Polyclonal antibodies were generated to the full-length (anti-FL-Hrs) and amino-terminal half (aa 1-376, anti-N-Hrs) of the recombinant protein. Western analysis of fly extracts using the anti-FL-Hrs antibody detects a major band of 110 kDa in wild-type animals, whereas no protein is detected in hrs third-instar larvae (L3) or white prepupae (WPP). However, the anti-N-Hrs antibody recognizes a 30 kDa band in mutant animals in addition to the 110 kDa band in wild-type animals, suggesting that a 270 amino acid truncated protein is expressed in mutant animals. Furthermore, the presence of full-length Hrs protein in late stage 17 Df embryos suggests that maternally deposited Hrs protein is very stable and may compensate for the loss of zygotic Hrs in embryonic development. Indeed, embryos produced by mothers homozygous for hrs in their germline cells (maternal knockout or mKO) lack full-length Hrs protein and die early in embryogenesis. Thus, the effects of loss of Hrs function may be analyzed in zygotic mutant third-instar larvae/early pupae or in germline mutant embryos (Lloyd, 2002).
Analysis of the protein expression of Hrs suggests that Hrs is ubiquitously expressed. To determine the subcellular localization of Hrs, expression was examined in garland cells and muscle cells of third-instar larvae. Anti-Hrs labels vesicles enriched in perinuclear regions of muscle cells, whereas labeled vesicles are predominantly in the periphery of garland cells. These staining patterns are specific; they are not seen in hrs mutant cells. Interestingly, overexpression of hrs leads to an enlargement or accumulation of Hrs-positive vesicles and a reduction in cell size (Lloyd, 2002).
Both Hrs antibodies also label type I synaptic boutons of the neuromuscular junction (NMJ). There is some colocalization of Hrs with the synaptic vesicle (SV) marker Synaptotagmin, but most staining appears to be outside SV-rich regions. However, electrophysiological analysis of wild-type and mutant neuromuscular junctions suggests that Hrs does not play an important role in regulating synaptic vesicle exocytosis. Furthermore, Hrs is not enriched in the synapse-rich neuropil of the larval brain, even when overexpressed in neurons using the elav-GAL4 driver (Lloyd, 2002),
To determine if Hrs functions in endocytosis, trafficking of internalized tracers was investigated in third-instar larval garland cells, large cells with a high rate of fluid-phase endocytosis. Wild-type garland cells show strong labeling of peripheral vesicles after a 5 min incubation with avidin-Cy3, indicating rapid internalization of dye into endosomes. Mutant cells are much larger than wild-type cells but show strong labeling of peripheral vesicles, suggesting that dye internalization is not significantly impaired. However, many labeled vesicles (endosomes) in mutant cells are much larger than those observed in wild-type cells. Furthermore, analysis of lysosomal markers suggest that while lysosomes are reduced in size in hrs mutant cells, smaller endosomes are capable of delivering avidin to low pH compartments at a rate similar to wild-type cells (Lloyd, 2002).
Next, hrs mutant endosomes were analyzed using transmission electron microscopy (TEM). Garland cells were incubated with HRP for 5 min, fixed, and sectioned for TEM. In wild-type garland cells, HRP labels the lumen and internal membrane of peripherally located endosomes. In hrs mutant larvae, mutant endosomes are dramatically enlarged, but do not show significant HRP labeling, despite their ability to internalize avidin dye. Rather, in mutant cells, HRP labels a vast tubulo-vesicular network at the periphery of mutant cells, which is not seen in wild-type cells. Finally, endosomes in wild-type garland cells undergo invagination of their limiting membrane to eventually collapse upon themselves. In hrs garland cells, there is a strong reduction in the relative number of invaginated endosomes (5-fold, P = 0.008) and collapsed endosomes (10-fold, P = 0.003). These data suggest that endosomes are enlarged in hrs mutant cells due to an inability of endosomes to invaginate their limiting membrane (Lloyd, 2002).
