spitz
The ventral midline provides the site for Spitz expression and processing. The Drosophila EGF receptor (DER) is activated by secreted Spitz to induce different cell fates in the ventral ectoderm. Processing of the precursor transmembrane Spitz to generate the secreted form is shown to be the limiting event. The ectodermal defects in single minded (sim) mutant embryos, in which the midline fails to develop, suggests that the ventral midline cells contribute to patterning of the ventral ectoderm. The Rhomboid and Star proteins are also expressed and required in the midline. The ectodermal defects of spitz, rhomboid or Star mutant embryos can be rescued by inducing the expression of the respective normal
genes only in the midline cells. Rho and Star thus function non-autonomously, and may be required for the production or processing of the Spitz precursor. Secreted Spitz is the only sim-dependent contribution of the midline to patterning the ectoderm, since the ventral defects observed in sim mutant embryos can be overcome by expression of secreted Spitz in the ectoderm. While ectopic expression of secreted Spitz in the ectoderm or mesoderm gives rise to ventralization of the embryo, increased expression of secreted Spitz in the midline does not lead to alterations in ectoderm patterning. A mechanism for adjustment to variable levels of secreted Spitz emanating from the midline may be provided by Argos, which forms an inhibitory feedback loop for DER activation. The production of secreted Spitz in the midline, may provide a stable source for graded DER activation in the ventral ectoderm (Golembo, 1996).
The formation or suppression of particular structures is a major change occurring in development and evolution. One example of such change is the absence of the seventh abdominal segment (A7) in Drosophila males. This study shows that there is a down-regulation of EGFR activity and fewer histoblasts in the male A7 in early pupae. If this activity is elevated, cell number increases and a small segment develops in the adult. At later pupal stages, the remaining precursors of the A7 are extruded under the epithelium. This extrusion requires the up-regulation of the HLH protein Extramacrochetae and correlates with high levels of spaghetti-squash, the gene encoding the regulatory light chain of the non-muscle myosin II. The Hox gene Abdominal-B controls both the down-regulation of spitz, a ligand of the EGFR pathway, and the up-regulation of extramacrochetae, and also regulates the transcription of the sex-determining gene doublesex. The male Doublesex protein, in turn, controls extramacrochetae and spaghetti-squash expression. In females, the EGFR pathway is also down-regulated in the A7 but extramacrochetae and spaghetti-squash are not up-regulated and extrusion of precursor cells is almost absent. These results show the complex orchestration of cellular and genetic events that lead to this important sexually dimorphic character change (Foronda, 2012).
The elimination of a part of an animal body is a major change occurring during morphogenesis and evolution. This study has analyzed the mechanisms required for one such change, the absence of the male seventh abdominal segment. The study shows that the suppression of this segment involves the interplay between Hox and the sex determining genes, which regulate targets implementing the morphological change. The reduction or suppression of this segment is also a sexually dimorphic feature characteristic of higher Diptera, so the mechanisms shown here may be relevant for the evolution of morphology (Foronda, 2012).
In early pupa, during the second phase of cell division, there is a reduction in the number of A7 histoblasts, both in males and females, but stronger in males perhaps because wg is not expressed in the male A7 histoblasts. It has been shown that fewer histoblasts result in a smaller adult segment. Therefore, the reduced number of A7 histoblasts may account in part for the reduced size of the A7 segment in females. The control of the second phase of cell division involves the EGFR pathway, and Abd-B was found to reduce the number of histoblasts in the A7 through down-regulation of EGFR activity. If this activity is eliminated in the male A7, an increase is observed the number of histoblasts, that many of these cells remain at the surface at the time of extrusion and that a small A7 forms in the adult. It was also previously reported that a small A7 is observed in the male adult when expressing vein, an EGFR ligand. It is possible that the high number of histoblasts obtained when over-expressing elements of the EGFR pathway makes many of them unable to be extruded by a 'titration' effect, that is, there may be 'too many' histoblasts for the invagination mechanism to extrude them at the correct time. However, the EGFR pathway may also hinder extrusion since lower levels are seen of emc-GFP and also many histoblasts remain at the surface after high EGFR activation (Foronda, 2012).
At later pupal stages (around 35-40 h APF) there is the extrusion of the male A7 histoblasts. It was observed, however, that a few histoblasts also invaginate in the female A7, suggesting the male intensifies a mechanism present in both sexes. The extrusion requires the activity of emc, and correlates with higher emc expression in the male A7 histoblasts at about the time of extrusion. The invagination of histoblasts superficially resembles that of larval cells, and it also requires myosin activity. This would suggest that, due to the higher levels of Abd-B and DsxM, male A7 histoblasts may have adopted a mechanism similar to that used by cuticular larval epidermal cells (LECs) for their elimination. Recent reports, however, suggest an alternative mechanism. It has been demonstrated that an excess of proliferation in the epithelium leads to cell death-independent cell extrusion. Since this study has observed that prevention of cell death in the male A7 does not cause the development of an A7 (although delamination is delayed), the mechanism driving extrusion may be more similar to that of an overproliferating epithelium than to that taking place in larval cells (Foronda, 2012).
The data are consistent with emc increasing the expression of spaghetti-squash to accomplish apical constriction and extrusion. However, high expression of emc may not be sufficient to effectively induce histoblast extrusion, suggesting other genes are required. Besides, a strong reduction of emc leads to a very small and poor differentiated male A7 segment, reflecting that this gene is required for several cellular functions, among them cell survival. Perhaps significantly, emc is also expressed in embryonic tissues preceding invagination of different structures in the embryo, suggesting a common requirement for invagination at different developmental stages. It is thought that emc forms part of complex networks that have, among other cellular functions, that of contributing to the extrusion of A7 histoblasts (Foronda, 2012).
Although regulation of the EGFR pathway and emc are two key events in controlling male A7 development, previous experiments have also shown the contribution of the wingless gene, absent in male A7 but present in male A6 and female A7, in the development of this segment. These results have been confirmed and it was also shown that a reduction in wg expression can partially suppress the Abd-B mutant phenotype. Absence of wg is probably required to reduce cell proliferation in the male A7 but the data suggest wg may also be needed to maintain high emc levels. Apart from the role of wg, it was also shown that some A7a cells are transformed into A6p cells, thus reducing the number of A7 cells that might contribute to the adult segment. Finally, the expression of bric-a-brac must also be down-regulated in male A7 histoblasts to eliminate this metamere. Thus, this suppression is a complex process using different genes and mechanisms (Foronda, 2012).
The suppression of the male A7 depends ultimately on the levels of Abd-B expression. The role of this Hox gene is probably mediated in part by dsx, since Abd-B regulates dsx transcription and dsx governs, in turn, the expression of genes required for cell proliferation and extrusion. That Hox genes regulate dsx expression has also been demonstrated in the male foreleg, suggesting that Hox genes specify the different parts of the body where sexual dimorphism may evolve. The different dsx isoforms (DsxF and DsxM) determine the outcome of this regulation. A significant difference between the activities of these two proteins in the A7 is the regulation of emc levels. In the female, emc expression is similar in the A7 and the A6 and, accordingly, histoblast extrusion in females is small and confined to the central dorsal region, a domain virtually absent in the adult tergite. By contrast, the DsxM isoform increases Emc expression to drive large extrusion of A7 cells and elimination of the segment (Foronda, 2012).
Only the male A7, but not anterior abdominal segments, is eliminated. Therefore, the increase in emc expression, and subsequent events observed in the A7, depends on the higher Abd-B expression in the A7 in relation to the A6. Several Hox loci, like Sex combs reduced, Ultrabithorax or Abd-B are haplo-insufficient, and relatively small differences in the amount of some of these Hox proteins can drive major phenotypic changes, suggesting some downstream genes can sense these slight differences and implement major changes in morphology (Foronda, 2012).
Previous studies have shown the cooperation of Abd-B and the sex determination pathway in controlling the pigmentation of the posterior abdomen. It is thought that Abd-B plays a dual role in regulating the morphology of the posterior abdomen. First, it regulates dsx expression, thus allowing the possibility to develop sexually dimorphic characters; second, it cooperates with Dsx proteins in establishing pattern. Part of the effect implemented by Abd-B may be mediated by the levels of expression of dsx (distinguishing male A6 from male A7), and from the nature of the Dsx proteins (male and female ones). Although there is no conclusive evidence that the different levels of dsx in the A6 and A7 play a role in development, it is noted that this difference correlates with that of Abd-B (and depends on it), that high levels of DsxM are sufficient to increase emc-GFP in the A7 of females and eliminate this segment, and that these same high levels similarly increase emc-GFP and partially rescue the Abd-B mutant phenotype in males. Hox genes, therefore, may provide a spatial cue along the anteroposterior axis to activate dsx transcription and allow the formation of sexually dimorphic characters, but they may also cooperate with Dsx proteins to determine different morphologies. This double control by Hox genes may apply to all the sexually dimorphic characters and be also a major force in evolution (Foronda, 2012).
Spitz, and spitz group genes are at the top of the regulatory hierarchy in the development of salivary ducts. The salivary primordium consists of two regions: a more dorsal pregland anlage and a ventral preduct anlage. Spitz signaling to ventral cells through the EGF-receptor acts to block forkhead expression in preduct cells, thereby restricting gland identity to more dorsal cells. Forkhead acts in dorsal pregland cells to block duct fate, specifically acting to repress Serrate, a duct specific gene as well as breathless and trachealess, also required for duct formation. The spitz group genes rhomboid and pointed are required for duct fate (Kuo, 1996).
Growth and patterning of the Drosophila wing disc depends
on the coordinated expression of the key regulatory gene
vestigial both in the dorsal-ventral (DV) boundary cells
and in the wing pouch. It is proposed that a short-range
signal originating from the core of the DV boundary cells
is responsible for activating Egfr in a zone of organizing
cells on the edges of the DV boundary. Using loss-of-function
mutations and ectopic expression studies, it has been shown
that Egfr signaling is essential for vestigial transcription
in these cells and for making them competent to undergo
subsequent vestigial-mediated proliferation within the wing
pouch (Nagaraj, 1999).
Third instar wing discs
stained with an antibody directed against the N-terminal
portion of the Spitz protein show a strong
expression of Spi along the DV boundary and weaker
expression throughout the disc. This elevated protein
level at the DV boundary is likely to reflect post-transcriptional
control, since work from several laboratories
has shown that SPI mRNA is expressed ubiquitously at low levels
throughout the wing disc. To determine if the growth promoting activity of the Notch pathway is
mediated by Egfr, the dpp-Gal4 driver system was used
to ectopically express an activated, secreted form
of Spi (sSpi) that would
activate Egfr along this boundary. This results in
an extensive outgrowth of the wing pouch. When these discs are stained with an a-Vg
antibody, the overproliferating cells are found to
express Vg. These results can either
imply that Vg expression is activated by the Egfr
pathway leading to cell proliferation or that Egfr
activation results in random proliferation of cells
within the pouch, which then secondarily express
the Vg protein. To distinguish between these
possibilities, the vg 1 mutant allele in
which Vg expression is reduced but not
eliminated, was used. In dpp-Gal4/UAS-sspi;vg 1/vg 1 flies
there is no expansion of the wing pouch. It is concluded that activation of
EGFR leads to expression of Vg, which functions
downstream of or parallel to the Egfr pathway
for the proper proliferation of cells in the pouch (Nagaraj, 1999).
Although there is a single EGF receptor in Drosophila, multiple ligands activate it. This
work explores the role of two ligands, Spitz and Vein, in the embryonic ventral ectoderm. Spitz is a
potent ligand, whereas Vein is an intrinsically weak activating ligand. Prior to gastrulation, vein is expressed in the
future neuroectoderm. This early phase of expression appears to cooperate with Spitz in the induction
of medial neuroblast cell fates. Following gastrulation at stage 9, vein expression in the
neuroectoderm becomes confined to three to four cell rows on each side of the midline. This pattern
gradually restricts, such that by stage 11 only one cell row on each side of the midline expresses vein. Secreted Spitz,
emanating from the midline, triggers expression of vein in the ventral-most cell rows, by inducing
expression of the ETS domain transcription factor Pointed P1. In the absence of Vein, lateral cell fates
are not induced when Spitz levels are compromised. The positive feedback loop of Vein generates a
robust mechanism for patterning the ventral ectoderm (Golembo, 1999).
Expression of vein is observed in positions adjacent to the ventral midline, where activation of the
Egfr pathway by secreted Spitz emanating from the midline is maximal. This raised the possibility that
vein expression may be triggered by Spitz. In rhomboid mutant embryos in which active Spitz molecules are
not produced, no expression of vein is observed at stage 11, demonstrating that Rho/Spitz are indeed
necessary for vein expression in the ventral ectoderm. To test if Egfr activation is sufficient to induce vein expression in the embryo, the pathway was
ectopically activated at two different stages. Rho was ectopically expressed at stage 9 in the central
domain of the embryo by Kruppel-Gal4, and this gives rise to ectopic vein expression in the same region. At stage 11 secreted Spitz was expressed in the engrailed domains, resulting in the induction of
vein expression in the same pattern. These experiments demonstrate that high levels of Egfr
activation are sufficient for inducing vein expression (Golembo, 1999).
Induction of gene expression by Egfr in the ventral-most rows of cells occurs
for the argos, orthodenticle, and tartan genes. Induction is obtained through inactivation
of Yan, an ETS domain transcriptional repressor, and induction of Pointed P1, an ETS domain
transcriptional activator. To test if induction of vein by Egfr is also mediated by Pointed P1, vein expression was examined in pointed mutant embryos. Traces of expression are observed in the
midline in stage 10 embryos only, whereas no expression is displayed by the ventral-most and lateral
ectodermal cell rows at stage 11, thus showing that vein induction is defective in pointed mutants.
Elimination of Pointed activity can also be obtained in the following manner: an activated form of Yan,
in which the inactivating MAP kinase phosphorylation sites have been mutated, blocks the activity of Pointed by competing for the same DNA-binding sites. Indeed, when activated, Yan is ectopically expressed in the Kruppel domain the endogenous
expression of vein is abolished in that region. To examine if Pointed P1 is sufficient for
induction, vein expression was examined in embryos in which Pointed P1 was expressed in the Kruppel
domain. Indeed, expression of vein in the same domain is observed. These results
demonstrate that under conditions of ectopic expression, Pointed P1 is necessary and sufficient for
vein expression (Golembo, 1999).
Two different nested cell fates are induced by the Egfr pathway in the ventral ectoderm, depending
on the distance of the cells from the midline. The cell rows closest to the midline assume the
ventral-most fate, as reflected by the expression of target genes such as otd and argos. Intermediate
levels of Egfr activation induce lateral cell fates, reflected by the expression of FasIII, which is observed
in five rows of cells on each side of the midline. Both ventral-most and lateral markers are eliminated in
mutants for Egfr, as well as in mutants that abolish Spitz or its processing (spitz, rho, or Star).
