InteractiveFly: GeneBrief

prominin/eyes closed: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - eyes closed

Synonyms - prominin (prom)

Cytological map position - 60D1

Function - enzyme cofactor

Keywords - eye morphogenesis, membrane biogenesis, cellularization, vesicles

Symbol - eyc

FlyBase ID: FBgn0259210

Genetic map position -

Classification - ubiquitin-like domain

Cellular location - cytoplasmic and possibly nuclear

NCBI link: Entrez Gene

eyc orthologs: Biolitmine

A mutation impacting photoreceptor morphogenesis, eyes closed (eyc), has been identified as a fly homolog of p47, a protein co-factor of the p97 ATPase implicated in membrane fusion. Temporal misexpression of Eyc during rhabdomere extension early in pupal life results in inappropriate retention of normally transient adhesions between developing rhabdomeres. Later Eyc misexpression results in endoplasmic reticulum proliferation and inhibits rhodopsin transport to the developing photosensitive membrane. Loss of Eyc function results in a lethal failure of nuclear envelope assembly in early zygotic divisions. Phenotypes resulting from eyc mutations have provided the first in vivo evidence of a role for p47 in membrane biogenesis (Sang, 2002).

Membrane fusion is fundamental to virtually all aspects of cell physiology, including vesicle-mediated transport through the secretory pathway and the postmitotic reconstitution of Golgi, endoplasmic reticulum (ER) and nuclear membranes from mitotic vesicles. Substantial evidence suggests fusion is mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) integral membrane proteins, assisted by a host of additional cytoplasmic proteins, including two ATPases, NSF (N-ethylmaleimide-sensitive factor: see Drosophila Comatose and NEM-sensitive fusion protein 2) and p97 (Acharya, 1995; Latterich, 1995; Rabouille, 1995). These ATPases disassemble unproductive cis-SNARE complexes, priming them for fusion by making SNAREs available to engage in trans with cognate SNAREs of target membranes and thereby promoting another round of fusion (Mayer, 1996; Rabouille, 1995). Although exceptions exist, NSF is typically targeted by alpha-SNAP (soluble NSF attachment protein alpha) to SNARE complexes that result from heterotypic membrane fusion, while p97 (Edwardson, 1998; Mellman, 1995) is targeted by p47 to complexes resulting from homotypic membrane fusion (Sang, 2002 and references therein).

p47 was first found complexed with p97 in an in vitro Golgi reassembly assay (Kondo, 1997). Loss of p47 results in the failure to reassemble Golgi stacks after their vesiculation during mitosis. Stoichiometry is a crucial determinant of p47/p97 activity; ratios above or below optimal decrease Golgi reassembly (Meyer, 1998). Co-operation between NSF and p97 pathways is suggested by the competition of alpha-SNAP and p47 for a common syntaxin-5 SNARE complex (Rabouille, 1998). Both pathways are required to rebuild Golgi cisternae from mitotic Golgi fragments in vitro; alone, each promotes vesicle fusion leading to morphologically distinct cisternae (Acharya, 1995; Rabouille, 1995). Fusion of ER membranes from yeast mutant for the p97 homolog, CDC48, is inhibited in an in vitro assay. Similarly, a rat liver microsome fusion assay also demonstrates a requirement for p97 in ER assembly. In addition to its role in membrane fusion, p97 also participates in other activities, including ubiquitin-dependent protein degradation, nuclear transport pathways and DNA unwinding. p97 activity is directed to different cellular pathways by the regulation of its co-factors. To date, a role for p47 has not been described in development and morphogenesis (Sang, 2002 and references therein).

Photoreceptor morphogenesis demands a high level of performance by mechanisms mediating directed membrane traffic. Rhabdomeres, like their vertebrate counterparts, the outer segments of rod and cone photoreceptors, are enormously amplified central subdomains of the photoreceptor apical membrane. Rhabdomere growth during pupal life is driven by delivery of copious photosensitive membrane to the developing rhabdomere. Defects in membrane traffic yield rhabdomere defects, which are plain against the precise, stereotyped and sizeable rhabdomeres of normal flies; Drosophila photoreceptor morphogenesis is a sensitive assay of membrane differentiation. The trapezoid of adult rhabdomeres has its origin early in pupal life with the establishment of a stereotyped set of contacts between photoreceptor apical faces, the future rhabdomeres. While maintaining the zonula adherens (z.a.) junctions formed during pattern formation, inpocketing of photoreceptor apices into the retinal epithelium by closure of the lens-secreting cone cells 'above' them, brings photoreceptors 'face-to-face' in a trapped apical cavity that is the precursor of the inter-rhabdomeral space (IRS) (see the movie at Rhabdomere extension and the origin of rhabdomere trapezoids). Contacts between photoreceptors R2, R4 and R7 occlude R3 from the center of the ommatidium, displacing its apical membrane to the future 'point' of the trapezoid. Between 37% and 55% of the way through the photoreceptor differentiation process (p.d.), photoreceptor apical surface amplification extends these contacts to the retinal floor, along a 'core' of partitions at the center of the ommatidium; photoreceptor apices meet in a manner resembling the sectors of an orange. During extension, the previously undifferentiated apical membrane is reorganized in center-surround domains dedicated to future development of rhabdomere and stalk membrane. By 55% of the way through the process, face-to-face contacts between photoreceptors are relinquished, opening the IRS (Sang, 2002).

In order to further characterize contacts between photoreceptor apical faces, developing eyes were stained between 37% p.d. and 55% p.d. using phalloidin and antibodies to the apical membrane protein, Crumbs (Crb), as well as antibodies against Armadillo (Arm, Drosophila ß-Catenin) and anti-Drosophila E-cadherin (DE-Cad: Shotgun) proteins associated with adhesive cell-cell contacts that are typically localized to z.a. junctions. Confocal images of wild type 37% p.d. eyes show Crb, Arm and DE-Cad across the entire apical membrane, in addition to strong staining in z.a. junctions. By 50% p.d., the stage when apical face contacts are released, Arm and DE-Cad have retreated from the apical surface and stain only the z.a junctions. These results suggest that the 'unsticking' of R cell apical faces by 50% p.d. may be due to reorganization of Arm and DE-Cad in the R cell apical membrane (Sang, 2002).