Inward budding of endosome membrane is believed to be the first step of multivesicular body (MVB) formation. To determine if MVB formation is impaired in hrs mutant animals, electron microscopy was performed at the NMJ. In wild-type synapses, large, classical MVBs are occasionally observed, and they are believed to be endosomal intermediates containing synaptic vesicle proteins destined to be delivered to somatic lysosomes. Much more frequently, though, 60-120 nm vesicles were observe that also appear to contain small internal vesicles. Remarkably, in hrs mutant synapses, there is a 5-fold reduction in the number of these small MVBs. These data suggest that Hrs regulate formation of MVBs at the synapse (Lloyd, 2002).
One proposed function of multivesicular bodies is to partition transmembrane proteins and lipids destined for delivery to the lysosome from those destined to be recycled back to the surface of the cell. While most cell surface proteins are recycled from endosomes, activated TKRs such as the EGFR are trafficked inside MVBs for degradation in the lysosome. Because Hrs is phosphorylated in response to TKR activation (Komada, 1995), the possibility that Hrs regulates TKR degradation and signaling was investigated in Drosophila (Lloyd, 2002).
The first TKR pathway analyzed was the Torso pathway, which functions very early in development to pattern the embryonic termini. The Torso receptor is present throughout the surface of the early embryo, but it binds ligand only at the poles. Ligand binding of the Torso receptor triggers the ras/MAPK cascade leading to the phosphorylation of ERK/MAPK. A diphospho-ERK (dpMAPK)-specific antibody has been used to investigate TKR signaling in the early embryo. In wild-type embryos, terminal dpMAPK staining initiates approximately 2 hr after egg laying (AEL), peaks about 30 min later, and then rapidly terminates during the initiation of gastrulation. In cellular blastoderm hrs maternal knockout (mKO) embryos, dpMAPK staining is enhanced and spatially broadened when compared to wild-type embryos. Furthermore, dpMAPK expression remains elevated at the anterior and posterior termini during early gastrulation, when compared to wild-type. Hence, Torso signaling is both spatially broadened and temporally prolonged (Lloyd, 2002).
MAPK activation induces the expression of the terminal gap genes huckebein (hkb) and tailless (tll), and the pattern of expression of these genes can be used as a quantitative readout for Torso receptor activation. In mutant embryos, both anterior and posterior domains of hkb are significantly expanded by 23% and 50%, respectively. Similarly, the anterior stripe of tll is moved posteriorly by 50%, and the posterior stripe is expanded by 25%. Thus, these data confirm that Torso signaling is enhanced and broadened both spatially and temporally in mutant embryos and demonstrate that Hrs negatively regulates signaling of the Torso TKR pathway (Lloyd, 2002).
Next, whether an increase in Torso signaling was due to the failure of receptor degradation was investigated. Total levels of the Torso receptor in early (0-4 hr) embryos are similar between wild-type and hrs mKO embryos. However, a 56 kDa band corresponding to the cytoplasmic domain of Torso (500 aa) is prevalent in mutant animals but barely detectable in wild-type embryos. These data suggest that a failure to degrade the active cytoplasmic portion of the receptor may underlie the increased Torso signaling observed in early embryos. To further examine degradation of the Torso receptor, the kinetics of receptor protein expression were examined in wild-type and mutant embryos. In wild-type embryos, Torso protein expression is maximal just prior to receptor activation (1-2 hr AEL) and is rapidly downregulated such that it is barely detectable by 5 hr AEL. In hrs mKO embryos, in contrast, Torso protein levels fail to decline until 9-10 hr AEL, at which time zygotic expression of hrs is abundant. These data demonstrate that hrs is essential for Torso TKR degradation (Lloyd, 2002).
During early gastrulation, hrs mKO embryos display numerous morphological defects. Some mKO embryos fail to complete posterior terminal cellularization, and their yolk appears to be extruded posteriorly during gastrulation. During germ band elongation, most hrs mKO embryos undergo highly abnormal development and exhibit aberrant folding or twisting. The vast majority of mutant embryos arrest morphological development prior to germband retraction, and few secrete cuticle. These phenotypes suggest that hrs may be required to regulate multiple early embryonic signaling pathways (Lloyd, 2002).
