From the
Mutations in the norpA gene of Drosophila
melanogaster severely affect the light-evoked photoreceptor
potential with strong mutations rendering the fly blind. The norpA gene has been proposed to encode phosphatidylinositol-specific
phospholipase C (PLC), which enzymes play a pivotal role in one of the
largest classes of signaling pathways known. A chimeric norpA minigene was constructed by placing the norpA cDNA behind
an R1-6 photoreceptor cell-specific rhodopsin promoter. This
minigene was transferred into norpA
Phosphatidylinositol-specific phospholipase C (PLC)
PLC comprises a large family of
enzymes. More than a dozen PLC-encoding genes have been identified and
the cognate cDNAs cloned (see Meldrum et al., 1991 for
references). The respective PLC enzymes have been classified into four
major groups (designated PLC-
A
PLC enzyme for which a function in vivo has been proposed is
encoded by the norpA gene of Drosophila melanogaster.
Strong mutations in the norpA gene of Drosophila have
long been known to abolish the light-evoked photoreceptor potential,
rendering the fly blind (Hotta and Benzer, 1970; Pak et al.,
1970). norpA mutants have been shown to be deficient in PLC
activity in head (Yohsioka et al., 1985), and molecular
cloning of the norpA gene has shown that it encodes a protein
that is similar in structure and amino acid sequence to vertebrate PLC
(Bloomquist et al., 1988). These data, as well as a growing
body of evidence suggesting that PLC is involved in invertebrate
phototransduction (reviewed by Payne, 1986; Pak and Shortridge, 1991),
have converged to suggest that the norpA gene encodes PLC that
is utilized in phototransduction in Drosophila. This is in
contrast to vertebrate phototransduction which is proposed to occur via
activation of cGMP phosphodiesterase (reviewed by Kaupp and Koch, 1986;
Stryer, 1986).
To determine whether the norpA gene indeed
encodes PLC activity that is required for phototransduction in
Drosophila, we constructed a chimeric norpA minigene
by fusing norpA cDNA to the ninaE gene promoter.
Since ninaE gene encodes R1-6 photoreceptor
cell-specific rhodpsin (O'Tousa et al., 1985; Zuker
et al., 1985), its promoter will drive expression of norpA RNA in R1-6 photoreceptor cells. The norpA minigene
chimera was transformed into norpA
Flies were grown at 21 °C in a 12 h light/12 h
dark cycle on Carolina Instant Medium (Carolina Biological)
supplemented with dry yeast or on cornmeal/sucrose/agar medium
(Roberts, 1986) supplemented with
Immunogold decoration of sites in photoreceptor cells that are
stained by the norpA antiserum was carried out using minor
modifications of the methods of Long et al.(1994). Briefly,
flies were decapitated manually and the heads fixed overnight at 4
°C in 2% paraformaldehyde, 0.2% picric acid, and 0.1 M
sodium cacodylate, pH 7.2. After rinsing in 0.1 M sodium
cacodylate, heads were incubated in a 0.5 M ammonium chloride,
0.1 M sodium cacodylate, at room temperature for 1 h to block
any remaining free aldehyde groups. The heads were washed twice in 50%
ethanol and twice in 70% ethanol at room temperature for 5 min each to
dehydrate the sample. Heads were then infiltrated and embedded with LR
white resin (London Resin Company, Ltd., Hampshire, United Kingdom)
according to manufacturer's directions. One-hundred-nm sections
were prepared and placed on Formvar-coated nickel grids for
immunolocalization. Grids were incubated in a blocking solution of 0.5%
BSA, 0.05% Tween 20, 1
The
chimeric norpA minigene was subcloned into the pCaSpeR4 transformation vector (Thummel and Pirrotta, 1991) and the
resulting DNA injected into a total of 533
norpA
To determine the position where the norpA minigene had
inserted into the genome of the T-15 and T-35 transformant lines,
labeled norpA cDNA was hybridized in situ to squashes
of salivary chromosomes prepared from the transformants. The insertion
site was at 46A in the T-15 transformant line and at 44A in the T-35
transformant, both on the right arm of the second chromosome (data not
shown).
