rhabdomere biogenesis was disrupted, with smaller rhabdomeres. In accordance with these observations, the TEM analysis of angur mutant eye clones showed no detectable rhabdomeres. Consistent with the defect in rhabdomere formation, no membrane depolarization was observed in angur mutant eye clones. The visual cascade in Drosophila begins with rhodopsin being converted to meta-rhodopsin by light, followed by a subsequent phosphorylation by GPRK1. The phosphorylated meta-rhodopsin binds to arrestins, where its activity is quenched. The rhodopsin-arrestin complex is endocytosed, and rhodopsin is degraded in the lysosomes. Thus, endocytosis regulates rhodopsin turnover in the photoreceptor cells, and blocking of the endocytic pathway components by mutations in Arr1, Arr2, or AP2 leads to retinal degeneration and photoreceptor cell death. This further establishes a strong link between endocytosis and retinal degeneration. Thus, it is likely that defective endocytosis in angur mutant eye clones affects meta-rhodopsin turnover, resulting into defective rhabdomeres and disorganized ommatidia. Moreover, endocytosis of several signaling receptors also has been shown to regulate morphogenesis during Drosophila eye development. The Notch signaling pathway has been studied extensively, and it has been shown that the Notch extracellular domain is transendocytosed during this process into the Delta-expressing cells. The localization of Notch and Delta is disrupted when endocytosis is acutely blocked, and this prevents internalization of Notch in the Delta-expressing cells. It is thus speculated that the endocytic defect in angur mutant clones affects turnover and internalization of signaling molecules that may lead to defective rhabdomere development (Choudhury, 2016).
The functional analysis of the σ2-adaptin mutants as well as of other subunits of AP2 reveals its requirement in maintaining basal synaptic transmission and regeneration of synaptic vesicles under high-frequency nerve firing at Drosophila NMJ synapses. The data are consistent with previous reports showing the requirement for Drosophila α-adaptin in SV recycling at NMJ synapses. However, in contrast to its role at mammalian central synapses, this study found that loss of AP2 at Drosophila NMJ synapses affects basal synaptic transmission and SV endocytosis, albeit not very strongly. Moreover, the kinetics of SV re-formation in σ2-adaptin mutants was only mildly affected. Consistent with the recent observation that loss of the AP2 complex at mammalian central synapses or its subunits in C. elegans affects synaptic vesicle biogenesis, compromised synaptic transmission and SV re-formation was observed after high-frequency stimulation, suggesting that at Drosophila NMJ synapses, AP2 function is required not only for SV trafficking but also for SV regeneration under high-frequency nerve stimulation. A direct estimation of vesicle pool size in σ2-adaptin mutants further supports its requirement in the regulation of SV pool size and is consistent with a recent report. Because loss of AP2 in Drosophila does not completely abrogate SV re-formation, it remains possible that other known synaptic clathrin adapters such as AP180, Eps15, and Epsin might partially compensate for loss of the AP2 complex. This is corroborated by the observation that even a 96% reduction in synaptic α-adaptin level caused only an ~50% reduction in clathrin, suggesting that other clathrin adapters might contribute to retention and/or stability of synaptic clathrin (Choudhury, 2016).
The striking physiological defect in σ2-adaptin mutants or neuronally reduced α-adaptin was their inability to recover from synaptic depression even after 4 min of cessation of high-frequency stimulation. Consistent with these observations, the endo-exo cycling pool did not recover to its initial value even after 4 min of rest following high-frequency nerve stimulation. This suggests that in addition to its requirement in synaptic vesicle endocytosis, AP2 complex functions at a relatively slower step downstream of membrane retrieval, possibly during membrane sorting from endosomes to regenerate fusion-competent synaptic vesicles (Gu, 2013; Kononenko, 2014; Choudhury, 2016 and references therein).