Distinct programs of membrane reorganization mark photoreceptor differentiation. Between 55% and 70% p.d., the apical membrane protein Crb is relocalized from the entire surface to the stalk. This redistribution occurs in eyc mutants (Sang, 2002).

A Drosophila strain, fliA4, mutant in alpha-actinin, contains a second mutation that degrades rhabdomere morphogenesis (R. L. Longley, PhD Thesis, Purdue University, 1994). Mutant rhabdomeres are fragmented and inappropriate adhesions join rhabdomeres to rhabdomeres and to stalks of other photoreceptors. In occasional planes, all rhabdomeres meet at the ommatidial center, resembling the closed rhabdoms common to most arthropods. The gene was consequently named eyes closed (eyc). It is notable that the external morphology of eyc1 eyes is completely normal (Sang, 2002).

eyc1 photoreceptors are indistinguishable from wild type at 37% p.d.. At this stage, R cell apical membranes contact each other in stereotyped combinations. R2, R4 and R7 contact 'in front of' R3, pre-figuring the trapezoid of the adult ommatidium. The z.a. junctions that join adjacent R cells appear normal. The apical membranes of wild type and mutant R cells are infolded, but do not show regional differentiation (Sang, 2002).

By 55% p.d., the eyc1 phenotype is plain. The IRS fails to open and R cells contact each other in rhabdomere tip-to-tip and rhabdomere-to-stalk contacts. Distinctive stalk and rhabdomere membranes are evident, but they are not organized in a simple center-surround. Islands of trapped stalk make loops in R cell cross-sections. Contacts between R cells in adult ommatidia commonly include those seen during rhabdomere extension, notably R2/R4/R7; the rhabdomere of R4 frequently bifurcates to contact R2 and R7. Strong R5/R6 contact, not seen between rhabdomeres during extension, is also common in the mutant (Sang, 2002).

In order to test the hypothesis that persistent contacts between mutant rhabdomeres are due to the inability of R cells to clear adhesive proteins from their apical faces, mutant eyes were examined using phalloidin, anti-Arm, anti-DE-Cad and anti-Crb antibodies. Confocal micrographs of eyc1 50% p.d. eyes show abnormal Arm staining in R cell apical membranes, often in patches localized to abnormal R cell contacts. DE-Cad staining is similarly localized to abnormal contacts. It is speculated that eyc1 R cells are unable to reorganize their apical membranes correctly at this crucial stage of morphogenesis (Sang, 2002).

Despite the severity of the rhabdomere phenotype, eyc1 eyes show the expression of rhodopsin in outer photoreceptors at the appropriate developmental stage; there is no obvious morphological phenotype beyond rhabdomere malformation (Sang, 2002).

Since the temporal misexpression of eyc coincides with the developmental stage at which the rhabdomere/stalk organization of the photoreceptor apical surface is established, it was hypothesized that Eyc misexpression at this stage might produce an eyc-like eye phenotype. In order to test this, the GAL4/UAS system was used to drive Eyc expression at mid-pupal stages, and its effect on rhabdomere development was examined (Sang, 2002).

Developmentally staged pupae carrying one copy of UAS-eyc and one copy of Hs-GAL4 were given six cycles of 45 minutes 37°C heat shock and 5 hours 15 minutes 25°C recovery, starting at 30% p.d. Eyes were dissected from freshly eclosed adults and examined using confocal and electron microscopy. eyc-like defects were found in the eyes of heat-shocked Hs-GAL4/UAS-eyc flies. In the EM, approximately 23% (21/89) of ommatidia showed abnormal contacts between rhabdomeres. Eyc misexpression that started later in pupal development did not generate an eyc eye phenotype. These results support the hypothesis that the eyc phenotype is due to mid-pupal Eyc misexpression (Sang, 2002).

More severe defects were observed when heat shocks were continued into later pupal stages; rhabdomeres displayed abnormal adhesions and their size was reduced in almost all R cells. Moreover, anti-Rh1 immunostaining using 4C5 showed these animals were also deficient in rhodopsin delivery to outer R cell rhabdomeres. Most contained little or no rhodopsin; instead, rhodopsin was concentrated in the photoreceptor cytoplasm. Control Hs-GAL4 flies given the same heat shock regimen did not show a phenotype. UAS-eyc driven by GMR-GAL4, which induces eyc transgene expression starting immediately behind the furrow in third instar larvae, also generated an eyc-like phenotype (Sang, 2002).

p97 has been shown essential for the budding of vesicles from transitional ER (Zhang, 1994) and the possibility was therefore examined that rhodopsin accumulation in the photoreceptor cytoplasm was related to ER defects. Parallel preparations were made for both anti-Rh1 immunostaining and electron microscopy. Hs-GAL4/UAS-eyc animals received three 1 hour heat shocks separated by 5 hours recovery starting at 70% p.d.; eyes were dissected and prepared for observation at approximately 85% p.d. In accordance with results obtained with earlier heat shocks, overexpression of Eyc inhibits Rh1 transport to the rhabdomere. Using electron microscopy, abundant ER accumulation was found in photoreceptor cytoplasm, which is a condition not seen in control animals (Sang, 2002).

