Eukaryotic genomes encode large families of deubiquitinating enzymes (DUBs). Genetic data suggest that Fat facets (Faf), a Drosophila DUB essential for patterning the compound eye, might have a novel regulatory function; Faf might reverse the ubiquitination of a specific substrate, thereby preventing proteasomal degradation of that protein. Additional genetic data implicate Liquid facets (Lqf), a homolog of the vertebrate endocytic protein epsin, as a candidate for the key substrate of Faf. Here, biochemical experiments critical to testing this model were performed. The results show definitively that Lqf is the key substrate of Faf in the eye; Lqf concentration is Faf-dependent, Lqf is ubiquitinated in vivo and deubiquitinated by Faf, and Lqf and Faf interact physically (Chen, 2002).
To detect Lqf protein levels in developing eyes, an antibody was generated to Lqf. Eye discs were double-labeled with anti-Lqf and antibodies to the endocytic protein Shibire. This shows that Lqf and Shi colocalize at cell membranes: Lqf and Shi are concentrated apically in cells within the morphogenetic furrow, an indentation that marks the onset of differentiation, and also in developing photoreceptors where their membranes meet. Similar results were obtained with antibodies to two other endocytic proteins (Dap160 and alpha-Adaptin [alpha-Ada]), and with phalloidin, which labels f-actin at cell membranes (Chen, 2002).
One prediction of the hypothesis that Faf activity prevents the degradation of Lqf is that in the developing eyes (larval eye discs) of faf null mutant flies, there should be less Lqf protein than in wild-type eyes. It was expected there would be less Lqf protein, as opposed to no Lqf protein, because the lqf null mutant eye phenotype is much more severe than the faf null mutant eye phenotype (Chen, 2002).
To test whether the level of Lqf is affected by faf+ gene activity, first the levels of Lqf were compared in adjacent groups of faf+ and faf- cells in the eye disc, using confocal microscopy. Clones of homozygous faf- cells were generated in faf+/faf- heterozygous eye discs, marked by the absence of ß-galactosidase (ß-gal) expression. The eye discs containing clones were triple-labeled with antibodies to ß-gal (to outline the clones), to Lqf (to detect the level of Lqf protein), and to Shi (as a negative control). It was found that throughout the eye disc, the level of Lqf protein, reflected in the strength of the signal from antibody labeling, is lower within the faf- clones than in the faf+/faf- heterozygous cells surrounding them. In contrast, the levels of Shi protein are the same within and outside the clone boundaries (Chen, 2002).
To quantify the difference in Lqf protein levels in faf+ and faf- cells, the levels of Lqf were assayed in eye disc protein extracts prepared from wild-type and faf- flies in Western blot experiments. Homozygotes for two different mutant faf alleles that behave genetically as strong loss-of-function mutations were used: fafBX4 is an inversion that makes no functional Faf protein, and fafFO8 encodes an Faf protein with histidine residue 1986, which is critical for DUB catalytic activity, changed to tyrosine. There is two- to three-fold less Lqf in eye disc protein extracts of faf- homozygotes than in wild-type extracts. faf+ transgene restores function back to faf- flies. The transgene containing faf+ genomic DNA, which when introduced into faf- homozygotes complements the mutant eye phenotype, results in a two- to three-fold increase in Lqf protein level in eye disc extracts. A nearly identical transgene that fails to complement the faf- mutant phenotype because it has a point mutation in the codon for cysteine 1677, which is critical to the DUB activity of Faf, fails also to increase the level of Lqf protein in eye disc extracts. It is concluded that faf+ activity results in an increase in the level of Lqf protein (Chen, 2002).
A second prediction of the model wherein Faf prevents proteolysis of Lqf by deubiquitinating it, is that there should be Lqf protein linked to Ub chains present in eye discs. Ubiquitinated proteins are usually detected on Western blots as ladders of protein bands of higher molecular weight than the protein in question, in increments of ~8 kD; each 'rung' on the ladder represents a protein species with a Ub chain that is one Ub residue longer than the previous rung. Proteins with Ub chains are rapidly degraded, and thus difficult to detect; usually, inhibition of proteasome and/or DUB activity is required to detect them. It this study, inhibition of the DUB activity of Faf, genetically, stabilizes ubiquitinated forms of Lqf (Chen, 2002).