Conversely, ectopic secreted Spitz or Rho is capable of expanding expression of both lateral and
ventral-most fates. To examine the role of Vein in the ventral ectoderm, vein null mutants were examined for the expression of marker genes. No defects in the expression of otd or FasIII were observed. These results indicate that at this level of resolution, the function of Vein is redundant. This is also consistent with normal levels of activated MAP kinase that are observed at stage 9 in vein mutant embryos. A role for Vein is revealed under conditions in which the level of Spitz is
compromised. Flies that are heterozygous for a null allele of Spitz are viable. Normal patterning of the
ventral ectoderm takes place in heterozygous spitz embryos, as monitored by the expression pattern of FasIII. Embryos were generated that are homozygous for the vein null allele and carry
only one functional copy of spitz. In these embryos, patterning of the ventral-most cells is normal, as
reflected by the expression of otd or FasIII. However, more lateral cells fail to
express FasIII. These results indicate that when Spitz levels are compromised in
heterozygous embryos, the cell row closest to the midline undergoes normal patterning. However, the
levels of Spitz may be too low to pattern the more lateral cells. Under these conditions, induction of
Vein appears to be critical to facilitate Egfr activation in these cells. Upon ectopic expression of Vein, the ventral-most markers are not induced at all or only intermittently,
thus reflecting the reduced inherent activity of Vein. To test directly the
biological activity of Vein in a controlled setting, it is necessary to eliminate endogenous signaling by
Spitz, as well as the presence of Argos. This can be achieved in embryos that are mutated for rhomboid, and
thus exhibit no expression of the ventral-most markers (such as argos) or lateral markers. Ectopic expression of Vein in rho mutant embryos is capable of restoring FasIII
expression, but did not induce otd expression. Thus, Vein is capable of
inducing the lateral cell fates in the absence of secreted Spitz (Golembo, 1999).
Two distinct mechanisms have been proposed for patterning by morphogens. In one case, a gradient of a single morphogen gives rise to distinct cell fates caused by the
induction of different levels of signaling in the same pathway. The complementary scenario involves a
relay mechanism: an initial induction by the primary morphogen induces the production of a relay factor
triggering another signaling pathway, to pattern the more distant cells. This work describes a
combination of the two models. Only the EGF receptor cascade patterns the ventral ectoderm.
However, the primary signal, Spitz, induces a relay mechanism by triggering expression of Vein,
another ligand of Egfr. Again, it is important to emphasize that although the restricted spatial
distribution of secreted Spitz is critical for correct patterning, Vein and Argos distribution may be more
uniform. Argos reduces the overall level of EGF receptor signaling, whereas Vein provides a lower
level of activation, capable of inducing only the lateral cell fates. vein expression is also induced by the EGF-receptor pathway in follicle cells within the dorsal-anterior corner of the egg chamber. It thus appears that Vein may provide a positive feedback loop in
several tissues that are patterned by EGF receptor activity (Golembo, 1999 and references).
Another facet of the activity of Vein that should be considered is its sensitivity to Argos inhibition. If
the lateral cells have not encountered sufficient levels of Spitz prior to the induction of Argos, their
capacity for activation in the presence of Argos is severely compromised, and relies on the continued
availability of secreted Spitz from the midline. The induction of Vein in the ventral-most cells, which
takes place in parallel to the induction of Argos, may help to overcome this problem. Vein is capable of
activating Egfr in the presence of Argos: in embryos heterozygous for spitz, the activity of Vein
induces the lateral fates. This takes place in a situation in which argos is expressed in the ventral-most cells. Vein itself is not capable of inducing expression of argos.
In conclusion, this work has revealed a powerful regulatory network, orchestrated by the sequential
utilization of Spitz and Vein, two ligands of the EGF receptor with different properties. Induction of
Vein takes place once the ventral-most cell fates already have been determined by high Spitz levels.
Vein expression prolongs the time window of activation of the Egfr pathway to ensure that the lateral
cell fates will be specified correctly. Vein is suited to this task, since it is a less potent ligand than Spitz,
capable of inducing only the lateral cell fates. Vein can activate Egfr even in the presence of Argos,
thus balancing the parallel negative-feedback loop of Argos (Golembo, 1999).
Signaling by the Drosophila EGF receptor (Egfr) is modulated by four known EGF-like proteins: the agonists Vein (Vn), Spitz (Spi), and Gurken (Grk) and the
antagonist Argos (Aos). Egfr is broadly expressed and thus tissue-specific regulation of ligand expression and activity is an important mechanism for controlling
signaling. The tissue-specific regulation of Vn signaling was investigated by examining vn transcriptional control and Vn target gene activation in the embryo and the
wing. The results show a complex temporal and spatial regulation of vn transcription involving multiple signaling pathways and tissue-specific activation of Vn target
genes. In the embryo, vn is a target of Spi/Egfr signaling mediated by the ETS transcription factor PointedP1 (PntP1). This establishes a positive feedback loop in
addition to the negative feedback loop involving Aos. The simultaneous production of Vn provides a mechanism for dampening Aos inhibition and thus fine-tunes
signaling. In the larval wing pouch, vn is not a target of Spi/Egfr signaling but is expressed along the anterior-posterior boundary in response to Hedgehog (Hh)
signaling. Repression by Wingless (Wg) signaling further refines the vn expression pattern by causing a discontinuity at the dorsal-ventral boundary. The potential for
vn to activate Egfr target genes correlates with its roles in development: vn has a minor role in embryogenesis and does not induce Egfr target genes such as aos and
pntP1 in the embryo. Conversely, vn has a major role in wing development and Vn/Egfr signaling is a potent inducer of Egfr target genes in the wing disk. Spi also
has the potential to induce Egfr target genes in the wing disk. However, the ligands appear to evoke specific responses that result in different patterns of target gene
expression. Other factors modulate the potential of Vn so that induction of Vn/Egfr target genes in the wing pouch is cell specific (Wessells, 1999).
Differences between Vn and Spi are apparent in the patterns of target
gene induction resultant from the ectopic expression of Vn and soluble Spitz (sSpi) in the
wing pouch. Effects have been noted for three Egfr target genes:
aos, pntP1, and kekkon-1 (kek1). In each case, a different
response to the ligands is seen. Both ligands induce ectopic aos
expression but Vn does so in a broader domain than sSpi, however, neither induces aos in the L3/L4
intervein region. In the embryo, the Egfr target
gene pntP1 mediates aos induction by sSpi. Likewise in the wing, ectopic sSpi induces pntP1
expression in cells that also expressed aos. However, following ectopic Vn no detectable
change in pntP1 expression is seen using in situ hybridization and
only very weak induction of pntP1-lacZ is seen in a domain that
does not correspond fully with ectopic aos expression. This suggests either that another transcription
factor mediates the induction of aos in response to Vn or
that PntP1 is capable of inducing aos, even when changes in
its own expression level are too low to be detected by
current methods. Both Vn and sSpi induce ectopic expression
of kek1, but predominantly in posterior cells rather
than throughout the domain of their ectopic expression. Thus the action of both ligands appears
limited by the presence or absence of some other factor in
anterior cells. There is also a difference in the level of
induction: Vn, which functions to induce kek1
expression in normal development, is a potent
inducer of high levels of ectopic kek1 expression,
whereas sSpi induces low levels of ectopic kek1 expression
and appears to reduce expression of endogenous kek1 (Wessells, 1999).
How repeating striped patterns arise across cellular fields is unclear. To address this the repeating pattern of Stripe (Sr) expression across the parasegment (PS) was examined in Drosophila. This pattern is generated in two steps. Initially, the ligands Hedgehog (Hh) and Wingless (Wg) subdivide the PS into smaller territories. Next, the ligands Hh, Spitz (Spi), and Wg each emanate from a specific territory and induce Sr expression in an adjacent territory. The width of Sr expression is determined by signaling strength. Finally, an enhancer trap in the sr gene detects the response to Spi and Wg, but not to Hh, implying the existence of separable control elements in the sr gene. Thus, a distinct inductive event is used to initiate each element of the repeating striped pattern (Hatini, 2001).
The repeating
pattern of Stripe (Sr) expression across the parasegment (PS) is generated by inductive
inputs from three spatially localized ligand sources. The ligands,
Hh, Spi, and Wg, emitted by En, Ve, and Wg territories, respectively,
control Sr expression in cells adjacent to each ligand source. There
are three notable features to this regulation: (1)
each ligand-producing territory induces Sr expression in the adjacent
territory; (2) the induction is asymmetric, either anterior or
posterior to each source; (3) the induction is initiated at the
high level of signaling achieved near the source, limiting expression
of Sr to a narrow row of cells. Because these same ligands act more
broadly in cuticle cell fate specification, these results also suggest
that the ligands and signaling territories operate in a fundamentally
distinct way in order to construct a repeating striped pattern. These
observations reveal a strategy used to generate a repeating striped
pattern across a cellular field that may be used generally (Hatini, 2001).
Each Sr row is initiated
adjacent to a different ligand source. The induction of Sr was limited to a narrow row of cells at each position. Manipulating either the ligand level, or the sensitivity of cells to a specific signaling pathway, leads to a broadened territory of Sr induction. Thus, local activity gradients of Hh, Spi, and Wg are each generated, and a threshold for activation of Sr is only surpassed in cells adjacent to each source. The gradient landscape of Spi and Wg is sculpted
using the inducible antagonists Argos and Naked, respectively. Although how the activity landscape for Hh is sculpted was not specifically addressed, the Hh pathway also makes use of an inducible antagonist. It is likely that Hh spread is limited by binding to the Hh receptor Ptc, which is upregulated by Hh input (Hatini, 2001).
To generate the repeating striped tendon
pattern, the Sr gene must be able to respond to each of three
different ligands. To account for this, it is expected that the Sr
promoter is modular, and each Sr row is induced via a separable,
cis-acting response element. An enhancer trap P-insertion in
the sr gene (sr03999) provides evidence
for this since it detects the response to Spi and Wg, but not to Hh,
implying that the P-insertion separates response elements in the
sr gene. A separable Sr promoter element
controlling Hh-dependent expression has been identified. Although
this element operates only in dorsal and lateral epidermis, and not
ventrally where Sr is expressed in repeating striped pattern, this observation strongly suggests that the control elements will be modular. Furthermore, in this dorsal/lateral
element, the presence of functional, consensus Cubitus interruptus
(Ci) DNA binding sites suggests direct regulation of Sr by the Hh
signaling pathway. The obvious analogy is to the modularity of
regulatory regions of certain pair-rule genes, which are able to
integrate non-periodic information in order to generate periodicity. Note that the induction of the
sr gene is limited to cells bordering each ligand source,
even though each of the signals can act across several cell
diameters. It is predicted that a given Sr response-element is configured to sense and respond only to a particularly high threshold level of each ligand (Hatini, 2001).
The ligands controlling Sr expression emanate from each
of three territories across the PS. These territories are established by the primary organizing signals, Wg and Hh. In the earliest step, cross regulation
between Wg and En/Hh-expressing cells stabilizes each ligand's
expression and consolidates these two territories. In addition, through negative
regulation, both Wg and Hh limit the expression of Ser to a central
territory within the PS. Finally, signals from the En/Hh
territory induce Ve expression in two cell rows just posterior to the
En/Hh territory. The exact width of the Ve-expressing
territory is adjusted as local input from the Ser territory induces a
third Ve-expressing cell row. Thus, Hh and Wg act indirectly by
defining and limiting each other's expression territory, as well as that of downstream ligands. All of these ligands then organize the
repeating pattern. Three ligands induce Sr expression at specific
positions across the PS, while the role of Ser reveals a particularly
interesting spatial cue. Although the second row of Sr is induced in
the anterior-most row of Ser-expressing cells, Ser expression is not
necessary for this. Rather, Ser dictates the spacing between Sr row 1
and 2, because it defines the breadth of the Ve territory and thereby
the position of the first non-Ve cell that can induce Sr in response
to Spi-Egfr signaling (Hatini, 2001).
Sr expression is induced asymmetrically relative to each ligand source. For instance, Hh induces Sr posterior to the En territory, but not in the En territory or anterior to it. Wg imparts asymmetry
to Hh/En signaling, and thereby prevents Ve expression anterior to
the En/Hh territory. In exactly the same way, via antagonism
of Hh signaling, Wg appears to block Sr expression anterior to the
En/Hh territory, because the removal of Wg function allows Sr
expression anterior to the En/Hh territory. The Wg signaling pathway imposes asymmetry to the
dorsal/lateral Sr regulatory element via consensus Pangolin DNA
binding sites. This principle is likely to extend to
the ventral control of Sr for the generation of one element of the
repeating striped pattern (Hatini, 2001).
One reason why Hh signaling cannot induce Sr expression in the En cells is that En represses expression of the Hh signal transducer, Ci. Nevertheless, it is still necessary to explain why
signals from both the Ve and Wg territories cannot induce Sr in the
En cells, even though each signal definitely acts on these cells to
specify cuticle fate. A clue comes from the observation that
when the En territory is not maintained Sr is induced symmetrically
relative to the Wg or to the Ve sources. Thus, it is proposed that the En protein prevents Sr induction by Wg or Egfr inputs by repressing Sr
expression. This is supported by the observation that activating Wg
signaling at high levels in En cells still does not lead to Sr
expression.
To explain why Sr is not induced by Spi in the Ve territory, nor by
Wg in the Wg territory, it is inferred that there is a specific block to autocrine signaling in each territory. Interestingly, this block is
specific to Sr induction, and not to other outcomes of signaling,
such as cuticle fate specification. It suggests a lack of an
activator essential to induce Sr expression or expression of a
repressor that blocks such a response in the Ve and Wg
territories (Hatini, 2001).
The same ligands establish strikingly distinct patterns across the same cellular field. While the cuticle pattern comprises a diversity of cell types, the Sr expression pattern reflects the near-periodic
specification of the same cell type. These distinct outcomes arise
because the same ligands act in a fundamentally different manner in
these two processes. As an example, Wg specifies smooth cuticle in a
broad region anterior to the Wg territory, in the Wg territory, and
posterior to the Wg territory (in anterior En/Hh cells). However, as is shown in this study, Wg induces Sr only
anterior to the Wg territory in a narrow region, and not in the Wg
territory or posterior to it. Also, Spi, through Egfr function,
induces denticles over a broad region, both in the Ve territory and
anterior to it in a subset of En/Hh cells. However, Egfr function
induces Sr only in a narrow region posterior to the Ve territory.
Thus, despite the broad effects of Wg and Egfr on cuticle pattern,
the effect of Wg and Egfr in building the repeating striped pattern
is constrained to a narrow region of cells. As a final example of the
distinction between control of denticle pattern and control of
repeating striped pattern, in the same row of cells, cuticle fate is
specified by Spi while tendon fate is specified by Hh input. The
unique effects of these ligands on Sr expression are crucial for the
establishment of the repeating striped pattern.
Thus, the information encoded in the signaling territories is decoded
in different ways to achieve both repeating pattern and cell-type
diversity across the same field (Hatini, 2001).
The generation of near-periodic Sr pattern across the PS is conceptually similar to the two-step process that is used in establishing the periodic body plan through pair-rule gene expression in syncitial embryos. Initially, primary pattern organizing centers are established at the boundaries of a field of naive nuclei or cells. In the first step, these centers establish patterned expression of secondary organizing
genes across the field, subdividing the field into distinct gene
expression territories. In the second step, the information encoded
in these territories is used to initiate a repeating striped gene
expression pattern. In the embryo, Bicoid together with Hunchback and
Nanos organize expression of the gap genes. Territories of gap gene expression are then used to establish the periodic pattern of primary pair-rule gene expression. In the PS, Wg and Hh organize overall
parasegmental pattern by first defining each other's territory and
then the territories of secondary regulatory genes, Ser and Ve. The
signaling territories are then used to establish near-periodic
expression of Sr. Note that although the conceptual similarity is
striking, the mechanisms generating these two repeating patterns are
distinct. The pair-rule gene expression pattern is established in a
unique, syncitial system by diffusion of transcriptional regulators
in a common cytoplasm, whereas Sr expression is established across an
epithelial monolayer by communication between cells via
inter-cellular signaling systems. In addition, while a balance
between diffusible activators and repressors determines pair-rule
gene expression at any point along the syncitial embryo, the unique
properties of the signaling territories across the PS determine Sr
expression. In particular, the juxtaposition of pairs of territories,
one that sends a signal with one that can initiate Sr expression in
response to the signal, is utilized to initiate Sr expression
adjacent to the boundaries between these territories (Hatini, 2001).