Several studies in cell culture support a model that indicates that the AP2 complex is an obligate heterotetramer for subunit stability and function. However, studies of AP2 function in C. elegans suggest that α/σ2 subunits and β2/μ2 subunits may constitute two hemicomplexes that can carry out the minimal function of the AP2 complex independent of one another. Does AP2 in Drosophila form hemicomplexes and contribute to vesicle trafficking? The results do not support this model for Drosophila. First, unlike C. elegans, in which individual mutants for subunits of the AP2 complex are viable, the Drosophila mutant for α-adaptin is early larval lethal. Consistent with these findings, neuronal knockdown of α-adaptin or β2-adaptin resulted in third instar lethality. Second, if the hemicomplexes were functional in Drosophila, one would observe significant levels of synaptic β2-adaptin in α-adaptin knockdown or σ2-adaptin mutants. However, it was observed that removing any of the subunits of the Drosophila AP2 complex in neurons resulted into an unstable AP2 complex and degradation of α- and β2-adaptins. Third, inhibiting AP2 complex assembly by reducing synaptic PI(4,5)P2 levels also resulted into an unstable AP2 complex. These observations strongly suggest that in contrast to C. elegans, all the subunits of AP2 in Drosophila are obligate partners for a stable AP2 complex for clathrin-dependent SV trafficking (Choudhury, 2016).
Mutations in genes implicated in regulating CME, BMP signaling, or actin cytoskeleton dynamics all show abnormal NMJ development in Drosophila, characterized by either supernumerary boutons or an increased number but smaller boutons. The NMJ phenotype due to loss of σ2-adaptin is consistent with that of other known endocytic mutants implicated in CME. The contrasting synaptic overgrowth observed in σ2-adaptin mutants suggests a role for AP2 in mediating presynaptic growth signaling. Such synaptic overgrowth also was observed when any of the subunits of AP2 were downregulated or its assembly was interfered with by downregulating neuronal PI(4,5)P2. This suggests a pathway in which PI(4,5)P2 and the AP2 complex interact obligatorily to regulate synapse growth. The NMJ phenotype in angur mutants is strikingly more severe from that of the other synaptic mutants and points toward multiple growth signaling pathways that are possibly affected by an AP2-dependent endocytic deficit (Choudhury, 2016).
The morphology of the synapse is a consequence of the neuronal cytoskeleton network that shapes the growing synapse. Futsch, a protein with MAP1B homology, regulates the synaptic microtubule cytoskeleton, thereby controlling synaptic growth at the Drosophila NMJ. The hypothesis that the microtubule organization could be altered was strengthened by the dramatic decrease in the number of Futsch-positive loops in the σ2-adaptin mutants. Futsch-positive loops have long been known to be associated with stable synaptic boutons, while the absence or disruption of these loops is indicative of boutons undergoing division or sprouting. The dramatic increase in the number of boutons in these mutants with a corresponding decrease in the number of loops correlates well with the fact that these boutons might be undergoing division. The phenotype associated with futsch mutants is fewer and larger boutons with impaired microtubule organization, which is an expected phenotype when bouton division is impaired. The σ2-adaptin mutants, however, show a larger number but smaller-sized boutons, indicating that bouton division is enhanced. One of the signaling events that has been shown to dictate this process is BMP signaling. Further, BMP signaling is also required during developmental synaptic growth. Consistent with this, elevated pMAD levels were found in σ2-adaptin mutants, indicating that BMP signaling is upregulated in these mutants. It has also been demonstrated that BMP signaling plays a role in maintenance of the presynaptic microtubule network. Drosophila spichthyin and spartin mutants have upregulated BMP signaling with significantly increased microtubule loops. It has been proposed that Futsch acts downstream of BMP signaling to regulate synaptic growth. The σ2-adaptin mutants also show upregulated BMP signaling, but in contrast to spichthyin and spartin mutants, σ2-adaptin mutants have fewer Futsch loops, which also appeared fragmented. This suggests that deregulated BMP signaling, whether upregulation or downregulation, impairs microtubule stability. dap160 mutants also show supernumerary boutons, with fragmented Futsch staining suggesting that the microtubule dynamics are misregulated in these mutants. Interestingly, Nwk levels are drastically altered in dap160 mutants, suggesting that Nwk levels are important for maintaining microtubule stability. Because σ2-adaptin mutants have normal Nwk levels, the observed synaptic overgrowth is independent of Nwk and suggests regulation of Futsch through Nwk-independent pathways (Choudhury, 2016).
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).
Cancer stem cells (CSCs) are postulated to be a small subset of tumor cells with tumor-initiating ability that shares features with normal tissue-specific stem cells. The origin of CSCs and the mechanisms underlying their genesis are poorly understood, and it is uncertain whether it is possible to obliterate CSCs without inadvertently damaging normal stem cells. This study shows that a functional reduction of eukaryotic translation initiation factor 4E (eIF4E) in Drosophila specifically eliminates CSC-like cells in the brain and ovary without having discernable effects on normal stem cells. Brain CSC-like cells can arise from dedifferentiation of transit-amplifying progenitors upon Notch hyperactivation. eIF4E is up-regulated in these dedifferentiating progenitors, where it forms a feedback regulatory loop with the growth regulator dMyc to promote cell growth, particularly nucleolar growth, and subsequent ectopic neural stem cell (NSC) formation. Cell growth regulation is also a critical component of the mechanism by which Notch signaling regulates the self-renewal of normal NSCs. These findings highlight the importance of Notch-regulated cell growth in stem cell maintenance and reveal a stronger dependence on eIF4E function and cell growth by CSCs, which might be exploited therapeutically (Song, 2011).
The differential cell growth rates observed between ectopic NBs and normal or primary NBs and the correlation between cell growth defects and NB fate loss prompted a test of whether slowing down cell growth might selectively affect the formation of ectopic NBs. Attenuation of TOR signaling, a primary mechanism of cell growth regulation, through NB-specific overexpression of TSC1/2, a strong allele of eIF4E antagonist 4EBP [4EBP(LL)s], or a dominant-negative form of TOR (TOR.TED) all partially suppressed ectopic NB formation in α-adaptin (ada) mutants without affecting normal or primary NBs. Interestingly, RNAi-mediated knockdown of eIF4E, a stimulator of oncogenic transformation and a downstream effector of TOR signaling, showed a better suppression than manipulating other TOR pathway components, suggesting that eIF4E might play a more important role in ectopic NB formation. Strikingly, the brain tumor phenotypes caused by overactivation of N signaling - as in lethal giant larvae (lgl) mutant, aPKCCAAX overexpression, or N overexpression conditions - were also fully suppressed by eIF4E knockdown. Furthermore, the brain tumor phenotypes of brat mutants were also completely rescued by eIF4E RNAi (Song, 2011).
In contrast, normal NB formation or maintenance was not affected by eIF4E knockdown. NBs with eIF4E knockdown remained highly proliferative, as evidenced by the mitotic figures, and displayed relatively normal apical basal cell polarity. There are several other eIF4E-like genes in the fly genome (Hernandez, 2005), which may play partially redundant roles in normal NB maintenance. eIF4E knockdown appeared to specifically block ectopic NB formation caused by the dedifferentiation of IPs in type II NB lineages, since it did not affect ectopic type I NB formation in cnn or polo mutants that are presumably caused by symmetric divisions of type I NBs. In addition, cell fate transformation induced by N overactivation in the SOP lineage was not affected by eIF4E RNAi, supporting the idea that eIF4E is particularly required for type II NB homeostasis. Supporting the specificity of the observed eIF4E RNAi effect, another eIF4E RNAi transgene (eIF4E-RNAi-s) also prevented ectopic NB formation. Moreover, a strong loss-of-function mutation of eIF4E also selectively eliminated ectopic NBs induced by N overactivation without affecting normal NBs, reinforcing the hypothesis that ectopic NBs exhibit higher dependence on eIF4E (Song, 2011).
To further support the notion that the ectopic NBs are particularly vulnerable to eIF4E depletion, a conditional expression experiment was carried out in which eIF4E-RNAi-s was turned on in brat mutants using the 1407ts system, after ectopic NBs had been generated. Whereas the brain tumor phenotype exacerbated over time in the brat mutants, 1407-GAL4-driven eIF4E-RNAi-s expression in brat mutants effectively eliminated ectopic NBs, leaving normal NBs largely unaffected (Song, 2011).
In normal type II NB lineage, eIF4E protein was enriched in the NBs. Ectopic NBs induced by N overactivation in ada mutants also expressed eIF4E at high levels, whereas spdo mutant NBs exhibited reduced eIF4E expression. Thus, eIF4E up-regulation correlates with N-induced ectopic NB formation in a dedifferentiation process that likely involves elevated cell growth (Song, 2011).
