A genetic study of inositol trisphosphate involvement in phototransduction using Drosophila mutants

Vol.

132,

October

BIOCHEMICAL

No. 2, 1985 30,

AND

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Pages

1985

513-519

A GENETIC STUDY OF INOSITOL TRISPHOSPHATE INVOLVEMENT IN PHOTOTRANSDUCTION USING DROSOPHILA MUTANTS Hiroko

Inouel

, Tohru

1 Department School

of Physiology, of Medicine,

2 Department University Received

July

23,

Yoshioka'

and Yoehiki

Hotta*

Yokohama City University, Yokohama, 232 Japan

of Physics, of Tokyo,

Faculty of Science, Tokyo 113, Japan

1985

Phoephatidylinositol 4,5-biephoephate phosphodiesterase activity was visual found to be almost absent in the compound eyes of Drosophila mutant, norpA (no receptor potentials A). We compared the enzyme activities among independently isolated norpA alleles, each having a different degree of the vision defect. A close correlation was found between the size of receptor potentials (electroretinogrsm), phototactic behavior and the enzyme activity. The correlation exists not only among in a single, temperature-dependent allele under alleles, but also kept at 18'c different temperature; the enzyme activity of flies (phototactic) was about five times higher than that of the ones kept at 28'~ (blind). These results suggest that hydrolysis of phoephatidylinositol 4,5-bisphosphate is involved in phototransduction process in 0 1985 Academic Press, Inc. Drosophila eyes.

It

has

involved

as In

cl,*).

been

proposed

a second order mutants

bisphoephate

(PtdInsP2) mutant

potentials, rhabdomere

the rhabdomere a

reduced

fracture

both almost

of the

electronmicroscopy

Abbreviations: phosphatidylinositol; DG, diacylglycerol;

trisphoephate invertebrate

in the hypothesis,

any defects

(no

of the most

number

inositol

(InsP3) phototransduction

we examined in

is

if

Drosophila

phosphatidylinoeitol

4,5-

hydrolysis.

norpA

are

this

have

although (4)

messenger

to test

transduction

The

that

receptor

potentials

morphology

(3)

normal. extreme

and rhodopsin

has

allele

We also

receptor of

described was found

of norpA% particles

no

content

We have previously

intramembraneoue (3).

A)

revealed

demonstrated

that

that

to

by the

the

have

freezenorpA

InsP inoeitol trisphosphate; PtdIns, P&InsP *, phosphatidylinoeitol 4,5-bisphosphate; ERG, electroretinogram 0006-291X/85 513

AN

Copyright 0 1985 rights qf reproduction

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by Academic Press, Inc. in any form reserved.

Vol.

132,

No. 2, 1985

mutants

harbor

inositol

(PtdIns)

BIOCHEMICAL

deficits

We report defective close

(6)

in

in the correlations

behavioral including

in the

metabolism;

phosphodiesterase

defects

AND

extremely

this

paper

norpA

mutant

that

between the

PtdInsP2

the mutant

phosphatidyl-

of

and PtdIns

(5)

eyes.

phosphodiesterase

enzyme deficit

a temperature-sensitive

COMMUNICATIONS

the

(DC) kinase

low in

degrees

RESEARCH

to

eyes of Drosophila.

Materials Materials: of Canton-S

enzyme related

diacylglycerol

being

and

BIOPHYSICAL

activity

Furthermore, electrophysiological

among

the

mutant

is

we show and alleles

strain.

and Methods

As a normal strain, an inbred (CS) was used, from which all

Drosophila melanogaster strain the mutants used were derived. norpA- JMll , norpA= and norpA-. KO50