A near-periodic striped pattern of veins is produced in the developing wing disc. Emerging evidence suggests that the two-step strategy may also apply to this system, and that unique properties of putative signaling territories are used to initiate wing veins adjacent to at least two territories across the wing blade. In the first step, combined action of Hh and Dpp establishes different territories of downstream regulatory genes across the A/P axis of the future wing blade. While Hh establishes a territory of Ptc expression adjacent to the compartment boundary, Dpp acts more broadly across the wing and establishes two nested territories of Spalt and Optomotor-blind expression. In the second step, veins are induced adjacent to at least two territories, suggesting that an unknown ligand emanates from one territory and induces the vein in the adjacent territory. The possibility that the two-step process described here for the PS is used across the wing blade suggests that this may be a general strategy for creating repeating striped patterns across other cellular fields (Hatini, 2001).
Addition of secreted Spitz (but not the membrane-associated form) to S2 Drosophila cells expressing EGF-R gives rise to a rapid tyrosine autophosphorylation of EGF-R. Following autophosphorylation, EGF-R associates with the DRK adapter protein. Consequently, activation of MAP kinase is observed. A dose response between the levels of Spitz and MAP kinase activity is also observed (Schweitzer, 1995).
Drosophila Spitz is an activating ligand for the EGF receptor (Egfr). It has been shown that Star is required for Spitz activity. Star is quantitatively limiting for Spitz production during eye development. Star and Spitz proteins colocalize in Spitz sending cells and this association is not coincident with the site of translation, consistent with a function for Star in either Spitz processing or transmission. Minimal sequences within both Spitz and Star have been defined
that mediate a direct interaction, and this binding can occur in vivo (Hsiung, 2001).
Genetic analysis has indicated that Star (with rhomboid) may
function upstream of the transmission of Spitz from sending
cells and Star protein has been shown to be localized to the nuclear
envelope and the early ER. Taken together, these data suggest that Star may
function in the translation, post-translational cleavage,
glycosylation, secretion or presentation of Spitz. To
approach this, Star and Spitz proteins and spitz mRNA were localized in a series of pair-wise double stains at the confocal level. Spitz and Star proteins do colocalize, but spitz mRNA does not. Furthermore Spitz and Star proteins appear together in granular structures that are perinuclear as well as apical in the cells. This suggests that the Spitz-Star interaction persists through much or all of the secretory pathway (Hsiung, 2001).
Controlling the quantity of Star directly
affects the quantity of Spitz antigen seen (in loss- and gain-of-function mosaic clones and by ectopic expression in the
entire eye). In short, less Star results in less Spitz and more
Star in more (and ectopic) Spitz. Consistent with this, it was
found that overexpression of unprocessed full length mSpitz alone has no phenotypic effect, but overexpression of Star does result in a moderate
rough eye suggesting that normally spitz RNA is in excess and
the quantity of the signal is limited by Star. Furthermore, overexpressing both mSpitz and Star together results in a synergistic effect and a grossly disordered eye, with a large excess of photoreceptors and a deficit of accessory cells. Since the main function of the Spitz/Egfr signal in the eye is to recruit cells to the developing clusters and specify them as photoreceptor
neurons, this phenotype is consistent with a great increase in
the quantity of this signal. Immuno-colocalization data
appear to suggest that there is more Star antigen than Spitz
in the developing eye. However, it is very difficult to
draw any conclusions as to the actual relative abundance of
these proteins : these experiments were not quantitative.
Taken together, all these data suggest that the quantity of
Star protein is the critical limiting factor for the Spitz/Egfr signal, at least during the normal development of the compound eye (Hsiung, 2001).
A series of GST-mediated in vitro binding experiments was used to define a single region, each in Spitz and Star, that mediates their direct interaction with one another. In Spitz, this 48-residue segment (SpitzH) is virtually
identical to the 'factor' domain -- that part of the protein that
contains the six cysteine residues and other features that
show homology to the small diffusible growth factors of
the TGF-alpha family. In Star, a 19-amino-acid GFBD responsible
for binding to Spitz has been identified. To confirm these results,
a test in vivo was conducted: SpitzH was fused to CFP as well as a dominant
nuclear localization signal (NLS). This CFP-SpitzH-NLS fusion when expressed in
HeLa cells is directed to the nucleus by virtue of the NLS
and a cyan-colored signal is detected there by confocal
microscopy. A fusion protein was also made in which an
evenly distributed yellow fluorescent protein protein kinase (YFP-PK) protein was fused to the GFBD from Star (YFP-Star7-PK). When YFP-Star7-PK
is expressed alone in HeLa cells, the yellow signal is evenly
distributed, but when coexpressed with CFP-SpitzH-NLS,
the yellow signal moves to the nucleus. This 'cargo' experiment confirms that the Spitz factor domain and the Star GFBD can bind in vivo. Taken together, the in vitro 'GST-pull down' experiments and the in vivo HeLa cell
'cargo' experiments are consistent with a direct interaction
in the living fly between the Spitz factor domain and the Star
GFBB. However, neither of these two experiments tested
this interaction in the secretory pathway (Hsiung, 2001).
It is interesting to note that the Spitz 'factor' domain is N-terminal to the Spitz trans-membrane domain, and thus
presumed to lie outside of the plasma membrane (or in the
lumen of the organelles of the secretory pathway). The
GFBD in Star lies C-terminal to its trans-membrane domain
and thus would appear to lie on the wrong side of the plasma
or organelle membranes to interact directly with the Spitz
factor domain. However, structural features of Star have led
others to suggest that Star is actually a type II integral
protein, with its C-terminus outside and its N-terminus
inside. This is therefore consistent with a direct interaction between the Spitz factor domain and the Star GFBD in vivo (Hsiung, 2001).
In summary, Spitz and Star proteins
associate in living cells in the developing Drosophila
compound eye, Star controls the quantity of Spitz signal,
and these proteins interact via the factor domain in Spitz
and the GFBD in Star. These data are consistent
with a role for Star in some stage or stages of Spitz signal
production subsequent to its translation. These conclusions
are very similar to those reached by others for Rhomboid
family proteins. While no firm conclusions can be drawn from these data, it is suggested that Star may be involved in a complex in the secretory pathway that
acts in the maturation of Spitz. Star could act before Rhomboid, because Star has been localized early in the pathway and Rhomboid has been localized to the
apical microvillae, or, they may act together. There is no evidence to suggest that either Star or Rhomboid are themselves proteases capable of cleaving
Spitz: perhaps they recruit one. Alternately Star may act
as a chaperone to route the pro-Spitz protein correctly
within the secretory pathway or it might be required for
the correct folding of Spitz or it may recruit glycosylation
enzymes. Indeed, the data suggest that Star can interact with
the Spitz factor domain in vitro in conditions in which it may not be correctly folded. In the developing eye, anterior to the furrow, Star appears to be quantitatively limiting on Spitz expression. It may be that Spitz pro-protein that is not correctly routed or cleaved may be unstable (Hsiung, 2001).
While there are several known Rhomboid proteins, Star
appears to be unique in the Drosophila genome. While
homologs of Rhomboid have been detected in vertebrates, no homolog of Star has been detected outside of Drosophila. Star is essential in Drosophila for the activation of an otherwise inactive growth factor homolog (Spitz). There
may be proteins with similar functions in vertebrates, which have conserved structure but which are too far diverged at the primary sequence level to be found with current computer searching algorithms (Hsiung, 2001).
Cleavage of the ubiquitously expressed transmembrane form of Spi (mSpi)
precedes EGF receptor activation. The Star and Rhomboid (Rho) proteins are necessary for Spi cleavage in Drosophila cells. Complexes between the Spi and Star proteins, as well as between the Star
and Rho proteins were identified, but no Spi-Star-Rho triple complex was detected. This observation suggests a sequential activity of Star
and Rho in mSpi processing. The interactions between Spi and Star regulate the intracellular trafficking of Spi. The Spi precursor is
retained in the periphery of the nucleus. Coexpression of Star promotes translocation of Spi to a compartment where Rho is present both
in cells and in embryos. A Star deletion construct that maintains binding to Spi and Rho, but is unable to facilitate Spi translocation, has lost
biological activity. These results underscore the importance of regulated intracellular trafficking in processing of a TGFalpha family
ligand (Tsruya, 2002).
To identify the domain(s) of Star required for its biological
activity, deletion constructs were generated. Because Star is a
novel protein with no defined domains (except for the transmembrane
domain), no clues were available for the generation of such constructs.
Searching the database, a Bombyx mori cDNA
sharing homology with Star was identified. Complete sequence of this cDNA identified a type II transmembrane protein of 315 amino acids, which was termed BmS. Notably, the amino-terminal cytoplasmic domain of this protein is only
74 residues long, and the major boxes of homology are located
immediately carboxy-terminal to the transmembrane domain. This pattern of homology raised the possibility that most of the amino-terminal domain of Star may be
dispensable for its function, and prompted an examination of constructs of
Star missing most of the amino- or carboxy-terminal domains. Two
truncated Star constructs were generated. NTM contains the entire
amino-terminal and transmembrane domains, and is truncated 16 residues after the transmembrane domain. Conversely, in TMC, the amino-terminal 259 residues were deleted, and an initiator methionine was added (Tsruya, 2002).
The biological activity of the constructs was tested in S2 cells. Only
the TMC construct was capable of promoting mSpi cleavage, but at a
lower efficiency than full-length Star. sSpi could be detected in the
medium of cells coexpressing mSpi, TMC and Rho, but not in the medium
of cells expressing only mSpi and TMC. Stable transfected lines expressing
mSpi and TMC provided a more sensitive assay and produced low levels of
sSpi. The lower levels of sSpi, however, indicate
that this construct is less potent than the full-length Star protein.
The NTM and BmS constructs are not active alone or even in the
presence of Rho. In addition, after sequencing the Star54 null allele, a termination codon was identified at position Q387 in the extracellular domain. This protein is 67 residues longer than NTM and yet is inactive, indicating that the carboxy-terminal region of Star is essential for its
biological function (Tsruya, 2002).
The TMC, NTM, and BmS constructs were also tested in flies heterozygous
for the Star mutation: such mutants display rough eyes due to
haplo-insufficiency. Again, only ectopic expression of the TMC
construct in eye discs (by GMR-Gal4) is capable of rescuing the phenotype, giving rise to wild-type eyes. High levels of expression of full-length Star (in Star heterozygous flies, as well as in wild-type flies) leads to the
appearance of rough eyes and to the formation of extra wing veins,
consistent with Egfr hyperactivation. The reduced biological activity of TMC is evident, since it is not capable of eliciting these hyperactivation phenotypes. It is concluded that, in cells and in flies, only the Star construct containing the carboxy-terminal domain retains the essential activities of Star, but is less potent than full-length Star (Tsruya, 2002).
Having shown that Star is essential for mSpi cleavage, it was asked
whether Star interacts directly with the mSpi protein. The
capacity of Star to form protein complexes with mSpi was examined. Cells were
cotransfected with constructs expressing mSpi-FLAG, Star-HA, and, as
a negative control, CD8-GFP. mSpi was immunoprecipitated by anti-FLAG, and
coprecipitation was followed by anti-HA and anti-GFP immunoblotting. Indeed, immunoprecipitation of mSpi gives rise to significant coprecipitation of Star. The robust interaction between mSpi and Star was
also demonstrated in the reciprocal experiment, where immunoprecipitation of Star-TAP results in coprecipitation of mSpi-GFP (Tsruya, 2002).
In view of the association between Spi and Star, whether
the biological activity of the Star deletion constructs correlates with
the capacity to bind Spi was examined. The NTM, TMC, or BmS constructs were
coexpressed with mSpi in S2 cells. Although only the TMC construct,
containing an intact carboxy-terminal domain, is active in cells and
flies, all three proteins coprecipitate mSpi with similar
efficiencies. The basis for loss of Star biological activity, in spite of the ability to bind Spi, is described below (Tsruya, 2002).
To identify the domain(s) of mSpi mediating the interaction with Star,
mSpi-GFP was constructed. GFP was inserted amino-terminal to the EGF
domain. This construct retains the capacity to undergo Star- and
Rho-dependent cleavage in S2 cells. It is also biologically active in
flies, since coexpressed mSpi-GFP and Star in wing discs give rise to
abnormal, wrinkled wings. mSpi-GFP and several
deletion constructs were tested for their ability to interact with
Star. A secreted Spi protein lacking the cytoplasmic and transmembrane
domains retains the capacity to bind Star, pointing to the
extracellular domain of Spi as the mediator of binding. In accordance
with this conclusion, an mSpi protein lacking the EGF domain shows only
residual levels of binding to Star. Further deletions show that the
EGF domain of Spi is necessary for the interaction, since secreted
constructs lacking this domain show only background association with Star. Thus, the EGF domain of Spi mediates the interaction with Star (Tsruya, 2002).
The interaction between Star and mSpi prompted an examination of whether
Star also associates with Rho. Star-HA was coexpressed with Rho-TAP
in S2 cells. Immunoprecipitation of Rho-TAP showed coprecipitation of
Star. In the reciprocal experiment, immunoprecipitation of Star-HA or Star-TAP coprecipitates Rho. To identify the regions of Star necessary for this interaction, the experiment was repeated with the three Star constructs. The NTM and BmS constructs retain the interaction with Rho, whereas the TMC construct shows no detectable interaction or only weak interaction. It appears that sequences within the amino-terminal cytoplasmic domain of Star that are not present in TMC mediate the binding to Rho (Tsruya, 2002).
The capacity of Star to bind both Spi and Rho, raises the
possibility that Star may function as a scaffold protein, to form a
trimeric protein complex. Examination of possible direct interactions between mSpi and Rho failed to detect any significant coprecipitation. If a trimeric complex is
formed, it would be expected that Star would promote coprecipitation of mSpi by Rho. However, no elevation in mSpi coprecipitation with
Rho was detected when Star was also expressed. Under these conditions, Star retains the capacity to bind mSpi, regardless of the expression of Rho. Therefore, it appears that whereas mSpi-Star or Star-Rho complexes are formed in the cells, no trimeric complex is present (Tsruya, 2002).
Because coexpression of Rho and Star significantly promotes processing
of mSpi, it is possible that short-lived trimeric complexes are formed
as a result of cleavage of mSpi. To circumvent this problem, the formation of complexes with a noncleavable mSpi precursor was examined.
An mSpi protein in which 16 amino acids between the EGF and
transmembrane domains were deleted, does not undergo cleavage in S2
cells. A similar construct fails to be cleaved in the
Xenopus assays. Like mSpi, the deleted
mSpi protein readily forms complexes with Star, but not with Rho. Again, even in the presence of Star, Rho is not capable of
coprecipitating this uncleavable Spi construct, arguing
against the presence of even a transient triple complex. This experiment suggests that Star and Rho function sequentially in mSpi processing (Tsruya, 2002).
In view of the apparent sequential roles of Star and Rho, attempts were made
to identify the cellular compartments in which they are active. The
subcellular localization of Star and Rho may provide a clue as to their
order of activity. Star protein has been reported to be in the periphery of
the nucleus, consistent with an ER localization. Immunostaining of S2 cells to follow Star overexpression confirms this localization pattern. Immunostaining of Rho in S2 cells overexpressing Rho shows a punctate distribution that does not
colocalize with a 120-kD integral Golgi membrane protein, as well as plasma membrane staining. This pattern is in accordance with previous observations in embryos showing punctate endogenous Rho distribution. Following overexpression, plasma membrane staining is also detected. Expression of Star and Rho shows only a restricted overlap. The distinct cellular distribution of these proteins suggests that a key feature in the regulation of mSpi processing may be its cellular trafficking. This possibility is in accord with the sequential activities of Star and Rho implied by the coprecipitation results (Tsruya, 2002).
