Calnexin 99A, the sole lineage specific gene in this group, is highly related to its mammalian counterpart, and there are four Drosophila isoforms, coded for by separate genes; only two isoforms have been characterized (Christodoulou, 1997). In the CNS Calnexin 99A expression is restricted to longitudinal and midline glia. Expression is also detected in cells lining the developing gut (Brody, 2002).
Calnexin is thought to play a role in the folding of a large number of proteins. Consistent with this notion, cnx is found by Northern blot to be expressed at all stages during development and in the adult. cnx transcript was detected in wt embryos, larvae, and adult heads and bodies. cnx transcript and Cnx protein were also detected in heads from flies lacking eyes (eya1), indicating that cnx expression is not restricted to the eyes. Despite the widespread expression pattern of cnx, it was not required for viability or fertility of the flies, since the mutants were homozygous viable. Although Rh1 requires Cnx for its expression, Cnx expression is normal in Rh1 mutants (Rosenbaum, 2006).
EP(3)3522 is located 800 bp upstream from Calnexin 99A (Cnx99A). There are three Calnexin homologs in Drosophila. The first Calnexin identified was mapped to 14D on the X chromosome (also known as CG9906). A second Calnexin-like molecule was mapped to 99A on the third chromosome (also known as CG11958) (Christodoulou, 1997). Upon examination of the Drosophila genome a third Calnexin-like molecule, CG1924, was found at 11A on the X chromosome. The Calnexins encode a calreticulin domain that consists of a low capacity, high affinity calcium binding domain, a globular domain and a high capacity, low affinity calcium binding domain, and a short transmembrane domain (Christodouloul, 1997). Calnexin homologs have been found in a variety of organisms including mammals, plants and yeast. In vertebrates, Calnexin is a membrane protein of the endoplasmic reticulum and is thought to function as a molecular chaperone aiding in the folding of polypeptides (Christodoulou, 1997). Furthermore, mutations in Calnexin have been implicated in a variety of human diseases in which key proteins are not folded properly (Chevet, 1999; McGovern, 2003 and references therein).
Misexpression of Cnx99A generated a fused commissure phenotype as revealed by BP102 immunohistochemistry. The two most medial longitudinal fascicles, as visualized with mAb 1D4, cross and recross the midline leading to a phenotype that is reminiscent of the Robo loss of function phenotype. In order to determine if Robo protein was present, an anti-Robo antibody was used on EP(3)3522/scaGAL4 embryos. Normal levels of Robo protein accumulation were detected: therefore, the inappropriate crossing of the midline by 1D4-positive fascicles is not due to the loss of Robo protein (McGovern, 2003).
To determine if the misexpression phenotype observed was due to changes in cell fate, a set of antibody markers was used to examine neurons and glia. The midline cells appeared wild type at all stages with mAb Sim. Conversely, when midline cells were examined with a riboprobe to NetA, it was found that RNA accumulation in the midline cells began to disappear from some cells at stage 15. NetA RNA became restricted to an even smaller number of midline cells at stage 16, and by stage 17 very few midline cells showed NetA RNA accumulation. This same phenotype was observed with a NetrinB (NetB) riboprobe as well as anti-Wrapper antibody, which identifies the midline glia that ensheathe the commissures. While the over expression of Calnexin99A does not appear to affect early CNS midline determination, it does result in the down-regulation of NetA, NetB and Wrapper at later stages (McGovern, 2003).
A riboprobe to an EST containing Cnx99A revealed strong RNA accumulation throughout the developing CNS at stage 9 in wild-type embryos. At stage 12, accumulation in the gut was observed. Weaker RNA accumulation in the CNS was observed to the end of stage 17. Because there is over 82% nucleotide identity between the three Drosophila Calnexin homologs, including high homology in the 5 and 3 UTRs, it is possible that this riboprobe recognizes all three Calnexin homologs (McGovern, 2003).
