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Annexin A1 Interaction with a Zwitterionic Phospholipid
Monolayer: A Fluorescence Microscopy Study
J. Alfredo Freites, Shahla Ali and Michael B. Dennin
Department of Physics and Astronomy and Institute for Surface and Interface Science
University of California, Irvine, CA 92697-4575
Anja Rosengarth1 and Hartmut Luecke1,2,3
1Department of Molecular Biology and Biochemistry, 2Department of Physiology and
Biophysics, 3Department of Information and Computer Science
University of California, Irvine, CA 92697-3900
January 26, 2004
1
Abstract
We present the results of a fluorescence microscopy study of the interaction
of annexin A1 with dipalmitoylphosphatidylcholine (DPPC) monolayers as a
function of the lipid monolayer phase and the pH of the aqueous subphase.
We show that annexin A1- DPPC interaction depends strongly on the do-
main structure of the DPPC monolayer and only weakly on the subphase pH.
Annexin A1 is found to be line active, with preferential adsorption at phase
boundaries. Also, annexin A1 is found to form networks in the presence of
a domain structure in the monolayer. Our results point toward an impor-
tant contribution of the unique N-terminal domain to the organization of the
protein at the interface.
2
Introduction
The annexins are a multigene family of proteins characterized by their
capacity of reversibly binding to anionic phospholipids in a Ca2+-dependent
manner.1,2 Their common folding motif is a disc-shaped C-terminal core
domain that contains four (eight in annexin A6) homologous repeats of five
α-helices each, with the calcium-binding sites located on the convex side
of the disc. In contrast, the N-terminal domain is variable and is thought
to confer specific properties to each annexin. Although no unambiguous
physiological role has been determined for this family, annexins have been
associated with various membrane-related phenomena, including membrane
organization, membrane trafficking, fusion and ion-channel formation.1–3
Annexin A1 is characterized by an N-terminal domain that comprises its
first 41, residues with residues 2 to 17 forming an amphipathic α-helix. As
is the case with other annexins with large N-terminal domains, annexin A1
exhibits membrane aggregation properties.2 Results from vesicle aggrega-
tion studies with annexin A1,4,5 annexin A1-A5 chimeras6,7 and truncated
annexin A1 mutants,8–10 as well as structural studies,5,11,12 suggest that an-
nexin A1 possesses two distinct membrane binding sites. One is the canonical
calcium-dependent binding site to anionic phospholipids that is part of the
core domain. The other one is calcium independent and non specific for an-
ionic lipids. Based on high-resolution structural studies of annexin A1 in
the presence12 and the absence11 of calcium, Rosengarth and Luecke have
proposed a two-step model for the annexin A1-membrane interaction lead-
3
ing to membrane aggregation. Starting with the protein in its inactive form,
the first step would be the calcium-mediated binding to anionic phospholipid
headgroups of one membrane. This process involves a change in conforma-
tion of the C-terminal core that results in the previously buried N-terminal
domain becoming solvent accessible. The second step would be the binding
of a second membrane via hydrophobic interactions with the now exposed
amphipathic N-terminal domain.
To evaluate the hypothesis of a direct interaction between lipid membranes
and annexin A1, Rosengarth et al.13 conducted a study of the interaction of
annexin A1 with DPPC, DPPS, and DPPC - 20 mol% DPPS monolayers.
Tensiometry measurements were carried out both in the presence and in
the absence of calcium ions. A monotonic increase in surface pressure as a
function of time was considered as an indication of protein penetration into
the phospholipid monolayer. It was shown in that study that annexin A1
is capable of penetrating phospholipid monolayers in the absence of calcium
and in the absence of calcium and anionic phospholipids. The penetration
process kinetics were found to be best described as first-order in the presence
of calcium and DPPS, and second-order in the absence of calcium. Similar
experiments conducted with annexin A5, an annexin with a short N-terminal
domain of only 16 amino acids, did not show any indication of penetration of
this protein into any of the monolayers. Also, no penetration into the DPPC
monolayer was found when a proteolytic fragment of annexin A1 lacking
the N-terminal domain was tested. These results demonstrated the possible
occurrence of a calcium-independent hydrophobic interaction between the
4
annexin A1 N-terminal domain and phospholipid membranes.
The emerging model of the cell membrane14 pictures an inhomogeneous
medium that is organized into well-defined domains. The domain structure
is dependent upon local composition and ordering. In this context, an al-
ternative point of view to conventional binding experiments is to consider
the influence of spatial organization on membrane-protein interaction. In
the case of calcium-mediated annexin interaction with model membranes,
microscopy studies of lipid monolayer systems at the micron15,16 and sub-
micron17 scales have revealed the formation of microstructural domains that
are specific, in distribution and morphology, to the calcium-mediated binding
event. Such behavior is manifested as formation of condensed domains in sin-
gle component fluid monolayers16 or formation of anionic lipid-rich domains
in mixed monolayers.17 A similar characterization has not been reported for
the calcium-independent annexin A1 membrane interaction.