The distribution of mSpi-GFP was followed in cells. mSpi-GFP shows a
peripheral nuclear localization in S2 cells, as well as diffuse
cytosolic staining, but it is not detected in the Golgi or plasma
membrane. Therefore, it appears that the mSpi protein is retained in the ER, a compartment in which it cannot undergo cleavage (Tsruya, 2002).
To identify the domain(s) of mSpi required for its ER retention, a
construct containing intact extracellular and transmembrane domains,
but lacking the intracellular domain was examined. This protein shows
only residual levels of peripheral nuclear localization and is
detected primarily in a punctate pattern, consistent with exit from the
ER. Therefore, the cytoplasmic domain of mSpi is necessary for its ER retention (Tsruya, 2002).
The peripheral nuclear distribution of Star and mSpi appears
similar, whereas the punctate localization of Rho suggests that it is
present in a more advanced compartment along the secretory network.
Because Rho is required for efficient mSpi cleavage, how does mSpi
reach the Rho compartment? One option is that Star forms a complex with
mSpi and alters its intracellular localization. The distribution of
mSpi-GFP was examined following coexpression with Star. Indeed, the
original peripheral nuclear distribution of mSpi-GFP disappears, and
instead it is found in a prominent punctate pattern, which
does not correspond to the Golgi and only partly overlaps with the
lysosomes and endosomes. The alteration in mSpi
distribution in the presence of Star was observed in all cells (Tsruya, 2002).
In contrast, expression of Rho with mSpi-GFP does not alter its
distribution, in agreement with a lack of coprecipitation.
However, coexpression of Rho with mSpi-GFP and Star reduces dramatically the levels of mSpi-GFP observed in the cells, in accordance with the results obtained in anti-Spi blots. The
residual mSpi-GFP is found in a punctate staining colocalizing with
Rho. These results show that Rho can alter the levels of
mSpi in the cells, but only in the presence of Star. They are
consistent with sequential activity of Star and Rho, where Star is
required first to transport mSpi from the ER to the compartment containing Rho, and Rho subsequently facilitates the cleavage and secretion (Tsruya, 2002).
To test the distribution of mSpi and the effect of Star in embryos,
the mSpi-GFP construct was expressed in embryos. After crossing
several independent transgenes of the UAS-mSpi-GFP construct to embryonic Gal4 drivers, only very weak fluorescence was seen, in
contrast with other GFP constructs such as UAS-CD8-GFP. This result may indicate a high turnover and low steady-state levels of the
noncleaved, mSpi precursor. Only in a small percentage of embryos could
the mSpi-GFP be visualized. Embryos expressing mSpi-GFP show a
peripheral nuclear distribution colocalizing with the nuclear membrane
protein Lamin. In contrast, embryos expressing mSpi-GFP and Star
show a punctate distribution. Although Star is normally
expressed in all embryonic cells, it is assumed that the overexpression of
mSpi-GFP alone generates an excess of this protein and thus essentially represents the mSpi distribution in the absence of Star (Tsruya, 2002).
In an alternative way to circumvent the high mSpi turnover,
the distribution of mSpi-GFP was followed after transient injection of the
UAS-mSpi-GFP construct to embryos carrying prd-Gal4
or actin-Gal4. Monitoring fluorescence several hours
after injection, a bright peripheral nuclear distribution of mSpi-GFP
was seen. Coinjection of mSpi-GFP and Star constructs
eliminates this distribution and leads to the accumulation of mSpi-GFP
in a punctate pattern, similar to the distribution in S2 cells. Conversely, coinjection of mSpi-GFP and Rho does not alter the
mSpi peripheral nuclear distribution. Finally, coinjection of mSpi-GFP, Star, and Rho also gives rise to the mSpi-GFP plaques, but their intensity is decreased. It is concluded that the effect of Star on mSpi localization observed in S2 cells also occurs in embryos and is likely to reflect the essential role of Star
in mSpi intracellular trafficking (Tsruya, 2002).
It was of interest to test whether the biological activity of Star indeed
corresponds to the capacity to promote mSpi trafficking. Although the
two Star deletion constructs retain the ability to bind mSpi, only the TMC construct containing an intact carboxy-terminal
domain is biologically active in cells and flies. Each of the
two Star constructs was coexpressed with the mSpi-GFP construct. In
the TMC expressing cells, mSpi-GFP displays the same punctate
distribution observed when coexpressed with full-length Star. In contrast, mSpi-GFP remains in the peripheral nuclear pattern in the presence of NTM and BmS. These results imply that the carboxy-terminal domain is required for the ability of Star to target mSpi to the proper compartment. Without this property, Star is not biologically active (Tsruya, 2002).
How is the cellular distribution of Star itself regulated? In cells,
Star is found in a peripheral nuclear distribution.
Examination of the distribution of the Star deletion constructs shows
that TMC retains the same distribution as full-length Star.
In contrast, NTM is not seen in the ER and is detected instead in a
weak punctate and prominent membrane staining. Thus, the
cellular distribution of Star itself is regulated and residues in the
carboxy-terminal domain may promote interactions with other proteins
that regulate the peripheral nuclear localization of Star (Tsruya, 2002).
In conclusion, this work shows that the intracellular localization and trafficking of mSpi is crucial for its regulated cleavage. mSpi is
normally retained in a peripheral nuclear compartment where it does not undergo cleavage and may be rapidly degraded. Star, which is
also enriched in this compartment, associates with mSpi and translocates it to a compartment in which Rho is enriched, to allow cleavage. The association between Star and Rho may allow Star to efficiently deliver mSpi. The cleavage process thus entails orchestration of trafficking and protein-protein associations to ensure tight regulation of mSpi processing and, hence, Egfr activation (Tsruya, 2002).
The spitz gene is required for photoreceptor determination. Mosaic analysis suggests that spitz produces a diffusible signal during ommatidial development. Rhomboid, another member of the spitz group and the EGF receptor also interacts with sevenless-rhomboid in a pattern that suggests a model in which Rhomboid acts as a mediator of a ligand-receptor interaction between Spitz and Egfr in the developing eye. These data suggest that photoreceptors other than R7 use a Ras1 signaling pathway activated by the Spitz/Egfr interaction, in a manner analogous to the Ras1 pathway activated by Boss/Sevenless in photoreceptor R7 (Freeman, 1994).
The rhomboid (rho) gene, which encodes a transmembrane protein, is a member of a small group of genes (ventrolateral genes) required for the differentiation of ventral epidermis in the Drosophila embryo. rho is the only member of the ventrolateral genes expressed in a localized pattern corresponding to cells requiring the activity of the ventrolateral pathway. Spatial localization of rho plays an analogous role in establishing vein pattern in the adult wing. Ectopic expression of rho during wing development leads to the formation of extra veins. Gene dosage studies among ventrolateral genes suggest that RHO may facilitate Spitz-EGF-R signaling, resulting in activation of RAS (Sturtevant, 1993).
Spatially restricted processing of Spitz may be responsible for EGF-R graded activation. On the basis of genetic interactions, it has been suggested that the Rhomboid (Rho) and Star proteins act as modulators of EGF-R signaling . No alteration in EGF-R autophosphorylation or the pattern of MAP kinase activation by secreted Spitz is observed when the Rho and Star proteins are coexpressed with EGF-R in S2 cells. The ventralizing effect of secreted Spitz is epistatic in embryos mutant for rho or Star, suggesting that Rho and Star may normally facilitate processing of the Spitz precursor (Schweitzer, 1995).
Activation of the Drosophila epidermal growth factor receptor (Egfr) by the transmembrane ligand, Spitz (Spi), requires two additional
transmembrane proteins: Rhomboid and Star. Genetic evidence suggests that Rhomboid and Star facilitate Egfr signaling by processing
membrane-bound Spi (mSpi) to an active, soluble form. To test this model, an assay based on Xenopus animal cap explants was used in
which Spi activation of Egfr is both Rhomboid and Star dependent. Spi is on the cell surface but is kept in an inactive state by
its cytoplasmic and transmembrane domains; Rhomboid and Star relieve this inhibition, allowing Spi to signal. Spi is
likely to be cleaved within its transmembrane domain. However, a mutant form of mSpi that is not cleaved still signals to Egfr in a Rhomboid and Star-dependent
manner. These results suggest strongly that Rhomboid and Star act primarily to present an active form of Spi to Egfr, leading secondarily to the processing of Spi into
a secreted form (Bang, 2000).
Rhomboid and Star-mediated Egfr signaling was analyzed by an assay in
which Xenopus animal cap explants were isolated from embryos
injected with in vitro synthesized Egfr, spi,
rhomboid, and Star mRNA. This assay is based
on the fact that Egfr activates the Ras pathway, which should lead to an
up-regulation in the expression of the Ras target gene Xenopus
Brachyury (XBra) in animal caps.
Animal caps from injected embryos were allowed to develop until sibling
embryos were late gastrulae (stage 11.5), when they were analyzed for
XBra expression by RNAse protection assay (RPA). Expression of XBra can be induced in animal caps by Egfr but
only under the same conditions that are required for the activation of
Egfr in Drosophila. Thus, expression of XBra
is not induced in animal caps that express Egfr alone, Egfr along with
mSpi, or Egfr along with just Rhomboid and Star. In contrast, a high level of XBra expression is
induced when animal caps express Egfr along with mSpi, Rhomboid, and
Star. The requirement for Rhomboid and Star for Egfr
activation can be overcome in the animal cap assay, as in
Drosophila, by expressing sSpi, an engineered form of Spi that
contains just the extracellular domain. In addition,
Egfr activation can be blocked, as in Drosophila, by introducing the
Egfr inhibitor, Argos (Bang, 2000).
By themselves, Rhomboid and Star each weakly
promote mSpi activation of Egfr, however, together they are strongly
synergistic. Thus, both Rhomboid and Star may be
required to achieve maximal levels of Egfr activation, but for lower
levels of signaling, either one alone may be sufficient. It is possible
that Rhomboid and Star are obligate cofactors but that there are
homologous proteins present in the animal cap that fulfill the role
of the missing component, albeit weakly. Alternatively, this result may
reflect a way in which various levels of receptor activation may be
achieved. In some settings, such as the Drosophila wing veins,
rhomboid and Star are codependent, whereas in the
eye, Star is sufficient and rhomboid function appears
to be dispensible (Bang, 2000).
Next, a determination was made whether Rhomboid and Star are required for Egfr
activity by acting in the signaling cell, the receiving cell, or in
both cells. To do this, activation of XBra was measured in
sandwiches that were made by combining an animal cap expressing Egfr
with another animal cap expressing mSpi, in the presence or absence of
Rhomboid and Star. When Rhomboid and Star are present in the
receptor-expressing cells, mSpi fails to activate Egfr. However, when Rhomboid and Star are present in the
ligand-expressing cells, mSpi strongly activates Egfr.
It has been suggested that Rhomboid and Star may act as cell adhesion
molecules to bring together the receptor and ligand into a cell surface
complex. To
test this idea, sandwiches were made in which rhomboid and
Star were expressed in both the sending and receiving cells.
Interestingly, this configuration attenuates the level of Egfr
signaling, with the strongest repression occuring when both Rhomboid
and Star are present on both sides of the sandwich. It is an intriguing possibility that an interaction between
Rhomboid and/or Star in trans may dampen the
level of signal received by Egfr, providing another possible mechanism
by which the level of Egfr activation could be finely tuned. Together,
these results argue against models in which Rhomboid and Star regulate
receptor function or act as cell adhesion molecules and support a model
in which Rhomboid and Star potentiate Egfr activation by acting in the
signaling cell (Bang, 2000).
It was asked whether Rhomboid and Star potentiate Egfr signaling by
changing the activity of its ligand, as suggested by the observation
that sSpi does not require Rhomboid and Star to activate Egfr, whereas
mSpi does. To address this question, a series of chimeras were made by
replacing portions of human TGF-alpha, a vertebrate homolog of Spi,
with the corresponding regions from mSpi. Human TGF-alpha alone strongly activates the human EGFR in the animal
cap assay. Strikingly, when the cytoplasmic (C) and
transmembrane (TM) domains of TGF-alpha are replaced with those of mSpi
(TGF-alpha/SpiTMC), the chimeric molecule activates the
human EGFR only when Rhomboid and Star are present. In contrast, chimeric molecules in which the TGF-alpha C or TM
domains are replaced separately with those of mSpi
(TGF-alpha/SpiC and TGF-alpha/SpiTM,
respectively) are constitutively active. Thus,
together the mSpi TM and C domains are sufficient to confer Rhomboid
and Star dependence on TGF-alpha. This result suggests that the C and
TM domains maintain Spi in an inactive state, and that their ability to
do so is transferrable to another EGFR ligand. As predicted by this
interpretation, a membrane-bound form of Spi that activates Egfr
signaling in the absence of Rhomboid and Star can be generated by
replacing the mSpi TM and C domains with those of TGF-alpha
(Spi/TGF-alphaTMC). In addition, SpiDelta53C, a Spi mutant in which 53 carboxy-terminal residues are deleted and 17 cytoplasmic residues remain, exhibits some Rhomboid and
Star-independent activity, providing further evidence that the C domain
plays an inhibitory role. Together these results
argue strongly that the C and TM domains of mSpi act to maintain an
inactive state, with ligand activation occuring upon interaction with
Rhomboid and Star (Bang, 2000).
One way in which Rhomboid and Star could lead to ligand
activation is by promoting proteolytic processing, thus converting mSpi into a form similar to sSpi. Given the possibility that only low levels of Spi are required to
activate Egfr, a more sensitive assay was used to determine whether
Rhomboid and Star promote proteolysis of Spi. Conditioned medium was
prepared from dissociated animal cap cells from embryos injected with
RNA encoding sSpi, or mSpi, or coinjected with RNAs encoding mSpi,
Rhomboid, and Star. Egfr-injected animal caps were incubated in the
conditioned medium and then analyzed for expression of XBra. The conditioned medium from animal caps expressing sSpi or
mSpi/Rhomboid/Star contains an activity
that activates Egfr, whereas that from animal caps expressing mSpi alone
does not. In addition, the conditioned medium activity is Egfr dependent, as it is ineffective on uninjected animal
caps. These results suggest that Rhomboid and
Star activate mSpi by promoting its cleavage and secretion (Bang, 2000).
Next, it was determined whether proteolytic processing is required for
Rhomboid and Star activation of mSpi. To do this, potential
sites for processing of mSpi were removed by deleting the sequences encoding the 15 amino acids (aa) between the Spi EGF and TM domains (Spi-15aa). This region was selected because cleavage of TGF-alpha is known to
take place within an analogous interval. When tested in the animal cap assay, Spi-15aa strongly
activates Egfr in a Rhomboid and Star-dependent manner. In contrast, conditioned medium prepared from animal caps
expressing Spi-15aa, Rhomboid, and Star does not contain any activity
that activates Egfr, indicating that Spi-15aa is not cleaved. Taken together, these results suggest that cleavage of mSpi
depends on the sequence deleted in the Spi-15aa mutant; however, mSpi
does not need to be cleaved to activate Egfr signaling. Thus, Rhomboid
and Star may act to present mSpi to Egfr and subsequently facilitate or
allow its cleavage (Bang, 2000).