Next, the effects of loss of Hrs function on the Egfr pathway were examined. Egfr signaling is required for a wide variety of cell fate decisions throughout Drosophila development. Secretion of the Egfr ligand, Spitz, in the ventral midline leads to graded activation of Egfr and the ras/MAPK cascade in the ventral ectoderm. dpMAPK staining is present in the ventral neuroectoderm and dorsal cephalic regions of the gastrulating embryo, and this staining pattern requires Egfr activation. dpMAPK staining in these regions is expanded in hrs mKO embryos, demonstrating that Egfr signaling is enhanced in the absence of Hrs (Lloyd, 2002).
Next to be examined was whether enhanced MAPK signaling in the ventral ectoderm results in an expansion of ventral fate. The transcription factor ventral nervous system defective (vnd) is a primary target of Egfr signaling in the ventral ectoderm, and genetic manipulations leading to enhanced Egfr signaling lead to an expansion of cells expressing vnd. In hrs mKO embryos, there is an expansion of the number of cells expressing Vnd from 1-2 in wild-type to 3-4 in the mutant. Furthermore, Fas III expression, known to be a downstream target of Egfr activation, is upregulated in hrs mKO embryos. Thus, a dorsal expansion of cells expressing dpMAPK and Vnd and an upregulation of Fas III expression suggest that Egfr signaling is enhanced in hrs mKO embryos (Lloyd, 2002).
To determine if enhanced Egfr signaling is due to increased receptor activity, tyrosine phosphorylation of the receptor was examined by immunoprecipitating Egfr from pupal lysates and Western blotting with a phosphotyrosine-specific antibody. Levels of tyrosine-phosphorylated receptor are increased 2- to 3-fold in hrs/hrs and hrs/Df animals when compared to wild-type (+/+) or heterozygous (hrs/+) animals. When these same membranes are stripped and reprobed with anti-Egfr antibody, total levels of the receptor are found to be decreased approximately 2-fold in hrs/hrs and hrs/Df mutant animals. Together, these data indicate that the relative levels of the active form of Egfr are increased approximately 5-fold in hrs mutant animals. Finally, levels of other ubiquitously expressed surface transmembrane proteins examined at this stage are unchanged or slightly decreased in mutant animals, suggesting that there is not a general defect in turnover of plasma membrane proteins (Lloyd, 2002).
Proteins that constitute the endosomal sorting complex required for transport (ESCRT) are necessary for the sorting of proteins into multivesicular bodies (MVBs) and the budding of several enveloped viruses, including HIV-1. The first of these complexes, ESCRT-I, consists of three proteins: Vps28p, Vps37p, and Vps23p or Tsg101 (see Drosophila Tsg 101) in mammals. A mutation was characterized in the Drosophila homolog of vps28. The dVps28 gene is essential: homozygous mutants die at the transition from the first to second instar. Removal of maternally contributed dVps28 causes early embryonic lethality. In such embryos lacking dVps28, several processes that require the actin cytoskeleton are perturbed, including axial migration of nuclei, formation of transient furrows during cortical divisions in syncytial embryos, and the subsequent cellularization. Defects in actin cytoskeleton organization also become apparent during sperm individualization in dVps28 mutant testis. Because dVps28 mutant cells contained MVBs, these defects are unlikely to be a secondary consequence of disrupted MVB formation and suggest an interaction between the actin cytoskeleton and endosomal membranes in Drosophila embryos earlier than previously appreciated (Sevrioukov, 2005).
In the Drosophila genome, a single gene, CG12770, exhibits significant homology to the yeast and mammalian Vps28 proteins. The cDNA GH04443 is derived from this locus and encodes a predicted protein of 210 amino acids that is 62% and 35% identical to its human (hVPS28) and yeast (ScVPS28) counterparts, respectively. There are no similarities to other protein sequence motifs in the database. An antibody raised against dVps28 recognizes a protein of the expected size that is widely expressed during Drosophila development and also in cultured Drosophila S2 cells. In S2 cells as well as in cells of the eye disc and in isolated spermatocysts, dVps28 protein was uniformly distributed throughout the cytosol with no obvious enrichment in any organelle (Sevrioukov, 2005).