In this work, fluorescence microscopy results are reported on the evolution
of the interaction process of annexin A1 with dipalmitoylphosphatidylcholine
(DPPC) monolayers as a function of the monolayer phase state and the com-
position of the aqueous subphase. DPPC is zwitterionic, and it has already
been established that a calcium-independent annexin A1 interaction with
DPPC exists.13 Central to this work is the possibility of testing the protein
behavior in a heterogeneous medium by exploiting the well-understood phase
behavior of DPPC monolayers. Both the zwitterionic nature of DPPC and its
domain structure are potentially relevant to the identified secondary protein
5
membrane binding site. The phase behavior of annexin A1 - DPPC mono-
layer system is found to be consistent with a mean-field theory proposed by
Netz et al.18 Annexin A1 is line active, and its phase behavior suggests that,
upon adsorption, it undergoes a form of self-assembly. These results appear
to be dependent on the nature of the secondary binding site.
Experimental
Dipalmitoylphosphatidylcholine (DPPC) was purchased from Avanti Polar
Lipids (Alabaster, AL). The monolayer fluorescent probe employed was chain-
labeled nitrobenzoxadiazole phosphatidylcholine (NBD-PC) from Molecular
Probes (Eugene, OR). Both lipids were specified more than 99% pure and
used as received. Spreading solutions contained less than 0.7 mol% (with re-
spect to total lipid) of fluorescent probe and were prepared with chloroform
(HPLC grade, EM Science, Gibbstown, NJ) in concentrations of 1.4-1.5 g/L.
Monolayer subphase buffers contained 50 mM MES/NaOH, 100 mM NaCl,
pH 6.0 adjusted by NaOH, or 50 mM Tris/HCL, 100 mM NaCl, pH 7.4 ad-
justed by HCl. Additionally, buffers contained either 1mM ethyleneglycol bis
(β-aminoethyl ether)-N, N, N ′, N ′-tetraacetic acid (EGTA) or 1mM CaCl2.
The water used throughout all the experiments was filtered using a Milli-Q
device (Millipore) and had a resistivity greater than 18 MΩ.
Expression and purification of full length porcine annexin A1 was per-
formed according to Rosengarth et al.19 Protein fluorescent labeling was
performed by incubating a mixture of 1- 5 mg of protein dialyzed against 0.1
6
M sodium carbonate/bicarbonate (pH 9.0), and 1 mg of Texas Red Sulfonyl
Chloride (Pierce, Rockford, IL) on ice for 1 hour. The mixture was then
dialyzed against 20 mM sodium phosphate (pH 7.5), 150 mM NaCl (three
times), and finally, against the buffer used in the monolayer experiment.
All the experiments were performed in a NIMA Technologies (Coventry,
UK) 601M Langmuir trough equipped with a PS-4 pressure sensor, and con-
trolled by a desktop computer. Trough temperature was controlled at 20.0
± 0.5 C. The trough and tensiometer were mounted on a Olympus BX-60
(Olympus America, NY) epifluorescence microscope. This was placed on a
vibration isolation table (Newport RS 3000, Irvine, CA). Microscopy images
were captured with a monochrome CCD camera (Cohu 5515, San Diego,
CA) connected to a video monitor and a videocassette recorder. Selected
video frames were digitized to 640 pixels x 480 pixels 8-bit grayscale images
with a desktop computer using a frame grabber, cropped to the desired size
and presented without further processing. Quantitative image analysis was
performed using ImageJ (National Institute of Health, Washington, DC).
The phospholipid solution was spread with a Hamilton microsyringe to
form a monolayer at the air/buffer interface. After spreading, 30 minutes
were allowed for solvent evaporation and overall system relaxation. Isotherm
experiments were conducted with a barrier speed of 4.0 A2/molecule · min.
Protein/lipid monolayer imaging experiments were conducted at constant
surface pressure. Initially, the monolayer was compressed to a specific trough
area, then the motion control of the trough barriers was set to maintain a
7
constant surface pressure. Once the surface pressure and the trough area had
reached stationary values, the annexin A1 solution was injected underneath
the monolayer at the bottom of the trough, using a bent long-needle Hamilton
syringe. This procedure was conducted without perturbing the monolayer.