As
shown in Fig. 3, norpA
In agreement with prior work (Zhu et
al., 1993), norpA antiserum stains retina of white strain
controls, but there is a definite lack of staining of retina of
norpA
norpA mutants have long been known to exhibit
defects in vision and, more recently, it has been proposed that the
norpA gene encodes a phospholipase C enzyme that is essential
for phototransduction (Bloomquist et al., 1988; Schneuwly
et al., 1991; Pak and Shortridge, 1991). The question
addressed here is whether expression of norpA protein in mutants is
sufficient to rescue the low amount of PLC activity in head of
norpA mutant concomitant with rescuing the phototransduction
defect. Indeed, our results show that expression of a norpA minigene in norpA mutant results in restoration of norpA
protein in retina as well as a concomitant partial restoration of PLC
activity and light-evoked responses of photoreceptor cells. This is the
best evidence thus far supporting the hypothesis that the norpA gene encodes a PLC that is utilized in phototransduction.
There
are several possible explanations why expression of the norpA minigene in mutant fails to fully rescue 100% of the amount of PLC
activity observed in wild-type retina. First, the ninaE promoter, which was used to drive the expression of
norpA, targets expression only in a subset of photoreceptor
cells while wild-type expression of norpA occurs in all of the
photoreceptor cells of the compound eye and ocelli (eg. Schneuwly et al., 1991). Second, the minigene was
constructed by combining parts of two heterologous genes (ninaE and norpA), and this may have a drastic affect on amounts
of viable RNA produced or amount of translated product that appears in
photoreceptor cells. Third, position effects of the inserted DNA on the
chromosome or presumptive changes occurring in the transposon during
insertion into the genome may adversely affect efficiency of expression
of the gene (Spradling, 1986). Considering that any or all of these
could lead to a reduction in the amount of norpA protein that is
expressed in transformed mutants when compared to that in wild-type
flies, the observed rescue is quite convincing.
More importantly,
the quantity of PLC activity exhibited by transformants appears to
correlate well with the extent to which photoreceptor cells respond to
light as well as the amount of norpA protein that appears to be
present. The T-35 transformant exhibits less PLC activity than found in
normal heads (27%) as well as less than T-15 transformant head
(Fig. 3). This correlates well with results of the ERG analyses
which show the response amplitude for T-35 transformant is less than
that of white mutant or T-15 transformant under conditions of
identical intensity stimulation (Fig. 6). Moreover, the amount of
norpA protein in head of T-35 transformant, as judged by the darkness
of staining of norpA protein in Western analyses, appears to be less
than the amount in white heads or the T-15 transformant head
(Fig. 2). Thus, there appears to be a positive correlation
between reduction in PLC activity, reduction in the amount of norpA
protein, and reduction in ERG amplitude of T-35 transformants, which
results argue for a direct relationship between norpA protein, PLC
activity, and photoreceptor cell responses.
The significance of the
present work is underscored by the fact that the norpA gene
product is a PLC, a pivotal enzyme in one of the largest classes of
signaling pathways known. Very little is known about the identity of
signaling pathways that utilize any of the subtypes of PLC. The
norpA-encoded PLC is one of the few for which a specific
receptor and signaling pathway has been identified. Moreover, rescue of
the phototransduction defect by expression of a norpA minigene
proves that the gene identified by Bloomquist et al.(1988) is
indeed the norpA gene as well as provides a means to carry out
structure-to-function analyses on the norpA-encoded PLC.
Alterations can now be made in norpA DNA prior to introducing
it into the germline and the results can be examined in vivo.
Furthermore, the significance of the conclusion that the
norpA-encoded PLC is essential for phototransduction in
Drosophila is strengthened by the recent identification of a
bovine homologue of norpA that is found in the retina
(Ferreira et al., 1993; Lee et al., 1993). This
bovine retinal PLC has been shown to localize in the outer segments of
cone cells, but not rods (Ferreira and Pak, 1994). Inasmuch as the
outer segments of cones are specialized structures for
phototransduction, there might be an inositol phosphate-based
phototransduction pathway in cones that is mediated by a
norpA-like PLC (Ferreira and Pak, 1994). Thus, further studies
designed to elucidate the function of the norpA-encoded PLC in
the Drosophila visual system may yield important clues needed
to develop models of an analogous phototransduction process in mammals.