In order to quantitate this difference, tangential sections approximately 25 µm below the corneal surface were collected and approximately 20 ommatidia were photographed. ER stacks in R cells were counted manually on prints. In Hs-GAL4/+ controls, the majority of R cells had two to three ER stacks, similar to normal photoreceptors at this stage. In Hs-GAL4/UAS-eyc flies, R cells showed more ER stacks, ranged from three to 10 stacks. It is speculated that the cytoplasmic rhodopsin staining observed in the confocal microscope results from disruption of vesicle traffic to the rhabdomere, possibly owing to rhodopsin becoming trapped in the ER (Sang, 2002).

The impact of Eyc overexpression on rhodopsin synthesis and maturation was investigated. In normal photoreceptors, rhodopsin synthesis and core glycosylation in the ER yields immature forms that are deglycosylated in the Golgi before transport of the mature 35 kDa form to the rhabdomere. If Eyc overexpression disrupts vesicle-mediated protein trafficking, an increase in immature rhodopsin might be expected in Hs-GAL4/UAS-eyc eyes. To test this, pupae were heat shocked as before and rhodopsin maturation was assessed using Western blots. Mature rhodopsin levels were decreased in Hs-GAL4/UAS-eyc retinas relative to Hs-GAL4 controls, consistent with the reduced stain in rhabdomeres observed using immunohistochemistry. Immature, higher molecular weight rhodopsin also increased relative to mature rhodopsin in these eyes, suggestive of defects in rhodopsin processing, potentially arising from disturbance of intracellular membrane traffic (Sang, 2002).

In order define the function of Eyc, imprecise excision of P1363 was used to generate loss-of-function eyc alleles. 250 lines were screened and 27 homozygous lethal excisions were recovered. Embryo development was observed in progeny of 10 balanced excision heterozygotes. In all 10 lines, approximately one quarter of the embryos, later confirmed as lacking the eyc ORF using PCR, arrested before cellularization. Three of the lines (eycl20, eycl27 and eycl39) were selected for molecular characterization and rescue experiments (Sang, 2002).

Thus, gain and loss of Eyc function results in developmental phenotypes that share a common focus of membrane biogenesis. The original eyc allele, recovered as a second hit in a strain selected as flightless in an EMS screen, is a gain-of-function mutation that results in Eyc misexpression at the time when photoreceptor apical surfaces must reorganize to relinquish the face-to-face contacts that guide rhabdomere extension to the retinal floor (see the movie at Rhabdomere extension and the origin of rhabdomere trapezoids) and to establish definitive rhabdomere and stalk subdomains. eyc mutants fail to release these contacts and to generate the normal center-surround organization of rhabdomere and stalk membrane. Temporal misexpression in the mutant coincides with a stage at which photoreceptor apical surfaces are undifferentiated, consistent with the participation of both stalk and microvillar membrane in adhesions. It is speculated that normally directed membrane traffic is necessary for photoreceptor membrane reorganization, including removal of proteins that promote adhesion, such as Armadillo and DE-Cadherin (Sang, 2002).

A requirement for Eyc in nuclear envelope reassembly adds a novel role for p47. The failure of nuclear envelope reassembly in eyc loss-of-function mutants, and the inhibition of nuclear divisions by anti-Eyc antiserum is consistent with observations that pretreatment of nuclear membrane vesicles with NEM, known to inactivate p97, prevents fusion (Macaulay, 1996). The continuity of outer nuclear membranes with the ER, where a role for p47/p97 in membrane fusion is well established, offers a plausible basis for a shared fusion mechanism (Sang, 2002).

One attractive possibility for the proliferation of ER in response to elevated Eyc is suggested by observations in yeast that excess Sec17p, the yeast alpha-SNAP homolog, inhibits membrane fusion by stabilizing unproductive cis-SNARE pairing. SNAREs nucleate assembly of the COPII coats mediates ER-Golgi transport. Since transport vesicle budding must endow the vesicle with SNAREs that are competent to mediate fusion, coat assembly may reject fusion-incompetent cis-SNARE complexes. Excess Eyc may shift the balance in favor of fusion-incompetent cis-SNARE complexes, hindering budding, increasing the size of the ER and disrupting normal rhodopsin traffic (Sang, 2002).

Alternate scenarios for an effect of Eyc overexpression include diversion of p97 activity from other tasks. For example, p97 also participates in ubiquitin-dependent protein degradation and nuclear transport pathways, and is targeted to these activities by a protein complex, Ufd1/Npl4, which competes with p47 for binding to p97 (Meyer, 2000). Excess Eyc might diminish the availability of p97 for these roles. p47/p97 activity in vitro is stoichiometry dependent (Meyer, 1998) and it is possible that excess Eyc produces a sub-optimal ratio, which compromises normal membrane fusion in vivo. Excess Eyc may also impact NSF-mediated pathways since alpha-SNAP and p47 compete (Rabouille, 1998) for Golgi syntaxin-5 SNARE complexes (Sang, 2002 and references therein).

Rhabdomere defects of eyc mutants are provocative in light of the report that anti-NSF and anti-alpha-SNAP antibodies do not inhibit MDCK apical membrane delivery. SNAREs participate in apical membrane transport and presumably require disassembly after fusion. p47/p97 is an attractive candidate to mediate such disassembly (Sang, 2002).

It is interesting to consider Drosophila photoreceptor morphogenesis against the background of Arthropod eye development generally. Higher Dipterans and some Coleopterans are unusual in having 'open' rhabdoms in which separate rhabdomeres individually face the IRS. Rhabdomeres of most insects and crustaceans adhere on the central axis to form a multicellular 'closed' rhabdom. As in Drosophila, closed rhabdoms develop 'down' from the distal, corneal surface of the eye to the retinal floor. Rhabdomere contacts of closed rhabdoms display diverse and intriguing patterns which may offer clues to the adhesive rules guiding rhabdomere extension. Perhaps only a small change in the 'machine language' of photoreceptor development, for example, regulation of targeted membrane delivery, might allow fly rhabdomeres to unstick after extension, opening the IRS (Sang, 2002).

