BIOLOGICAL OVERVIEW

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 (eyc^l20, eyc^l27 and eyc^l39) 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).

GENE STRUCTURE

cDNA clone length - 1112

Exons - 1

Bases in 3' UTR - 46

PROTEIN STRUCTURE

Amino Acids - 353

Structural Domains

Conceptual translation of the longest Eyc ORF predicts a 353 amino acid protein with 46% similarity and 31% identity extending over the entire protein to rat p47, a co-factor that regulates the activity of the AAA ATPase, p97 (Patel, 1998). Two additional p47 homologs, human p47 and yeast SHP1 have been reported. In addition to eyc, the Drosophila Genome Project findings predict a potential p47 homolog, CG11139, localized to 43C4-5; no mutant phenotype or characterization of this gene has been reported (Sang, 2002).

date revised: 20 February 2002

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