COP9 complex homolog subunit 5
The COP9 signalosome is an essential multi-subunit repressor of light-regulated development in plants. It has also been identified in mammals. This complex is similar to the regulatory lid of the proteasome and eIF3 and several of its subunits are known to be involved in kinase signaling pathways. In order to reveal the developmental function of the COP9 signalosome in animals, Drosophila genes encoding eight subunits of the COP9 signalosome have been isolated. Co-immunoprecipitation and gel-filtration analysis shows that these proteins are components of the Drosophila COP9 signalosome. Yeast two-hybrid assays indicated that several of these proteins interact, some through the PCI domain. Disruption of the CSN5 subunit by either a P-element insertion or deletion of the gene causes lethality at the late larval or pupal stages. This lethality is probably a result of numerous pleiotropic effects. These results indicate that the COP9 signalosome is conserved in invertebrates and that it has an essential role in animal development (Freilich, 1999).
Since most CSN5 homozygotes die during larval or pupal development (Freilich, 1999), it seemed likely that embryos receive a maternal contribution of CSN5 RNA or protein. In situ hybridization confirms this expectation, showing that CSN5 RNA accumulates in the nurse cells beginning in the germarium and continuing through most of oogenesis. During stage 10, CSN5 RNA is transferred to the oocyte along with the bulk of the nurse cell cytoplasm. In embryos, uniformly distributed maternal RNA is evident until gastrulation begins. The earliest zygotic expression is in an anterior stripe during cellular blastoderm. During gastrulation, zygotic expression becomes evident in the ventral furrow, the cephalic furrow, and both the anterior and posterior midgut invaginations (Doronkin, 2002).
To enable an analysis of early embryonic requirements for CSN5, homozygous, CSN5-mutant germline clones were induced. These clones revealed requirements for CSN5 during oogenesis as well as embryogenesis. In ovarian germline clones the level of CSN5 mRNA is dramatically reduced, but still detectable, indicating that the P-element-induced allele, CSN5L4032, is hypomorphic. Depending on the paternal allele, embryos derived from the germline clones show either a reduced amount of CSN5 RNA in the zygotic pattern or no detectable CSN5 RNA (Doronkin, 2002).
Flies carrying CSN5 germline clones lay eggs with a range of abnormal phenotypes that are affected by temperature. Flies grown at 25°C laid eggs with phenotypes closest to normal. The most frequent defects at 18°C are different from those at 29°C. At 18°C many of the defective eggs have fused dorsal appendages. At 29°C there is an increasing frequency of properly separated but short dorsal appendages. These results suggest that aberrations in patterning the follicular epithelium predominate at 18°C, while defects in follicle cell migration predominate at 29°C (Doronkin, 2002).
Because the eggshell phenotypes were only partially penetrant, it is possible that they are caused by somatic, rather than germline, CSN5 clones. To test this possibility, somatic clones were induced in the ovary by using the follicle cell driver E22c-GAL4 to induce expression of UAS-FLP. Under these conditions, there were no eggshell defects at any temperature, indicating that this requirement for CSN5 function is limited to the germline (Doronkin, 2002).
In addition to the eggshell defects, the viability of CSN5 mutants also depends on temperature. At 29°C the original P-element mutation is lethal during early development with fewer than 1% of the mutant larvae becoming prepupae. By contrast, at 18°C 90% of the mutant larvae pupariate and 1-2% escape as adults. Mobilization of the original P-element insertion confirms that it is responsible, not only for lethality, but also for the eggshell defects; precise excisions are viable and have normal dorsal appendages (Doronkin, 2002).
Some mutations that disrupt the DV patterning of the eggshell also affect the patterning of the embryo. To look for effects on the embryonic DV fate map, the expression of three zygotic genes was used as markers: decapentaplegic (dpp), rhomboid (rho) and twist (twi). dpp is expressed on the dorsal side of the embryo as well as its anterior and posterior ends. rho is expressed in two, eight-cell-wide ventrolateral domains and later also in a narrow stripe on the dorsal side of the embryo. twi, a marker for the mesoderm, is expressed ventrally in the embryo (Doronkin, 2002).
For all three of the markers, many of the CSN5-mutant embryos appear to be ventralized. In these embryos dpp expression on the dorsal side is reduced or absent. The dorsal rho stripe is reduced and the lateral stripes are moved dorsally. twi expression appears to expand dorsally about halfway around the embryo. Some embryos show stronger ventralization at their anterior or posterior ends. Infrequently, there were also embryos that appeared to be dorsalized (Doronkin, 2002).
To characterize CSN5 mutants further, the spatial localization of the RNAs were examined for two determinants of AP polarity, bicoid (bcd) and oskar (osk). The localization of bcd RNA to the anterior pole of the oocyte is crucial in the establishment of AP polarity. In CSN5 mutant oocytes and embryos, bcd mRNA is abnormally expressed in 10%-15% of oocytes. In these abnormal oocytes, the bcd mRNA is diffusely distributed and sometimes accumulates near the center of the oocyte. In mutant embryos, the bcd RNA often shifts toward the dorsal side of the embryo (Doronkin, 2002).
The posterior pole of the egg chamber is defined by the tight, posterior localization of osk RNA. Although most CSN5-mutant oocytes and embryos are nearly normal, osk RNA in 10%-15% of mutant oocytes and embryos is reduced or mislocalized. In the abnormal oocytes, the osk RNA is typically diffuse or concentrated in the center of the oocyte. Only small amounts were localized at the posterior pole. In the abnormal embryos only a small amount of osk RNA at the posterior pole remains. In these embryos the osk RNA appears to be shifted slightly dorsally from its normal position at the extreme posterior end (Doronkin, 2002).