The simGAL4 driver was used to investigate the consequence of misexpressing Cnx99A in the CNS midline cells. The BP102 phenotype observed with simGAL4 misexpression was quite similar to the phenotype observed with scaGAL4 misexpression. Misexpression of Cnx99A in all neurons and glia or in CNS midline cells alone resulted in a fuzzy, fused commissure phenotype. Furthermore, NetA, NetB, and Wrapper accumulation were down-regulated with both drivers. Loss of wrapper has previously been shown to result in a fuzzy fused commissure phenotype. In wrapper mutants the midline glial cells develop and migrate properly but at stage 16 they fail to ensheathe the commissural axons and die. In Cnx99A misexpression embryos the BP102 phenotype observed is likely due in part to the down-regulation of Wrapper at later stages. Misexpression of Cnx99A could be affecting CNS midline cell survival directly or indirectly by altering the expression of genes required for CNS midline cell survival such as Wrapper (McGovern, 2003).
Since Cnx99A contains calcium-binding domains the possibility was considered that the inappropriate midline crossing of the 1D4-positive fascicles was due to calcium sequestration in axons. Previous studies have shown that the disruption of Ca2+-calmodulin (CaM) signaling, by sequestering free Ca2+ in the growth cone, results in inappropriate crossing of the midline by the MP1 neuronal pathway. In order to investigate the consequence of misexpressing Cnx99A in midline cells verses neurons the simGAL4 driver was used. If overexpression of Cnx99A in all neurons and midline cells results in reduced calcium levels in the growth cone, it would be expected that restricting expression to just the CNS midline cells would generate a less severe phenotype. This in fact was the case, since only the most medial 1D4 positive fascicle was found to cross the midline when Cnx99A was misexpressed with simGAL4. This is in stark contrast to the severe 1D4 phenotype produced when Cnx99A was misexpressed in all neurons with scaGAL4. The few 1D4-positive fascicles that do cross the midline when Cnx99A was misexpressed with simGAL4 could be a result of reduction in midline guidance cues such as Slit, NetA, and NetB at later stages of CNS development (McGovern, 2003).
Two putative loss of function alleles of Cnx99A were generated. Since Cnx99A is lethal when misexpressed with scaGAL4, EP(3)3522 males were mutagenized and a reversion of the lethality phenotype was sought when scaGAL4 was present. Each putative loss of function allele was placed in trans to a deficiency for the region spanning Cnx99A. No embryonic loss of function phenotypes were observed in the CNS upon examination with mAbs BP102 or 1D4 (McGovern, 2003).
By screening the Zuker collection of EMS-mutagenized Drosophila lines, two independent mutants were identified that displayed a severe reduction in Rh1 compared to wild-type (wt). The two mutants were established to be allelic, since the transheterozygotes displayed reduced levels of Rh1. Furthermore, both alleles were shown to be recessive (Rosenbaum, 2006).
To identify the mutant locus responsible for the phenotype, deficiency mapping was used to narrow the cytogenetic location to 99A7 on the third chromosome, corresponding to four genes. This small region was sequenced and mutations were identified in the coding region of calnexin (Christodoulou, 1997) in both alleles. One allele harbored a C to T transition at nucleotide position 544, causing a premature stop codon at glutamine182, and that the other allele harbored a G to A transition at nucleotide position 356, causing a premature stop codon at tryptophan119. Based on cytology, the two alleles were designated cnx99A1 (cnx1) and cnx99A2 (cnx2), respectively. Drosophila calnexin99A (Cnx) displays 49% amino acid identity with human calnexin (chromosome 5q) (Rosenbaum, 2006).
To provide further evidence that mutations in cnx were responsible for the severe reduction in Rh1, the wt cnx gene was introduced into the cnx mutants by using a duplication for 99A that was translocated to the X chromosome [Dp(3;1) B152]; the introduced gene was confirmed to restored the normal function. Consistent with the presence of the stop codons, the cnx mutants displayed severely reduced levels of cnx transcript. In addition, the Cnx protein was absent in both of the cnx mutants, while it was detected in wt flies. Lane 4 confirms that the mutants were allelic. Cnx protein was not detected in the cnx1 and cnx2 mutants when they were crossed to a deficiency (Df) that eliminated 99A (Df(3R)Ptp99A[R3]). These data support the hypothesis that the mutations were in the cnx gene (Rosenbaum, 2006).