We thank Dr. Linda Hall for providing helpful advice,
Dr. Guoping Feng for technical help in microinjection of Drosophila embryos, and Alan Siegal for technical help in
immunohistochemistry.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
mutant by
P-element-mediated germline transformation to determine whether it
could rescue the phototransduction defect concomitant with restoring
PLC activity. Western blots of head homogenates stained with norpA
antiserum show that norpA protein is restored in heads of transformed
mutants. Moreover, transformants exhibit a large amount of measurable
PLC activity in heads, whereas heads of norpA
mutant
exhibit very little to none. Immunohistochemical staining of tissue
sections using norpA antiserum confirm that expression of norpA protein
in transformants localizes in the retina, more specifically in
rhabdomeres of R1-6 photoreceptor cells, but not R7 or R8
photoreceptor cells. Furthermore, electrophysiological analyses reveal
that transformants exhibit a restoration of light-evoked photoreceptor
responses in R1-6 photoreceptor cells, but not in R7 or R8
photoreceptor cells. This is the strongest evidence thus far supporting
the hypothesis that the norpA gene encodes phospholipase C
that is utilized in phototransduction.
hydrolyzes phosphatidylinositol 4,5-bisphosphate to yield two
preeminent second messenger molecules, inositol trisphosphate
(IP
) and diacylglycerol, in one of the cornerstone
signaling pathways of cellular communication (Rhee et al.,
1989; Meldrum et al., 1991; Rhee and Choi, 1992a, 1992b).
IP
elicits the release of calcium from intracellular
stores, and diacylglycerol has been shown to activate protein kinase C.
These, in turn, affect other cellular processes which, eventually,
result in a cellular response.
, PLC-
, PLC-
, and
PLC-
) based on molecular weight, immunological, and structural
differences (Rhee et al., 1989). The PLC-
, -
, and
-
enzymes have been further divided into subgroupings (PLC-
1
through PLC-
4, PLC-
1 through PLC-
2, and PLC-
1
through PLC-
3) based on differences in amino acid sequence and
that each subtype is encoded by a separate gene (Meldrum et
al., 1991; Rhee and Choi, 1992a, 1992b). Observations that the
different subtypes of PLC differ in tissue distribution have led to the
idea that they are coupled to different receptors and are utilized in
different cellular processes (Rhee et al., 1989; Fain, 1990;
Meldrum et al., 1991). However, very little is known about the
identity of signaling pathways that utilize the enzymes or how they
function in vivo. Even for the PLC enzymes that have been
purified or extensively characterized biochemically (Banno et
al., 1986; Hakata et al., 1982; Hofmann and Majerus,
1982; Homma et al., 1988; Manne and Kung, 1987; Nakanishi
et al., 1988; Meldrum et al., 1989; Rebecchi and
Rosen, 1987; Takenawa and Nagai, 1981; Wang et al., 1986), the
functions of the enzymes in vivo remain poorly described.
mutant by P-element-mediated germline transformation (Spradling
and Rubin, 1982; Rubin and Spradling, 1982; Spradling, 1986).
Transformed flies were examined to see whether the expression of the
norpA protein in R1-6 photoreceptor cells is sufficient to rescue
the phototransduction defect and the accompanying lack of PLC activity
exhibited by norpA mutants.
Drosophila Strains
The D. melanogaster white (w ) mutant was used in all
experiments as a control group because its genetic background is most
similar to norpA
mutant.
norpA
mutants were chosen for
transformation experiments because they are strong mutants which
completely lack detectable amounts of norpA protein (Zhu et
al., 1993) and express severely reduced amounts of norpA mRNA as well (Bloomquist et al., 1988; Zhu et
al., 1993).
-carotene at 0.125 mg/ml.
Separate experiments verified that Carolina Instant Medium has
sufficient vitamin A to mediate normal vision (Lee, 1994). One- to
4-day-old (after eclosion) adults were used for Western blot analysis,
PLC activity assays, and immunohistochemistry. Four-day-old adults were
used for most electrophysiological analyses although adults of various
ages were also tested.