Since the localization of osk and bcd RNAs depends on polarization of the microtubule lattice, a reporter for the motor protein kinesin was used to examine microtubule organization in CSN5 germline clones. Kinesin moves toward the plus ends of microtubules, and in stage 8-9 wild-type egg chambers kinesin-ß-gal localizes to the posterior of the oocyte. However, in some CSN5-mutant oocytes kinesin-ß-gal staining was diffuse or mislocalized (Doronkin, 2002).
In addition to its role in determining the AP axis, CSN5 may have a distinct role in pole cell development. In normal embryos, the pole cells form as a tight, contiguous cluster at the posterior end of the embryo. As gastrulation and germ band extension begin, somatic epithelial cells at the posterior end of the embryo form a shallow cup that will eventually become the posterior midgut invagination. The pole cells adhere to this cup and remain tightly clustered on its surface as they are conveyed over the dorsal side of the embryo and then into its interior. In CSN5-mutant embryos the number of pole cells is often reduced, as might be expected because of the inefficient localization of oskar RNA. In addition, the pole cells are occasionally found in a loose, non-contiguous group near, but not tightly associated with, the posterior end of the embryo. This is an unusual phenotype, not seen in other mutants that impair the formation of pole plasm. Thus, in addition to its role in oskar RNA localization, CSN5 may have a separate role in organizing the pole cell cluster (Doronkin, 2002).
Since CSN5 germline clones cause defects in both the AP and DV axes, it seemed possible that grk signaling is compromised. grk is unusual among axis-determining genes in being required for both axes. To assess the role of CSN5 in grk signaling, reporters for either the posterior or the dorsal Grk signal were used. In the absence of the posterior Grk signal, the posterior follicle cells appear to adopt the anterior follicle cell fate and express markers that are characteristic of the border cells. Two such markers, an enhancer trap called PZ6356 and a slbo-lacZ enhancer trap, were used to monitor whether CSN5 is required for the early Grk signal. For both markers, loss of CSN5 from the germline causes lacZ expression in the posterior follicle cells of many egg chambers, suggesting a reduction in Grk signaling. To monitor Egfr signaling to the dorsal follicle cells at stages 9 and 10, a kekkon (kek)-lacZ reporter construct was used. Because the kek gene acts downstream of the Egfr pathway in the follicle cells, it can serve as a sensitive indicator of grk activity coming from the oocyte. At 18°C kek expression is abnormal in about a third of CSN5-mutant egg chambers at stage 10 (but only 3%-4% at 25°C). In most of these egg chambers, expression in the dorsal anterior follicle cells over the oocyte was reduced or, rarely, absent. A small number of egg chambers show broader expression of kek in the follicle cells, probably reflecting the small number of dorsalized embryos arising from these mutant egg chambers. It is concluded that in most egg chambers both posterior and dorsal Grk signaling are impaired in CSN5-mutant germline clones (Doronkin, 2002).
Further evidence that CSN5 affects Grk signaling comes from testing for genetic interactions between CSN5 and either grk or Egfr. Females heterozygous for strong grk alleles lay eggs with fused or partially fused dorsal appendages. This dominant phenotype provides a sensitive background for detecting interactions. With the exception of a precise P-element excision, all CSN5 alleles show strong enhancement of the dominant grk phenotype. In addition, CSN5L4032 weakly enhances the dominant eggshell phenotype of a loss of function Egfr allele, Egfrf2 (Doronkin, 2002).
These results suggested that production of grk RNA or protein might be affected in CSN5 germline clones. In situ hybridization using a grk probe showed normal or nearly normal localization of grk RNA in most CSN5-mutant stage 10 oocytes. In some of these mutant oocytes the messenge was improperly localized, probably because the oocyte nucleus was no longer located at the dorsal corner of the oocyte. Interestingly, in these oocytes the 'dorsal' follicle cells are often columnar as though the nucleus had been properly localized at an earlier stage. A Northern blot showed nearly normal amounts of grk mRNA in ovaries carrying CSN5-mutant germline clones, consistent with the strong signals seen by in situ hybridization in most oocytes (Doronkin, 2002).
Immunostaining of egg chambers using anti-Grk antibodies showed a more extreme effect. Grk protein is strongly reduced in CSN5 mutants compared with controls, although the residual protein usually appears to be properly localized. This reduction was confirmed by western blot analysis. There were also a few cases of Grk protein mislocalization, sometimes being present all along the anterior end of the oocyte (Doronkin, 2002).
The reduction in Grk protein appeared to be most extreme at early stages in oogenesis. Grk expression begins in region 2a in wild-type germaria. The signal appears in several cells per cyst in regions 2a and 2b and then becomes concentrated in the oocyte cytoplasm by region 3. In viable, hypomorphic combinations such as CSN5ex21/CSN5L4032, Grk expression could not be detected in the germarium. With this combination Grk does become detectable from stage 2-3 onwards, suggesting that a reduction in CSN5 causes a delay in the beginning of Grk accumulation. Taken together these results show that the major effect of CSN5 mutations appears to be on grk RNA translation or on stability of the protein (Doronkin, 2002).
Because of the similarity between the CSN5 and spindle-class phenotypes, a connection between CSN5 and the meiotic checkpoint mediated by mei-41 was tested. The viable hypomorphic combination CSN5ex21/CSN5L4032 causes a reduction in Grk protein level, especially during the early stages of oogenesis. Five to fifteen percent of eggs laid by these transheterozygotes had fused dorsal appendages, indicating a partial reduction of Grk. When CSN5ex21/CSN5L4032 flies were also homozygous-mutant for mei-41, however, the normal Grk protein level was restored, and the eggshell phenotype was rescued (Doronkin, 2002).