Although Rh1 protein levels are severely reduced in the cnx mutants, Rh1 transcript levels are normal in both. These data suggest that Cnx functions post-transcriptionally and are consistent with a role for Cnx as a chaperone in Rh1 biosynthesis (Rosenbaum, 2006).
To investigate the role of Cnx in Rh1 biosynthesis, the kinetics of Rh1 maturation in the cnx1 mutant were assessed. Transgenic flies carrying the Rh1 gene under the control of a heat-shock promoter (hs) were used and tagged with an epitope corresponding to 12 amino acids at the C terminus of bovine rhodopsin (hs-Rh1-bov transgene). The transgene was introduced into wt, cnx1, and ninaAP269 mutant flies. To initiate Rh1 biosynthesis, the transgenic flies were given a 1 hr heat-pulse at 37°C, shifted back to 22°C, and assayed at specific time points. The fate of the heat-induced Rh1 protein was followed by using a monoclonal antibody (1D4) directed to the epitope tag (bov). No Rh1 expression was detected in the flies prior to heat-pulse. In wt flies, Rh1 was initially detected as immature high-MW forms that were converted to the mature low-MW form by 18 hr. In the cnx1 mutant, Rh1 was also initially detected as immature high-MW forms but was significantly reduced by 24 hr. By 48 hr, very little Rh1 was detected, suggesting that most of the Rh1 was degraded (Rosenbaum, 2006).
The failure of Rh1 to mature in the cnx1 mutant was similar to the fate of Rh1 in the ninaA mutant. NinaA is a chaperone specifically required for Rh1 biosynthesis and maturation. As in the cnx1 mutant, Rh1 is initially detected as immature high-MW forms in the ninaA mutant. In contrast to the cnx1 mutant, where most of the Rh1 was degraded, Rh1 accumulates in the immature high-MW form in the ninaA mutant (Rosenbaum, 2006).
The subcellular localization of Rh1 was followed in pulse-chase experiments. In wt flies, by 8 hr following the heat-pulse, Rh1 immunolocalized to the ER in a perinuclear fashion, was detected in a punctate pattern consistent with transport vesicles, and was detected in the rhabdomeres. By 12 hr, more Rh1 was detected in the rhabdomeres, and by 24 hr, mature Rh1 localized solely to the rhabdomeres. This represents the normal progression for Rh1 maturation and transport through the secretory pathway. In the cnx1 mutant, by 8 hr, Rh1 was detected predominantly in the ER. By 12 hr, Rh1 was still most noticeable in the ER. By 24 hr, Rh1 labeling was detected in both the ER and rhabdomeres, but was significantly fainter than wt. These results show that in the cnx mutant, while most Rh1 was degraded, some Rh1 successfully evaded the quality-control mechanisms and was transported to the rhabdomeres. In the ninaA mutants, at 8, 12, and 24 hr, Rh1 was detected primarily in the ER. A very small amount of Rh1 was detected in the rhabdomeres of ninaA mutants, again indicating that a small amount of Rh1 evaded the ER's quality-control system (Rosenbaum, 2006).
It is possible that Cnx and NinaA are part of a protein-processing pathway, ensuring proper folding and quality control of Rh1 during biosynthesis. To gain insights into the epistatic relationship between the two chaperones, mutant flies were created that were defective in both cnx and ninaA. The ninaAP269;cnx1 double mutant displayed severely reduced levels of Rh1, comparable to those seen in the cnx1 mutant alone. These data demonstrate that in the double mutant, Rh1 was effectively degraded rather than accumulating in the ER (as in ninaA). Therefore, cnx is epistatic to ninaA, as the phenotype of the cnx mutation overrides the phenotype of the ninaA mutation in the double mutant (Rosenbaum, 2006).