Construction and Transformation of the norpA Minigene
Chimera
Standard molecular biological techniques (Sambrook
et al., 1989) were used to excise a 1.7-kilobase
SpeI/BglI restriction fragment that contains the
ninaE open reading frame from ninaE genomic DNA
(O'Tousa et al., 1985). The excised ninaE DNA
fragment was replaced with a 3.8-kilobase BglI/SalI
restriction fragment that contains the norpA open reading
frame derived from the norpA cDNA (nucleotides 519-4331 in
Bloomquist et al., 1988) to create a chimeric
ninaE/norpA minigene (Fig. 1). This norpA minigene chimera was subcloned into the pCaSpeR4 transformation vector (Thummel and Pirotta, 1991) for
transformation into the germline of
norpA /white mutant using the
procedure decribed by Spradling(1986). Surviving adults were crossed
back to norpA
mutants. Transformed
flies were identified in the progeny of this cross by their red eye
color caused by expression of the mini-white gene which is
contained within the pCaSpeR4 transformation vector.
Figure 1:
Schematic
illustration of construction of a chimeric norpA minigene. A
ninaE/norpA chimeric minigene was constructed by
removing a fragment containing the ninaE open reading frame
from ninaE genomic DNA and replacing it with a cDNA fragment
that contains the norpA open reading frame (represented by the
cross-hatched box at the top). The arrow at the top
represents the transcription start site of the ninaE gene.
Positions of translation start and stop codons in the norpA open reading frame are also labeled. The chimeric norpA minigene was cloned into the KpnI site in the polylinker
region (black box) of the pCaSpeR4 P-element
transformation vector (represented by the circle) (Thummel and
Pirotta, 1991). Stipled boxes labeled 5`P and 3`P in the pCaSpeR4 vector represent the 5` and 3` P-element
inverted repeats. DNA sequences in the pCaSpeR4 transformation
vector that are bacterial plasmid derived (pUC) or comprise the
Drosophila mini-white gene (large semicircular
arrow) are indicated.
For
identification of the insertion site of the norpA minigene
chimera in the genome, chromosome squashes were prepared from salivary
glands of transformed flies as described previously (Shortridge et
al., 1991), except that the cDNA probe was prepared by labeling
with digoxygenin-dUTP (Boehringer Mannheim) according to
manufacturer's instructions. Visualization of the
digoxygenin-labeled DNA after hybridization was by immunostaining in
sheep anti-digoxygenin antibody (Boehringer Mannheim) followed by
incubation with 0.4 mM 4-nitro blue tetrazolium chloride, and
0.4 mM 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP)
according to the manufacturer's instructions.
Western Blot Analysis
Tissues from 0- to 4-day-old
flies were homogenized in 50 mM Tris-HCl, pH 7.4, 250
mM KCl, 0.05% sodium deoxycholate, and 0.1 mM
phenylmethylsulfonyl fluoride in 1.5-ml microfuge tubes using Teflon
pestles. Homogenates were centrifuged briefly at 12,000 g to remove particulate matter. These crude homogenates were
fractionated by 7% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (PAGE), electroblotted onto a nitrocellulose membrane,
stained using antiserum generated against the major gene product of
norpA using conditions described by Zhu et al.(1993).
Phospholipase C Activity Assays
PLC activity
assays were carried out by incubating a 0.1 ml volume of 50 mM
Tris-Cl, pH 7.5, 10M CaCl
, 0.1
mg/ml BSA, 0.2 mM phosphatidylinositol (Sigma), 44,000
disintegrations/min phosphatidyl-[
H]inositol
4,5-bisphosphate (New England Nuclear), and Drosophila tissue
extract for 5 min at room temperature essentially as described Zhu
et al.(1993). Crude tissue extracts were prepared by grinding
tissue in a buffer of 50 mM Tris-Cl, pH 7.5, 250 mM
KCl, 0.05% sodium deoxycholate, 0.1 mM DTT, and 0.1
mM phenylmethylsulfonyl fluoride, using a Teflon pestle in a
1.5-ml microfuge tube on ice. These homogenates were then centrifuged
briefly at 12,000
g to remove particulate matter.