To test whether the homology of Vps28 proteins extends to their biochemical activity, its binding to Vps23p/Tsg101 was examined. The Drosophila homolog dTsg101 is encoded by cDNA GH09529. Because antibodies are not yet available against endogenous dTsg101 protein, epitope-tagged versions of dTsg101 and dVps28 were coexpressed to test their interaction in S2 cells. Immunoprecipitation of dVps28 from S2 cells resulted in the coprecipitation of expressed HA-dTsg101, which was increased after coexpression of Myc-dVps28. These results indicated that, like its yeast and mammalian orthologs, dVps28 binds specifically to dTsg101 (Sevrioukov, 2005).
Consistent with ESCRT's conserved function from yeast to mammalian cells, loss of dVps28 function causes morphological changes in MVBs and developmental defects in the compound eye that indicate a subtle misregulation of cell signaling molecules. However, for the ligands and receptors, no significant changes were detected in their cell surface levels or their delivery to lysosomes in dVps28 cells, indicating that any changes were too subtle to be detected by the immunofluorescence methods used (Sevrioukov, 2005).
One possible explanation for this observation is that some Vps28 functions are partially fulfilled by another protein. It is not likely that this hypothetical protein is similar to Vps28p in sequence because no another Vps28-like molecule in the completed genome sequences of Drosophila melanogaster. Another possibility is that the dVps28l(2)k16503 allele does not completely inactivate dVps28 function. Although this possibility cannot be ruled out, it is unlikely for two reasons: (1) the dVps28l(2)k16503 allele is a strong mutation that causes lethality early in development at the transition from first to second instar, much earlier than a null mutation in the hrs gene, which regulates the sorting of receptors into MVBs and (2) the lethal phase and the loss of dVps28 protein were indistinguishable between larvae homozygous for dVps28l(2)k16503 and those that were hemizygous over a deficiency of the region. This indicates that the dVps28l(2)k16503 allele removes most if not all of dVps28 function (Sevrioukov, 2005).
Another possibility for the relatively mild phenotypes is the perdurance of dVps28 protein. This is suggested by the dramatic phenotypes after removal of the maternal contribution in mKO dVps28 embryos: many remained unfertilized and the remaining embryos were arrested in their development before reaching the cellular blastoderm. It is interesting to compare this phenotype to that of embryos lacking any maternal Hrs contribution. Hrs binds to mono-ubiquitinated membrane proteins, whose sorting into MVBs is thought to be mediated by Hrs binding to ESCRT-1. Consistent with this notion, mKO hrs embryos exhibit a reduced down-regulation of cell surface receptors and a resulting enhanced activation of the MAP kinase pathway. Importantly, these defects were observed after cellularization had been completed and during gastrulation. The finding that mKO dVps28 embryos exhibit major defects before completion of cellularization indicates functions of dVps28 in addition to the down-regulation of membrane proteins mediated by the interaction of ESCRT-I with Hrs (Sevrioukov, 2005).
Surprisingly, the earliest defect found in fertilized eggs was an uneven distribution of nuclei. In Drosophila, the first 13 nuclear divisions occur without accompanying cell divisions. The first five of these syncytial divisions occur close to the center of the embryo, with nuclei subsequently spreading out along the anterior-posterior axis. This process, referred to as axial expansion, depends on the function of the actin cytoskeleton. In mKO dVps28 embryos, this process often is disorganized resulting in an uneven distribution of nuclei (Sevrioukov, 2005).
Other dVps28 phenotypes emerged during cellularization. After axial expansion, the nuclei move to the embryo's cortex where the last four of the syncytial nuclear divisions occur. The resulting nuclei are surrounded by invaginating membranes in a process that requires the actin-myosin network. At this stage, two defects were evident in embryos lacking dVps28. Many of the cells that formed were droplet shaped instead of the usual cuboidal shape. The actin cytoskeleton is required for normal cellularization and interfering with is function, by cytochalasin, or altering the activity of the Rho or Cdc42 GTPases, interferes with normal cellularization. Furthermore pole cells, usually the first cells to form, were absent in most embryos. The lack of pole cells has previously been observed in embryos in which axial expansion is perturbed upon interference with the actin cytoskeleton (Sevrioukov, 2005).