The results obtained with the Spi-15aa deletion mutant suggest that
mSpi, like TGF-alpha, is processed to generate a soluble form. To
examine the nature of this processing further, the
possibility was tested that the processing includes a cleavage within the transmembrane domain
of mSpi. This possibility is suggested by the results obtained with the
Spi/TGF-alpha chimeras, showing that the Spi
transmembrane domain is important for Rhomboid and Star-dependent
activation. Moreover, another multimembrane-spanning protein,
Presenilin-1, mediates proteolyis of the beta-amyloid precursor
protein and Notch, both of which are cleaved within their transmembrane
domains. If processing does lead to a cleavage in the
membrane, it was reasoned that this would release the intracellular domain
of Spitz in a Rhomboid/Star-dependent manner. To detect this cleavage, a chimeric molecule was generated in which the
mSpi C domain is replaced with the myc-tagged, intracellular domain of
the Xenopus Notch receptor (Spi/NICD). The endogenous, gamma-secretase-dependent Notch cleavage site is not present in the
Spi/NICD chimeric molecule.
If proteolytic processing of this molecule occurs within the Spi TM
domain in a Rhomboid/Star-dependent manner, NICD may be
released, translocate to the nucleus, and activate target genes. As a Notch target gene Xenopus Enhancer-of-split-related-1 (Esr-1) was analyzed in animal caps that were coinjected with the neuralizing factor noggin, because
Esr-1 is normally expressed in neural tissue and its induction
by NICD is more robust in a noggin background (Bang, 2000).
When tested in the animal cap assay, Spi/NICD activates
Egfr, but only in the presence of Rhomboid and Star, indicating that the
Spi/NICD chimeric molecule still exhibits Rhomboid and
Star-dependent Spi activity. This result
also indicates that the myc-tagged Xenopus NICD can
effectively replace the Spi C domain, suggesting that the ability of
the C domain to maintain Spi in an inactive state depends more on its
structure than on its primary sequence. Significantly,
Spi/NICD also activates the Notch target gene, Esr-1, in a Rhomboid and Star-dependent manner. This result suggests that Rhomboid and Star promote a proteolytic processing event within the Spi-TM domain that releases NICD. In addition, because Esr-1 induction is Rhomboid and Star dependent in the absence of Egfr, Rhomboid and Star can function independent of Egfr (Bang, 2000).
By analogy to the beta-amyloid precursor protein and Notch, whose
activites are regulated by multiple, interdependent cleavage events, the possibility was tested that the 15 amino acids between the Spi EGF and TM domains that are required for
production of soluble Spi are also required for the cleavage of the
Spi/NICD chimeric molecule within its TM domain. Thus, the sequence encoding these 15 amino acids was depleted in the
Spi/NICD chimera to produce Spi-15aa/NICD. This deletion mutant still strongly activates Egfr in a Rhomboid and Star-dependent manner, but no longer
induces Esr-1, indicating that NICD is not released, and thus
cleavage of this mutant does not occur. Thus,
these results provide further independent evidence for the contention that Rhomboid and Star-dependent cleavage of mSpi requires the amino
acids deleted in the Spi-15aa mutant, but mSpi need not be cleaved to
activate Egfr signaling. Finally, these results suggest that there is a
Rhomboid and Star-dependent cleavage event of mSpi within its TM
domain. One possible explanation for these observations is that mSpi is
cleaved both within the TM domain and within the 15 amino acids between
the TM and EGF domains. Alternatively, a single cleavage of mSpi could
occur within its TM domain that depends on the 15 amino acid interval (Bang, 2000).
Several models could account for the Rhomboid and Star-dependent
effects observed. One model is that Rhomboid and Star are
required to direct mSpi to the proper compartment for signaling to
occur. The results from the biotinylation experiments suggest strongly
that Rhomboid and Star are not required for transport of mSpi to the
cell surface, but it remains a possibility that Rhomboid and Star could
play a role in localizing mSpi to specific cell surface microdomains. An alternative class of
models is that mSpi is at the cell surface and ready to signal, but
that Rhomboid and Star are required for bringing mSpi into an active
conformation. One version of this model is that Rhomboid and Star
activate mSpi by promoting its oligomerization. However, this idea is
difficult to reconcile with the observation that sSpi is active and
either does not require oligomerization or oligomerizes independently
of Rhomboid and Star. In addition, soluble EGF, which is similar to
sSpi, binds as a monomer to the extracellular domain of the EGFR in a
1:1 ratio, suggesting that membrane-bound EGFR ligands may also
bind the receptor as monomers. For these reasons, an alternative model is favored in which mSpi is present at the membrane
in an inactive dimeric or oligomeric complex. Rhomboid and Star would
be required to either prevent formation of this complex or to alter its
conformation such that mSpi could be presented as an active form. This
model is precedented by observations suggesting that a number of
receptor tyrosine kinases exist as inactive dimers that are activated
when specific inter-subunit conformational changes occur upon ligand
binding. Thus, formation of an inactive mSpi
complex would be mediated by its C and TM domains and inhibited by an
interaction between these domains and Rhomboid and Star. This model
explains both why removal of these domains relieves the requirement for
Rhomboid and Star, and transfer of these domains to TGF-alpha confers
Rhomboid and Star dependence. Such a model also predicts that sSpi
would be Rhomboid and Star independent (Bang, 2000).
How do Rhomboid and Star promote cleavage of mSpi? Rhomboid
and/or Star could play a passive role by making mSpi
accessible to proteolysis upon presentation. Alternatively, Rhomboid
and/or Star may actively facilitate Spi proteolysis
either by activating or recruiting a protease or transporting Spi to
the appropriate subcellular compartment. It is also possible that Rhomboid
and/or Star could themselves have proteolytic activity, as has been proposed for Presenilin-1. A protease responsible for Spi cleavage has yet to be identified. Finally, although
this study strongly suggests that presentation of Spi is inhibited by
its C-domain, the question of whether proteolysis
of Spi is also affected by the C-domain has not been addressed. For instance, proteolytic
release of the extracellular domain of membrane bound neuregulin is
dependent on its cytoplasmic domain. Future
experiments will be aimed at determining whether Rhomboid and Star play
a passive or an active role in the proteolysis of mSpi (Bang, 2000).
The membrane proteins Star and Rhomboid-1 have been genetically defined as the primary regulators of EGF receptor activation in Drosophila, but an understanding of their molecular mechanisms has remained elusive. Both Star and Rhomboid-1 have been assumed to work at the cell surface to control ligand activation. This study demonstrates that they control receptor signaling by regulating intracellular trafficking and proteolysis of the ligand Spitz. Star is present throughout the secretory pathway and is required to export Spitz from the endoplasmic reticulum to the Golgi apparatus. Rhomboid-1 is localized in the Golgi, where it promotes the cleavage of Spitz. This defines a novel growth factor release mechanism that is distinct from metalloprotease-dependent shedding from the cell surface (Lee, 2001).
In the absence of Star, Spitz is retained in the ER. This explains why the domain of EGF receptor activation is much narrower than the expression pattern of Spitz, and why ectopic expression of full-length Spitz does not activate the receptor. Star, a protein with a single TMD, chaperones Spitz into the Golgi apparatus and the subsequent secretory pathway. The principal interaction between Spitz and Star occurs between the lumenal domains of the two proteins. Two models can be envisaged: Star could specifically block the ER retention signal; alternatively, Star could actively export Spitz from the ER, and in doing so, counteract retention (Lee, 2001).
Drosophila genetics indicate that Star and Rhomboid-1 are both prime regulators of EGF receptor activity: they both appear to be necessary and they cannot replace each other. It has not been possible to separate their functions. The results described here explain their codependency and synergy, and also provide a clear mechanistic distinction between them. An important issue is whether Star is necessary for Rhomboid-1-dependent proteolysis itself, as an enzymatic cofactor. This possibility can be ruled out, based on the Spi:TGFalpha-C chimera: it leaves the ER independently of Star and is efficiently cleaved by Rhomboid-1 in the absence of Star, implying that the primary function of Star is to export Spitz from the ER, thereby allowing it access to Rhomboid-1. Note, however, that the Rhomboid-1-dependent cleavage of the Spi:TGFalpha-TMC chimera suggests a possible secondary, nonessential role for Star as an adaptor, delivering substrate to Rhomboid-1. The data also do not rule out a role for Star in promoting efficient Spitz secretion (Lee, 2001).
The data clearly show that Rhomboid-1 is a Golgi-localized protein that triggers the proteolytic cleavage of Spitz. Rhomboid-1 could therefore be a novel protease, or it could recruit an unidentified protease; detailed biochemical analyses will be needed to resolve this. Star and Rhomboid-1 are sufficient to cleave Spitz in all cells tested, suggesting that they may be the only components required. The analysis also rules out the involvement of metalloproteases that are responsible for the release of TGFalpha and many other growth factors, further supporting the idea that Rhomboid-1 may itself be a protease. The absence of a genetically identified candidate protease, other than Rhomboid-1, despite much genetic screening, is also consistent with this hypothesis. The principal objection to this parsimonious model is Rhomboid-1's lack of identifiable protease domains. However, there are two recent precedents for multiple transmembrane domain proteins without recognizable protease domains being discovered to be novel proteases: presenilin-1 and SREBP site 2 protease (Lee, 2001).
Despite the distinctions between Spitz and TGFalpha processing, the similarities between flies and mammals may be greater than is at first apparent. For example, mature TACE (an ADAM family metalloprotease that acts on TGFalpha) is predominantly localized in intracellular compartments, suggesting that the cell surface may not be the only location of TGFalpha cleavage. Additionally, there is evidence for TACE-independent TGFalpha processing. Furthermore, TGFalpha also undergoes regulated transport through the secretory pathway, albeit by a distinct mechanism dependent on PDZ domain proteins. Finally, it is worth pointing out
that while TGFalpha appears to be the mammalian ligand most similar to Spitz, there are several other analagous human EGF receptor ligands whose regulation is still poorly understood (Lee, 2001).
The polytopic membrane protein Rhomboid-1 promotes the cleavage of the membrane-anchored TGFalpha-like growth factor Spitz, allowing it to activate the Drosophila EGF receptor. Until now, the mechanism of this key signaling regulator has been obscure, but this analysis suggests that Rhomboid-1 is a novel intramembrane serine protease that directly cleaves Spitz. In accordance with the putative Rhomboid active site being in the membrane bilayer, Spitz is cleaved within its transmembrane domain, and thus is the first example of a growth factor activated by regulated intramembrane proteolysis. Rhomboid-1 is conserved throughout evolution from archaea to humans, and these results show that a human Rhomboid promotes Spitz cleavage by a similar mechanism. This growth factor activation mechanism may therefore be widespread (Urban, 2001).
Although Rhomboid-1 does not contain any obvious sequence homology domains, it has the characteristics of a serine protease. (1) Four of its six essential residues parallel the residues required for a serine protease catalytic triad charge-relay system (S217, H281, and N169) and an oxyanion stabilization site (consisting of a glycine two residues away from the active serine, and the serine itself; G215 and S217). These are the two active site determinants of serine proteases, and these four essential residues account for all of the amino acids known to participate directly in the serine protease catalytic mechanism. (2) These residues are absolutely conserved in all Rhomboids, and their mutation to even very similar residues (i.e., G215A, S217T, and S217C) abolishes Rhomboid-1 activity. These are hallmarks of active site residues. (3) The location of the essential residues is highly suggestive of a serine protease active site; both G215 and S217 occur in the conserved GASGG motif, which is remarkably similar to the conserved GDSGG motif surrounding the active serine of 200 different serine proteases. Furthermore, the essential residues N169 and H281 occur at the same height in their transmembrane domains (TMDs) as the GASGG motif, consistent with the proposal that they associate with S217 to generate a catalytic triad. Finally, Spitz processing is directly inhibited by the specific serine protease inhibitors DCI and TPCK, and Rhomboid-1 itself becomes limiting in their presence, suggesting that Rhomboid-1 is their direct target and thus the serine protease responsible for Spitz cleavage (Urban, 2001).
The proposed Rhomboid-1 catalytic triad is unusual, as it contains an asparagine rather than the more common aspartate. The central importance of this aspartate, however, is uncertain, since serine proteases with catalytic dyads of only serine and histidine have been identified, and even in those enzymes with catalytic triads, the aspartate is 100-fold less sensitive to mutation than the serine or histidine. In the case of Rhomboid-1, although N169 is essential in the assay presented in this study, there is evidence that in other contexts, its mutation leaves residual Rhomboid-1 activity. Further support for the possibility that N169 replaces an aspartate in a catalytic triad comes from the mechanism of some cysteine proteases, whose catalytic mechanisms are identical to serine proteases: they use catalytic triads with an asparagine to orient the histidine. Overall, the idea is favored that N169 does form part of the catalytic triad, but without a structural analysis of the active site, the possibility remains that it instead could be involved in oxyanion stabilization. In summary, although several mechanistic questions remain, these results strongly suggest that Rhomboid-1 is a serine protease that catalyses proteolysis in a membrane bilayer. Since no intramembrane serine protease is listed in either the MEROPS protease or EC enzyme databases, Rhomboid-1 and RHBDL2 appear to be the first examples of this kind of enzyme (Urban, 2001).
It is not clear how Rhomboid-1 functions within the lipid bilayer. Proteases catalyze hydrolysis of the peptide bond and thus require water to be accessible to their active sites. Although the Rhomboid-1 active site is situated within the membrane bilayer, helical packing among the Rhomboid-1 TMDs could provide an aqueous environment surrounding its active site. Consistent with this idea, there is a conserved helical repeat of charged and/or polar residues in TMD II that contributes the putative catalytic triad residue N169; this could form an aqueous environment around the active site. This polar face is also likely to mediate associations with other TMDs. TMD VI, which contributes the putative catalytic triad residue H281, is also predicted to contribute to TMD interactions: it contains two tandem GxxxG motifs known to mediate strong associations between transmembrane helices of the same orientation. The only helices in Rhomboid-1 of the same orientation as TMD VI are TMDs II and IV, which contribute the active site residues N169 and S217, respectively. Thus, TMDs II, IV, and VI might associate to generate the putative catalytic triad while the polar face of TMD II could provide the local aqueous environment required for catalysis (Urban, 2001).
Proteases in general cannot cleave folded proteins, and most TMDs adopt a helical conformation, with the amino acid side chains facing outward, sterically hindering access to the peptide backbone. The putative aqueous cavity of the Rhomboid-1 active site could force the hydrophobic Spitz TMD to change conformation, allowing cleavage. Alternatively, it has been proposed that intramembrane proteases function by partially unfolding their helical substrates, extending them into the cytosol and thus simultaneously unwinding the helix and providing an aqueous environment for proteolysis to occur. The essential residues W151 and R152 in the large lumenal loop of Rhomboid-1 could be involved in substrate unwinding from the lumenal side. Note that the helical packing and substrate unwinding models are not necessarily mutually exclusive, and various aspects of each model may prove to be important for intramembrane proteolysis by Rhomboid-1. Ultimately, direct biochemical analysis of purified Rhomboid-1 protein activity will be required to answer many of these questions, but this has not yet been achieved for any intramembrane protease, despite intensive study. However, the observations that Rhomboid-1 does not require endoproteolytic activation or other Drosophila cofactors suggests that these important goals may be achievable (Urban, 2001).
Although many other membrane-bound growth factors are activated by proteolytic release, Spitz is unusual, because it is cleaved within its TMD. Regulated intramembrane proteolysis (RIP) has recently emerged as a novel mechanism for controlling several important signaling pathways, including Notch receptor activation and cholesterol biosynthesis, by the release of cytoplasmic transcription factor domains from membrane-anchored proteins. As with other known RIP proteases, Rhomboid-1 is a polytopic membrane protein that is a member of a large protein family with homologs in many species. However, previously described RIP proteases have been limited to either aspartyl or metalloproteases, while Rhomboid is a serine protease. Beyond this mechanistic distinction, there are two major differences between the pathways involved in current examples of RIP and Spitz cleavage by Rhomboid-1 (Urban, 2001).