Protein concentration in homogenates were determined using the BCA
protein assay (Pierce) with BSA as a standard and an appropriate amount
of extract (amount empirically determined to yield linear results with
respect to time) added to the reaction mixture. Reactions were stopped
by precipitating in 5% trichloroacetic acid and quantifying emissions
in the supernatant by liquid scintillation.
Immunocytochemistry
Drosophila heads were
embedded in O.C.T. compound (Tissue-Tek) and frozen on dry ice. Ten
µm thick tissue sections were cut on a Reichert cryostat and
transferred onto gelatin-subbed slides (Gall and Pardue, 1971). The
sections were fixed for 30 min in a freshly made solution of 150
mM sodium phosphate, pH 7.0, 75 µM lysine, 10
µM sodium metaperiodate, and 2% paraformaldehyde (McLean
and Nakane, 1974). Following tissue fixation, sections were washed two
times in PBS for 5 min each. The sections were blocked, incubated with
antibodies, washed, and developed in the same way as Western blots
mentioned above except primary antibody incubations were carried out
overnight at 4 °C.
Electron Micrograph Immunohistochemistry
The
primary antibody used in the immunohistochemical preparations was
affinity purified from norpA antiserum by cross-linking a bacterially
expressed norpA fusion protein (Zhu et al., 1993) to cyanogen
bromide-activated Sepharose beads (Pharmacia) according to
manufacturer's instructions followed by passing the crude
antiserum across the cross-linked beads packed loosely in a column. The
column with bound antibodies was washed in 1 PBS and the
antibodies eluted in glycine-Cl buffer, pH 2.3 (Harlow and Lane, 1988).
PBS, supplemented with 2.5% normal goat
sera for 15 min prior to incubation with affinity purified norpA
antibody. Primary antibody staining was done by incubating the grid
preparations for 30 min at room temperature in blocking solution
containing affinity purified primary antibody at a final concentration
of 20 µg/ml. Samples were washed four times for 30 min each in a
washing solution of 0.5% BSA, 0.1% Tween 20, 1
PBS. Primary
antibody was detected with a 1:20 dilution of 15 nm gold Auroprobe
(Amersham) according to manufacturer's instructions. After
washing the preparations in washing solution, as described above, the
sample was stained with uranyl acetate and lead citrate. Specimens were
observed with an electron microscope (H500:Hitachi Ltd, Tokoyo)
operating at 75 kV.
Electrophysiological Analyses
Electroretinograms
(ERGs) and prolonged depolarizing afterpotential (PDA) analyses were
carried out on adult flies essentially as described by Chen et al. (1992). The fly's compound eye was carefully located at the
focal plane of an optical stimulator using 625-nm light at an average
intensity of 16.43 log quanta/cm
s. A glass
micropipette was inserted into the retinal cell layer under 625-nm
light. After the fly was dark adapted for 40 min, the eye was
stimulated by 470-nm light and the ERG was recorded, amplified, and fed
into a MacLab/2e-Macintosh LC II computer system for storage, viewing,
and analysis. PDA was induced by two stimuli of 470 nm at about 16.04
log quanta/cm
s for 2 s each and followed by two of
570 nm stimulation at about 16.39 log quanta/cm
s
for 2 s each.
Expression of norpA Minigene in Photoreceptor Cells of
norpA
A chimeric norpA minigene
was constructed by replacing the open reading frame of the
Drosophila ninaE gene (O'Tousa et al., 1985;
Zuker et al., 1985) with that of the norpA gene
(Bloomquist et al., 1988) such that expression of the norpA
protein would come under control of the ninaE gene promoter
(Fig. 1). Since the ninaE gene encodes the major form of
rhodopsin in R1-6 photoreceptor cells, the expression of the
norpA protein, derived from the chimeric minigene, should occur in the
retina, more specifically in R1-6 photoreceptor cells.
Mutant
mutant embryos. Forty-five of the
injected embryos developed into fertile adults, and of these, two
different transformant lines were isolated (designated T-15 and T-35).