It is concluded that in eye discs, Lqf is ubiquitinated, and subsequently either deubiquitinated by Faf or degraded. The observation that considerable amounts of nonubiquitinated Lqf protein remain in faf- eye discs indicates either that only a fraction of the Lqf protein in the eye disc is ubiquitinated, and/or that DUBs other than Faf also deubiquitinate some Lqf protein (Chen, 2002).
A third prediction of the model wherein Lqf is the substrate of Faf is that the proteins should, either directly or indirectly, interact. Anti-Lqf was used to immunoprecipitate Lqf from protein extracts prepared from embryos, and tested for the presence of Faf in the immunoprecipitates on Western blots. Embryos were used because sufficient protein could not be obtained from eye discs. In addition, to facilitate detection of Faf, the embryos were transformed with a P{hs-myc-faf+} transgene, which expresses a fully functional, myc-tagged Faf protein upon heat shock, that can be detected on Western blots with anti-myc. myc-Faf was detected in the anti-Lqf immunoprecipitate of the protein extract from heat-shocked transformant embryos (Chen, 2002).
It is concluded that myc-Faf and endogenous Lqf proteins interact physically in Drosophila embryos. Bacterially produced or in vitro translated partial Faf and full-length Lqf proteins do not bind to each other in GST pull-down assays. One possible explanation is that only full-length Faf can bind to Lqf in these assays. Alternatively, Faf and Lqf may require other proteins for their interaction (Chen, 2002).
These experiments provide critical biochemical evidence for a model in which a DUB called Faf specifically deubiquitinates Lqf protein, thereby preventing its proteolysis. There is less Lqf protein in the developing eye in the absence of catalytically functional Faf protein, that Lqf is ubiquitinated and subsequently deubiquitinated by Faf, and that Faf and Lqf interact physically. Taken together with previous genetic evidence that provides strong support for the model, it is concluded that Faf is a substrate-specific regulator of ubiquitination, a novel function for a DUB (Chen, 2002).
The eyes of faf null or lqf hypomorphic mutants have more than the normal complement of eight photoreceptor cells in each facet, owing to the failure of a cell communication pathway early in eye development. The Faf/Lqf interaction is essential in only a small number of cells in the eye disc, which must be particularly sensitive to the levels of Lqf, and in these cells, Lqf presumably controls the frequency or specificity of endocytosis. Although the precise mechanism of epsin function is unknown, vertebrate epsin binds to the endocytosis complex and also to PIP2 at the cell membrane, and is required for endocytosis (Chen, 1998; Itoh, 2001). Apparently, appropriate endocytosis in this small group of cells is essential for successful communication with their neighbors; increased Lqf levels either enables these cells to send a signal to their neighbors that inhibits neural determination, or else prevents them from sending their neighbors a positive differentiation signal (Chen, 2002).
Through a variety of mechanisms, endocytosis is proposed to regulate ligand/receptor interactions during development. How Lqf and endocytosis regulate faf+-dependent cell signaling remains to be determined. Since faf has vertebrate homologs, this mode of regulation is likely to be conserved. The finding that Lqf is the key substrate of Faf in the Drosophila eye shows not only that a DUB can regulate ubiquitination and thus proteolysis, but also that an endocytosis complex protein can be a target for the control of a cell communication event critical to cell determination (Chen, 2002).
Ligands of the Delta/Serrate/Lag2 (DSL) family must normally be endocytosed in signal-sending cells to activate Notch in signal-receiving cells. DSL internalization and signaling are promoted in zebrafish and Drosophila, respectively, by the ubiquitin ligases Mind-bomb (Mib) and Neuralized (Neur). DSL signaling activity also depends on Epsin (Liquid facets), a conserved endocytic adaptor thought to target mono-ubiquitinated membrane proteins for internalization. Evidence is presented that the Drosophila ortholog of Mib (Dmib) is required for ubiquitination and signaling activity of DSL ligands in cells that normally do not express Neur, and can be functionally replaced by ectopically expressed Neur. Furthermore, both Dmib and Epsin are required in these cells for some of the endocytic events that internalize DSL ligands, and the two Drosophila DSL ligands Delta and Serrate differ in their utilization of these Dmib- and Epsin-dependent pathways: most Serrate is endocytosed via the actions of Dmib and Epsin, whereas most Delta enters by other pathways. Nevertheless, only those Serrate and Delta proteins that are internalized via the action of Dmib and Epsin can signal. These results support and extend the proposal that mono-ubiquitination of DSL ligands allows them to gain access to a select, Epsin-dependent, endocytic pathway that they must normally enter to activate Notch (Wang, 2005).