Because the two chaperones are required for Rh1 biosynthesis, the levels of NinaA protein were examined in the cnx mutants. NinaA levels in cnx were indistinguishable from wt levels, suggesting that the defects in Rh1 were the result of a lack of Cnx, rather than a lack of NinaA (Rosenbaum, 2006).
The association of Cnx with Rh1 was assessed by coimmunoaffinity experiments. Cnx was isolated in a stable complex with Rh1, but did not bind to or elute from the immunoaffinity column in the absence of Rh1. These data indicated that Cnx and Rh1 physically associate in a protein complex, consistent with a role for Cnx as a molecular chaperone for Rh1 (Rosenbaum, 2006).
Examination of the cnx mutants revealed that they displayed an age-related retinal degeneration. Photoreceptor cells in 1-day-old cnx mutants displayed diminished rhabdomere size as compared to wt. They also displayed accumulations of rough ER membranes, dilated Golgi, and various types of deposits. These secretory pathway defects were consistent with a failure in Rh1 maturation. The identity of the deposits is unknown, but they may consist of degraded material within the cells. At 1 month, cnx mutants displayed a dramatic loss of rhabdomeres in the R1-6 photoreceptor cells, while the R7 and R8 photoreceptor cell rhabdomeres remained. The Drosophila compound eye is made up of approximately 750 individual eye units called ommatidia. Each ommatidium contains eight photoreceptor cells. Only R1-6 photoreceptors express Rh1, whereas R7 and R8 cells express a variety of different opsins (Rh3-6). The finding that only R1-6 rhabdomeres degenerated, and not R7 and R8, suggested that the opsins located in R7 and R8 are expressed normally (Rosenbaum, 2006).
To confirm normal expression of the minor rhodopsins in R7 and R8 cells, immunocytochemical analysis of Rh3, Rh4, and Rh5 opsins was performed in the cnx mutants. All three opsins were found to be correctly localized to the rhabdomeres of the R7 and R8 cells, confirming that while Cnx was required by Rh1, it was not required by the R7 and R8 opsins (Rosenbaum, 2006).
Whether ER processing of other photoreceptor proteins was defective in the cnx mutant was examined. Immunocytochemical analysis revealed that another membrane protein, chaoptin, was present at its normal location in the rhabdomeres of the R1-8 photoreceptor cells in both cnx mutants. In addition, immunoblotting analysis was performed to confirm that a number of key phototransduction proteins, including the G protein α subunit (Gqα), the TRP and TRPL channels, PLC-β (norpA), arrestin 1 (Arr1), and arrestin 2 (Arr2) were all expressed at levels indistinguishable from wt. These results demonstrated that while Cnx was required for processing of Rh1, it was not required for expression of these other photoreceptor cell proteins (Rosenbaum, 2006).
To determine the expression pattern for Cnx, polyclonal antibodies were generated that recognized a 97 kDa band in wt flies that was not present in the mutants. Cnx localized to the ER of all eight photoreceptor cells, often to ER cisternae that were tightly associated with the nuclear envelope. The labeling pattern for Cnx was compared to the ER proteins, InsP3R (inositol-1,3,5-trisphosphate receptor) and NinaA. All three proteins were expressed in the ER, but were absent from the rhabdomeres. Although the rhabdomeres of the central R7 photoreceptor cells are labeled by the InsP3R antibody, this labeling is nonspecific. While Cnx protein was uniquely required by Rh1 in the R1-6 cells, it was detected in the ER of all eight photoreceptor cells (Rosenbaum, 2006).
To assess whether the retinal degeneration observed in the cnx mutants is enhanced by light activation of the phototransduction cascade, the cnx mutants were reared for 1 month in constant darkness. These flies displayed a less severe retinal degeneration compared with cnx mutants grown for 1 month on a 12:12 light-dark cycle. Therefore, activation of phototransduction by light enhances the retinal degeneration in the cnx mutants. This result is contrasted to other known mutants defective in Rh1 maturation, such as ninaA, in which the retinal degeneration is light independent. In addition, the ninaA mutants degenerate more slowly compared to the cnx mutants (Rosenbaum, 2006).