Presence of norpA Protein in Head of Transformed
Mutants
Antiserum generated against the major gene product of
norpA has been previously described (Zhu et al.,
1993). As shown in Fig. 2, norpA antiserum detects the 130-kDa
norpA protein in head homogenates of the white strain (which
is wild-type for norpA). This protein is missing in head
homogenates of norpA mutant, but is
present in head homogenates of norpA
mutants after they have been transformed with the norpA minigene chimera. This 130-kDa protein is not detectable in head
homogenates of white-eyed siblings of norpA
transformants (data not shown), which eye color indicates that
the particular flies do not express the mini-white gene, and
thus do not harbor norpA mini-gene inserts on their
chromosomes. These data demonstrate that the norpA mini-gene
chimera, when transformed into heads of norpA
mutant, is capable of directing the synthesis of norpA protein.
Figure 2:
Immunodetection of norpA protein on
Western blots. Lanes were loaded with protein from two heads. Blots
were stained with antiserum generated against the major gene product of
the norpA protein (Zhu et al., 1993) which detects the 130-kDa
norpA protein (arrow) that is absent from head of norpA mutant. Primary antibody binding was detected using an alkaline
phosphatase-conjugated secondary antibody with NBT/BCIP as chromogen
(Zhu et al., 1993). The norpA protein is present in head of
two progeny lines of norpA mutant (designated T-15 and T-35)
which have been transformed with the chimeric norpA minigene.
The intensity of staining of norpA protein in T-35 transformant head is
consistently less in Western blots than that in T-15 transformant or
white mutant. Numbers on the left indicate molecular
mass (in kDa) and relative positions of migration of protein size
standards.
Rescue of Phospholipase C Activity in Head of Transformed
Mutants
Drosophila heads contain a high amount of PLC
activity that is severely reduced by norpA mutations (Yoshioka
et al., 1985; Inoue et al., 1988; Zhu et
al., 1993; McKay et al., 1994). To determine whether PLC
activity is rescued in heads of norpA transformants, head homogenates were tested for their ability to
cleave PIP
in an in vitro activity assay.
mutants
exhibit less than 1% of PLC activity that is measured in white strain (control) heads. The amount of PLC activity in white heads has been determined to be comparable to that found in
wild-type heads.
(
)norpA
transformants exhibit
64% (T-15) and 27% (T-35) of the amount of PLC activity found in
white heads (Fig. 3), demonstrating that the severe
reduction of PLC activity exhibited by norpA mutants is
capable of being at least partially reversed by transforming the
norpA minigene chimera into the germline.
Figure 3:
Phospholipase C activity in head
homogenates. Head homogenates of white (w) mutant, norpA mutant, and T-15 and T-35 transformants were assayed for the
ability to cleave [H]PIP
in an in
vitro biochemical reaction. Results are expressed as specific
activity of the homogenate (pmol of [
H]PIP
cleaved/5 min/mg of protein). Data shown are averaged from four
or more determinations with error bars indicating standard deviation.
PLC activity is severely reduced in the head of the norpA mutant, in comparison to that found in head of white controls, a strain selected because it matches the genetic
background of norpA mutants used in this study. Transformation
of the chimeric norpA minigene into the genome of the
norpA mutant partially restores PLC activity in head.
Transformants T-15 and T-35 exhibit 64 and 27%, respectively, of the
amount PLC of activity found in white mutant.
Spatial Localization of norpA Protein in Head
To
localize the expression of norpA protein in tissues, norpA antiserum
was used to immunostain the norpA protein in tissue sections of heads.
Since the expression of norpA RNA in transformants is driven
by the ninaE gene promoter, which gene normally encodes
R1-6 photoreceptor cell-specific rhodopsin (O'Tousa et
al., 1985; Zuker et al., 1985), norpA protein would be
expected to localize in retina, more specifically in R1-6
photoreceptor cells.
mutant (Fig. 4). The retina
of norpA
mutants that have been
transformed with the norpA minigene stain darkly by norpA
antiserum, demonstrating that expression of the norpA minigene
chimera occurs in the retina, as expected.
Figure 4:
Immunocytochemical staining of norpA
protein in tissue sections of head. Frontal sections of heads were
stained using antiserum generated against the norpA protein. Primary
antibody binding was detected using an alkaline phosphatase-conjugated
secondary antibody with NBT/BCIP as chromogen (Zhu et al.,
1993). Staining of the norpA protein can be clearly seen in the retina
of white controls (A), but no staining is seen in
retina of the norpA mutant (B). Staining of retina of
norpA transformants, T-15 (C) and T-35 (D),
is clearly visible. Staining in transformants also appears over the
lamina, which is potentially due to the additional presence of the
norpA protein in axons of photoreceptor cells and likely caused by
expressing norpA as a heterologous minigene chimera in the
transformants.