Interestingly, checkpoint activation leads to modification of the Vasa protein, as shown by a slightly reduced mobility during SDS polyacrylamide gel electrophoresis. This result is relevant to the spindle-class and CSN5 phenotypes because Vasa regulates translation of Gurken and, as a consequence, axial patterning. This Vasa modification is checkpoint dependent since it is present in spn-B mutants but absent in mei-41 spn-B double mutants (Doronkin, 2002 and references therein).
A similar reduced mobility of Vasa protein is detected in CSN5 mutants. For viable CSN5 mutants there were two Vasa bands: one corresponding to Vasa from wild-type ovaries and a second with lower mobility. In stronger mutant combinations, most of the Vasa protein was modified, while in weaker combinations most Vasa had normal mobility. The shift in Vasa mobility was suppressed by mei-41 mutations. Interestingly, removal of one dose of mei-41 completely restores normal Vasa mobility for a weak CSN5 combination. For stronger CSN5 mutants, full restoration of Vasa mobility requires removal of both mei-41 genes (Doronkin, 2002).
The gene mei-W68 is required for the initiation of meiotic recombination in Drosophila ovaries and is likely to induce DNA double strand breaks (DSBs) as recombination begins (McKim, 1998). Mutations in mei-W68 rescue spindle-class defects, including Grk protein accumulation, eggshell morphology and Vasa modification. These results suggested that since DSBs are not formed in the absence of mei-W68, DNA repair by the spindle-class genes is not required. A similar interaction is seen between mei-W68 and CSN5. Hetrerozygosity for mei-W68 is sufficient to suppress the phenotypes of both strong and weak CSN5 allelic combinations (Doronkin, 2002).
These data demonstrate that absence of CSN5 function during meiosis activates a DNA-damage checkpoint that is mediated by Mei-41. Because the reduction in DSBs in mei-W68 heterozygotes removes the requirement for CSN5, it is likely that CSN5 promotes DNA repair, as do the spindle-class genes (Doronkin, 2002).
The R1-R6 subclass of photoreceptor neurons connects to the first optic ganglion of the optic lobe, the lamina, and relies upon glial cells as intermediate targets. Conversely, R cells promote glial cell development, including migration of glial cells into the target region. The CSN5 subunit of the COP9 signalosome complex is expressed in R cells, accumulates in the developing optic lobe neuropil, and is required in R cells to induce lamina glial cell migration. In a set of CSN5 missense alleles, R1-R6 targeting is disrupted. Genetic analysis of protein null alleles also reveals that the COP9 signalosome is required at an earlier stage of development for R cell differentiation. Whether these two effects are independent, or represent a single function for CSN5, is as yet undetermined (Suh, 2002).
A single loss-of-function mutation, initially designated quo1, was identified in an ethyl methane sulfonate (EMS) screen for defects in R cell connections. Two additional EMS-induced alleles were identified, quo2 and quo3, from a collection of lethal mutations generated in the 89C/D region. quo1 mutant animals die as larvae or pupae, while quo2 and quo3 phenotypes are less severe with a lethal phase later during pupal development. In some cases, quo2/quo3 heterozygotes survived to adulthood.These animals are morphologically normal, but are uncoordinated and sluggish, and die a few days after eclosion (Suh, 2002).
In quo1, quo2, and quo3 mutant larvae, many R1-R6 axons fail to terminate in the lamina but instead project through this region into the medulla. This defect in target specificity was revealed using Ro-taulacZ, a marker for a subset of R1-R6 axons (i.e., R2-R5). In contrast to wild-type, approximately 60% of the R2-R5 neurons in quo1 project into the medulla. Mistargeting in homozygous quo1 mutants and in quo1 in trans to a deficiency is indistinguishable. quo2 and quo3 phenotypes are qualitatively similar to, but less severe than, quo1 (Suh, 2002).
Meiotic recombination and deficiency mapping localized quo to the cytological region between bands 89C7 and 89D1. Complementation tests placed quo between the proximal breakpoint of Df(3R)RK6-3 and the distal breakpoint of Df(3R)Sbd104. A region of some 200 kb was isolated through positional cloning and approximately 20 kb of that region, defined by the two aforementioned breakpoints, demarcated the region containing the mutations. A panel of fragments was introduced into flies by P element-mediated transformation and tested for rescue of the quo connectivity defects and lethality. A genomic fragment of 6.6 kb rescues the mutant phenotype. A single open reading frame of 327 amino acids was identified within the fragment and cDNAs corresponding to it were isolated. A heat shock cDNA transgene rescued both lethality and the R cell targeting phenotype. NCBI Blast search results show that this cDNA encodes a protein 65% and 74% identical to Jun-activation-domain binding protein1 (JAB1) (Claret, 1996) and subunit 5 of the Arabidopsis COP9 signalosome (CSN5) (Kwok, 1998), respectively. Each of the three EMS alleles leads to specific amino acid substitutions in the JAB/MPN domain (as defined in the SMART program [EMBL]) in residues conserved between plant, fly, and human. The quo gene (and its alleles) is referred to as CSN5 and the Quo protein as JAB1/CSN5 (Suh, 2002).
To determine whether CSN5 is required in R cell afferents or the brain, genetic mosaic analyses were undertaken. CSN51 mutant patches in the retina, in otherwise heterozygous animals, were created by X-ray-induced mitotic recombination or by using FLP/FRT-mediated recombination driven by eye-specific expression of the FLP-recombinase. R1-R6 targeting in adult tissue was assessed using a marker specific for the projections of R1-R6 axons, Rh1-lacZ. Of 22 mosaic animals, 15 exhibited an R1-R6 mistargeting phenotype. These results were confirmed using genetic mosaic analyses in developing eye-brain complexes by generating eye tissue homozygous for CSN51 while the target was wild-type. These projections were assessed using a pan-R cell specific marker, mAb24B10. All animals of this genotype exhibited abnormal projections. These results and the finding that the CSN5 mutant phenotype is rescued by neuron-specific expression of a full-length cDNA are consistent with CSN5 acting in R cells to regulate R1-R6 targeting (Suh, 2002).