The alleles of norpA gene used were KO50 norpAis a temperature-sensitive allele and can produce receptor potentials when it is kept at lower temperature (less than 25'C), while it lacks the photoresponse at higher temperature (more than 28’C) (7). A mutant, sine oculis (without compound eyes) were also used to test -whether the enzyme is localized in the eyes. 32 P-labelled Preparation of 32 P-labelled PtdInsP : PtdInsP2 was -32 P labelled Drosophila -------El extracted from y the method described by Hauser and El berg (8) with a slight modification. Flies were fed for 24 hours $9 P labelled inorganic phosphate (0.1 mCi/O.5 ml of 1% sucrose). WhP labelled flies were homogenized by microhomogenizer and mixed with to extract phospholipids other than chloroform/methanol (l:l, v/v) The mixture was centrifuged chloroform/methanol/ PtdInsP . and v/v) was added to the precipitate. concentgated IX1 (200:100:0.75, 0.2 volume of 1 N HCl was added to the After the centrifugation, supernatant and the lower phase was washed with chloroform/methanol/l N Extracted phospholipids were separated by thin Hcl (3:48:47, v/v). layer chromatography on a silica gel plate (Art. 5631, Merck), developed chloroform/acetone/methanol/acetic acid/water (40:17:15:12:8, with were impregnated in 1% potassium oxalate solution v/v). The Yaates before use. P-labelled phospholipids were detected by autoradiography and the corresponding area of PtdInsP was scraped off and eluted with as described by chloroform/methanol/water/HCl (145:24$:115:1, v/v) Irvine et al. (9). This eluate was added with 0.25 volume of chloroform and the same volume of 1N HCl. The lower phase was taken and was neutralized with Tris.

Assay of PtdInsP2 phosphodiesterase: PtdInsP2 phosphodiesterase was assayedwith a react containing 50 mM Tris HCl (pH 7.01, 0.5 9n mixture f P PtdInsP2 (about 10,000 cpm) and head homogenate mM EDTA and 0.2 mM (0.17 mg protein) in a final volume of 0.1 ml. Concentration of the substrate was adjusted by adding non-radioactive PtdInsP2 extracted from bovine brain kindly supplied by Dr. Hayashi of Kobe University. Incubation was carried out for 5 min at room temperature (22.C) and the was terminated by adding 0.1 ml of 10% trichloroacetic acid reaction After centrifugation, followed by an addition of serum albumin. supernatant was washed with diethyl ether and neutralized with 1 N NaOH. They were then loaded on Dowex 1x8 column (formate form) and separated 514

Vol.

132,

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No. 2, 1985

AND

BIOPHYSICAL

as described by Downes and Michell (10). was taken and radioactivity was counted.

RESEARCH

An aliquot

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of each

fraction

Electroretinogram (ERG) measurement: ERG was obtained by placing one electrode on a compound eye and a reference electrode inside the body The cornea1 electrode was a fine cotton wick, kept moist with (11). 0.9% NaCl in a capillary tube. The reference electrode was a glass pipette with 30-50 um tip, filled with 0.9% NaCl. This electrode was inserted dorsally through soft thoracic-abdominal junction. Assay of phototactic behavior: For the assay of phototactic behavior, about one hundred flies were placed in two 6 cm-long, transparent plastic tubes connected mouth to mouth. Flies were tapped to one end, and the tube was placed 3 cm away and perpendicular to a 15 Wfluorescent lamp. Flies were left undisturbed under the condition until their spatial distribution became quasi-stable (about one minute). The phototactic index is calculated as number of flies in the lighter half Phototactic Index = log number of flies in the darker half The index is about 1.0 + 0.2 for normal strain under the condition The near-zero phototactic index indicates absence of described above. If flies phototaxis. are positively or negatively phototactic, the phototactic index becomes positive or negative, respectively.

Results PtdInsP2

phosphodiesterase

Fig.

1

products iS

of

anion

shows

[32P]PtdInsP2

extremely

reduced

intermediate

peak

also

in Fig

observed Although

the

homogenate, highly of

we

localized

--sine

oculis

could

demonstrate

within

the

(a mutant

the

with

eyes while

the marked alleles

A similar

is

the

evident

32 P-labelled

that

while

reduction

enzyme activities

compound

norpA

It

of

an InaP

norpA-

in InsP2

JMll

peak

showed

production

is

1.

isolated

Therefore,

chromatography

hydrolysis.

height.

in comparison

activity,

exchange

in the case of norpA=,

activity

enzyme

activity

indicate

that

compound

PtdInsP2

eyes.

without

eyes)

that

of normal

from

freeze-dried

isolated reductions that

were measured

the

brain

with

enzyme. 515

head

contains

only

fly.