Regulation of gene expression and RNA editing in Drosophila adapting to divergent microclimates

Determining the mechanisms by which a species adapts to its environment is a key endeavor in the study of evolution. In particular, relatively little is known about how transcriptional processes are fine-tuned to adjust to different environmental conditions. This study examined Drosophila melanogaster from 'Evolution Canyon' in Israel, which consists of two opposing slopes with divergent microclimates. Several hundred differentially expressed genes and dozens of differentially edited sites were identified between flies from each slope; these changes were correlate with genetic differences, and CRISPR mutagenesis was used to validate that an intronic SNP in prominin regulates its editing levels. It was also demonstrated that while temperature affects editing levels at more sites than genetic differences, genetically regulated sites tend to be less affected by temperature. This work shows the extent to which gene expression and RNA editing differ between flies from different microclimates, and provides insights into the regulation responsible for these differences (Yablonovitch, 2017).

This study analyzed the genomes, transcriptomes, and editomes of individual fly lines from the two slopes of Evolution Canyon, building upon previous studies examining Drosophila from this canyon1. Evolution Canyon is uniquely suited for studying the biodiversity, evolution and adaptation of organisms that live in relatively close proximity to each other, and is a potential model for incipient sympatric speciation. Although genetic and gene expression differences have been discovered previously in flies from different environments, most studies have examined fly populations at different latitudinal clines, and many signatures of adaptation found so far may be related to migration out of Africa. This is the first study to systematically examine gene expression and RNA editing differences in flies from different microclimates (Yablonovitch, 2017).

Several candidate genes were identified whose expression may play an adaptive role in the Evolution Canyon flies. The most striking of these are the Glutathione S-transferase genes, which show global under-expression in the NFS1 (north facing slope 1) flies compared to the SFS (south facing slope), and tend to be under-expressed in the NFS2 flies compared to the SFS as well. These detoxification enzymes metabolize antioxidants, and their decreased expression in the NFS1 and NFS2 relative to the SFS flies may be related to the decreased sun exposure, as there is 200–800% more solar radiation on the SFS18. It is interesting to note that a previous study examining flies from Evolution Canyon showed enrichment of glutathione metabolism and transferase activity in genomic regions with evidence of inter-slope differentiating selection. In addition, some Glutathione S-transferase genes have been shown to have significantly decreased expression in European flies compared to African flies in brain tissue. Other genes involved in pigmentation, stress response, digestion, and chitin-related processes also showed significant gene expression differences between flies from the two slopes. Future studies will be needed to address the potential adaptive role of the expression of these genes, and any regulatory mutations that are responsible for these gene expression changes (Yablonovitch, 2017).

A strong connection was shown between the genetic differences and the gene expression and RNA editing differences of the flies from the two slopes. This implies that, despite the flies being collected from Evolution Canyon years prior to these experiments, genetic regulation of gene expression and RNA editing still persists in the isofemale lines. In particular, it was confirmed that an intronic SNP regulates the editing levels of two sites in the prominin transcript, although the exact amount of editing level regulation contributed by the SNP could not be determined, since a CRISPR-associated PAM mutation was made in the same mutant. Since both the editing sites and the intronic SNP are conserved in many Drosophila species, and since most of the NFS1 lines contain the mutant allele that causes a decrease in editing, it is possible that the less stressful NFS environment decreased the strength of selection against this mutation. Although an editing-related prominin phenotype has not been identified, previous studies examining RNA editing evolution in different Drosophila species have demonstrated evidence of selection, especially for conserved, non-synonymous site. As the sites in prominin dealt with in this study are likewise conserved and code for non-synonymous amino acid changes, it's still possible that they play an adaptive role (Yablonovitch, 2017).

For RNA editing, both population genetic differences and environmental differences were capable of regulating editing between flies from the two slopes, and that the regulation seems to act through changing the structural stability of the RNA editing substrate. A change in environment regulates editing to a large extent for dozens of sites, most likely by affecting the stability of many RNA structures simultaneously. In contrast, genetic regulation is more site-specific, likely due to particular SNPs nearby editing sites which change the stability of the RNA structure containing those sites. This result is also supported by previous studies that examined editing level differences between and within Drosophila species. Population genetic differences in editing tend to be maintained regardless of the environment in which they were measure, suggesting that genetic regulation may be more influential than environmental regulation of these sites. One 3' UTR site in the falafel transcript was found that exhibits a genotype-environment interaction between the NFS1 and SFS fly populations, as well as several non-synonymous sites in the cacophony transcript between the NFS2 and SFS fly populations. Future studies will be needed in different populations and environments to determine whether these trends in editing happen universally (Yablonovitch, 2017).

To conclude, this study found surprising connections between genetics, gene expression, and RNA editing in flies from the distinct microclimates of Evolution Canyon. By sequencing individual lines, it was possuble to show a clear correspondence between genotype and gene expression differences between flies from the two opposing slopes, some of which may be important for adaptation. In addition, both genetic and environmental regulation of RNA editing was observed in these flies, though the two modes of regulation seem to operate mostly independently of each other. This study sets the stage for future examinations of the regulation of adaptive gene expression and RNA editing differences, not only in other fly populations, but in other species as well (Yablonovitch, 2017).


Ter94, the partner of Eyes closed in membrane biogenesis

A Drosophila homolog of the membrane fusion protein CDC48/p97 has been cloned. The open reading frame of the Drosophila homolog encodes an 801 amino acid long protein (TER94), which shows high similarity to the known CDC48/p97 sequences. The chromosomal position of TER94 is 46 C/D. TER94 is expressed in embryo, in pupae and in the adult, but is suppressed in larva. In the adults, the immunoreactivity is exclusively present in the head and in the gonads of both sexes. In the head the most striking staining is observed in the entire neuropil of the mushroom body and in the antennal glomeruli. Besides TER94, sex-specific forms are also detected in adult gonads: p47 in the ovaries and p98 in the testis. TER94/p47 staining is observed in the nurse cells and often in the oocytes, while TER94/p98 staining is present in the sperm bundles. On the basis of the TER94 distribution it is suggested that TER94 functions in the protein transport utilizing endoplasmic reticulum and Golgi derived vesicles (Pinter, 1998).