All of these early phenotypes are consistent with a role of dVps28 in directly or indirectly organizing the actin cytoskeleton. A role for endosomes in actin remodeling during cellularization has previously been established in embryos mutant for nuf or rab11. The small GTPase Rab11 localizes to recycling endosomes, and Nuf is a homolog of Arfo2 that directly binds Rab11 and acts in recycling endosomes. Mutants eliminating either of these proteins cause gaps in which actin fails to be recruited to the furrows during cortical nuclear divisions, similar to the observations in mKO dVps28 embryos. In all three of these mutants, the actin defects may be a consequence of the failure to recruit Discontinuous actin hexagon (Dah: a membrane-associated protein that localizes to invaginating furrows in syncytial blastoderm embryos and during cellularization) to invaginating furrows. Dah has significant similarity to dystrobrevin and dystrophin. Dystrophin plays a critical role in anchoring the actin cytoskeleton to membranes, and consistent with a similar function for Dah, embryos lacking maternally contributed Dah fail to properly assemble the actin cytoskeleton at furrows (Sevrioukov, 2005 and references therein).
A subset of the defects in rab11 mutant embryos has been linked to a requirement for trafficking through the recycling endosome during cellularization. Consistent with this notion, defects in rab11 and nuf embryos only become apparent during cortical divisions. In mKO dVps28 embryos, by contrast, defects were detected in the distribution of nuclei, long before cellularization initiates. This indicates a function of dVps28 in actin remodeling independent of recycling to the cell surface and independent of Rab11 and Nuf. This is consistent with the finding that in mKO dVps28 embryos, recycling endosomes are not affected, as judged by Rab11 and Nuf localization, suggesting that dVps28 is required for furrow localization of Dah down-stream or in parallel to Rab11 and Nuf's function in recycling endosomes. Such a model is difficult to reconcile with the canonical function of Vps28 as an ESCRT-1 subunit involved in targeting proteins into the interior vesicles of late endosomes (Sevrioukov, 2005 and references therein).
It is unlikely that Dah mediates all effects of dVps28 on the actin cytoskeleton. No defects are observed in embryos lacking Dah before cycle 10, long after defects have become apparent in mKO dVps28 embryos. Furthermore, Dah is not required during spermatogenesis, another developmental context in which effects of dVps28 on the organization of the actin cytoskeleton were observed. Spermatogenesis in dVps28 mutant testis progresses until bundles of 64 syncytial spermatids are formed. These spermatids are separated by a process called individualization that requires complex membrane rearrangements. At the site of these rearrangements, syncytial membrane, cytoplasm, and vesicles accumulate in the cystic bulge. In wild-type testis, an actin-dependent process drives the cystic bulge away from the 64 spermatid nuclei toward the distal end. The loss of synchrony in the movement of the cystic bulge is the first detectable defect during spermatogenesis in dVps28 mutant testis. Because dah mutants have no phenotype in males, these results indicate an independent connection between dVps28 function and the actin cytoskeleton (Sevrioukov, 2005 and references therein).
Actin acts at many stages in the endocytic pathway. In yeast, the initial internalization step requires actin polymerization, and in mammalian cells, early endosomes move on the tip of actin tails. Additionally, late endocytic organelles require the actin cytoskeleton for fusion in yeast and mammalian cells. Furthermore, a screen of the yeast genome for mutations interfering with protein sorting to the vacuole identified several regulators of the actin cytoskeleton (Sevrioukov, 2005 and references therein).
Importantly, several of these mutants identified on the basis of a vacuolar sorting phenotype also exhibited defects in the organization of the actin cytoskeleton. For example, aberrant actin patches and a reduction of actin cables were observed in yeast lacking Vps36p, one of the subunits of the ESCRT-II complex. It will be interesting to see whether a similar functional connection between the actin cytoskeleton and the ESCRT-I complex may underlie the enigmatic phenotypes of Tsg101 mutations in mice that cause cell cycle arrest and early embryonic lethality (Sevrioukov, 2005 and references therein).