10%

of

Secondly,

flies

is

the head homogenate

(6) not

eyes of norpA

enzyme manually

contained

(data

of the enzyme activity compound

whole

phosphodiesterase

Firstly,

did

the

high

not

shown).

in the

several

essentially

lack

Vol. 132, No. 2, 1985

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

a

b C

12

”

Elution

volume

(ml)

Fig. 1 Separation of radioactive product hydrolyzed from [32P] PtdInsP on Dowex 1x8 column (formate form). Elution was with (a) 0.1 M fox& acid/O.2 M ammonium formate (for inorganic phosphate and inoaitol (b) 0.1 M formic acid/O.4 M ammonium formate (for monophosphate); inositol bisphosphate); (c) 0.1 M formic acid/l.0 M ammonium formate (for inositol trisphosphate). 10 ml of each solution were run through Note at constant flow (0.3 ml/min) and the fraction volume was 1 ml. that the inositol trisphosphate peak is almost absent in es, but X411 small peak is present in norpA=. A; e-, 0: normal, EE5 l ;norpA--.

ERG, phototactic

two

index ---2

b, behavioral

In Fig

2a,

norpA

alleles

extreme

On

receptor

small

the

is

in Fig

in

parallel

other

2c.

and

It with

with

those

no receptor

hand,

activities

phosphodiesterase summarized

have

potentials

phosphodiesterase

and electrophysiological

are compared norpA%,

allele,

phototactic.

and PtdInsP

normal

of

potentials

a milder

partially

allele

fly.

the

mutants

Is clear

that

degree

behavioral

The

The

described of the

and

of the

and entirely JMll of norpA-

phototactic.

of

the

syndromes

most nonshows

PtdInsP2

above

are

enzyme deficiency

electrophysiological

abnormalities. PtdInsP, We allele,

phosphodiesterase

activity

-of temperature-sensitive

the enzyme activity of a assayed KO50 This mutant produces norpA-. 516

temperature-sensitive receptor

potentials

mutant

E norpA

and

is

Vol.

132,

No. 2, 1985

BIOCHEMICAL

(a)

AND

( I 1

ERG

BIOPHYSICAL

RESEARCH

( II 1

( 111 1

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L-----b

(b)

Fig 2 Electropbysiological, behavioral and biochemical abnormalities of norpA mutants; Index and (c) relative (a) ERG, (b) phototactic phosphodieaterase activity for PtdInaP2. I, II and III represent normal, norpA= and-=, respectively. Note close correlations between the electrophysiological, behavioral PtdIneP2 and phosphodiesterase defects among the alleles. The values of the enzyme expressed as nmol/mg protein/5 min for normal, activity g&l-land norpA= are 39.6 + 5.7 (n=lD), 3.2 + 0.6 (n=4) and 1.0 + 0.6 (n=6), respectively. The error bare in the figure represents S.D. For details of experimental conditions, 886 Materials and Methods.

phototactic when

when it

shifted

to

essentially

no

temperature wa8 also

norpA=

28-c.

shown

norpA-

KO50 (Table

at

On the

difference

was altered

The effect the

is raised

while

18-C,

other

hand,

in ERG nor from

18

it

becomes

28

‘C.

blind

strain

showed

the normal

in phototaxis

'C to

entirely

when

The blindness

the

breeding

of norpA=

to be temperature-independent. of temperature head homogenate 1).

The

on the PtdInsP2 was compared enzyme

activity 517

hydrolyzing with was

that

of

markedly

activities

of

normal

and

reduced

in

Vol.

132,

No.