(1) Previously characterized examples of RIP result in the cytoplasmic release of either membrane-tethered transcription factors or proteins that are required for the activation of transcription factors. Conversely, Spitz cleavage releases a growth factor into the lumen of the Golgi apparatus, which is then secreted as an active signal for the EGF receptor in neighboring cells. (2) There is also a clear distinction between the mechanisms regulating intramembrane cleavage. All other known RIP proteases are widely expressed and have broad substrate specificity. Intramembrane cleavage is regulated by a prior cleavage that removes the bulk of the lumenal or extracellular portions of the target protein. Only after this cleavage takes place can the intramembrane proteases recognize and cleave their substrates. Conversely, Rhomboid-1 activity is regulated primarily by its transcription; Rhomboid-1 expression is tightly regulated and precisely prefigures EGF receptor signaling during Drosophila development. Furthermore, Rhomboid-1 is site-specific in its cleavage ability, since it specifically cleaves Spitz but not similar proteins such as TGFalpha. The human Rhomboids, too, show specificity, since RHBDL2 but not RHBDL cleaves Spitz (Urban, 2001).
Since Drosophila Rhomboid-1 is the prototype of a family consisting of over 75 proteins, both in prokaryotes and eukaryotes, understanding its mode of action has implications beyond Drosophila signaling and development. Furthermore, conservation of a proteolytic function and biochemical mechanism in a human Rhomboid, coupled with the absolute conservation of the putative catalytic residues, suggests that all Rhomboids are intramembrane serine proteases. Although their physiological roles remain unclear, it is notable that most, but not all, organisms have Rhomboids. This is consistent with a role in important but not essential processes, for example, intercellular signaling. Intriguingly, recent analysis supports this notion. Only one Rhomboid member outside Drosophila has been studied. In the human pathogenic bacterium Providencia stuartii, the Rhomboid-like AarA protein is involved in promoting the release of an unknown factor that regulates virulence in response to cell population size (Rather, 1999; Gallio, 2000). Gram-positive bacteria like Providencia use peptide-based factors as quorum sensing signals, and in some cases these are proteolytically released from precursors. Thus, although the current evidence is very limited, it is notable that even in a bacterium, Rhomboid-dependent proteolysis may be involved in signal production during cell communication. Proteases control many aspects of cell regulation; they also have substantial clinical significance. Defining the substrates of other Rhomboids should thus reveal their physiological and perhaps pathological roles in humans and other species (Urban, 2001).
Drosophila has three membrane-tethered epidermal growth factor (EGF)-like proteins: Spitz, Gurken and Keren. Spitz and Gurken have been genetically confirmed to activate the EGF receptor, but Keren is uncharacterized. Spitz is activated by regulated intracellular translocation and cleavage by the transmembrane proteins Star and the protease Rhomboid-1, respectively. Rhomboid-1 is a member of a family of seven similar proteins in Drosophila. Four of the rhomboid family members have been examined: all are proteases that can cleave Spitz, Gurken and Keren, and all activate only EGF receptor signaling in vivo. Star acts as an endoplasmic reticulum (ER) export factor for all three. The importance of this translocation is highlighted by the fact that when Spitz is cleaved by Rhomboids in the ER it cannot be secreted. Keren activates the EGF receptor in vivo, providing strong evidence that it is a true ligand. These data demonstrate that all membrane-tethered EGF ligands in Drosophila are activated by the same strategy of cleavage by Rhomboids, which are ancient and widespread intramembrane proteases. This is distinct from the metalloprotease-induced activation of mammalian EGF-like ligands (Urban, 2002).
Star regulates Spitz cleavage by Rhomboid-1 by transporting Spitz to the Golgi apparatus. Strikingly, although Star was essential for ligand secretion into the culture medium in each case, it does not affect the ability of Rhomboids 2, 3 and 4 to catalyse Spitz cleavage. The extensive O-linked glycosylation that is diagnostic of transit through the Golgi apparatus (and which increases the apparent molecular weight of Spitz) was not present in cell lysates. Therefore, in contrast to Rhomboid-1, Rhomboids 2, 3 and 4 causes the accumulation of an intracellular cleaved Spitz that is not transported past the trans-Golgi network and thus not secreted (Urban, 2002).
The ability of Rhomboids 1-4 to catalyse Spitz cleavage in the tissue culture assay suggested that all may be involved in activating the EGF receptor in vivo. This has been clearly demonstrated for Rhomboid-1, was genetically determined in the case of Rhomboid-3, and has been proposed for Rhomboid-2. To investigate this further, the potential activity of Rhomboids 2-4 in vivo was compared by overexpressing them in developing Drosophila tissues. In all cases examined, Rhomboids 2-4 cause similar phenotypes to Rhomboid-1, consistent only with EGF receptor hyperactivation. When expressed in the developing wing, for example, all core Rhomboids produce ectopic and thickened vein phenotypes similar to those observed for Rhomboid-1. This phenotype was modified predictably by mutations in other members of the EGF receptor pathway. Furthermore, as in cell culture assays, all four Rhomboids are synergistic with the co-expression of Star. In all cases, UAS Rhomboids 1 and 3 produce consistently strong wing phenotypes, whereas Rhomboids 2 and 4 are weaker. Similar results were obtained in the eye, follicle cells of the ovary and the embryo. Importantly, no other phenotypes were observed in eyes, wings or embryos expressing Rhomboids, suggesting that they do not affect any other pathways. If, for example, the previously uncharacterized Rhomboids 2 or 4 caused the activation of other signaling pathways, their ectopic expression would lead to additional phenotypes. These observations confirm that Rhomboids 2-4 contain the same proteolytic activity as Rhomboid-1; furthermore, the absence of phenotypes associated with other pathways strongly suggests that Rhomboids 1-4 are all dedicated to regulating EGF receptor signaling. These data demonstrate that Rhomboids 1-4 all share proteolytic activity against the ligand Spitz (Urban, 2002).
A new Spitz-like gene was identified as a cDNA submitted to GenBank by the Berkeley Drosophila cDNA sequencing project. With the subsequent completion of the Drosophila genome sequence, this gene has been annotated as Keren and is the only previously unknown membrane-tethered EGF-like molecule identified by the Drosophila genome project. Keren has been referred to previously as Spitz-2 and Gritz. Like Spitz and Gurken, Keren has a single extracellular EGF repeat and a single transmembrane domain. The amino acid sequence of Keren is more closely related to Spitz than to Gurken (49% identity, 55% similarity to Spitz; 30% identity, 37% similarity to Gurken). While all three ligands were predicted to have N- and O-linked glycosylation signals, Spitz contains a 10 residue insert in its N-terminus, which contains an additional O-linked glycosylation site. Consistent with this, Spitz is the only ligand to be hyperglycosylated in the presence of Star, although deletion of the insert does not fully abolish hyperglycosylation. Rhomboids 1-4 all cleaved Keren in a mammalian tissue culture assay. There was, however, an interesting distinction between Keren and Spitz: Star was not essential for Keren secretion in every case. A significant amount of Keren was secreted in the presence of Rhomboids 3 and 4 alone. Despite this, Star always enhanced the secretion of cleaved Keren, implying that it can interact with Keren (Urban, 2002).
Star's role in Spitz activation is to export Spitz from the ER to the Golgi apparatus where it encounters the proteolytic activity of Rhomboid-1. Star also promotes the release of Keren into the medium, suggesting its role is similar to that in Spitz processing. This relocalization of Keren is very similar to the relocalization observed for Spitz, suggesting that Keren also needs to be relocalized to the Golgi apparatus for efficient processing and secretion (Urban, 2002).
A striking variety of glycosylation occur in the Golgi complex in a protein-specific manner, but how this diversity and specificity are achieved remains unclear. This study shows that stacked fragments (units) of the Golgi complex dispersed in Drosophila imaginal disc cells are functionally diverse. The UDP-sugar transporter Fringe-Connection (Frc) is localized to a subset of the Golgi units distinct from those harboring Sulfateless (Sfl), which modifies glucosaminoglycans (GAGs), and from those harboring the protease Rhomboid (Rho), which processes the glycoprotein Spitz (Spi). Whereas the glycosylation and function of Notch are affected in imaginal discs of frc mutants, those of Spi and of GAG core proteins are not, even though Frc transports a broad range of glycosylation substrates, suggesting that Golgi units containing Frc and those containing Sfl or Rho are functionally separable. Distinct Golgi units containing Frc and Rho in embryos can also be separated biochemically by immunoisolation techniques. Tn-antigen glycan is shown to be localized only in a subset of the Golgi units distributed basally in a polarized cell. It is proposed that the different localizations among distinct Golgi units of molecules involved in glycosylation underlie the diversity of glycan modification (Yano, 2005).
The pattern of glycosylation is extremely diverse, yet is highly specific to each protein. How can this specificity (and diversity) be achieved? There are >300 glycosylenzymes in humans and >100 in Drosophila, but is their enzymatic specificity sufficient to explain the precise modification of all substrates? One possible mechanism that might also contribute to the specific (and diverse) pattern of glycosylation would be the localization/compartmentalization of glycosylenzymes (Yano, 2005).
The Golgi complex, where protein glycosylation takes place, has been regarded as a single functional unit, consisting of cis-, medial-, and transcisternae in mammalian cells. However, the three-dimensional reconstruction of electron microscopic images of the mammalian Golgi structure has suggested the existence of more than one Golgi stack, with the individual stacks being connected into a ribbon by tubules bridging equivalent cisternae. Furthermore, during mitosis, the Golgi cisternae of mammalian cells become fragmented without their disassembly. In Drosophila, Golgi cisternae are stacked but are not connected to form a ribbon at the embryonic and pupal stages even during interphase, although there has been no evidence to date to indicate functional differences among the Golgi fragments (Yano, 2005).
A Drosophila UDP-sugar transporter, Fringe connection (Frc) transports a broad range of UDP-sugars that can be used for the synthesis of various glycans, including N-linked types, GAGs, and mucin types. Interestingly, despite its broad specificity, loss-of-function studies have revealed that Frc is selectively required for Notch glycosylation, but not for GAG synthesis. This observation prompted a study at Frc localization; in this study, it was found that Frc is localized only to a subset of Golgi fragments in Drosophila discs and embryos (Yano, 2005).
Frc, Sfl, a glycosylenzyme of GAGs, and Rho, a processing enzyme of Spi glycoprotein, are localized to distinct Golgi fragments, which are referred to as 'Golgi units,' in Drosophila cells. frc mutants do not exhibit defects in the glycosylation and function of Spi nor do they exhibit defects in glycosylation or function of GAG core proteins. Moreover, biochemically separated distinct Golgi units containing Frc and Rho were isolated by immunoisolation technique. This study clearly shows that there are functionally distinct Golgi units in a Drosophila cell (Yano, 2005).
The Golgi complex is a stack of cis-, medial-, and transcisternae in mammalian cells. In contrast, Golgi markers often do not overlap with each other in Saccharomyces cerevisiae, in which the Golgi cisternae are not stacked but disassembled. The Golgi cisternae of Drosophila are stacked but are not connected to form a ribbon at the embryonic and pupal stages even during interphase. To determine whether Drosophila imaginal disc cells have assembled or disassembled Golgi cisternae, the localizations were compared of the cis-cisternal marker dGM130, the transcisternal marker dSyntaxin16 (dSyx16), and the Golgi-tethered 120-kDa protein, which is commonly used to detect the Golgi complex in Drosophila. The 120-kDa protein was identified by immunoaffinity purification and protein sequencing as a Drosophila homolog of the vertebrate 160-kDa medial Golgi sialoglycoprotein (MG160), which resides uniformly in the medial-cisternae of the Golgi apparatus in vertebrate cells. An antibody specific for the 120-kDa protein also stained numerous Golgi fragments in imaginal disc cells. More than 80% of immunoreactivity for the 120-kDa protein colocalizes with both dGM130 and dSYX16, suggesting that 120-kDa protein-positive fragments of the Golgi complex indeed comprise assembled cisternae; these fragments will be referred to as 'Golgi units'. The distributions of the 120-kDa protein, dGM130, and peanut agglutinin (PNA), another transcisternal marker, also shows that the markers are closely apposed but not identical, suggesting that the Golgi units are polarized. Interestingly, most of the PNA-positive transcisternae are oriented toward the basal side of the cell, within the Golgi complex, whereas most of the GM130-positive cis-cisternae are oriented toward the apical side of the cell. The cis-to-trans polarity of each Golgi unit thus appears to be correlated with the apico-basal polarity of the disc cells (Yano, 2005).
Drosophila mutant larvae defective in the UDP-sugar transporter Frc manifest a highly selective phenotype: the lack of Notch glycosylation in the presence of normal GAG synthesis (Goto, 2001). This limited phenotype was unexpected, given that Frc exhibits a broad specificity for UDP sugars used in the synthesis of various glycans including N-linked types, GAGs, and mucin types. However, given that the frcR29 allele studied previously (Goto, 2001) is hypomorphic, whether the selective glycosylation defect might be a consequence of partial loss of Frc activity was examined. With the use of imprecise excision, a new allele, frcRY34, was generated the presence of which results in the death of most larvae during the second-instar stage, much earlier than the death induced by frcR29. Real-time PCR analysis revealed that the amount of frc transcripts in the second-instar larvae of frcRY34 or frcR29 mutants was 4.2% and 24.4% of that in the wild type, respectively. About 1 kb of the gene, including the transcription initiation site, was deleted in the frcRY34 allele. Together, these observations suggest that frcRY34 is essentially a null allele (Yano, 2005).
Clonal cells of the frcRY34 mutant exhibit normal levels of GAGs, as detected by immunostaining with the 3G10 antibody, whereas the amount of GAGs was reduced in clones of tout-velu (ttv) mutant cells. Given that GAGs are required for signaling by Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp),the expression was examined of corresponding target genes [patched (ptc) for Hh signaling and Dll for Wg and Dpp signaling] in the wing discs of the frcRY34 mutant. Expression of ptc and that of Dll in the ventral compartment of the wing discs were unaffected in the mutant clones, suggestive of normal GAG function (Yano, 2005).
Given that Notch glycosylation by Fringe (Fng), a fucose-specific ß1,3-N-acetylglucosaminyltransferase, requires Frc activity, Notch glycosylation was examined in the frcRY34 mutant. The frcRY34 mutant clones in the dorsal compartment, but not those in the ventral compartment, of the wing discs induce wg expression at their borders, as has been observed with fng mutant clones, suggesting that Notch glycosylation is impaired in the frcRY34 mutant. The ectopic expression of Wg induced by the frcRY34 mutant clones is likely responsible for the observed induction of Dll expression in the dorsal compartment (Yano, 2005).
To determine why the loss of a UDP-sugar transporter with a broad specificity selectively affects Notch glycosylation, the subcellular localization of Frc was examined. Frc tagged with the Myc epitope was expressed in imaginal discs under the control of the arm-Gal4 driver. The Gal4-induced expression of Frc-Myc rescues the frc mutant phenotype, suggesting that Frc-Myc is functional and properly localized. Immunostaining of imaginal discs of wild-type larvae expressing Frc-Myc with antibodies to Myc and to the 120-kDa protein revealed that Frc localizes to only a small subset of Golgi units. Thus, it is hypothesized that the Golgi units might be functionally heterogeneous, and that those containing Frc might modify some proteins, including Notch, but not others (Yano, 2005).