To localize norpA
expression at the subcellular level in retina, immunogold labeling of
norpA protein in tissue sections of retina were visualized using
electron microscopy. As shown in Fig. 5, staining of retina in
transformants by norpA antiserum occurs over the rhabdomeres of
R1-6 photoreceptor cells, but not R7 or R8 photoreceptor cells.
This is what is expected, since the expression of norpA is
being driven by the promoter of the ninaE gene, which gene
encodes R1-6 photoreceptor cell-specific rhodopsin.
Figure 5:
Subcellular localization of norpA protein
in retina of transformed norpA mutant. Staining of the norpA
protein in retinal tissue was visualized by immunogold decoration (15
nm diameter) and electron microscopy (60,000 magnification).
Shown is a cross-section of the rhabdomeric region of a single retinal
ommatidium of the T-35 transformant. Immunogold decoration of the norpA
protein (shown by the presence of dense particles) appears over the
rhabdomeric membranes of the peripheral (R1-6) photoreceptor
cells, but is absent over the rhabdomeric region of the central
photoreceptors (R7 and R8).
Electrophysiological Analysis Shows Rescue of
Phototransduction
ERG and PDA analyses were performed to
determine whether transformants exhibit light-induced depolarizing
responses in the retina. white mutant exhibits a normal ERG
and PDA while norpA mutant exhibits no
ERG or PDA (Fig. 6). norpA
transformants indeed exhibit electrophysiological responses to
light as measured by ERGs (Fig. 6), demonstrating that expression
of the norpA minigene is sufficient to rescue the lack of
light-induced photoreceptor response exhibited by
norpA
mutant. ERG tracings of T-15
transformant resemble that of a wild-type response to light stimuli,
while those of T-35 transformant take longer to return to base line
after high intensity light stimulation than do wild-type or T-15
transformant (Fig. 6). There are no observable differences in
ERGs between male, and females (data not shown). Moreover, ERG
sensitivity does not appear to change with age (not shown).
Figure 6:
ERG and PDA analysis. Electroretinographic
waveforms (left column) and prolonged depolarizing after
potentials (right column) were recorded from white mutant, norpA mutant, T-15 transformant, and T-35
transformant. Shown are typical results from 4-day-old (after eclosion)
adult females, although no difference in ERG or PDA was observed
between males and females (data not shown). All ERG stimuli were 470 nm
(duration = 1 s) with relative intensities of -7.39,
-6.43, -5.16, and -3.50 for white and
-6.43, -5.16, -3.50, and -2.60 for T15 and T35.
PDA stimuli (duration = 2 s) were either 470 or 570 nm as
indicated along the bottom. white mutants show normal
wild-type ERG and normal PDA, while norpA mutants completely
lack electrophysiological responses to light. T-15 transformants
exhibit an almost normal ERG while T-35 exhibit lower amplitudes and
takes longer to return to base line. Both transformants (T-15 and T-35) exhibit no responses or very small responses
induced by the second (blue, 470 nm) or the third (yellow, 570 nm)
stimuli in the PDA waveforms.
PDA
tracings of both T-15 and T-35 transformants resemble that of control
flies except that there is no or very little response induced by a
second (blue light) or third (orange light) stimuli (Fig. 6). The
observed responses to the second and third stimuli in control PDA
tracings are known to be derived from depolarization of R7 and R8
photoreceptor cells (eg. Stark and Zitzmann, 1976), indicating
that the R7 and R8 photoreceptor cells are not functioning in
transformants. These data correlate exactly with the
immunohistochemical data, shown above, which demonstrate the presence
of norpA protein in rhabdomeres of R1-6 photoreceptor cells, but
its absence from R7 and R8 photoreceptor cells (Fig. 5). This
finding was also expected, since the ninaE gene should drive
expression of the norpA coding sequence only in R1-6
photoreceptor cells.
,
phosphatidylinositol 4,5-bisphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.