R cell differentiation and pattern formation are normal in CSN51, CSN52, and CSN53 alleles. The targeting defect in CSN51, CSN52, and CSN53 does not result from transformation of R1-R6 neurons into R7 and/or R8 neurons that normally project into the medulla. The mistargeted neurons continue to express the R1-R6-specific markers Rh1-LacZ and Ro-taulacZ. In the developing eye, Bar (expressed specifically in R1 and R6 neurons), Prospero (expressed in R7 neurons and nonneuronal cone cells), and Boss are expressed in patterns indistinguishable from wild-type. These results are consistent with plastic sections of homozygous adult mutant patches. Of some 704 ommatidia scored from 10 independent mosaic patches, the number, organization, and morphology of CSN51 mutant R cells in some 697 ommatidia were indistinguishable from wild-type. In the remaining seven ommatidia, a single R cell was missing. Small numbers of missing R cells have been observed in other connectivity mutants (Suh, 2002).
Developmental studies have indicated that R1-R6 axons initially recognize lamina glia as intermediate targets in the developing lamina prior to making connections to specific lamina neurons several days later. Based on morphological studies, it has been proposed that the establishment of precise patterns of R1-R6 projections relies on interactions between R1-R6 afferents and lamina glia cells. The notion that lamina glia, not lamina neuronal precursors, are intermediate targets for R1-R6 afferents is supported by phenotypic analyses of nonstop and hedgehog mutants. Defects in glial cell induction are not an indirect effect of R1-R6 mistargeting, since glial cells develop normally in other genetic backgrounds in which R1-R6 axons mistarget (Suh, 2002).
To assess whether CSN5 is required in R cells for the development of lamina glia, target development was examined using the glial-specific anti-Repo antibody, both in homozygous CSN51 and in genetic mosaics in which mutant R cell axons project into a wild-type brain. While R cell-dependent lamina precursor cell proliferation and neuronal differentiation occurs normally, lamina glial cell development is disrupted in CSN51 homozygotes. This mutant phenotype is also observed in wild-type targets innervated by CSN51 mutant R cells. In wild-type, three layers of glia (epithelial, marginal, and medulla glia) surround the lamina plexus where R1-R6 axons terminate. In CSN51 homozygotes and mosaic animals in which the eye is mutant and the target is wild-type, there is a marked reduction in lamina glial cell number (approximately 46% of that in wild-type), and the remaining cells form disorganized rows (Suh, 2002).
The effects of CSN51 on lamina epithelial and marginal glia were compared to the effects on medulla glia by analyzing mutant animals carrying enhancer trap markers for these two populations. The lamina glial cells were visualized using 1.3D2 enhancer trap line, and the medulla glial cells with MZ97. The number of marginal and epithelial cells are markedly reduced, and the cells are highly disorganized. In contrast, there is no appreciable effect on the number of medulla glia in CSN51 mutants. While the medulla glial cell layer was disrupted in some cases, in most preparations a continuous row of medulla glia forms (Suh, 2002).
Defects in lamina glial cell development could be due to a defect in glial cell migration or differentiation. To distinguish between these two possibilities, the distribution of glial cells was analyzed in the developing optic lobe using anti-repo antibody as a marker. Lamina glial cells are derived from groups of cells flanking the lamina plexus; glial cells generated in these regions then migrate into the target. In wild-type, glial cells express Repo as they migrate into the R cell projection field. In CSN51, there is an increase in the number of cells that accumulate at the lateral edges. In wild-type, there are 12.1 ± 2.0 cells, whereas in homozygous CSN51 mutants there are 21.9 ± 3.4 cells and in CSN51 mutant eyes projecting into a wild-type target, there are 19.0 ± 3.1 cells. This accumulation is consistent with a failure of many glial cells to migrate in from the margin and parallels the decrease in the number of marginal and epithelial glial cells in the lamina. Based on these observations, it is concluded that lamina glial cell migration is disrupted in CSN51mutants (Suh, 2002).
These defects in lamina glial cell development and in R1-R6 targeting are similar to those observed in nonstop mutants. In contrast to CSN5, nonstop is required in lamina glial cells, and not in R cell afferents, for glial cell differentiation. The R1-R6 hyperinnervation phenotype in nonstop is similar to that in CSN5 mutants. These data argue that CSN5 functions in R cells to promote normal cell migration and development of lamina glial cells. It remains possible that CSN5 plays a dual function in R cells being required in a non-cell-autonomous fashion to induce glial cell development and in a cell-autonomous function in R1-R6 growth cone targeting. This is thought unlikely, since R1-R6 mutant neurons in a small patch of mutant retinal tissue target to the lamina plexus in a fashion similar to wild-type, while mutant neurons show marked mistargeting when imbedded in large mutant patches (Suh, 2002).