During animal development, Wnt/Wingless (Wg) signaling is required for the patterning of multiple tissues. While insufficient signal transduction is detrimental to normal development, ectopic activation of the pathway can be just as devastating. Thus, numerous controls exist to precisely regulate Wg signaling levels. Endocytic trafficking of pathway components has recently been proposed as one such control mechanism. This study characterizes the vesicular trafficking of Wg and its receptors, Arrow and DFrizzled-2 (DFz2), and investigates whether trafficking is important to regulate Wg signaling during dorsoventral patterning of the larval wing. A role for Arrow and DFz2 in Wg internalization has been demonstrated. Subsequently, Wg, Arrow and DFz2 are trafficked through the endocytic pathway to the lysosome, where they are degraded in a hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-dependent manner. Surprisingly, Wg signaling is not attenuated by lysosomal targeting in the wing disc. Rather, it is suggested that signaling is dampened intracellularly at an earlier trafficking step. This is in contrast to patterning of the embryonic epidermis, where lysosomal targeting is required to restrict the range of Wg signaling. Thus, signal modulation by endocytic routing will depend on the tissue to be patterned and the goals during that patterning event (Rives, 2006).
During patterning and growth of the wing imaginal disc, cells along the D/V axis interpret positional information and, hence, their fate, from the concentration of Wg ligand. The graded distribution of Wg, with high levels near the source at the D/V boundary and low levels toward the edges of the wing pouch, is therefore crucial for normal wing development. Lysosomal targeting of Wg and its receptors has been proposed as a mechanism for shaping the Wg gradient and attenuating signal transduction. To address this model, both trafficking to the lysosome and lysosome function was interfered with using genetic and pharmacological means (Rives, 2006).
In Drosophila, the hrs loss of function allele is a valuable tool for interrupting vesicular traffic to the lysosome. Hrs functions in late endosome invagination, a process that separates endocytic cargo to be recycled from cargo destined for the lysosome. Trafficking of the EGFR and Torso RTKs into the late endosome/MVB is an important step in signal attenuation; hrs mutant embryos experience elevated tyrosine kinase signaling due to the persistence of active receptors. Likewise, in the wing disc and the ovarian follicle cell, Hrs is required for downregulation of Tkv levels and dampening of the Dpp signal. Thus, Hrs activity is required to attenuate multiple developmental signals (Rives, 2006).
The fact that RTK and Dpp signaling levels are elevated in hrs mutant cells implies that active receptor complexes continue to signal inside the cell from an endocytic compartment. Although receptor internalization may turn off signaling by preventing ligand-receptor interaction, it is clear that many receptors remain active on endosomal membranes. For instance, activated EGFR can be detected in association with downstream signaling effectors on early endosomes, suggesting that signaling persists after endocytosis. This study reports dramatic intracellular accumulation of Wg, Arrow, and DFz2 in hrs or deep orange (dor) mutant wing discs; dor encodes a yeast VPS homologue required for delivery of vesicular cargo to lysosomes. Similar observations have been made for Wg and for Wg and Arrow. Given this dramatic intracellular accumulation of ligand, receptors, and a signal transducer, Wg signaling levels are expected to be elevated in hrs mutant cells. However, based on antibody stains for three Wg targets, no altered Wg signaling was detected in mutant cells. This was true for large null mutant clones, induced early in development, as well as in discs from larvae bearing a null hrs allele. The attenuation of Wg signaling, therefore, appears to be regulated differently from the attenuation of RTK and Dpp signaling (Rives, 2006).