2, 1985

BIOCHEMICAL

AND

Table Phosphatidylinoaitol

BIOPHYSICAL

RESEARCH

1

4,5-bisphosphate

phosphodiesterase

Breeding

Temperature

18-c normal

COMMUNICATIONS

28-C

37.8 + 6.6

(5)

41.3 + 4.7

(6)

nor-&=

4.1 + 1.1

(4)

0.9 + 0.3

(5)

m=

1.3 + 0.1

(3)

0.8 + 0.1

(3) The the

The assay conditions are described in the legend of Fig. 1. is expressed as nmol/mg protein/5 min. Values are activity means + S.D. Number of experiments is give in the parenthesis.

KO50

IlorpA-

when it

activity

was found

breeding

raised

at

norpA- KO50

the

of

change

is

in the

temperature

On the

28-c.

kept

at 18'~

enzyme activity

other

was

hand,

the

significantly

of normal

and

enzyme

high.

No

norpA=,

when

was altered.

Discussion In

the

activity

present

is

study,

localized

correlation

is

in the

present

abnormality

that

exists

single,

correlation

temperature.

was observed The the

metabolism of

compound

eyes of fly

among norpA

not

only

the

messenger possibility

primary in

in

defect

the eye.

mutant

will the

that

nor

of such a close

If

this

shed light signal the

norpA

is

the

gene

effect in the

function 518

alleles

case,

and showed

also

in a

different

enzyme activity

strain. a

near

possibility the

the more detailed

PtdInsP2 analysis

of InsP

However, is

but

suggests in or

close

activity

under

norpA%

is

a

We also

on the

on the hypothesis

transduction.

enzyme

norpA-, KO50

mutants

and

mutants.

correlation

of norpA

heads,

among mutant

allele,

in the normal

phosphodiesterase

of the

Such a temperature

neither

presence

PtdInsP2

the reduction

temperature-sensitive

breeding

that

that

between

electrophysiological this

we found

essential

as a second 3 we cannot exclude a for

the

general

Vol.

132,

No. 2, 1985

phospholipid since

BIOCHEMICAL

metabolism

the mutant

has abnormalities

PtdIns

phosphodiesterase

the

experiment

of InsP

or

by chemical

BIOPHYSICAL

in the membrane,

and

mutant

AND

3

injection analysis

especially

in other (5,6).

Direct into

RESEARCH

in PtdIns

enzymes,

turnover,

such as DG kinase

evidence

will

the photoreceptor

of intracellular

COMMUNICATIONS

InsP

be given

cells 3

in the

of

norpA

compound

eyes of Drosophila.

Acknowledgment form

This work was supported by Grants-in-Aid No. the Ministry of Education, Science and Culture

58124027 and 59123006 of Japan.

References 1.

Fein, A., Payne, R., Corson, D. W., Berridge, M. J. and Irvine, R. F. (1984) Nature 311, 157-160. A. J., Tarver, A. P., 2. Brown, J. E., Rubin, L. J., Ghalayini, Irvine, R. F., Berridge, M. J. and Anderson, R. E. (1984) Nature 311, 160-163. K. and Hotta, Y. (1982) The Structure of The Eye ted. 3. Hirosawa, Hollyfield, J. G.) pp. 45-53, Elsevier Biomedical, New York. Genetic Approach to the Nervous 4. Pak, W. L. (1979) Neurogenetics: System (ed. Breakefield, X. 0.) pp. 67-99, Elsevier, North-Holland. T., Inoue, H. and Hotta, Y. (1984) Biochem. Biophys. 5. Yoshioka, Res. Commun. 119, 389-395. T., Inoue, H. and Hotta, Y. J. Biochem. (Tokyo) (1985) 6. Yoshioka, 97, 1251-1254. Y. and Benzer, S. Proc. Natl. Acad. Sci. U. S. A. (1970) 7. Hotta, 67, 1156-1163. 8. Hauser, G. and Eichberg, J. Biochim. Biophys. Acta (1973) 326, 201-209. R. F., Letcher, A. J. and Dawson, R. M. C. (1984) Biochem. 9. Irvine, J. 218, 177-185. 10. Downes, C. P. and Michell, R. H. (1981) Biochem. J. 198, 133-140. 11. Hotta, Y. and Benzeer, S. (1969) Nature 222, 354-356.

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