The JAB1/CSN5 protein could act directly in R cell growth cones to mediate interaction with target cells or alternatively, it could act indirectly, for instance, in R cell nuclei to control gene expression required for R1-R6 targeting. To gain insight into how JAB1/CSN5 regulates R cell differentiation and interaction between R cell growth cones and lamina glial cell targets, the subcellular distribution of JAB1/CSN5 was determined. To assess the JAB1/CSN5 expression pattern in the developing visual system, a Myc-epitope tag was inserted into the C terminus of the CSN5 open reading frame in a genomic construct (myc-CSN5) and introduced into flies. Four independently generated transgenic lines were analyzed. In each case, the tagged transgene rescued the CSN5 lethality and connectivity phenotypes. This supports the view that the expression pattern observed using a Myc-tagged genomic construct accurately reflects the endogenous expression of JAB1/CSN5. Third instar eye-brain complexes of animals carrying the transgene were stained with anti-Myc antibody. Anti-Myc staining is predominantly localized to the cytoplasm throughout the developing eye disc. In the developing optic ganglia, anti-Myc staining is prominent in the lamina plexus, which at this stage in development largely comprises R cell axons and growth cones. Myc immunoreactivity also was enriched in the medulla neuropil. This is the region to which the R7 and R8 cells, as well as lamina and medulla neurons, send their axons. The expression of the Myc-tagged genomic construct in each of four transgenic lines was identical. The JAB1/CSN5 expression pattern is similar to other proteins previously shown to be required for signaling in growth cones (e.g., Dock and Pak) during R cell axon guidance and targeting (Suh, 2002).
Since the CSN51 homozygous phenotype is indistinguishable from the CSN51 over a deficiency, it seems likely that this allele is a strong loss-of-function or a null allele. Nevertheless, since all three CSN5 alleles are missense mutations, attempts were made to identify mutations that delete CSN5 coding sequence to unambiguously establish the null phenotype. Protein null mutations were generated by imprecise excision of a weak CSN5 mutant that carries a P element within the promotor region of the CSN5 locus. Six CSN5 protein null alleles were isolated. In contrast to the pupal lethality observed in animals carrying CSN5 missense mutations (i.e., CSN51, as well as CSN52 and CSN53 ) or the late larval and pupal lethality of partial loss-of-function mutations in CSN5 (Freilich, 1999), protein null homozygotes die in early larval stages. A heat shock-driven CSN5 cDNA transgene rescues the lethality. One null allele, designated CSN5N, was chosen for further phenotypic analysis. It is the result of a deletion of the entire open reading frame as demonstrated through both PCR analysis and Southern blots (Suh, 2002).
Since CSN5N larvae do not survive to third instar, the eye phenotype was assessed in genetically mosaic animals. CSN5N eye tissue was generated using FLP recombinase expressed under the control of the eyeless promoter (ey-FLP) to promote eye-specific FRT-mediated mitotic recombination between wild-type and CSN5N chromosomes. Under these conditions, more than 50% of the cells within the eye disc are homozygous mutant. Plastic sections of adult mutant eye tissue stained with toluidine blue have revealed that both the position of R cells and their morphology are abnormal. Abnormalities in differentiating R cells were observed in CSN5N clones in developing third instar eye discs. Expression of Elav, an early neuron-specific nuclear protein, and Chaoptin, a later appearing R cell surface protein, is greatly reduced in the mutant clones. However, Futsch, a neuronal microtubule-associated protein that appears at an early stage of R cell differentiation, and Boss, an early R8-specific protein, were expressed normally. Expression of other markers including Dachshund, Cubitus interruptus (Ci), Delta, and Atonal was similar to wild-type. Cell cycle progression in the eye disc appeared normal as assessed using BrdU incorporation. The lack of a cell cycle phenotype as assessed with BrdU incorporation is consistent with the observation that the size of homozygous CSN5N mutant clones is similar to sister clones homozygous for the wild-type allele (Suh, 2002).
These data indicate that JAB1/CSN5 is critical for neuronal differentiation, while earlier fate specification, patterning, and cell cycle events appear to proceed largely as in wild-type. Cone cell development is also disrupted in CSN5N since the expression of the cone cell markers, Cut and Sparkling, is reduced. Since R cells induce cone cells, it is possible that cone cell defects are due to an indirect effect of loss of JAB1/CSN5 in R cells (Suh, 2002).
In both plant and animal cells, JAB1/CSN5 assembles into a complex called the COP9 signalosome (Kwok, 1998; Freilich, 1999). While the function of this complex in animal cells is not understood, in plant cells, though highly pleiotropic, it plays a crucial role in regulating light-dependent gene expression. To address whether the CSN5 null phenotype reflects the loss of function of the COP9 signalosome function, the role of another subunit, CSN4, in eye development was assessed. A CSN4-null mutant strain that harbors a deletion of part of the CSN4 coding region was acquired. In Arabidopsis, all reported subunit mutations led to a loss of the COP9 signalosome (Karniol, 2000). Similarly, in both CSN4N and CSN5N fly mutants, an intact COP9 signalosome does not form. CSN4N clones in the eye contain R cells with markedly abnormal cellular morphologies similar to R cells in CSN5N mutant clones. CSN4N and CSN5N mutant phenotypes in developing eye discs are also similar, with a marked reduction of Elav and Chaoptin expression. These data are consistent with the CSN5 null mutant phenotype reflecting the loss of the COP9 signalosome function in the developing eye (Suh, 2002).
R cell axons engage in a complex dialogue with developing lamina cells. R cell growth cones produce Hedgehog and Spitz, which induce the final cell division of lamina neuronal precursors and the differentiation of lamina neurons, respectively. R cell axons have also been proposed to produce a third signal that induces lamina glial cell development, although the molecular identity of this signal remains unknown. Lamina glial cells, but not lamina neurons, are essential for R1-R6 targeting, since genetic ablation of lamina glial cells results in R1-R6 mistargeting to the medulla. JAB1/CSN5 protein is also required for the development of lamina glial cells and is enriched in regions in the optic lobe neuropil containing R cell growth cones. Since these glial cells act as intermediate targets in the lamina, the R1-R6 neurons mistarget to the medulla. In JAB1/CSN5 mutants, lamina glial cells accumulate at the lateral edges of the developing lamina neuropil where they contact R cell growth cones. The accumulation of lamina glia at these sites and the genetic requirement in R cells are consistent with the conclusion that JAB1/CSN5 is required for signaling between R cell growth cones and lamina glial cells (Suh, 2002).