To test this hypothesis, the localizations of various molecules involved in protein modification in the Golgi complex were compared with that of Frc. It was found that Sfl is also restricted to a subset of Golgi units, but that its distribution does not overlap with that of Frc. This differential localization of Sfl and Frc might thus explain the observation that frc mutant clones in wing discs do not show any defect in GAG synthesis by Sfl (Yano, 2005).
The Spi-processing enzyme Rho was also localized to a subset of Golgi units distinct from those containing Frc, in addition to its presence in other compartments. This result indicates the existence of at least two types of Golgi units, those containing Rho and those containing Frc. To determine whether these two types of Golgi units differ functionally, the glycosylation state and function of Spi were examined in frc mutants (Yano, 2005).
Given that the extent of Notch glycosylation, as detected by wheat germ agglutinin (WGA), is markedly reduced in frc mutants compared with that in the wild-type background (Goto, 2001), whether the WGA-reactive glycan of Spi is also affected by frc mutation was examined. Myc epitope-tagged Spi was expressed in the wild type or the frcRY34 mutant. Spi-Myc was then precipitated from larval homogenates with antibodies to Myc and was examined for its glycosylation by SDS/PAGE and subsequent blot analysis with WGA. The reactivity of the Spi glycan with WGA was similar in the frc mutant and in the wild type. Whether the frcRY34 mutation affects the Spi glycan was examined by mobility shift analysis. The electrophoretic mobility of glycosylated Spi from the wild type was also similar to that from the frc mutant. Deglycosylation of Spi by neuraminidase, peptide-N-glycosidase (PNGase) F, and O-glycanases also increased its mobility to the same extent in wild-type and frc mutant larvae, suggesting that the core protein is not affected by the frc mutation. Together, these results indicate that the function of Frc is not necessary for formation of the Spi glycan (Yano, 2005).
Spi function was evaluated by examining developmental processes such as photoreceptor recruitment and bract formation, both of which require Spi activation. During eye development, although Spi is not necessary for the primary induction of the photoreceptor R8, it is required for the subsequent recruitment of R1 to R7. Given that photoreceptors R1 to R8 express ELAV and that R1 and R6 express Bar, the expression of these proteins was examined in frc mutants. In mutants harboring the hypomorphic allele frcR29, all photoreceptors are normally induced, although their direction is irregular as seen in fringe or Notch mutants. Similar results were obtained by clonal analysis of frcRY34 mutants. Spi function in photoreceptor recruitment thus did not appear to be impaired in the frc mutants. The frcR29 mutant also formed normal bracts on malformed legs. Tests were performed for genetic interaction between rho and frc mutations in wing vein formation. The rhove1 mutant is viable but shows partial loss of L3-5 veins. This phenotype is also apparent in rhove1, frcRY34/rhove1, frc+ flies, suggesting that Frc does not affect Rho function. From these results, it is concluded that the function of the Rho-Spi pathway is not affected by frc mutation (Yano, 2005).
To confirm that the Golgi units containing Frc and those containing Rho are distinct, whether these Golgi units could be selectively isolated was tested by using antibodies to Myc (for Myc-tagged Frc) or HA (for HA-tagged Rho). Because it is difficult to collect enough of the imaginal discs, the starting material was switched to embryos, and whether Frc and Rho are also localized to distinct Golgi units in embryos was examined. Frc-Myc and Rho-HA were coexpressed in the embryos by the arm-Gal4 driver; immunostaining with antibodies to Myc and to HA revealed that the Golgi units containing Frc-Myc (45.4% of total Golgi units) and those containing Rho-HA (43.0% of total Golgi units) are largely distinct: only 11.6% of total Golgi units were positive for both Frc-Myc and Rho-HA. Immunoisolation was attempted from embryonic lysates by using either antibody to Myc or HA and how much Frc-Myc and Rho-HA were coisolated in each immunoisolate was examined. When Frc-Myc was immunoisolated with an antibody to Myc, the recovery of Frc-Myc was 5.7 times greater than that of Rho-HA. Moreover, when Rho-HA was immunoisolated with an antibody to HA, the recovery of Rho-HA was 18.3 times greater than that of Frc-Myc. The immunoblot analysis of these immunoisolates with the anti-120-kDa antibody confirmed that the Golgi units were concentrated in these immunoisolates. These results support the notion that Frc-Myc-containing fraction is distinct and can be separated from Rho-HA-containing fraction (Yano, 2005).
Whether these distinct Golgi units contain different constituents was examined. Fringe (Fng) is one of the candidate molecules that may be colocalized with Frc. Therefore, expression of ectopically expressed Fng was examined in Rho- and Frc-containing immunoisolates. It was found that expression of Fng in Frc-containing immunoisolates was 26 times greater than in Rho-containing immunoisolates, supporting the idea that Fng is localized in the Frc-positive Golgi units rather than the Rho-positive Golgi units. It was also confirmed by immunostaining analysis that Fng colocalizes mostly with Frc (88.1% of the FNG-positive Golgi units), but not with Rho (16.6% of the Fng-positive Golgi units), by immunostaining analysis (Yano, 2005).
The data suggest that different Golgi units perform different functions, a notion that is also supported by the observation that Tn antigen (O-linked N-acetylgalactosamine) was detected in only a subset of Golgi units in imaginal eye disc cells. In addition, it was found that most of these Tn antigen-positive Golgi units are distributed in the basal region of the disc cells, suggesting that the differential distribution of Golgi units might contribute to the apicobasal polarity of glycan distribution (Yano, 2005).
In contrast to the larval stage, Frc is required for GAG synthesis at the early embryonic stage (Goto, 2001; Selva, 2001). To determine why the Frc requirement for GAG synthesis differs between the embryonic and larval stages, embryos expressing Frc-Myc were stained with antibodies to Sfl and to Myc. Sfl was found to be colocalized with Frc, likely explaining the importance of Frc for GAG synthesis at the embryonic stage. In addition, this embryonic requirement of Frc for GAG synthesis excludes the possibility that the selective defects in Notch and not in GAG synthesis observed in frc mutant larvae are caused by the selective Frc-dependent transport of a subset of UDP-sugars used only for glycosylation of Notch but not for GAGs synthesis (Yano, 2005).
It summary, these results provide evidence for the existence of functionally distinct Golgi units in Drosophila cells. Such functional heterogeneity of Golgi units is likely responsible for the diversity of protein glycosylation. At least two types of Golgi units containing either Frc or Sfl are present in larval disc cells. Two distinct sets of proteins, exemplified by Notch and GAG core proteins, might thus be selectively transported to Frc- or Sfl-containing Golgi units, respectively, where they undergo glycosylation by different sets of molecules (Yano, 2005).
The variety of Golgi units might be established by separate transport of secretory proteins and glycosylenzymes from the endoplasmic reticulum (ER) to the distinct Golgi units. In yeast, glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in vesicles distinct from those containing other secretory protein. Given that the GAG core protein Dally in Drosophila is anchored to the membrane by GPI, it is possible that Dally and Notch are loaded into distinct vesicles as they exit the ER (Yano, 2005).
Combinations of glycosylenzymes and transporters, such as Sfl and Frc, contained in Golgi units of Drosophila differ not only between embryos and larval disc cells but also among cell types. For example, Frc is localized to all Golgi units in salivary gland cells at the larval stage. It has also been shown that all of the Golgi complexes dispersed in oocytes may have the ability to process the Gurken precursor protein, which is usually cleaved in a subset of the Golgi complexes residing in the dorso-anterior region. The Golgi units may thus be altered in a manner dependent on development, cell type, and signaling processes (Yano, 2005).
The functional diversity of Golgi units also might contribute to the polarized distribution of glycans along the apicobasal axis of cells. It was found that Tn antigen is synthesized in the basal Golgi units of larval disc cells. Furthermore, certain types of glycans are distributed along the apicobasal axis of pupal ommatidia. These glycans might thus be synthesized differentially in the Golgi units that are asymmetrically distributed along the apicobasal axis and then be secreted at either the apical or basal cell surface (Yano, 2005).
Whereas Golgi units are dispersed throughout Drosophila cells, the Golgi complex in mammalian cells is thought to be a single entity that is located in the pericentriolar region through its association with the microtubule-organizing center in interphase and which is fragmented at the onset of mitosis. The Golgi fragments apparent in mammalian cells during mitosis are highly similar to the Golgi units of Drosophila cells in both electron and confocal microscopic images. The mammalian Golgi complex during interphase may therefore be comprised of functionally distinct units that are associated with the microtubule-organizing center and connected with each other (Yano, 2005).
Spitz (Spi) is the most prominent ligand of the Drosophila EGF receptor. It is produced as an inactive membrane precursor that is retained in the endoplasmic reticulum (ER). To allow cleavage, Star transports Spi to the Golgi, where it undergoes cleavage by Rhomboid. Since some Egfr phenotypes are not mimicked by any of its known activating ligands, an additional ligand (Keren) was identified by database searches. Krn is a functional homolog of Spi since it can rescue the spi mutant phenotype in a Rho- and Star-dependent manner. In contrast to Spi, however, Krn also possesses a Rho/Star-independent ability to undergo low-level cleavage and activate Egfr, as evident both in cell culture and in flies. The difference in basal activity correlates with the cellular localization of the two ligands. While Spi is retained in the ER, the retention of Krn is only partial. Examining Spi/Krn chimeric and deletion constructs implicates the Spi cytoplasmic domain in inhibiting its basal activity. Low-level activity of Krn calls for tightly regulated expression of the Krn precursor (Reich, 2002).
In order to identify additional Egfr ligand(s), databases searches were performed for new Spi homologs. Two expressed sequence tags (ESTs) representing the same gene were found to be highly homologous to Spi (LD34470 and LD34429), mapping to 74E/F. The complete sequence of both ESTs revealed an open reading frame of 217 amino acids (Reich, 2002).
In the three activating Egfr ligands identified to date, the homology was restricted to the EGF domain. In contrast, the similarity between Spi and the new putative ligand, extending throughout the coding region, is 56%. The length and domain organization of both proteins are similar. Homologous stretches are concentrated mainly in the extracellular domain, with significant identity in the EGF domain (65%), while limited homology can be found in the cytoplasmic domain. Because of the similarity to Spi, this protein was named Keren, which is the Hebrew word for an antler, or a sharp object (Reich, 2002).
The ESTs of Krn are comprised of two exons, where the entire coding region is located in the second exon. Interestingly, the 5' non-coding exon is also found in ESTs of a coding sequence located more 3' to the gene encoding Krn. This coding sequence is homologous to novel mouse and human proteins. The splicing of the 5' non-coding exon to the TGFa transcript was confirmed by RT-PCR of RNAs extracted from adult flies and S2 cells. According to the RT-PCR, Krn is expressed during the stages of embryonic and larval development, and in adults. Attempts were made to examine the Krn expression pattern using RNA in situ hybridization and antibody stainings on wild-type embryos or imaginal discs, but the signal was below detection level (Reich, 2002).
Spi is active only as a cleaved moiety (sSpi), which diffuses to neighboring cells and activates the receptor. Following this paradigm, the secreted form of Krn (sKrn) was overexpressed in various tissues. Indeed, in all tissues, the phenotypes of sKrn resembled the ectopic phenotype of sSpi. In the wing, MS1096-Gal4 driving sKrn gave rise to lethality, but several survivors had short bloated stumps for wings. In the ovary, expression of sKrn in the anterior follicle cells using 55B-Gal4 resulted in the formation of extra dorsal appendages. Ectopic sKrn driven by 69B-Gal4 in the embryonic ectodermal cells causes lethality, with the cuticle showing wider and square-edged denticle belts characteristic of Egfr hyperactivation. It is evident that sKrn can activate MAPK, as revealed by dpERK antibody) wherever it is expressed in the embryo, or in the wing disc. These results show that sKrn can mimic sSpi activity and activate the Egfr pathway (Reich, 2002).
Having demonstrated the biological activity of sKrn, it was of interest to determine whether its processing is regulated in a similar manner to Spi. Triggering of Egfr by Spi at stage 10 generates two prominent domains of activation, in the tracheal placodes and ventral ectoderm. These domains correspond to the sites of Rho expression in the tracheal placodes and midline, respectively. dpERK can be readily detected in these domains, while in spi mutant embryos no dpERK is observed at this stage. The capacity of the Krn precursor to rescue spi mutant embryos was examined. When Krn was ubiquitously expressed in the ectoderm of spi- embryos (using the 69B-Gal4 driver), complete rescue of the dpERK pattern was observed. Induction of the pathway by Krn at the sites of Rho expression in the midline and tracheal placodes indicates that, like Spi, processing of Krn is dependent upon Rho. To test whether Krn cleavage requires Star, Krn was expressed in Star mutant embryos. No rescue of the phenotype was observed, as monitored by dpERK. It is thus concluded that, like Spi, processing of Krn is Rho and Star dependent (Reich, 2002).
While Spi is an extremely potent ligand in its secreted form, when expressed in the precursor form, even at high levels, it shows no ectopic activity. Following this paradigm, the rescue of spi mutant embryos by high levels of expression of Krn shows detectable levels of dpERK only at the tissues where Spi cleavage normally takes place. Surprisingly, in contrast to Spi, Krn is capable of eliciting phenotypes in a variety of tissues following overexpression. For example, in the wing, Krn overexpression gives rise to bloated wings, similar to those obtained upon moderate activation of Egfr with the lambda-top construct. Over-expression in the eye leads to rough eyes and in the follicle cells to excess dorsal appendage material. In the embryo, expression of Krn gives rise to embryonic lethality, with cuticles displaying expansion of head structures, but otherwise normal in appearance (Reich, 2002).
A clearer understanding of Spi cleavage has been gained by studies in cells. Efficient cleavage of Spi occurs only in cells in which both Star and Rho are expressed. Spi is retained in the ER through its intracellular domain. Star binds Spi and translocates it from the ER to the Golgi, where Rho functions as a protease and cleaves Spi. Krn-GFP in Drosophila S2 cells shows partial release from retention, manifested by a vesicular distribution, indicating exit from the ER. The functional implication of this was observed through the ectopic phenotypes in various tissues. For instance, whereas ectopic Spi driven by ubiquitous GAL4 in embryos does not prevent hatching of larvae, ectopic Krn leads to lethality. This highlights the importance of retention, since it allows expression of high amounts of Spi protein in the cell, yet controls Spi activity by preventing Spi from reaching further compartments where cleavage occurs (Reich, 2002).
Chimeric and deletion constructs identify the cytoplasmic domains of Spi and Krn as the domains responsible for their different cleavage profiles. This is a result of different levels of retention in the ER. The mechanism of Spi and Krn retention is not yet clear. Spi has also been shown to be retained in a heterologous system of mammalian cells, implicating the action of conserved molecules or an intrinsic property of the protein. In one model, association of the Spi cytoplasmic domain with an additional protein(s) could mediate retention. In that case, it would be expected that Krn would have lower affinity to this protein(s). In another model, the Spi C-terminus itself could have an intrinsic inhibitory capability through protein folding that sterically prohibits association to proteins -- this would carry Spi further in the secretory pathway. In this case, Krn would be expected to possess a higher affinity to such chaperones, that would allow it to exit the ER without total dependence on Star (Reich, 2002).
Compared with Spi or Krn, the cytoplasmic domain of Grk, the third Egfr ligand with a transmembrane domain, is shorter (only 24 amino acids). Deletion of its cytoplasmic domain did not influence signaling by Grk. Like Krn, overexpression of the full-length Grk protein causes ectopic wing phenotypes. It would be interesting to see to what extent Grk is retained in the ER of S2 cells (Reich, 2002).