To date, two E3 ubiquitin ligases have been implicated in DSL signaling: zebrafish Mind-bomb (Mib) and Drosophila Neuralized (Neur). Both proteins have been shown to promote DSL ubiquitination, endocytosis and signaling. Moreover, loss-of-function mutations in zebrafish mib and Drosophila neur cause essentially the same hallmark phenotype exemplifying a failure of DSL-signaling in their respective organisms; namely, a dramatic hyperplasia of the embryonic nervous system at the expense of the epidermis. These observations have led to the suggestion that zebrafish Mib and Drosophila Neur are functional homologs. Yet, the two proteins show only limited sequence homology; moreover they appear to be members of distinct Mib and Neur ubiquitin ligase families, each having true orthologs in both vertebrate and invertebrate genomes. As a consequence, the relative roles of Mib and Neur are not known in any animal system and this uncertainty complicates the use of mutations in these genes to assay the role of ubiquitination in DSL endocytosis and signaling (Wang, 2005).
Using newly isolated mutations in dmib, evidence has been found that Dmib and Neur constitute functionally related ubiquitin ligases that are normally required for DSL signaling in different developmental contexts. In the developing Drosophila wing disc, Dmib is required for inductive signaling across the D-V compartment boundary, as well as for the refinement of wing vein primordia, both contexts in which Neur is normally not required or expressed. However, Dmib plays only a modest role in specifying sensory organ precursor (SOP) cells, and little or no role in the subsequent segregation of distinct cell types that form each sensory organ. Instead, Neur appears to provide the essential ubiquitin ligase activity required for DSL signaling in these latter two contexts. Similarly, in the embryo, where Neur is required for most DSL signaling events, Dmib has little or no apparent role. Indeed, embryos devoid of Dmib activity hatch as viable first instar larvae; moreover they develop into pharate adults that show only a limited subset of Notch-related mutant phenotypes, each of which appears to reflect the failure of a particular DSL signaling event that does not, normally, depend on Neur. Thus, it is inferred that Dmib and Neur share a common ubiquitin ligase activity that is essential for DSL ligands to signal (Wang, 2005).
Le Borne (2005) has published similar findings indicating a role for Dmib in DSL signaling, and the capacity of ectopic Neur to substitute for Dmib during wing development. The results differ, however, in that this analysis of clones of dmib- cells that express Ser, Dl, or the DlR+ chimera appears to indicate an absolute requirement for Dmib in sending both Drosophila DSL signals, Dl and Ser. By contrast, Le Borgne interprets his data as evidence for a regulatory rather than an obligatory role of Dmib in sending DSL signals, as well as for a lesser role of Dmib in Dl signaling compared with Ser signaling. Differences in experimental design, particularly in the means used to define or infer the identity of DSL signaling and Dmib-deficient cells, could account for the different conclusions reached (Wang, 2005).
It is noted that if Mib and Neur ligases have overlapping molecular functions in all animal systems, as they have in Drosophila, there is no compelling reason why the ligases would need to be deployed in the same way in different animal species. Instead, any given DSL-signaling process might depend on Mib in one animal system but on Neur in another, as appears to be the case for neurogenesis in zebrafish and Drosophila (Wang, 2005).
In contrast to the selective requirement for Dmib and Neur in overlapping subsets of DSL signaling contexts, Epsin is required for most or all DSL signaling events. This difference is expected if ubiquitination of DSL ligands by either Dmib or Neur is normally a prerequisite for Epsin-mediated endocytosis, and hence for signaling activity (Wang, 2005).
Do Dmib and Neur directly bind and ubiquitinate DSL ligands and thereby confer signaling activity by targeting them for Epsin-mediated endocytosis? Although ectopic Dmib and Neur activity are both associated with enhanced DSL ubiquitination, endocytosis and signaling, there is still no compelling evidence that either ligase directly binds and ubiquitinates DSL proteins, or that Dmib/Neur-dependent ubiquitination of DSL ligands confers signalng activity. However, this study shows that the obligate requirement for Dmib for Dl-signaling by wing cells can be bypassed by replacing the cytosolic domain of Dl with a random peptide, R+, that may serve as the substrate for ubiquitination by an unrelated ubiquitin ligase. This result provides in vivo evidence that Dmib/Neur-dependent ubiquitination of DSL ligands is normally essential to confer signaling activity. Moreover, the failure of the chimeric DlR+ ligand to bypass the requirment for Epsin, supports the interpretation that ubiquitination of DSL ligands confers signaling activity because it targets them for Epsin-mediated endocytosis (Wang, 2005).