Does JAB1/CSN5 play a direct role in regulating intercellular signaling pathways in R cell growth cones necessary for the induction of lamina glial cells? Recent studies in mammalian cells provide a precedent for JAB1/CSN5 function in contact-dependent intercellular interactions. JAB1/CSN5 has been shown to physically interact with LFA1, an integrin critical for the interaction between T cell receptors and antigen-presenting cells (Bianchi, 2000). Interaction between the T cell receptor and the MHC-peptide complex is converted into a high-affinity complex through the recruitment of additional adhesion molecules linking the cells together. This includes LFA1, an integrin that directly interacts with I-CAM, an Ig domain-containing protein expressed on the surface of the antigen-presenting cell. The costimulatory interaction between T cell and antigen-presenting cells creates a specialized contact area termed an ''immunological synapse.'' Transport and clustering of signaling components at the interface between these two cells leads to further amplification of intercellular signaling mediating the communication between them. It is attractive to envision that JAB1/CSN5 may contribute to the construction and/or maintenance of a structure supporting reciprocal interactions between the lamina glial cells and R cell axons (Suh, 2002).
Since the JAB1/CSN5 protein is also localized to R cell bodies, it is possible that it regulates lamina development indirectly, for instance, by controlling the expression of signals necessary for lamina glial cell induction. Indeed, studies in mammalian cells in culture and in plant cells provide a precedent for JAB1/CSN5 function in transcriptional regulation (reviewed in Chamovitz, 2001). Overexpression of mammalian JAB1/CSN5 stimulates Jun-dependent transcriptional activation (Claret, 1996), and the binding of JAB1/CSN5 to diverse proteins affects AP1 mediated transcription. Further support for the notion that JAB1/CSN5 regulates AP1-dependent transcription comes from studies in which the level of the COP9 signalosome, including CSN5, was elevated in cells by overexpressing another COP9 signalosome component, CSN2 (Naumann, 1999). These data raise the intriguing possibility that JAB1/CSN5 and the COP9 signalosome (more generally) regulate Jun-dependent intracellular pathways. However, since Jun mutations in the fly eye do not disrupt R1-R6 targeting, it is unlikely that the R cell innervation and glial cell induction defects reflect disruption of Jun regulation. Therefore, JAB1/CSN5 must be impinging on other signaling pathways to regulate R1-R6 targeting. Identifying these targets is an important future goal (Suh, 2002).
A role for JAB1/CSN5 in regulating gene expression is consistent with studies in Arabidopsis. Like true loss-of-function mutations in other subunits of the COP9 signalosome, antisense RNA inactivation of CSN5 in Arabidopsis derepresses a light-dependent developmental pathway in the dark. The COP9 signalosome facilitates the accumulation of the COP1 protein in the nucleus. COP1, probably acting as an E3 ubiquitin-ligase, in turn promotes degradation of the light-dependent transcriptional activator HY5. Hence, in the dark, the COP9 signalosome inhibits HY5-dependent transcription by regulating the cellular levels of HY5 itself. Through an unknown mechanism, light inactivates the COP9 signalosome function, export of COP1 from the nucleus, and stabilization of HY5. These findings raise the intriguing possibility that signaling events at the R cell growth cone could lead to changes in gene expression necessary for interactions between the growth cone and glia cell targets that are dependent upon the COP9 signalosome (Suh, 2002 and references therein).
A role for JAB1/CSN5 in photoreceptor cell differentiation was revealed through analysis of protein null alleles. Cells within null mutant clones express reduced levels of two neuronal differentiation markers: Elav, an RNA binding protein required for neuronal differentiation and Chaoptin, an R cell-specific, cell surface protein. Another neuronal marker, Futsch (a neuron-specific, microtubule-associated protein) is expressed normally. Consistent with a role for JAB1/CSN5 in differentiation, null mutant R cells survive into the adult and exhibit disrupted cellular morphologies. Interestingly, R cell neurons lacking Elav exhibit similar morphological defects. Defects are not restricted to R cell differentiation, however, since the expression of two cone cell homeodomain proteins (Cut and Sparkling) is also markedly reduced in CSN5 null mutants. Since R cells induce cone cell development, it remains unclear whether this reflects a role for CSN5 in cone cells. Early eye patterning genes such as Cubitis interruptus, dachshund, and atonal are expressed normally. Hence, JAB1/CSN5 plays a crucial role in R cell and cone differentiation but is largely dispensable for early patterning in the eye disc (Suh, 2002).
Since JAB1/CSN5 is found in Arabidopsis and Drosophila in two forms (a multisubunit complex, the COP9 signalosome, and in a smaller complex-independent form) (Freilich, 1999; Kwok, 1998), defects in eye development in CSN5 mutants may reflect the function of the JAB1/CSN5 monomer or the COP9 signalosome. To address this issue, the role of another component of the COP9 signalosome, CSN4, in eye development was analyzed. In Drosophila CSN4 mutants, the CSN complex does not form, but the JAB1/CSN5 monomer remains. Since CSN4 and CSN5 null phenotypes are indistinguishable, it is concluded that they reflect the essential function of the COP9 signalosome in eye development (Suh, 2002).
As opposed to the general R cell differentiation defects caused by the CSN4 and CSN5 null mutations, three different CSN5 missense mutations isolated in two independent screens specifically disrupt interactions between R cell afferents and lamina glial cells but do not lead to defects in R cell differentiation. These mutations are clustered within the JAB/MPN domain and lead to nonconservative substitutions in amino acids shared between plant, fly, and human JAB1/CSN5 (Suh, 2002).