The Drosophila fusome is a germ cell-specific organelle assembled from membrane skeletal proteins and membranous vesicles. Mutational studies that have examined inactivating alleles of fusome proteins indicate that the organelle plays central roles in germ cell differentiation. Although mutations in genes encoding skeletal fusome components prevent proper cyst formation, mutations in the bag-of-marbles gene disrupt the assembly of membranous cisternae within the fusome and block cystoblast differentiation altogether. To understand the relationship between fusome cisternae and cystoblast differentiation, attempts have been made to identify other proteins in this network of fusome tubules. Evidence is presented that the fly homolog of the transitional endoplasmic reticulum ATPase (TER94) is one such protein. The presence of TER94 suggests that the fusome cisternae grow by vesicle fusion and are a germ cell modification of endoplasmic reticulum. Fusome association of TER94 is Bam-dependent, suggesting that cystoblast differentiation may be linked to fusome reticulum biogenesis (Leon, 1999).

Antisera raised against a TER94 internal peptide reacts with bands of 94,000 Da in wild-type ovarian extracts and 57,000 Da in Escherichia coli cells expressing a fragment of TER94 as a GST-fusion protein. Both Cdc48p and vertebrate TERs oligomerize to form homohexameric complexes. When ovarian extracts were analyzed on native sucrose gradients, the peak of TER94 from flies sedimented was Mr ~500,000, which is close to the expected size (Mr ~530,000) for a homohexameric complex (Leon, 1999).

TER94 protein is present in both ovarian germ cells and somatic cells. TER94 is largely cytoplasmic in follicle and germ cells. Significantly, germ cells often contain one or several especially intense fluorescent signals, suggesting that TER94 is distributed unevenly in the cytoplasm. In cystocytes in germarial Region 1, these are usually somewhat diffuse bright regions, whereas in more mature cystocytes the bright spots are more sharply defined (Leon, 1999).

The number and positions of the TER-enriched regions suggest that they might correspond to fusomes. Stem cell fusomes in germ cells nearest the anterior tip appear as a single dot of intense staining, whereas those in a more posterior position (i.e. more mature cysts) contain elongated, branched fusomes. Precise colocalization of TER94 and Hu-li tao shao is strongest in Region 1 germ cells and declines in regions containing mature cysts. Because a fraction of TER is nuclear in yeast and mammals, Drosophila nuclei were examined closely. Most germ cell nuclei are faintly TER94 positive. Many examples of strong nuclear and perinuclear staining have been found in nonovarian somatic cells in larvae and adults (Leon, 1999).

Fusomes are the primary site of ER-like cisternae in young germ cells. If TER94 enrichment in fusomes represents accumulation at the fusome reticulum, TER94 distribution might be altered when the reticulum is not properly assembled. Bam is a fusome-associated protein and bam mutant fusomes are deficient in cisternae. The distribution of TER94 protein was examined in bam germ cells; it is distributed uniformly without signs of enrichment at the site of fusomes as is observed in wild-type germaria. Indeed, when the bam stem cell fusomes are visualized with Hts antibodies, it is clear that TER94 is no more abundant within or near stem cell fusomes than in any other cytoplasmic regions. Consistent with this conclusion, the merged images of TER94 and Hts distributions do not show immunofluorescent overlap, indicating that bam fusomes do not accumulate detectable TER94 (Leon, 1999).

TER94 is also enriched at a few sites that do not correspond to fusomes. It is speculated that these may be sites of Golgi bodies or transport vesicles, although unambiguous identification requires additional reagents as markers. These extrafusome sites of TER94 enrichment are also abolished in bam mutant cells (Leon, 1999).

The observation that TER94 fusome association is linked to Bam function can be explained by either a direct or indirect Bam dependent mechanism. Although loss of bam function might block fusome reticulum assembly before TER94 arrival, it is also possible that Bam recruits TER94 to the reticulum as part of the assembly process. This hypothesis has been difficult to test because Bam is a low-abundance protein in ovaries, and in vitro assays for Bam and TER94 interaction have produced inconsistent results. The interaction of Bam and TER94 as two-hybrid partners supports the hypothesis of in vivo interaction. Finding the Drosophila homolog of the S. cerevisiae protein Ufd3p as a second Bam interacting protein strengthens the significance of the Bam-TER94 interaction. Ufd3p and the yeast TER (i.e., Cdc48p) interact with one another directly. Ufd3p is required for efficient organelle vesicle fusion (Leon, 1999 and references therein).

A genetic screen was carried out in Drosophila to identify mutations that disrupt the localization of Oskar mRNA during oogenesis. Based on the hypothesis that some cytoskeletal components that are required during the mitotic divisions will also be required for Oskar mRNA localization during oogenesis, a screen was carried out for P-element insertions in genes that slow down the blastoderm mitotic divisions. A secondary genetic screen was used to generate female germ-line clones of these potential cell division cycle genes and to identify those that cause the mislocalization of Oskar mRNA. Mutations were identified in ter94 that disrupt the localization of Oskar mRNA to the posterior pole of the oocyte. Ter94 is a member of the CDC48p/VCP subfamily of AAA proteins which are involved in homotypic fusion of the endoplasmic reticulum during mitosis. Consistent with the function of the yeast ortholog, ter94-mutant egg chambers are defective in the assembly of the endoplasmic reticulum. A test was carried out to see whether other membrane biosynthesis genes are required for localizing Oskar mRNA during oogenesis. Ovaries that are mutant for syntaxin-1a, rop, and synaptotagmin are also defective in Oskar mRNA localization during oogenesis (Ruden, 2000).

