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33561–33568, 2001
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Stephen A. Waschuk‡, Elyssa A. Elton‡, Audrey A. Darabie‡, Paul E. Fraser‡§, and JoAnne
McLaurin‡¶储
From the ‡Centre for Research in Neurodegenerative Diseases, Departments of §Medical Biophysics and ¶Laboratory
Medicine and Pathology, University of Toronto, Toronto, Ontario M5S 3H2, Canada
Alzheimer’s disease pathology has demonstrated ation with membrane structures. Recent studies have demon-
amyloid plaque formation associated with plasma strated that plaque formation may be initiated in a plasma
membranes and the presence of intracellular amyloid- membrane form (3, 4) and that A deposition in aged dogs is
(A) accumulation in specific vesicular compartments. associated with the extracellular leaflet of the plasma mem-
This suggests that lipid composition in different compa- brane (5). Furthermore, the intracellular accumulation of A in
rtments may play a role in A aggregation. To test this lysosomal or late endosomal vesicles in vitro suggest that these
hypothesis, we have isolated cellular membranes from compartments may be involved in neurotoxicity (6 –9).
human brain to evaluate A40/42-lipid interactions. Pla- A is generated from the proteolytic cleavage of the amyloid
sma, endosomal, lysosomal, and Golgi membranes were precursor protein in the endoplasmic reticulum to generate
isolated using sucrose gradients. Electron microscopy A42 and the trans-Golgi network to generate A40 (10 –14). It
demonstrated that A fibrillogenesis is accelerated in
ing that mellitin decreases the fluidity of plasma membranes Golgi lipid bilayers demonstrate the characteristic red excita-
as it inserts into membranes to create pores. tion at 340 nm and blue excitation at 380 nm, whereas the
The structural dependence of A effects on membrane fluid- emission spectra indicates a single maximum at 430 nm indic-
ity of plasma membrane and Golgi lipid bilayers was examined ative of blue emission (Fig. 3). The red excitation band intensity
by comparing the random structured peptide with A40 and increases in polar solvents, and in hydrogen-bonding solvents,
A42 which exhibit -structure. In contrast to the random the red excitation corresponds to the blue emission population
structured peptides, seeded A40 decreased the membrane and is especially intense in gel phase lipid bilayers where little
fluidity of both plasma and Golgi membranes (Table I). Similar relaxation occurs. The addition of A to laurdan containing
results were detected for A42. This result suggested to us that membranes does not change the shape of either the excitation
A interactions with lipid bilayers is not only dependent on the or emission spectra but affects the intensity of laurdan fluores-
composition of the lipid bilayer but also on the structural char- cence in both plasma and Golgi membranes (Fig. 3). The ratio
acteristics of the peptide. of the blue to red components in the excitation reflects the
Dynamics of Lipid Head Groups and Interface—Besides the polarity of the probe. Addition of A40 to plasma membrane
packing of the lipid acyl chains, the dynamics of the polar head bilayers results in an increase in the blue/red excitation ratio
groups and the polarity of the lipid interface are relevant to the from 1.03 to 2.2, indicating that the environment sensed by
interaction of molecules, i.e. A, with the membrane surface. In laurdan becomes more hydrophobic after interaction of A with
order to obtain an insight into these properties, laurdan and membranes (Fig. 2). These results suggest that A produces a
N-⑀-dansyl-L-lysine probes were used. Laurdan naphthalene displacement of water molecules from the hydration shell of the
ring is located at the glycerol backbone and is anchored in the membrane, as a result of the promotion of lateral phase sepa-
bilayer by the lauroyl moiety, thereby imparting fluorescence ration and a higher degree of plasma membrane organization.
characteristics that are dependent on the polarity of its envi- Increasing the concentration of A does not further alter the
ronment (25, 41). The advantages of laurdan are that it is blue/red excitation ratio suggesting that the bilayer has a finite
completely non-fluorescent in aqueous environments, is inde- ability to accommodate A. The generalized polarization emis-
pendent of pH between 4 and 10, and independent of lipid sion (GPem) for plasma membranes was calculated to be 0.48,
polar head group; therefore fluorescence readings reflect only which increases to 0.57 in the presence of initially random
the polarity of the probe associated with the bilayer. The structured A40 and A42. The interaction of seeded A40/42
spectral properties of laurdan have been described by the demonstrates the same shift of the GPem to 0.56 and 0.54,
general polarization equation for both excitation and emis- respectively, indicating that in the presence of A the mem-
sion spectra, which render information about the lipid phase, brane becomes more structured at the head group-fatty acyl
polarity, and co-existence of multiple lipid phases within a chain interface.
single bilayer (25, 41). In contrast, both Golgi (Fig. 3B) and endosomal (data not
Laurdan excitation spectra in the presence of plasma and shown) bilayer blue/red excitation intensities do not change
Cellular Membrane-associated A Fibrillogenesis 33565
after addition of A suggesting that A binding does not alter
the phase of these lipids. Increasing concentrations of A still
did not induce a change in the ratio of excitation intensities
confirming that these bilayers do not undergo a concentration-
dependent phase transition. The endosomal lipid bilayer exhib-
its similar fluidity as the plasma membrane as illustrated by
DPH studies, yet has different laurdan fluorescence character-
istics suggesting that bilayer-specific lipid composition may
alter the resultant A-lipid interactions. In contrast to the
excitation properties of laurdan, the GPem values of Golgi and
endosomal membranes, 0.49 and 0.54, are only affected by the
addition of high concentrations of A demonstrating a decrease
to 0.42 and 0.44 for A40. It is interesting that A42 has a
lesser effect on the GPem than A40 with only modest alter-
ation of the GPem values to 0.47 and 0.51, respectively. These
results suggest that interaction of A42 with both Golgi and
endosomal membranes does not alter the micropolarity or hy-
dration of the interfacial region of the lipid bilayer.
To examine the specificity of the changes in laurdan fluores-
cence properties due to A40/42 interactions with Golgi and
plasma membranes, we examined the effects of A-(1–28) and
mellitin. Addition of A-(1–28) and mellitin to both bilayers
does not alter the blue/red excitation ratio. Furthermore, A-
TABLE III