Given the coincidence of nucleolar size change with ectopic NB formation, the involvement of the growth regulator dMyc was tested. dMyc protein levels were up-regulated in normal or N overactivation-induced ectopic NBs, but were down-regulated in spdo mutant NBs. Furthermore, dMyc transcription, as detected with a dMyc-lacZ transcriptional fusion reporter, was also up-regulated in both normal and ectopic NBs in ada mutants. A previous study in Drosophila S2 cells identified dMyc as a putative N target. In vivo chromatin immunoprecipitation (ChIP) experiments were carried out to assess whether dmyc transcription is directly regulated by N signaling in NBs. Using chromatin isolated from wild-type larval brains and a ChIP-quality antibody against the N coactivator Suppressor of Hairless [Su(H)], specific binding was demonstrated of Su(H) to its putative binding sites within the second intron of dmyc (dmyc-A). No binding to an internal negative control region proximal to the first exon of dmyc (dmyc-B) or to the promoter region of the rp49 gene was detected. N signaling thus directly activates dMyc transcription in the NBs. Similar to eIF4E RNAi, knockdown of dMyc strongly suppressed ectopic NB formation induced by Brat or Ada inactivation or N overactivation. Intriguingly, the strong tumor suppression effect of eIF4E knockdown was partially abolished by dMyc overexpression. Furthermore, dMyc function, as reflected by its promotion of nucleolar growth in IPs, was attenuated by eIF4E RNAi, although eIF4E RNAi alone had no obvious effect. Different from the reported eIF4E regulation of Myc expression in mammalian cells (Lin, 2008), dMyc promoter activity or protein levels remained unaltered under eIF4E RNAi conditions, suggesting that eIF4E may modulate dMyc activity without altering its expression. One possibility is that eIF4E may enter the nucleus to interact with Myc and promote its transcriptional activity. To test this hypothesis, HEK293T cells were transfected with Flag-tagged human eIF4E alone or in combination with HA-tagged dMyc. Indeed, both Drosophila dMyc and endogenous human c-Myc specifically coimmunoprecipitated with human eIF4E from nuclear extracts, indicating a conserved interaction between eIF4E and Myc within the nuclei of proliferating cells. Consistent with these biochemical data, dMyc transcriptional activity within NBs, which could be monitored with an eIF4E-lacZ reporter, was drastically reduced upon eIF4E knockdown (Song, 2011).
In contrast, eIF4E transcription, as detected with an eIF4E-lacZ transcriptional fusion reporter, as well as eIF4E protein levels detected by immunostaining were up-regulated upon dMyc overexpression and down-regulated by dMyc RNAi. It is unlikely that the changes in eIF4E-lacZ activity were due to global increases or decreases in β-galactosidase (β-gal) translation caused by altered dMyc levels, since lacZ expression from a dMyc-lacZ reporter was unaffected under similar conditions. Furthermore, like dMyc protein, eIF4E-lacZ reporter expression was up-regulated in normal NBs or ectopic NBs in ada mutants, further supporting the notion that dMyc may up-regulate eIF4E transcription. Moreover, ChIP experiments using chromatins isolated from wild-type larval brains and a ChIP-quality antibody against dMyc demonstrated specific binding of dMyc to an eIF4E promoter region harboring a cluster of adjacent noncanonical E boxes, supporting a direct regulation of eIF4E transcription by dMyc. dMyc and eIF4E thus appeared to form a regulatory feedback loop that promoted NB growth and renewal. Consistent with this model, while knocking down either dMyc or eIF4E had no noticeable effect on type II NB maintenance and only a mild effect on NB nucleolar size in the case of dMyc RNAi, their simultaneous knockdown led to a significant reduction in nucleolar size, premature neuronal differentiation, and loss of NBs (Song, 2011).
If the dMyc-eIF4E axis of cell growth control is a crucial downstream effector of N signaling in regulating NB maintenance, its up-regulation might be able to rescue the type II NB depletion phenotype resulting from reduced N signaling. Indeed, the loss of NBs associated with reduced Notch signaling was preventable when cell growth was boosted by dMyc overexpression. Thus, while N-IR directed by 1407-GAL4 led to complete elimination of type II NBs, the coexpression of dMyc, but not CD8-GFP or Rheb, an upstream component of the TOR pathway, resulted in the preservation of approximately half of type II NBs with apparently normal cell sizes, cell fate marker expression, and lineage composition. A similar effect was observed when dMyc was coexpressed with N-IR using the conditional 1407ts system, with transgene expression induced at the larval stage. While both dMyc and Rheb promote cell growth, they do so through distinct mechanisms, with the former increasing nucleolar size and the latter expanding cytoplasmic volume. These results thus provide compelling evidence that control of cell growth, particularly nucleolar growth, is a critical component in the maintenance of NB identity by N signaling (Song, 2011).