All the microscopy results on protein/lipid monolayer systems presented
were obtained during dual-label experiments, in which both the protein and
the phospholipid monolayer contained a fluorescent probe. Imaging was car-
ried out with two different sets of excitation-observation fluorescent cubes
that were manually switched during the experiment. For NBD-PC imaging,
an Olympus U-MWB fluorescent cube (exictation filter: wide-band blue 450-
480 nm; long-pass barrier filter 515 nm) was employed, herein identified as
wide-band filter. Texas Red imaging was performed with a band-pass filter
set (Omega O-5732; excitation filter: 560 ± 20 nm; excitation filter: 635 ±
27.5 nm), herein identified as Texas Red filter.
DPPC monolayer imaging was conducted during isotherm experiments (i.e.
under compression). Experiments on protein adsorption to the bare air/buffer
interface were conducted at constant trough area by injecting the annexin A1
solution at the bottom of the trough.
Results
This section is organized as follows. We will first present the phase behavior
and kinetics of the DPPC monolayer and annexin A1 separately. We will then
8
focus on the interaction between the annexin A1 and the LE-LC coexistence
phase of DPPC. This is the central result of the paper, and we will report on
the results for two different pH values. To support our interpretation of the
LE-LC results, we will discuss separately the interaction between annexin A1
and the pure LC phase of the monolayer and the interaction between annexin
A1 and the pure LE phase of the DPPC monolayer.
Surface Activity of Individual Components. The results of the phase
behavior of DPPC- 0.7 mol% NBD-PC at 20 C are shown in Fig. 1 and
Fig. 2. Both the Π vs. A isotherm and the corresponding fluorescence mi-
crographs are in good agreement with those reported in the literature for
DPPC monolayers.20–23 As a function of surface pressure, DPPC monolayers
exhibit three phases at room temperature: a low density gas-like phase (G);
a liquid isotropic phase, known as liquid expanded (LE); and a hexatic phase
with a tilted director, known as liquid condensed (LC). NBD-PC localizes
preferentially in the LE phase, as it is excluded from the LC phase due to
the acyl chain ordering. It is non fluorescent in an aqueous environment.
Therefore, it only produces a significant signal in the LE phase. The onset
of the monophasic LE region occurs at values of specific area in the range of
90 − 100A2/molecule, as indicated by the appearance of a uniformly bright
featureless image under the fluorescent filter. After further compression, a
first-order transition occurs form LE to LC, between 4.5 and 5.0 mN/m, as
indicated by the isotherm plateau and the observation of dark domains on the
fluorescence microscopy image (Figs. 2a,b). These curved, multilobular LC
domains are characteristic of monolayers of enantiomeric phospholipids.24 In
9
phospholipid monolayers, for a given cycle of compression and expansion, the
nucleation and growth of the LC phase depend on a series of factors: the com-
position of the subphase dominates the nucleation and early stages of growth;
whereas the subsequent domain shape evolution and growth are primarily de-
termined by the compression rate history.22,23,25,26 The LC monophasic region
appears in fluorescence microscopy images with the LC domain boundaries
flattened and in contact with each other (see Figs. 2c,d). The fluorescent
probe is segregated to the interboundary regions. As the pressure increases,
the fluorescent probe is increasingly excluded from the air-water interface.
The overall microstructure formation of phospholipid monolayers in a con-
densed biphasic state and the morphology of LC domains have been success-
fully described by a simple phenomenological model24,27 where mesoscopic
phenomena emerge from the interplay between electrostatic, interfacial and
chiral effects. Domain arrangement and the lack of secondary nucleation
events are considered to be due to a long-range repulsion between collinear
effective electric dipoles of neighboring LC domains. These electric dipoles
represent the net electrostatic effect arising from the ordering of the lipid acyl
chains. Domain morphology and growth are understood in terms of a balance
between the line tension associated with the LE/LC boundaries, that favors
compact shapes of low perimeter to area ratio, and the long-range dipolar
repulsion within LC domains, that favors more extended morphologies. It
has been shown24 that in the case of enantiomeric lipids both aspects are
governed by molecular chirality.
10
The surface-active character of annexin A1 was first reported by Rosen-
garth et al.13 Here, the tensiometry characterization is complemented with
fluorescence microscopy. Figure 3a shows the evolution of the surface pres-
sure after injection of annexin A1 into pH 6.0 buffer solution. A non zero
surface pressure is observed after a time lag of 1500 s. After a transient
period, a stationary value of surface pressure of around 10 mN/m seems to
emerge 6000 s after injection. The presence of protein at the surface was
first observed 200 s after injection. During the induction period and during
the first half of the transient period fluorescence microscopy reveals extended
condensed phase protein domains arranged in a foam-like pattern, as shown
in Fig. 4a. