Phospholipid vesicle fusion monitored by fluorescence energy transfer

BIOCHEMICAL

Vol. 88, No. 1, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 135-140

May 14, 1979

PHOSPHOLIPID

VESICLE

FUSION MONITORED BY FLUORESCENCE ENERGY TRANSFER

George

March

and Leslie

M. Loew'

Department of Chemistry University of New York at Binghamton Binghamton, New York 13901

State

Received

A. Gibson

19, 1979

SUMMARY A method is presented which allows the observation of phospholipid vesicle fusion by the occurrence of Fijrster resonance energy transfer between the amphiphilic probes dansyldipalmitoylphosphatidylethanolamine and 3-[4-(~-N,N-didecylaminostyryl)-l-pyridinium]-propylsulfonate. This method is applied to the Ca++ mediated fusion of phosphatidyl serine vesicles. INTRODUCTION Fluorescent research

but

fusion

probes

have been only

(l-4).

procedure of resonance procedure

for

The methodology studying energy

should

study

of long-range

cells

(5).

MATERIALS

are among the most occasionally

applied

outlined

in this

phospholipid transfer

provide

the

important

vesicle

between basis

interactions

work

extension

between

the

of membrane

provides

a simple

by monitoring

probes.

for

in membrane

to studies

fusion

unlike

tools

The success of the methodology

membranes

of vesicles

the

extent

of this to the and

AND METHODS

Dansyl-dipalmitoyl.phosphatidyl ethanolamtne was prepared as previously descriked (6); p-(N,N-dtdecylamino)styrylpyndlnlum propylsulfonate (DI-lOASPPS) was the gift of Dr. H. Parkins of this department. Bovine brain phosphatidylserine was purchased from Sigma Biochemicals and egg phosphatidylBoth were used as obtained. The choline was obtained from Calbiochem. buffer system was 50 mM tris (Gold Label, Aldrich Chemical Co.) which was adjusted to pH 7 by addition of HCl in double distilled water. 'Author

to whom correspondence

should

be addressed.

2 Abbreviations

used in this work: DI-lo-ASPPS = 3-[4-(e-N,N-didecylaminostyryl) 1-pyridiniuml-propylsulfonate; R.E.T. = resonance energy transfer; DDPPE = dansyldipalmitoylphosphatidylethanolamine; PS = phosphatidyl serine. 0006-291X/79/090135-06$01.00/0 135

Copyright All rights

@ 1979 by Academic Press, Inc. in any form reserved.

of reproduction

Vol. 88, No. 1, 1979

BIOCHEMKAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vesicles were prepareo as follows. Solutions containing the lipid and dyes were evaporated in a cylindrical vial under a stream of nitrogen. Buffer was added to the resulting film and the sample was sonicated for 15 min at 0" under a stream of NZ. Sonication was done at 30% power using a Branson Model iy W-185 sonicator. All spectrofluorometric measurements were done using a Perkin-Elmer MPF-44B spectrofluorometer interfaced to a Bascom-Turner Model # 8110 recorder. This recorder has data storage and processing capabilities which allowed generation of the difference spectra displayed in this work. Kinetic runs using phosphatidylserine vesicles were done in a thermostatted cuvette holder at 39°C.

DISCUSSION AND RESULTS Briefly,

the experimental

populations probe

of vesicles,

according

interact

via

tightly

a Fiirster

of the

ate

acceptor

fusion

distance

Satisfaction demonstrated volume tion

with

so this

bound

a half

to allow the

specific

spectrum

alter

satisfies

the

vesicles

equilibration experimental

for

are

must

must

are slow

bind on the

into

first

as a function

criterion. was explicitly

strong

that

(final

concentradispersion

of time. yield

in fluorescence

binding and free

A small

quantum

derivitization

Since than intensity

of phosphatidyl-

to the membrane probe.

we used

appropri-

transfer

egg lecithin

increase

that

136

the

a critical

fluorescence

a steady

system

using

of DI-lo-ASPPS

was observed

of bound

and DI-lo-ASPPS

DI-lo-ASPPS

a 2 mg/ml

We felt

probe

at room temperature.

solution

its

equation gives

has a much smaller

not

probes

that

pair

criterion

ethanolic

of 10 hr.

would rapid

pair

binding

dye we observe

life

ethanolamine

probe

M) was injected

DI-lo-ASPPS

membrane

of F'drster

this

egg lecithin

of 5 x low6

aqueous

in

for

of concentrated

kinetics

donor

The probes

probes

uses DDPPE as the donor

Evaluation

of the

and the fluorescence

with

of probes

of 36 ii;

fluorescent

probe.

and the

of two

process.

parameters

(R,)

bound

and acceptor

(7),

disscciation

establishment

acceptor

donor

mechanism

with

probe.

spectral

the

R.E.T.

One such system as the

bound

to two criteria,

the

a membrane

a similarly

to the membrane

timescale

involves

one containing

and one containing

chosen

design

in this

This

sufficiently

was borne

work.

out

BIOCHEMICAL

Vol. 88, No. 1, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fiqure 1. Emission spectra XE = 325 nm [PS] = 0.01 mg/ml. A sharp increase in intensity is observed as we approach the excitation wavelength in the second order of the monochromator. This results in occasional distortion in spectra (Figures 2,3,4) in this region.

We have chosen described

phosphatidyl

have shown in the pared

that

presence

of Ca++.

Comparing R.E.T.

B, if

with R.E.T.

which

and compare

the in

sum of individual both

the -donor

1).

the emission spectra,

vesicle

on the well

investigators

(8,9)

periods,

rapidly

suspensions

spectrum

will

both

mixing

remain

1% DI-lo-ASPPS

spectrum

obtained

we see (Figure

nm) and acceptor

13.7

fuse

were

pre-

2% DDPPE, and one (6) of equal identical

volumes

of the probes

stable

of

to i spectrum

2) we can see no evidence

mixture

contain

long

A mixture

or diffusive (Figure

(515

mg/ml)

an emission

At 39“C this

If we make vesicles

for

(A) containing

Figure

no fusion

so obtained

behavior.

sonication

give

studies

Several

stable

one

(see

should

spectra

usually

buffer,

fusion

system.

Two PS (0.01

2% DI-lo-ASPPS

A + 2 spectrum

initial

(PS)

PS vesicles,

suspensions

our

serine

in 50 mM tris.HCl

containing these

to perform

for

occurs. of Fijrster

over

10 hr.

and 1% DDPPE by cofrom 3) clear

this

dispersion

evidence

(600 nm) fluorescence.

of

Vol. 88, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 2. Difference between an emission spectrum of a mixture of separately labeled donor and acceptor conta ining vesicles and l/2 of the sum of spectrum A and spectrum B in Figure 1.

Figure 3. Difference between an emission spectrum of vesicles containing 1% DDPPE and 1% DI-lo-ASPPS, and l/2 of the sum of spectrum A and spectrum B in Figure 1.

138

BIOCHEMICAL

Vol. 88, No. 1, 1979

After with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

10 min Incubation 2mM Ce Cl2

400nm

65Onm

Figure 4. The emission spectrum of a mixture of separately labeled donor and acceptor containing vesicles incubated with 2 mM Cat+ was obtained and Subtracted from this was l/2 of the sum of the spectra of the stored. individually labeled vesicles which had also been treated with Cat+. The above difference spectrum clearly shows fluorescence energy transfer.

When donor fuse

in the

strated

containing

presence

by the

effect

will

appearance

appearance It

is

Cat+

system

raised

(9)

potential

of this

cell-cell

aggregation

this

of the

regarding

contain

probes).

We plan resolve

is described

the

study

139

of the vesicle

probes. with

concomitant

DDPPE and DI-lo-ASPPS. in the

absence

to extend

our

some of the

of PS vesicle

paper.

of fusion study

recently

fusion.

of vesicle-vesicle

in the accompanying

to

is demon-

of the

different

occur

caused

lipids

part

does

the mechanism for

only

between

not

are

The magnitude

transfer

to help

method

surface

shown to proceed

energy

timescales

general

is

behavior

4).

3 since

which

fusion

resonance

diffusion

to faster questions

vesicles

vesicles

of the

(Figure

to Figure

induced

shown that

by passive

(i.e., this

between

of Fijrster

further

of R.E.T.

compared

occur

In summary,

containing

of 2 mM CaC12 mixing

is diminished

fusions

and acceptor

The and

of

Vol. 88, No. 1, 1979

BIOCHEMICAL

AND EIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENTS We are grateful to the

New York

Cancer

Institute

to Dr.

State

Health

(CA 23838)

Helen

Parkins

Research for

support

for

Council of this

preparing

DI-lo-ASPPS

(# 362)

and the National

and

research.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Keller, P.M., Person, S., and Snipes, W. (1977) J. Cell Sci. 28, 167. Blumenthal, R., Weinstein, J.N., Sharrow, S.O., and Henkart, P. (1977) Proc. Nat. Acad. Sci. USA 74, 5603. Collard, J.G., DeWaldt, A., and Inbar, M. (1978) FEBS Lett. 90, 24. Wreschmer, D.H., and Gregoriadis, 6. (1978) Biochem. Sot. (London) Trans. 6, 922. Gibson, G.A., and Loew, L.M. (1979) Biochem. Biophys. Res. Comm., following paper; Biophys. J. 25, 260a. Stryer, L., and Waggoner, A.S. (1971) Proc. Nat. Acad. Sci. (U.S.) 67, 579. FSrster, von T. (1949) Z. Naturforsch. 4a, 321; Forster, van T. (1959) Disc. Faraday Sot. 27, 7. Papahadjopoulos, D., Vail, W.J., Newton, C., Nir, S., Jacobson, K., Poste, G., and Lazo, R. (1977) Biochem. Biophys. Acta 465, 579 and references cited therein. Ginsberg, L. (1978) Nature 275, 758.

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