The differential responses of normal and tumor-initiating stem cells to functional reduction of eIF4E prompted a test of whether chemicals that specifically inhibit eIF4E function might have therapeutic potential in preventing CSC-induced tumorigenesis. Indeed, the brain tumor phenotypes induced by N overactivation or ada loss of function were effectively suppressed by feeding animals with fly food containing Ribavirin, an eIF4E inhibitor that interferes with eIF4E binding to mRNA 5' caps and promotes the relocalization of eIF4E from the nucleus to the cytoplasm (Kentsis, 2004; Assouline, 2009) (Song, 2011).
The CSC hypothesis was initially developed based on studies in mammalian systems. Various studies have supported the notion that CSCs share many functional features with normal stem cells, such as signaling molecules, pathways, and mechanisms governing their self-renewal versus differentiation choice. However, the cellular origin of CSCs and the molecular and cellular mechanisms underlying their development or genesis remain poorly understood. It has been proposed that CSCs could arise from (1) an expansion of normal stem cell niches, (2) normal stem cells adapting to different niches, (3) normal stem cells becoming niche-independent, or (4) differentiated progenitor cells gaining stem cell properties. This study has showen that in the Drosophila larval brain, CSCs can arise from the dedifferentiation of transit-amplifying progenitor cells back to a stem cell-like state. Importantly, eIF4E was identified as a critical factor involved in this dedifferentiation process. More significantly, it was shown that reduction of eIF4E function can effectively prevent the formation of CSCs without affecting the development or maintenance of normal stem cells. This particular dependence on eIF4E function by CSCs appears to be a general theme, as reduction of eIF4E function also effectively prevented the formation of CSCs, but not normal GSCs, in the fly ovary. These findings may have important implications for stem cell biology and cancer biology, in terms of both mechanistic understanding and therapeutic intervention (Song, 2011).
This study also offers mechanistic insights into the cellular processes leading to the dedifferentiation of progenitors back to stem cells. In Drosophila type II NB clones with overactivated N signaling, ribosome biogenesis within ectopic NBs appears to be faster than in normal NBs, as shown by the fact that the ratio of nucleolar to cellular volume of the ectopic NBs is approximately fivefold higher than that of normal NBs. The faster growth rate is accompanied by the up-regulation of dMyc and eIF4E and appears to be essential for transit-amplifying progenitors to undergo complete dedifferentiation back to a stem cell-like state. When the function of cell growth-promoting factors such as eIF4E is attenuated, the faster cell growth of ectopic NBs can no longer be sustained and the dedifferentiation process stalls. As a result, brain tumor formation caused by uncontrolled production of ectopic NBs is suppressed. In contrast, normal NBs, which presumably have relatively lower requirements for cell growth and hence eIF4E function, maintain their stem cell fate and development under similar conditions. Therefore, a potential key to a successful elimination of CSC-induced tumors would be to find the right level of functional reduction in eIF4E, which causes minimal effects on normal stem cells but effectively obliterates CSCs. An ongoing clinical trial with Ribavirin in treating acute myeloid leukemia (AML) (Assouline, 2009), a well-characterized CSC-based cancer, demonstrated exciting proof of principle that such a strategy is feasible. The current version of Ribavirin, however, has certain limitations, such as its poor specificity and the high dosage (micromolar range) required for effective treatment. Thus, more specific and effective eIF4E inhibitors are urgently needed. The drug treatment experiments with Ribavirin validated Drosophila NBs as an excellent CSC model for searching further improved drugs. More importantly, the nuclear interaction between eIF4E and Myc unraveled by this biochemical analysis not only provides a new mechanistic explanation for the synergistic effects of eIF4E and Myc in tumorigenesis (Ruggero, 2004; Wendel, 2007), but also sheds new light on how to rationally optimize drug design and therapy for treating CSC-based cancer (Song, 2011).