What could be the biochemical basis for this specific connectivity phenotype? JAB1/CSN5 may be required for a single function; a reduction in its activity in the missense mutations may disrupt only the most sensitive process (e.g., lamina glial cell induction). Alternatively, JAB1/CSN5 may serve multiple functions. The missense mutations may selectively disrupt a subset of them that are dependent upon specific residues within the JAB/MPN domain, while the null mutation abolishes all JAB1/CSN5 functions. It is conceivable that these different functions are inherent to the different cellular forms of JAB1/CSN5 (e.g., complexed versus noncomplexed forms). In Arabidopsis, the CSN5 in the COP9 signalosome is primarily nuclear, while the CSN5 monomer is primarily cytoplasmic (Kwok, 1998). Therefore, the cytoplasmic localization of JAB1/CSN5 reported here could suggest that noncomplexed forms of JAB1/CSN5 are involved in R cell growth cone glial cell interactions, similar to the cytoplasmic form of JAB1/CSN5 that interacts with LFA1 (Suh, 2002).
Recent studies suggest that the multiple roles attributed to JAB1/CSN5 and the COP9 signalosome may reflect their function in regulating protein degradation. The COP9 signalosome positively regulates SCF complexes through the removal of Ned8 modifications from the cullin subunits (Lyapina, 2001), allowing for E3 activity. Mutations in the COP9 signalsome lead to the accumulation of multiple neddylated cullins in fission yeast (Zhou, 2001). Mutations in the Arabidopsis COP9 signalosome lead to the accumulation of ubiquitinated proteins (Peng, 2001). While the exact role of the COP9 signalosome in these processes is still obscure, it is proposed that the function of the COP9 signalosome and JAB1/CSN5 in R cells involves the regulation of protein degradation, probably through its interaction with specific E3 ligases. The specific missense mutations in CSN51, CSN52, and CSN53 may selectively disrupt the interactions with a subset of neddylated substrates involved in controlling interactions between R cell growth cones and lamina glial cells. Further biochemical and genetic analyses are required to determine the relationship between the structure of JAB1/CSN5 and its function in R1-R6 targeting, glial cell induction, and R cell differentiation (Suh, 2002).
The COP9 signalosome (CSN) is an eight-subunit complex that regulates multiple signaling and cell cycle pathways. The CSN has been linked to the degradation of Cyclin E, which promotes the G1-S transition in the cell cycle and then is rapidly degraded by the ubiquitin-proteasome pathway. Using CSN4 and CSN5/Jab1 mutants, it has been shown that the CSN acts during Drosophila oogenesis to remove Nedd8 from Cullin1, a subunit of the SCF ubiquitin ligase. Overexpression of Cyclin E causes defects similar to those caused by mutations in CSN or SCFAgo subunits -- extra divisions or, in contrast, cell cycle arrest and polyploidy. Because the phenotypes are so similar and because CSN and Cyclin E mutations reciprocally suppress each other, Cyclin E appears to be the major target of the CSN during early oogenesis. Genetic interactions among CSN, SCF, and proteasome subunits further confirm CSN involvement in ubiquitin-mediated Cyclin E degradation (Doronkin, 2003).
To investigate cyst formation and differentiation in CSN5 germaria, wild-type and CSN5 ovaries were stained with anti-Hts antibody to highlight the fusome that connects all the cells of a cyst through the ring canals. Fusome development is essential for germline cyst formation. In CSN5 mutant germaria the fusome was often less branched, and sometimes there were more individual fusomes than in wild-type germaria. Furthermore, spherical spectrosomes (fusome precursors) are frequently found in more posterior regions of the germaria, probably indicating retarded fusome development. CSN5 null mutant clones eventually cease mitotic divisions and often become enormously polyploid. Along with the increase in DNA, these cells often contain oversized spectrosomes or structures similar to a fragmented fusome, indicating dramatic changes in fusome development. Some mutant clones lacked spectrosomes/fusomes. Usually, these clones were found a significant time after heat shock and were localized in ovarioles with no subsequent germline development. CSN4N mutant clones show similar undifferentiated cysts with enlarged cell nuclei and defective fusome development. These data suggest that the intact CSN complex is required for proper cyst divisions and fusome development. The polyploid, nondividing germ cells may be the germline stem cells. More than three of these large polyploid cells are never seen in a particular germarium, and they retain contact with somatic cells that probably correspond to the basal and terminal filament cells of normal germaria (Doronkin, 2003).
The Drosophila F box protein Archipelago (Ago) has been proposed to target Cyclin E for ubiquitin-mediated degradation in imaginal discs. The hypomorphic alleles ago1, ago3, and ago4 were used to test for a similar role in Cyclin E degradation during oogenesis. Immunostaining shows that ago mutant clones marked by lack of GFP persistently accumulate Cyclin E at high levels. With one addition, these clones showed a similar range of phenotypes as those seen in CSN5 or CSN4 mutants or after overexpression of Cyclin E. Some mutant cysts had extra nurse cells and some had fewer than normal, and many were degenerating. Some cysts had been arrested after the stem cell division and some of these single-cell cysts were polyploid. Prominent in the ago clones was a phenotype that had not been previously noticed. Cyclin E accumulation in ago clones correlates with significantly DAPI-bright regions in nurse cell nuclei. Because these regions are likely to include heterochromatic sequences that are usually underreplicated during endoreplication, their enlargement may indicate a more complete replication of both heterochromatic and euchromatic sequences in ago clones. Although enlarged heterochromatin-rich regions are occasionally seen in CSN5 mutants and after Cyclin E overexpression, this phenotype is stronger in ago mutants, possibly suggesting a more specific role for ago in regulation of late replication (Doronkin, 2003).
In addition to the similar phenotypes between ago mutants and CSN, Nedd8, or cullin1 mutants, dominant interactions were found between ago and CSN mutants. CSN5/ago and CSN4/ago double heterozygotes show familiar ovarian defects: extra cystocyte divisions, fewer divisions but higher ploidy, and apoptotic egg chambers. These defects are very similar to the CSN5 mutant phenotype and to defects in oogenesis induced by Cyclin E overexpression. In addition, these double heterozygotes have enlarged heterochromatic regions in nurse cell nuclei, suggesting mutual CSN-ago control of late replication (Doronkin, 2003).
The regulatory lid of the proteasome is an eight-subunit complex that is closely related to the CSN. It appears to be necessary for the removal of ubiquitin side chains from the target protein as it is fed into the barrel of the proteasome for proteolysis. A mutation in the RPN6 subunit of the regulatory lid was tested for genetic interactions in oogenesis with CSN4 and CSN5 mutations. Both double heterozygotes show a strong interaction and the full range of CSN5-like ovarian defects, including apoptosis, incorrect number of mitotic divisions, and fusions of neighboring egg chambers (Doronkin, 2003).
The effect of the CSN on the activity of the SCF complex has been controversial. Although Nedd8 modification of Cullin1 stimulates SCF activity, the opposite process, deneddylation, has also been shown to be important for SCF function and cell cycle progression. For example, point mutations in the JAMM domain of the S. cerevisiae CSN5 homolog Rri abolish its deneddylation activity and enhance the growth defect shown by ts alleles of SCF genes. These results have led to the proposal that repeated cycles of neddylation and deneddylation are required for the sustained activity of the SCF. However, a recent gain-of-function analysis suggests that deneddylation by the CSN inhibits degradation of the SCF target p27kip1 (Doronkin, 2003).
The results of this study strongly support the idea that deneddylation of Cullin1 by the CSN is necessary for activity of the SCF complex. CSN mutations have the same, not opposite, effects on oogenesis as do Nedd8, cullin1, or ago mutations. CSN5 and CSN4 mutations also interact dominantly with cullin1 and ago mutations, further suggesting that the CSN works along with the SCF to promote Cyclin E degradation. These requirements for the CSN appear to demand its deneddylase activity, because the CSN5quo2 mutation, with a single amino acid substitution in the metalloprotease domain, behaves similarly to a CSN5 null (Doronkin, 2003).
Cycles of neddylation and deneddylation might control the association of an F box protein with an E3 ubiquitin ligase core complex or the association of a ubiquitin-loaded E2-conjugating enzyme with the E3 complex. Neddylation might also affect Cullin1 stability as suggested by the Cullin1 accumulation that is seen in CSN5 mutants and its reduction in Nedd8 mutants (Doronkin, 2003).
Conjugation of Nedd8 to cullins may regulate not only their activity, but also their subcellular distribution. Shuttling between the nucleus and cytoplasm has been proposed as a regulatory mechanism for E3 ubiquitin ligases when the target protein is ubiquitinated in the nucleus. The results showing that in CSN mutants, Nedd8-modified Cullin1 accumulates in the cytoplasm suggest that neddylation may be one way to regulate shuttling. Neddylation might favor nuclear export of Cullin1, and nuclear CSN would be required to remove Nedd8 and prevent export. Alternatively, neddylation might prevent Cullin1 nuclear import, and recycling of SCF into the nucleus would require cytoplasmic CSN. On either model, the CSN would be an important regulator of SCF activity. For example, modulation of SCF nuclear shuttling might affect the timing of Cyclin E degradation and entry into S phase of the cell cycle (Doronkin, 2003).
One of the important results of the current work is the demonstration that the CSN regulates the cell cycle in ovaries primarily through the turnover of Cyclin E. The apparent perdurance and gradual dilution of wild-type CSN5 protein in genetically null germline clones shows that reduced Cyclin E degradation affects both cell division and DNA replication. Slight reductions cause an extra division of the cystocytes. In contrast, continuous or strong accumulation of Cyclin E in null CSN5 mutants is able to reduce or stop cell divisions though often allowing endoreplication to continue. This switch from overproliferation to inhibition of cell divisions is sometimes visible in a single CSN5 mutant ovariole as the wild-type CSN5 protein is diluted by stem cell divisions. These observations support the view that different Cyclin E levels can lead to distinct and sometimes opposite effects (Doronkin, 2003).
Mutations in Drosophila ago, the C. elegans gene cul1, or the F box-encoding lin23 have been shown to cause increased cell proliferation, suggesting a critical role for SCF in regulating cell divisions. Extra cell division is found to be a frequent phenotype produced by mutations in CSN5, CSN4, cullin1, ago, or by overexpression of Cyclin E. However, SCF and CSN mutations have also been shown to cause the opposite effect on the cell cycle. In null mutant clones of cullin1 or Nedd8, cell proliferation in Drosophila eye discs is arrested. Similarly, loss of CSN5, CSN4, Cullin1, or ago inhibits and finally stops cell proliferation and often leads to enlarged nuclei. The abundance of Cyclin E and giant polyploid nuclei are also present in mice that are mutant for cul1 (Doronkin, 2003).
Elevated levels of Cyclin E that may give cells a proliferative advantage are found in many human tumors. In many of these tumors the Cyclin E gene itself is amplified. However, among breast and ovarian cancer cell lines that overexpress Cyclin E protein without amplification, several lines have mutations in hCDC4, the human homolog of archipelago, suggesting that SCF[hcdc4] acts to suppress tumor formation. The results suggest that the CSN might have a similar effect (Doronkin, 2003).
In summary, these genetic and functional relationships between the CSN, the SCF, and the proteasome link these complexes in the regulation of Cyclin E degradation during normal development. When either the CSN or SCF are disrupted, the periodic degradation of Cyclin E is prevented, and cell cycle deregulation ensues (Doronkin, 2003).
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date revised: 17 January 2008
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