During wing development, Ser and Dl both serve as unidirectional signals that specify the 'border' cell fate in cells across the D-V compartment boundary, and both are found to be equally dependent on Dmib and Epsin function for signaling activity. However, the two ligands differ in the extent to which they accumulate on the cell surface, and to which they are cleared from the surface as a consequence of Dmib and Epsin activity. Specifically, most Ser accumulates in cytosolic puncta rather than on the cell surface, whereas the reverse is the case for Dl. Furthermore, removing either Dmib or Epsin activity results in a dramatic and abnormal retention of Ser on the cell surface, whereas it has no detectable effect on the surface accumulation of Dl. Similar results for Dmib were also obtained by Le Borgne (2005). Thus, it appears that most Ser is efficiently cleared from the cell surface by the actions of Dmib and Epsin, whereas most Dl remains on the cell surface, irrespective of Dmib and Epsin activity. This unexpected difference provides two insights (Wang, 2005).
(1) In a previous analysis of the role of Epsin, focus was placed almost exclusively on Dl endocytosis and signaling and failed to obtain direct evidence that Epsin is required for normal DSL endocytosis, despite the obligate role for Epsin in sending both Dl and Ser signals. Instead, such a requirement could be detected only in experiments in which surface clearance of over-expressed Dl was abnormally enhanced by ectopically co-expressing Neur, or could only be inferred from experiments in which the requirement for Epsin was bypassed by replacing the cytosolic domain of Dl with the well-characterized endocytic recycling signal from the mammalian low density lipoprotein (LDL) receptor. By contrast, the different endocytic behavior of Ser has now allowed direct evidence to be obtained that Dmib and Epsin are both required for normal DSL endocytosis (Wang, 2005).
(2) It was found that even though bulk endocytosis of Ser depends on both Dmib and Epsin activity, neither requirement appears absolute. Instead, the accumulation of Ser can still be detected in cytosolic puncta in both Dmib- and Epsin-deficient cells. Moreover, a difference is detected in the abnormal cell surface accumulation of Ser in Dmib-deficient versus Epsin-deficient cells; significantly more Ser appears to accumulate in the absence of Dmib than in the absence of Epsin. It is inferred that both Dl and Ser are normally internalized by multiple endocytic pathways, only some of which depend on ubiquitination of the ligand, and only a subset of these that depends on Epsin. However, the two ligands normally utilize these pathways to different extents, most Ser being internalized by ubiquitin- and Epsin-dependent pathways, and most Dl being internalized by alternative pathways. It is presumed that this difference reflects the presence of different constellations of internalization signals in the two ligands, especially the presence of signals in Delta, but not Ser, that target the great majority of the protein for internalization pathways that do not depend on ubiquitination or Epsin. Nevertheless, only those molecules of Ser and Dl that are targeted by ubiquitination to enter the Epsin-dependent pathway have the capacity to activate Notch; all other routes of entry that are normally available appear to be non-productive in terms of signaling. These results reinforce previous evidence that endocytosis of DSL ligands, per se, is not sufficient to confer signaling activity; instead, DSL ligands must normally be internalized via the action of Epsin to signal (Wang, 2005).
Why must DSL ligands normally be internalized by an Epsin-dependent endocytic mechanism to activate Notch? Two general classes of explanation are considered. In the first, Epsin confers signaling activity by regulating an early event in DSL endocytosis that occurs before internalization. For example, Epsin might cluster DSL ligands in a particular way or recruit them to a select subset of coated pits or other endocytic specializations. Alternatively, Epsin-mediated invagination of these structures might control the physical tension across the ligand/receptor bridge linking the sending and receiving cell, creating a sufficiently strong or special mechanical stress necessary to induce Notch cleavage or ectodomain shedding. In the second class of models, Epsin acts by regulating a later event in DSL endocytosis that occurs after internalization. For example, Epsin might direct, or accompany, DSL proteins into a particular recycling pathway that is essential to convert or repackage them into ligands that can activate Notch upon return to the cell surface. In both cases, internalization of DSL ligands via the other endocytic routes normally available to them would not provide the necessary conditions, even in the extreme case of Dl, which appears to be internalized primarily by these other pathways (Wang, 2005).