The data suggest that Wg signaling is attenuated prior to Hrs-mediated lysosomal targeting of the receptor complex. In this case, removal of hrs prevents receptor and ligand degradation but has no bearing on signal output. Following endocytosis, internalized receptor-ligand complexes may be deactivated by physical dissociation in the increasingly acidic environment as they move through the endocytic compartment or by targeting to the lysosome for degradation. A model is favored in which the active Wg receptor complex is attenuated by dissociation earlier in the endocytic pathway; perhaps, this complex is more sensitive to pH levels in early endosomes, whereas, for example, a Dpp receptor complex is only uncoupled at the lower pH of later endosomal compartments (Rives, 2006).
Alternatively, there may be residual lysosomal degradation in hrs cells sufficient to effectively terminate signaling despite the accumulation of Wg, Arrow, and DFz2. It is not certain that Hrs is obligatory in targeting endocytic cargo to the lysosome. Internalized avidin, an endocytic tracer, still localizes to a low pH compartment in hrs mutant garland cells, suggesting that some trafficking to the lysosome continues in the absence of Hrs. Perhaps, this residual trafficking is sufficient to dampen Wg signaling levels but not RTK or Dpp levels (Rives, 2006).
In contrast to the genetic removal of hrs, treatment of wing discs with the lysosomal protease inhibitors chloroquine or NH4Cl leads to expansion of SOPs, a Wg gain-of-function phenotype. While this result agrees with the previous finding that chloroquine-treated embryos generate excess smooth cuticle, indicative of enhanced Wg signaling, it is surprising that disruption of lysosome function can affect signaling. Once internalized, receptors are sorted into inner MVB vesicles, they are presumably sequestered from intracellular effectors and thereby deactivated. If mild bases, such as chloroquine, solely affect lysosomal protease function, a step subsequent to MVB sorting, this should not affect Wg signaling output in embryos or in imaginal discs. As all endocytic compartments maintain an acidic environment that is crucial to their function, it is unlikely that alkalizing agents solely inhibit the lysosome. In a caution to their use, pharmacological reagents such as chloroquine and NH4Cl almost certainly disturb earlier pH-dependent trafficking steps as well, resulting in the accumulation of active receptor complexes. It is hypothesized that chloroquine- and NH4Cl-mediated alkalization prevents the dissociation of Wg from its receptor(s), thereby resulting in prolonged signaling (Rives, 2006).
Consistent with the excess SOP specification in chloroquine- and NH4Cl-treated discs, RNAi knockdown of Rab5 in cultured cells causes an increase in Wg-dependent reporter activation. These findings suggest that Wg signaling is normally attenuated at a trafficking step after internalization from the plasma membrane, but prior to Hrs-mediated lysosomal targeting. Such findings should be interpreted cautiously, however, as S2 cells are reported to be macrophage-like, and, thus, any effects on signaling output in these cells might not compare to that in wing disc cells in vivo. Nevertheless, attempts were made to define more precisely the trafficking step involved by treating cultured S2 cells with Shi dsRNA. So far the results have been ambiguous, since two trials demonstrated increased reporter activation while two other trials exhibited no such increase. Unfortunately, due to the compromised viability of endocytosis-defective cells in the wing disc, the DRab5 or Shi cell culture results could not be varified in vivo. However, in agreement with the data, a recent report shows enhanced Wg signaling, as evidenced by accumulation of the signal transducer Armadillo, in cells expressing a temperature-sensitive dominant negative variant of Shi. The viability issue was circumvented by transiently expressing dominant negative Shi with a 3-h upshift to the non-permissive temperature. Interestingly, no change was observed in Wg target gene expression under these conditions, suggesting that cell viability becomes compromised before such changes can occur (Rives, 2006).
While no evidence was found that lysosomal targeting modulates Wg signal output in the developing wing, it is clear that Wg, Arrow, and DFz2 are trafficked to the lysosome by Hrs. Hrs contains a conserved ubiquitin-interacting motif (UIM) and binds ubiquitin in vitro, suggesting that it regulates MVB sorting via direct interaction with ubiquitinated receptors. Monoubiquitination of cell surface receptors is emerging as an important signal for internalization and lysosomal sorting. It will be of interest to determine whether Arrow, Fz, and DFz2 undergo signaling-dependent monoubiquitination, and whether this has a consequence for Wg signaling output (Rives, 2006).