Expression of Krn in S2 cells allows the mechanism of low-level cleavage, which is Star and Rho independent, to be followed. What is the protease responsible for this cleavage? The sensitivity of Krn cleavage to inhibitors of serine proteases indicates that cleavage may be mediated by a protease of this family. Unlike Rho, which is expressed in a spatially and temporally regulated manner, the protease is expected to be ubiquitously expressed, since ectopic Krn causes abnormal phenotypes wherever it was expressed. This further elaborates the need for tight transcriptional control on Krn expression (Reich, 2002).
Co-expression of Star with Krn in S2 cells raises the amounts of secreted sKrn in the medium. This is a result of the efficient export of Krn from the ER by Star. Since higher levels of cleavage can be obtained by co-expressing both Rho and Star, it would seem to indicate that the protease involved in low-level cleavage is less efficient than Rho (Reich, 2002).
High-level cleavage of Krn was followed in embryos through the detection of dpERK. The activation profile followed the restricted expression of Rho, since Star is broadly expressed. Only in cell culture could Rho enhance cleavage of Krn without co-expression of Star. This probably occurs because Krn can 'leak' out of the ER, reach compartments where Rho is present and undergo cleavage by Rho independent of Star (Reich, 2002).
Sequence conservation within the transmembrane domains of the Rho protein have suggested that the cleavage site of Spi would reside within the transmembrane domain. There is also evidence to indicate that Rho cleaves Spi within the membrane. The transmembrane regions of Spi and Krn are conserved, and show 50% sequence identity. The transmembrane domain of Krn may thus possess the same recognition sites as that of Spi (Reich, 2002).
In view of the two modes of Krn cleavage, where could it have biological roles? The capacity of Krn to undergo Rho- and Star-dependent high-level cleavage suggests that it could provide the missing ligand in tissues where the rho phenotype is more severe than that of spi. This includes the formation of veins in the wing, generation of correct R8 spacing and inhibition of apoptosis after the morphogenetic furrow in the eye disc. In the embryo, the spi phenotypes are similar to those of rho or Star mutants, indicating that Krn is not likely to be required at this stage (Reich, 2002).
Could the low-level cleavage of Krn also play a physiological role in activating Egfr? In the eye imaginal disc, generation of clones of Egfr mutants either anterior or posterior to the morphogenetic furrow was not possible, due to cell lethality. The known rho family genes are not expressed anterior to the furrow. It is thus possible that the low-level cleavage of Krn may provide the residual levels of secreted ligand necessary to trigger Egfr anterior to the furrow, and allow cell survival. The low levels of Egfr activation anterior to the furrow are consistent with the non-detectable levels of dpERK in this domain. Sufficient levels of secreted ligand would need to be produced to elicit a biological response, in spite of the low level of krn expression. In the Krn misexpression assays presented in this work, high levels of expression were required to detect this low activity (Reich, 2002).
In order to examine the biological roles of Krn, krn double-stranded (ds) RNA was carried out. Efforts were focussed in the eye and wing imaginal discs, where Krn activity is expected. Uniform expression of krn dsRNA in these tissues, even when combined with spi dsRNA, did not yield a noticeable phenotype. The possible roles of Krn in these tissues may be confirmed only when mutations for krn become available and their phenotypes are tested, alone or in combination with mutants for spi (Reich, 2002).
In conclusion, Krn was found to be the functional homolog of Spi. Unlike Spi, Krn is capable of undergoing inefficient Star- and Rho-independent cleavage in flies and in cell culture. This is due to differences between the intracellular domains of Krn and Spi, which allow Krn to evade retention in the ER and reach further along in the secretory pathway. This calls for tight transcriptional control of Krn expression, in contrast to Spi, which can be ubiquitously and abundantly expressed (Reich, 2002).
Lipid modifications such as palmitoylation or myristoylation target intracellular proteins to cell membranes. Secreted ligands of the Hedgehog and Wnt families are also palmitoylated; this modification, which requires the related transmembrane acyltransferases Rasp (alternative name: Sightless) and Porcupine, can enhance their secretion, transport, or activity. rasp is also essential for the developmental functions of Spitz, a ligand for the Drosophila epidermal growth factor receptor (EGFR). In cultured cells, Rasp promotes palmitate addition to the N-terminal cysteine residue of Spitz, and this cysteine is required for Spitz activity in vivo. Palmitoylation reduces Spitz secretion and enhances its plasma membrane association, but does not alter its ability to activate the EGFR in vitro. In vivo, overexpressed unpalmitoylated Spitz has an increased range of action but reduced activity. These data suggest a role for palmitoylation in restricting Spitz diffusion, allowing its local concentration to reach the threshold required for biological function (Miura, 2006).
This study shows that the acyltransferase Rasp promotes palmitoylation of the Spi in addition to its previously reported substrate Hh. rasp mutants show phenotypes similar to spi mutants, and rasp is required for the activity of ectopic sSpi produced either by cleavage of endogenous Spi or by expression of a truncated protein. Rasp is also necessary for the hydrophobic character of Spi expressed in S2 cells. Palmitoylation of Spi by Rasp can be reproduced in COS cells, which do not contain any endogenous Spi palmitoyltransferase activity; either these cells do not express a Rasp homolog, or the homolog is too divergent to recognize Drosophila Spi. Mutation of the predicted active site histidine of Rasp blocks palmitate incorporation into Spi, suggesting that Spi may be a direct target of Rasp. However, the possibility cannot be excluded that other proteins present within COS cells contribute to the acyltransferase activity (Miura, 2006).
The basis for substrate recognition by Rasp is not obvious. There is little sequence homology between Hh and Spi following the palmitoylated cysteine, although both proteins have several basic amino acids in the vicinity; basic amino acids follow the palmitoylation site of some classes of intracellular proteins. Myc-tagged Skn, the mouse homolog of Rasp, has been reported to localize to the endoplasmic reticulum (ER) in CHO cells; in S2 cells, colocalization has been seen of HA-Rasp with markers of the Golgi apparatus. If Hh and Spi are palmitoylated in the same cellular compartment, they later follow different paths; Hh is released from the cell through the activity of the membrane protein Dispatched, whereas Spi requires Star for export from the ER and is then activated by Rho-mediated cleavage. Because sSpi can be palmitoylated, cleavage by Rho is not a prerequisite for palmitoylation. However, the effect of palmitoylation on secretion is more dramatic for full-length Spi than for sSpi, suggesting that palmitoylation may be more efficient when Spi undergoes its normal processing (Miura, 2006).
rasp is also required for processes mediated by EGFR ligands other than Spi. The observation that lack of rasp in the germline causes ventralization of the follicle cells suggests that Grk might be palmitoylated. Consistent with this possibility, it was observed that the extracellular domain of Grk also fractionates into the Triton X-114 layer when expressed in S2 cells, although to a lesser extent than Spi. The rasp phenotype is relatively mild compared to loss of grk, suggesting that Grk has a less stringent requirement for palmitoylation than Spi (Miura, 2006).
Wing vein development, which is affected in rasp mutants, requires both Rho and Vein, but not Spi. Since Vein is not synthesized as a transmembrane precursor, the requirement for Rho may suggest the involvement of Krn, a ligand closely related to Spi. Grk and Krn have cysteine residues immediately following the signal peptide, making them likely substrates for Rasp, but Vein does not, consistent with the observation that rasp is not required for the expression of the Vein target gene mirr. It is unclear whether vertebrate EGFR ligands undergo a similar palmitoylation, since none of the known ligands has an N-terminal cysteine residue; TGF-α is palmitoylated on two cysteines in the cytoplasmic domain of the transmembrane precursor, but this is likely to involve a different mechanism. It will be interesting to determine whether EGFR signaling is affected in mice mutant (Chen, 2004) for the rasp homolog Skn (Miura, 2006).
Both the acyltransferase Rasp and cysteine 29 are essential for the activity in vivo of endogenous or overexpressed full-length Spi, and both significantly enhance the activity of overexpressed truncated Spi. By contrast, in vitro studies with sSpiCS clearly argue that loss of palmitoylation has no effect on EGFR binding or activation, or on Argos binding. Thus, it is likely that palmitoylation defines biologically critical spatial or temporal aspects of Spi distribution, rather than affecting its inherent binding properties. Indeed, mutating cysteine 29 in either full-length or truncated Spi allows greater recovery of secreted Spi from cell culture media. In addition, wild-type tagged sSpi shows strong membrane localization both in S2 cells and in imaginal discs, while unpalmitoylated sSpi is not membrane associated in S2 cells and can reach and act on distant cells in vivo. It is therefore suggested that palmitoylation is required to maintain a high local concentration of Spi, perhaps by directly tethering Spi to the plasma membrane or allowing it to form a complex with other factors that restrict its diffusion (Miura, 2006).
Palmitoylation might have additional effects on Spi signaling; its strong effect on secretion of mSpi could be partially due to an inhibitory effect on Spi cleavage, although this would be unlikely to lead to increased Spi activity. It is also possible that palmitoylation contributes to endocytosis and recycling of Spi, a mechanism that has been reported to enhance Wg signaling. Palmitoylation is unlikely to affect ER retention of sSpi, as this occurs in both COS cells and S2 cells. In addition, no effect of palmitoylation has been observed on the intracellular distribution of tagged sSpi in S2 or COS cells (Miura, 2006).
Spi acts as a short-range signal in vivo, in part due to its induction of the secreted feedback inhibitor Argos. Palmitoylation of Spi does not affect its binding to Argos, as expected because this binding is mediated by the EGF domain of Spi. In addition, rasp is required for Spi function even in the complete absence of argos. It is therefore suggested that a high concentration of Spi is necessary simply to reach the level of EGFR activation required for biological function, irrespective of the presence of Argos. The results suggest that palmitoylation is the mechanism used to achieve this local accumulation of Spi (Miura, 2006).
Although Hh, Wg, and Spi all carry palmitate modifications essential for their function, palmitoylation appears to have different effects on each molecule. Wg, though not Wnt3a, requires palmitoylation for its secretion. Shh requires palmitoylation for incorporation into a lipoprotein complex that enhances its transport; Wg is also found in a similar complex. In addition to its effects on transport, palmitoylation enhances Hh activity in assays that do not require transport. It is noted that sSpiCS does not show the dominant-negative effects described for HhC84S, suggesting that palmitoylation does not affect Spi activity in the same way (Miura, 2006).
Palmitoylation of intracellular proteins frequently promotes membrane association, though it usually does so in conjunction with a second lipid modification. This raises the possibility that palmitoylated Spi is associated with the plasma membrane, rather than binding to lipoprotein particles like those that transport Hh and Wg. If so, it will be interesting to learn whether membrane-tethered sSpi can directly bind the EGFR. Full-length transmembrane Spi, in which the EGF domain is adjacent to the membrane, is inactive in the absence of Rhomboid, but membrane association of sSpi through its N-terminal palmitate group would place the EGF domain at a distance from the membrane. If membrane-bound Spi cannot activate the EGFR, Spi may be released from the membrane by depalmitoylation. Cycles of palmitoylation and depalmitoylation have been shown to regulate the intracellular localization of Ras. However, the N-terminal palmitate modification is likely to form a stable amide linkage as in Hh, rather than a labile thioester bond. Alternatively, release of Spi could be accomplished by proteolytic processing. Interestingly, it was found that the sequence of wild-type sSpi released into the media from S2 cells begins at methionine 45, whereas sSpiCS begins with the serine at position 29, immediately after the signal peptide (Miura, 2006).
The observation that palmitoylation of Spi is essential in vivo extends the importance of this modification of extracellular secreted proteins to a third class of ligands. However, its function appears to vary between different molecules and across species. Further study of membrane-bound palmitoyltransferases and their substrates is likely to yield new insights into the regulation of ligand secretion, transport, and activity (Miura, 2006).
Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes. Inappropriate activation of these receptors is a key feature of many human cancers, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signalling, called Argos, was identified in Drosophila. Argos functions by directly binding (and sequestering) growth factor ligands that activate EGFR. This study describes the 1.6-Å resolution crystal structure of Argos bound to Spitz, an EGFR ligand. Contrary to expectations, Argos contains no EGF-like domain. Instead, a trio of closely related domains (resembling a three-finger toxin fold) form a clamp-like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF. The individual Argos domains share unexpected structural similarities with the extracellular ligand-binding regions of transforming growth factor-β family receptors. The three-domain clamp of Argos also resembles the urokinase-type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF-like module of uPA. These results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented in this study define requirements for the design of artificial EGF-sequestering proteins that would be valuable anti-cancer therapeutics (Klein, 2008).
Drosophila is a well recognized model of several human diseases, and recent investigations have demonstrated that Drosophila can be used as a model of human heart failure. Previously, Optical coherence tomography (OCT) can be used to rapidly examine the cardiac function in adult, awake flies. This technique provides images that are similar to echocardiography in humans, and therefore it is postulated that this approach could be combined with the vast resources that are available in the fly community to identify new mutants that have abnormal heart function, a hallmark of certain cardiovascular diseases. Using OCT to examine the cardiac function in adult Drosophila from a set of molecularly-defined genomic deficiencies from the DrosDel and Exelixis collections, an abnormally enlarged cardiac chamber was detected in a series of deficiency mutants spanning the rhomboid 3 locus. Rhomboid 3 is a member of a highly conserved family of intramembrane serine proteases and processes Spitz, an epidermal growth factor (EGF)-like ligand. Using multiple approaches based on the examination of deficiency stocks, a series of mutants in the rhomboid-Spitz-EGF receptor pathway, and cardiac-specific transgenic rescue or dominant-negative repression of EGFR, it was demonstrate that rhomboid 3 mediated activation of the EGF receptor pathway is necessary for proper adult cardiac function. The importance of EGF receptor signaling in the adult Drosophila heart underscores the concept that evolutionarily conserved signaling mechanisms are required to maintain normal myocardial function. Interestingly, prior work showing the inhibition of ErbB2, a member of the EGF receptor family, in transgenic knock-out mice or individuals that received herceptin chemotherapy is associated with the development of dilated cardiomyopathy. These results, in conjunction with the demonstration that altered ErbB2 signaling underlies certain forms of mammalian cardiomyopathy, suggest that an evolutionarily conserved signaling mechanism may be necessary to maintain post-developmental cardiac function (Yu, 2010; Full text of article).
ErbB receptors, including the epidermal growth factor receptor (Egfr), are activated by EGF ligands to govern cell proliferation, survival, migration and differentiation. The different EGF-induced cell responses in development are regulated by deployment of multiple ligands. These inputs, however, engage only a limited number of intracellular pathways and are thought to elicit specific responses by regulating the amplitude or duration of the intracellular signal. The single Drosophila Egfr has four ligands: three of the TGF-alpha-type and a single neuregulin-like called vein (vn). This study used mutant combinations and gene replacement to determine the constraints of ligand specificity in development. Mutant analysis revealed extensive ligand redundancy in embryogenesis and wing development. Surprisingly, it was found that the essential role of vn in development could be largely replaced by expression of any TGF-alpha ligand, including spitz (spi), in the endogenous vn pattern. vn mutants die as white undifferentiated pupae, but the rescued individuals showed global differentiation of adult body parts. Spi is more potent than Vn, and the best morphological rescue occurred when Spi expression was reduced to achieve an intracellular signaling level comparable to that produced by Vn. These results show that the developmental repertoire of a strong ligand like Spi is flexible and at the appropriate level can emulate the activity of a weak ligand like Vn. These findings align with a model whereby cells respond similarly to an equivalent quantitative level of an intracellular signal generated by two distinct ligands regardless of ligand identity (Austin, 2014).
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