The present results do not distinguish between these models. However, recent studies of Epsin-dependent endocytosis in mammalian tissue culture cells suggest that Epsin may direct cargo proteins to different endocytic specializations or pathways, depending on their state of ubiquitination. They also suggest that interactions between Epsin and components of the core Clathrin endocytic machinery normally regulate where and how Epsin internalizes target proteins. Both properties might govern how DSL proteins are internalized, allowing the ligands to gain access to the select endocytic pathway they need to enter to activate Notch (Wang, 2005).
Endocytosis regulates Notch signaling in both signaling and receiving cells. A puzzling observation is that endocytosis of transmembrane ligand by the signaling cells is required for Notch activation in adjacent receiving cells. A key to understanding why signaling depends on ligand endocytosis lies in identifying and understanding the functions of crucial endocytic proteins. One such protein is Epsin (Drosophila Liquid facets), an endocytic factor first identified in vertebrate cells. This study shows in Drosophila that Auxilin, an endocytic factor that regulates Clathrin dynamics, is also essential for Notch signaling. Auxilin, a co-factor for the ATPase Hsc70, brings Hsc70 to Clathrin cages. Hsc70/Auxilin functions in vesicle scission and also in uncoating Clathrin-coated vesicles. Like Epsin, Auxilin is required in Notch signaling cells for ligand internalization and signaling. Results of several experiments suggest that the crucial role of Auxilin in signaling is, at least in part, the generation of free Clathrin. These observations in the light of current models for the role of Epsin in ligand endocytosis and the role of ligand endocytosis in Notch signaling (Eun, 2008).
A role for Clathrin in Notch signaling cells was originally inferred from the observation that Chc mutants are strong dominant enhancers of lqf hypomorphs. Since Epsin has both Ubiquitin- and Clathrin-binding motifs, and also binds the plasma membrane, the simplest scenario imaginable for Clathrin and Epsin function in Delta internalization is for Epsin to act as a Clathrin adapter that recognizes ubiquitinated Delta, and brings Clathrin to the membrane for CCV formation. However, in light of evidence that Epsin-dependent endocytosis of ubiquitinated transmembrane proteins such as Delta may not occur through formation of CCVs, it has become unclear how to interpret the Chc/lqf genetic interaction. The results presented in this study point to a crucial role for Clathrin in Notch signaling cells. One intriguing possibility is that Delta internalization depends on Clathrin not because Delta is endocytosed in CCVs, but because Clathrin is a positive regulator of Epsin function. More experiments are required to test this idea (Eun, 2008).
Why do tissues that lack Epsin or Auxilin display Delta-like phenotypes, rather than phenotypes indicating failure of many signaling pathways or even cell death? One possibility is that the apparent specificity of both Epsin and Auxilin might simply reflect the usual redundancy of endocytic protein functions, and an unusual dependence of Notch signaling on efficient endocytosis. Alternatively, a special function of Epsin might be crucial to Notch signaling cells. Two kinds of models have been proposed to explain why Notch signaling requires ligand endocytosis by the signaling cells. One idea (the 'pulling model') is that after receptor binding, ligand endocytosis generates mechanical forces that result in cleavage of the Notch intracellular domain (Notch activation), either by exposing the proteolytic cleavage site on the Notch extracellular domain, or by causing the heterodimeric Notch receptor to dissociate. Alternatively, ligand internalization prior to receptor binding might be required to process the ligand endosomally, and recycle it back to the plasma membrane in an activated form (the 'recycling model'). Epsin might generate an environment particularly conducive to either pulling or recycling, and Auxilin might be required specifically by Notch signaling cells because it activates Epsin, perhaps by providing free Clathrin. Alternatively, Auxilin might be needed to provide free Clathrin because Delta is internalized through CCVs. In this case, if Auxilin is required in Notch signaling solely to provide free Clathrin, the implication would be that efficient CCV uncoating is not important for generating uncoated Delta-containing vesicles per se, which are prerequisite for travel through an endosomal recycling pathway. Further understanding of the role of Auxilin in Notch signaling cells might be key to understanding the role of ligand endocytosis (Eun, 2008).
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