The results offer new information on how N signaling helps specify and maintain NSC fate. N signaling regulates stem cell behavior in various tissues of diverse species. However, it remains unclear how differential N signaling determines distinct cell fate within the stem cell hierarchy. This study demonstrates that N signaling maintains Drosophila NSC fate at least in part through promoting cell growth. The following evidence supports that cell growth, but not cell fate, change is the early and primary effect of N signaling inhibition in type II NBs: (1) Pros expression is not immediately turned on in spdo mutant NBs with reduced cell sizes. Instead, it gradually increases during the course of spdo mutant NB divisions. (2) Up-regulation of Pros is not the cause of stem cell fate loss in spdo mutant NBs, as shown by spdo pros double-mutant analysis. (3) Cell growth defects precede the up-regulation of Ase expression in aph-1 mutant NBs. (4) Promotion of cell growth, and particularly nucleolar growth, by dMyc is sufficient to prevent NB loss caused by N inhibition. At the molecular level, N signaling appears to regulate the transcription of dMyc, which in turn up-regulates the transcription of eIF4E. Such a transcriptional cascade and feedback regulation of dMyc activity by eIF4E may help to sustain and amplify the activity of the Notch-dMyc-eIF4E molecular circuitry. Hence, differential N signaling within the lineage can lead to different cell growth rates, which partially determine differential cell fates. Consistent with this notion, knockdown of both eIF4E and dMyc results in defects of NB cell growth and loss of stem cell fate (Song, 2011).
While many signaling pathways and molecules have been implicated in the maintenance of stem cell identity, the question of how a stem cell loses its 'stemness' at the cellular level remains poorly understood. A stem cell may lose its stem cell fate by undergoing a symmetric division to yield two daughter cells that are both committed to differentiation or through cell death. Earlier studies provided intriguing hints that cell growth and translational regulation could influence stem cell maintenance in the Drosophila ovary. This study usded detailed clonal analyses of NSCs over multiple time points to provide direct evidence that a NSC with impaired N signaling will gradually lose its identity due to a gradual slowing down of cell growth and loss of cell mass. Remarkably, such loss of stem cell fate can be prevented when cell growth is restored by dMyc, but not Rheb, overexpression, demonstrating the functional significance of regulated cell growth, particularly nucleolar growth, in stem cell maintenance. More importantly, this information offers clues on how to specifically eliminate tumor-initiating stem cells. These studies suggest that a stem cell, normal or malignant, has to reach a certain growth rate in order to acquire and maintain its stemness, presumably because when the stem cell grows below such a threshold, its proliferative capacity becomes too low, whereas the concentration of differentiation-promoting factors becomes too high to be compatible with the maintenance of stem cell fate. Consistent with this notion are the strong correlation between the expression of ribosomal proteins and cellular proliferation (van Riggelen, 2010) as well as the correlation between the reduction of NB sizes and the up-regulation of differentiation-promoting factor Pros or Ase in different developmental contexts (Song, 2011).
The results also provide new insights into how the evolutionarily conserved tripartite motif and Ncl-1, HT2A, and Lin-41 (TRIM-NHL) domain proteins regulate stem cell homeostasis. The TRIM-NHL protein family, to which Brat and Mei-P26 belong, include evolutionarily conserved stem cell regulators that prevent ectopic stem cell self-renewal by inhibiting Myc. However, the downstream effectors of the TRIM-NHL proteins remain largely unknown. This study identified eIF4E as such a factor. NB-specific knockdown of eIF4E completely suppresses the drastic brain tumor phenotype caused by loss of Brat. Interestingly, eIF4E knockdown is even more effective than dMyc knockdown in this regard. N signaling and Brat have been proposed to act in parallel in regulating Drosophila type II NB homeostasis. However, at the molecular level, how deregulation of these two rather distinct pathways causes similar brain tumor phenotypes remain largely unknown. The current results suggest that these two pathways eventually converge on the dMyc-eIF4E regulatory loop to promote cell growth and stem cell fate. N overactivation and loss of Brat both result in up-regulation of eIF4E and dMyc in transit-amplifying progenitors, accelerating their growth rates and helping them acquire stem cell fate. Consistent with a general role of eIF4E and dMyc in stem cell regulation, it was shown that partial reduction of eIF4E or dMyc function in the Drosophila ovary effectively rescues the ovarian tumor phenotype due to the loss of Mei-P26. The vertebrate member of the TRIM-NHL family, TRIM32, is shown to suppress the stem cell fate of mouse neural progenitor cells, partially through degrading Myc. Whether eIF4E acts as a downstream effector of TRIM32 in balancing stem cell self-renewal versus differentiation in mammalian tissues awaits future investigation (Song, 2011).
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