Signaling ligands are commonly internalized by receptor-mediated endocytosis, during which a ligand–receptor complex accumulates in coated pits on the plasma membrane and enters the cell in clathrin-coated vesicles. In the embryonic epidermis, endocytosis of Wg is thought to be receptor-mediated; expression of DFz2-GPI, which presumably lacks an endocytic signal, binds Wg but does not cause internalization. A similar model is predicted in the wing imaginal disc, where expression of DFz2-GPI stabilizes Wg to a greater extent than full length DFz2, most likely due to an inability to internalize Wg. Consistent with these views, it was found that extracellular Wg accumulates on the surfaces of arrow and fz−dfz2− mutant cells. This striking accumulation cannot be explained by ectopic wg gene expression and likely results from impaired Wg internalization. In support of this conclusion, Wg and Arrow can colocalize in endosomes. It was still possible to detect residual Wg internalization into arrow mutant cells and fz−dfz2− mutant cells. Yet, given the striking excess of extracellular Wg on receptor-deficient cells, a large increase was expected in the number of intracellular Wg puncta if Wg is internalized at a normal rate. This was not observed and led to a suggestion that Arrow, Fz, and DFz2 function as endocytic receptors for Wg. Since Fz does not contain an obvious endocytic signal, it is presumed that Arrow and DFz2 play more prominent roles. The residual intracellular Wg in receptor-deficient cells might be explained by a functional redundancy of Arrow and DFz2 in ligand internalization, in which case an absolute defect could only be observed by producing arrow-dfz2− doubly mutant cells. While this manuscript was in preparation, Piddini (2005) also reported that both DFz2 and Arrow contribute to Wg trafficking and degradation. A model was proposed in which DFz2 is important for Wg binding and internalization, while Arrow targets the Wg-DFz2 complex for degradation in the lysosome (Rives, 2006).
Contrary to hypothesis, recent evidence suggests that the accumulation of extracellular Wg on arrow and fz−dfz2− mutant clones is due to upregulation of the glypican Dally-like protein (Dlp) (Han, 2005). That study also observed an increase in the level of extracellular Wg on arrow and fz−dfz2− mutant clones. However, Wg accumulation was reduced if the mutant cells were compromised for the ability to make HSPGs by additional removal of sulfateless (sfl), an enzyme required for heparan sulfate biosynthesis, or brother of tout-velu (botv), a heparan sulfate copolymerase required for HSPG biosynthesis. This suggests that some of the build-up of extracellular Wg is due to trapping by excess HSPGs, rather than to a defect in endocytic trafficking (Rives, 2006).
In the process of evaluating endocytosis-defective cells for changes in Wg signaling levels, cells were frequently observed undergoing apoptosis. This is not surprising, since endocytosis is an important means for the cell to acquire macromolecules essential for viability as well as to gauge the growth needs of the tissue in which it resides. The results are troubling, though, given the widespread use of shits, DRab5DN and ShiDN in the Drosophila community. Thus, it is necessary to monitor cell viability and assay for expression of control genes when using these reagents in order to draw accurate conclusions about signaling levels (Rives, 2006).
One notable question that was not addressed experimentally is whether endocytosis of Arrow or DFz2 is induced by Wg stimulation or proceeds continuously, independent of ligand. Some evidence for Wg-induced endocytosis of DFz2 has recently been presented (Piddini, 2005). Signal-induced endocytosis is well established, especially for RTK signaling, and plays an important role in controlling signal duration. Constitutive endocytosis and recycling provide a more general means of regulating receptor concentration at the cell surface but may also be used to downregulate signaling by clearing activated receptors, as suggested for the Tkv receptor in the developing wing. Future investigation of this issue will provide insight into the regulation of Wg signaling by endocytosis (Rives, 2006).
Reference names in red indicate recommended papers.
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Devergne, O., Ghiglione, C. and Noselli, S. (2007). The endocytic control of JAK/STAT signalling in Drosophila. J. Cell Sci. 120(Pt 19): 3457-64. Medline abstract: 17855388
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date revised: 17 January 2008
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