The finding that light enhances the retinal degeneration in the cnx mutant led an investigation of whether Ca2+ influx through the light-sensitive channels contributes to the retinal degeneration. Null mutations in the gene encoding the eye-enriched PLC (norpA) eliminate the light-induced Ca2+ influx. norpA;cnx double mutants were generated and it was found that norpA slows down the onset and progression of the retinal degeneration in the cnx mutants. The finding that the retinal degeneration in the cnx mutants is light enhanced and slowed by norpA, in combination with previous findings that calnexin binds Ca2+ (Tjoelker, 1994), prompted a determination of whether Cnx plays a role in modulating Ca2+ in photoreceptor cells (Rosenbaum, 2006).
Current models of phototransduction suggest that virtually all aspects of excitation and adaptation are mediated within the microvilli. Because Cnx is located in the ER, it was predicted that mutations in cnx would not affect the basic light responses, but might cause defects in Ca2+ buffering in the cell body. The basic properties of the light-induced current (LIC) were investigated by using whole-cell patch-clamp recordings of photoreceptors from dissociated ommatidia to record the elementary responses (quantum bumps) representing the response to single-photon absorptions. In addition to wt flies, ninaA mutants were used as controls, because they express low (<1%) levels of functional Rh1 comparable to cnx, but unlike Cnx, the NinaA protein has no predicted Ca2+-binding domains. The most obvious phenotype in both cnx and ninaA was the great reduction in quantum capture (~100- to 200-fold), confirming the reduction in functional rhodopsin. The quantum bump amplitude and waveform in ninaA and cnx were indistinguishable from each other, but both showed a significant (~50%) increase in quantum bump amplitude compared to wt. A similar increase in quantum bump amplitudes has been described in Rh1 hypomorphs. Otherwise, the quantum bump waveform in both cnx and ninaA is indistinguishable from wt. Macroscopic responses to brief test flashes and modest light steps in cnx and ninaA were also indistinguishable from each other, but greatly reduced in sensitivity compared to wt. These results suggested that Cnx does not play a significant role in the basic light response and that, apart from the reduction in Rh1, all other key components of the phototransduction cascade are functional in both cnx and ninaA (Rosenbaum, 2006).
In order to measure Ca2+ levels in the cell body during illumination, the low-affinity Ca2+ indicator dye Fluo-4FF, loaded via the patch pipette, was used. Wild-type, ninaA, and cnx photoreceptor cells were illuminated with the same intensity of 485 nm light, equivalent to ~108 effectively absorbed photons/s in wt flies (though only ~106 photons/s in ninaA and cnx because of the ~100-fold reduction in quantum catch). Even in cnx and ninaA, this corresponds to ~40 effectively absorbed photons/s per microvillus and appeared to be nearly saturating, since a 7.5-fold brighter stimulus only induced slightly larger Ca2+ signals. Fluorescence was measured from the entire cell, where the dominant contribution appears to come from the cell body. After an ~10-20 ms latent period, which allows an estimate of Fmin, the fluorescence increased to a peak after 200-300 ms and then declined to a steady-state plateau. The absolute initial levels reached during this brief latent period in cnx and both wt and ninaA controls were indistinguishable, indicating that there was no systematic difference in dye loading or resting Ca2+ concentrations. However, both the maximum level reached and the plateau in cnx were approximately 2- to 3-fold higher than in either wt or ninaA controls. The comparison with wt is particularly striking, because the effective intensity of illumination was 100-fold times greater in wt flies. A reduction in the sacro-endoplasmic reticulum Ca2+ ATPase (SERCA) or the Na+/Ca2+ exchanger (CalX) was ruled out as being responsible for the increased Ca2+ since both proteins were expressed at wt levels in the cnx mutants. The striking differences in the cytosolic Ca2+ signals between cnx and wt (and ninaA) indicate that Cnx plays an important role in buffering Ca2+ in the cell body (Rosenbaum, 2006).
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date revised: 20 May 2006
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