In order to identify new genes required for OSK mRNA localization, OSK localization defects in egg chambers were sought in mutants for cell division cycle (CDC) genes that had been isolated in a 'mitotic delay-dependent survival' (MDDS) genetic screen. The rationale for this is that many cytoskeletal proteins required for mitotic divisions may also be required for mRNA localization. The advantage of studying the function of CDC genes during oogenesis, in which all of the mitotic divisions occur in region 1 of the germarium, is that later in oogenesis one can analyze the biological functions of the CDC genes independent of their mitotic functions. For example, Klp38B, a chromatin-binding kinesin-like-protein isolated in the MDDS genetic screen, is required not only for chromosome segregation during the meiotic and mitotic divisions, but also for the proper development of the oocyte, possibly by localizing mRNA or protein in the oocyte (Ruden, 2000 and references therein).

Based on the phenotypes of syx-1a, ter94, rop and syt mutant egg chambers, a three-step genetic pathway is proposed for the role of membrane fusion proteins on OSK mRNA localization during oogenesis. (1) Syx-1a is required in stage 1 egg chambers to get OSK mRNA to the oocyte. Syx was originally identified as a Drosophila homolog of a human tSNARE that is required for synaptic vesicle fusion in neurons. Interestingly, Syx5 in humans has recently been shown to be required for TERA-mediated (the human Ter94 ortholog) assembly of Golgi cisternae from mitotic Golgi fragments in vitro (Rabouille, 1998). (2) Ter94 is required to localize OSK mRNA within the oocyte. It is speculated that OSK mRNA might be transported in membranous particles because both the endoplasmic reticulum and OSK mRNA form particulate complexes in ter94-mutant egg chambers. (3) The final step in OSK mRNA localization is anchoring the mRNA to the posterior pole of the oocyte. It is proposed that Rop and Syt are required for this process because rop and syt mutant egg chambers have poorly formed cytoplasmic membranous structure in the oocytes, and, possibly as a result, OSK mRNA fails to remain localized at the posterior pole. Rop is a Drosophila homolog of yeast Sec1 and vertebrate n-Sec1/Munc-18 proteins and is a negative regulator of neurotransmitter release in vivo. Syt controls and modulates synaptic vesicle fusion in a Ca2+ dependent manner. It is concluded that many synaptic vesicle fusion proteins also function during other cellular processes such as OSK mRNA localization during oogenesis (Ruden, 2000 and references therein).


The actomyosin machinery is required for Drosophila retinal lumen formation

Multicellular tubes consist of polarized cells wrapped around a central lumen and are essential structures underlying many developmental and physiological functions. In Drosophila compound eyes, each ommatidium forms a luminal matrix, the inter-rhabdomeral space, to shape and separate the key phototransduction organelles, the rhabdomeres, for proper visual perception. In an enhancer screen to define mechanisms of retina lumen formation, Actin5C was identifed as a key molecule. The results demonstrate that the disruption of lumen formation upon the reduction of Actin5C is not linked to any discernible defect in microvillus formation, the rhabdomere terminal web (RTW), or the overall morphogenesis and basal extension of the rhabdomere. Second, the failure of proper lumen formation is not the result of previously identified processes of retinal lumen formation: Prominin (Eyes closed) localization, expansion of the apical membrane, or secretion of the luminal matrix. Rather, the phenotype observed with Actin5C is phenocopied upon the decrease of the individual components of non-muscle myosin II (MyoII) and its upstream activators. In photoreceptor cells MyoII localizes to the base of the rhabdomeres, overlapping with the actin filaments of the RTW. Consistent with the well-established roll of actomyosin-mediated cellular contraction, reduction of MyoII results in reduced distance between apical membranes as measured by a decrease in lumen diameter (see Model for Drosophila retinal lumen formation). Together, these results indicate the actomyosin machinery coordinates with the localization of apical membrane components and the secretion of an extracellular matrix to overcome apical membrane adhesion to initiate and expand the retinal lumen (Nie, 2014; Pubmed).


Cloning of the eyc gene was initiated from an eyc P-element allele (P1363), which failed to complement eyc1. Plasmid rescue of P1363 recovered ~16 kb genomic DNA flanking the P-element. A ~2 kb genomic probe (P3), 3' to the P-insertion site revealed a ~1.1 kb transcript which is elevated in eyc1 and P1363/Df(2R) Px2 flies relative to wild type at 30% p.d.. Eyc protein levels are likewise elevated relative to wild type at this stage. P3 was then used to screen a Drosophila adult head cDNA library and it identified a single class of cDNA clone, representing a transcript of 1.1 kb. This 1112 bp cDNA was sequenced on both strands to obtain the gene sequence. Genomic sequence of a P1 clone (DS02336) indicates eyc is intronless (Sang, 2002).

That eyc1 and eycP are recessive appears at odds with a hypothesis that suggests transcript elevation causes the eye phenotype: should they not be dominant? Regulation of transcription by interallelic interactions, known in Drosophila and elsewhere, could account for the current observations. The possibility is considered that a 3' suppressor sensitive to epigenetic influences contributes to eyc regulation. Analogous to the trans activity of the yellow enhancer, pairing a wild-type homolog with an eyc mutant may allow recruitment of a suppressor that downregulates transcription of both copies, over-riding decreased binding of a repressive factor by 3' changes in eyc1 and eycP. Further genetic and molecular analysis of eyc will be required to explore this possibility (Sang, 2002).

Since p97 is sensitive to alkylation by N-ethyl-maleimide (NEM), and since in vitro reassembly of Xenopus nuclear membrane is NEM sensitive (Macaulay, 1996; Marshall, 1997), it was speculated that p47/Eyc might be essential for nuclear envelope fusion. Indeed, interference with Eyc function in syncytial blastoderm embryos disrupts nuclear morphology and cell cycle progression. Embryos of eycl39/CyO inter se crosses were stained with an antibody to Lamin, a nuclear envelope protein that has been shown to play an essential role in nuclear envelope assembly. During the first half hour after egg deposition, no differences were observed in the nuclear envelopes of embryos from wild-type or eycl39/CyO crosses, suggesting early nuclear division in eyc nulls proceeds normally using maternally supplied Eyc. At 1.5 to 2 hours, wild-type embryos stained with anti-Lamin show two staining patterns: Lamin is either concentrated around the separated M-phase chromosomes, consistent with an association between Lamin and chromatin, or the interphase nuclear envelope is highlighted. In eycl39/CyO-derived progeny, approximately one quarter of the embryos show no nuclear envelopes at the same stage; occasional embryos showed Lamin aggregates, which may be similar to the annulate lamellae observed in Drosophila lamin Dm0 (Sang, 2002).

Concomitant with loss of the nuclear envelope, the normally dense nuclear DNA seen in zygotic nuclei becomes irregularly fragmented and dispersed. It seems likely that absence of the nuclear envelope results in abnormal chromosome segregation. Probably as a result of destructive mitosis and chromosome dispersal, eyc null nuclei do not migrate to the surface of the embryo and organize the normal hexagonal actomyosin staining pattern at the embryo membrane; cellularization fails in mutant embryos (Sang, 2002).

To further examine Eyc loss-of-function phenotypes, anti-Eyc antiserum was microinjected into the posterior of stage 2-3 embryos and its effects on nuclear division were observed using confocal microscopy 1 hour after injection (~ stage 4-5). A gradient of cortical nuclear organization was found across injected embryos: nuclei were dense and regularly arrayed at anterior ends; nuclei were sparse and poorly organized at the posterior. Occasional clustered nuclei could be found in the affected region (Sang, 2002).

Side views reveal disruption of cellularization in the posterior of anti-Eyc injected embryos. In normal stage 4-5 embryos syncytial nuclei are closely packed and cylindrical. By contrast, the posterior region of anti-Eyc injected embryos shows fewer nuclei; those that are there appear to have a spherical shape, presumably a more relaxed shape in the less crowded environment. It is speculated that the nuclear divisions are slowed in the Eyc-inhibited cytoplasm, perhaps by delays of postmitotic nuclear envelope reassembly (Sang, 2002).

These results indicate Eyc is fundamental to cell cycle progression during embryogenesis. To determine whether Eyc is generally used at different developmental stages and tissues, the EGUF/hid method was applied to generate homozygous eyc null eyes. Eyes are absent in flies in which eycl39 is homozygosed early in the development of the eye primordium, supporting a cell-essential role for Eyc (Sang, 2002).

A cell-essential role for p47/p97 is also suggested by failure to obtain ter94 (a Drosophila p97 homolog) null eye clones, as well as the failure to obtain ter94 loss-of-function germline clones (Sang, 2002).


At least two distinct ATPases, NSF and p97, are known to be involved in the heterotypic fusion of transport vesicles with their target membranes and the homotypic fusion of membrane compartments. The NSF-mediated fusion pathway is the best characterized, many of the components having been identified and their functions analysed. In contrast, none of the accessory proteins for the p97-mediated fusion pathway has been identified. The first such component, a protein of relative molecular mass 47,000 (p47), has now been identified. p47 forms a tight, stoichiometric complex with cytosolic p97 (one trimer of p47 per hexamer of p97). It is essential for the p97-mediated regrowth of Golgi cisternae from mitotic Golgi fragments, a process restricted to animal cells. Since a homolog of p47 exists in budding yeast, this indicates that it might also be involved in other membrane fusion reactions catalysed by p97, such as karyogamy (Kondo, 1997).

The highly conserved ATPase p97, a member of the AAA-ATPases, is found in a complex with its co-factor p47 in rat liver cytosol. Previously it had been shown that p97-mediated reassembly of Golgi cisternae from mitotic Golgi fragments requires p47, which mediates the binding of p97 to a Golgi t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment factor receptor), syntaxin 5. p47 also suppresses the ATPase activity of p97 by up to 85% in a dose-dependent and saturable manner suggesting that it has other roles in the membrane fusion cycle (Meyer, 1998).

AAA ATPases play central roles in cellular activities. The ATPase p97, a prototype of this superfamily, participates in organelle membrane fusion. Cryoelectron microscopy and single-particle analysis reveals that a major conformational change of p97 during the ATPase cycle occurs upon nucleotide binding and not during hydrolysis, as previously hypothesized. Furthermore, six p47 adaptor molecules bind to the periphery of the ring-shaped p97 hexamer. Taken together, these results provide a revised model of how this and possibly other AAA ATPases can translate nucleotide binding into conformational changes of associated binding partners (Rouiller, 2000).

The AAA-ATPase, p97/Cdc48p, has been implicated in many different pathways ranging from membrane fusion to ubiquitin-dependent protein degradation. Binding of the p47 complex directs p97 to act in the post-mitotic fusion of Golgi membranes. Another binding complex comprising mammalian Ufd1 and Npl4 is reported in this study. Yeast Ufd1p is required for ubiquitin-dependent protein degradation whereas yeast Npl4p has been implicated in nuclear transport. In rat liver cytosol, Ufd1 and Npl4 form a binary complex, which exists either alone or bound to p97. Ufd1/Npl4 competes with p47 for binding to p97 and so inhibits Golgi membrane fusion. This suggests that Ufd1/Npl4 is involved in another cellular function catalysed by p97, the most likely being ubiquitin-dependent events during mitosis. The fact that the binding of p47 and Ufd1/Npl4 is mutually exclusive suggests that these protein complexes act as adapters, directing a basic p97 activity into different cellular pathways (Meyer, 2000).

p47 is the major protein identified in complex with the cytosolic AAA ATPase p97. It functions as an essential cofactor of p97-regulated membrane fusion, which has been suggested to disassemble t-t-SNARE complexes and prepare them for further rounds of membrane fusion. The high-resolution NMR structure of the C-terminal domain from p47 is reported in this study. It comprises a UBX domain and a 13 residue long structured N-terminal extension. The UBX domain adopts a characteristic ubiquitin fold with a ßßalphaßßalphaß secondary structure arrangement. Three hydrophobic residues from the N-terminal extension pack closely against a cleft in the UBX domain. The p97 interaction surface has been identified using NMR chemical shiftperturbation studies (Yuan, 2001).

Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized. NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion. p97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly. Formation of a closed NE requires the p97-Ufd1-Npl4 complex, not previously implicated in membrane fusion. Subsequent NE growth involves a p97-p47 complex. This study provides the first insights into the molecular mechanisms and specificity of fusion events involved in NE formation (Hetzer, 2001).


Search PubMed for articles about Drosophila prominin/eyes closed

Acharya, U., Jacobs, R., Peters, J. M., Watson, N., Farquhar, M. G. and Malhotra, V. (1995). The formation of Golgi stacks from vesiculated Golgi membranes requires two distinct fusion events. Cell 82: 895-904. 7553850

Edwardson, J. M. (1998).Membrane fusion: All done with SNAREpins? Curr. Biol. 8: R390-R393. 9635186

Hetzer, M., et al. (2001). Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly. Nat. Cell Biol. 3(12): 1086-91. 11781570

Kondo, H., Rabouille, C., Newman, R., Levine, T. P., Pappin, D., Freemont, P. and Warren, G. (1997). p47 is a cofactor for p97-mediated membrane fusion. Nature 388: 75-78. 9214505

Latterich, M., Frohlich, K. U. and Schekman, R. (1995). Membrane fusion and the cell cycle: Cdc48p participates in the fusion of ER membranes. Cell 82: 885-893. 7553849

Leon, A. and McKearin, D. (1999). Identification of TER94, an AAA ATPase protein, as a Bam-dependent component of the Drosophila fusome. Mol. Biol. Cell 10(11): 3825-34. 10564274

Longley, R. L. (1994). Drosophila retinal morphogenesis during pupation and the role of integrins in retinal development. Ph.D. Thesis, Purdue University, Indiana 222pp

Macaulay, C. and Forbes, D. J. (1996). Assembly of the nuclear pore: Biochemically distinct steps revealed with NEM, GTP gammaS, and BAPTA. J. Cell Biol. 132: 5-20. 8567730

Marshall, I. C. B. and Wilson, K. L. (1997). Nuclear envelope assembly after mitosis. Trends Cell Biol. 7: 69-74

Mayer, A., Wickner, W. and Haas, A. (1996). Sec18p (NSF) driven release of sec17p (alpha-SNAP) can precede docking and fusion of yeast vacuoles. Cell 85: 83-94. 8620540

Mellman, I. (1995). Enigma variations - protein mediators of membrane fusion. Cell 82: 869-872. 7553845

Meyer, H. H., Kondo, H. and Warren, G. (1998). The p47 co-factor regulates the ATPase activity of the membrane fusion protein, p97. FEBS Lett. 437: 255-257. 9824302

Meyer, H. H., Shorter, J. G., Seemann, J., Pappin, D. and Warren, G. (2000). A complex of mammalian Ufd1 and Npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways. EMBO J. 19: 2181-2192. 10811609

Patel, S. and Latterich, M. (1998). The AAA team: related ATPases with diverse functions. Trends Cell Biol. 8: 65-71. 9695811

Pinter, M., et al. (1998). TER94, a Drosophila homolog of the membrane fusion protein CDC48/p97, is accumulated in nonproliferating cells: in the reproductive organs and in the brain of the imago. Insect Biochem. Mol. Biol. 28(2): 91-8. 9639875

Rabouille, C., Levine, T. P., Peters, J. M. and Warren, G. (1995). An NSF-like ATPase, p97, and NSF mediate cisternal regrowth from mitotic Golgi fragments. Cell 82: 905-914. 7553851

Rabouille, C., Kondo, H., Newman, R., Hui, N., Freemont, P. and Warren, G. (1998). Syntaxin 5 is a common component of the NSF- and p97-mediated reassembly pathways of Golgi cisternae from mitotic Golgi fragments in vitro. Cell 92: 603-610. 9506515

Rouiller, I., et al. (2000). A major conformational change in p97 AAA ATPase upon ATP binding. Mol. Cell 6(6): 1485-90. 11163220

Ruden, D. M., et al. (2000). Membrane fusion proteins are required for oskar mRNA localization in the Drosophila egg chamber. Dev. Biol. 218: 314-325.

Sang, T. K. and Ready, D. F. (2002). Eyes closed, a Drosophila p47 homolog, is essential for photoreceptor morphogenesis. Development. 129(1): 143-54. 11782408

Yablonovitch, A. L., Fu, J., Li, K., Mahato, S., Kang, L., Rashkovetsky, E., Korol, A. B., Tang, H., Michalak, P., Zelhof, A. C., Nevo, E. and Li, J. B. (2017). Regulation of gene expression and RNA editing in Drosophila adapting to divergent microclimates. Nat Commun 8(1): 1570. PubMed ID: 29146998

Yuan, X., et al. (2001). Solution structure and interaction surface of the C-terminal domain from p47: a major p97-cofactor involved in SNARE disassembly. J. Mol. Biol. 311(2): 255-63. 11478859

Zhang, L., Ashendel, C. L., Becker, G. W. and Morre, D. J. (1994). Isolation and characterization of the principal ATPase associated with transitional endoplasmic reticulum of rat liver. J. Cell Biol. 127: 1871-1883. 7806566

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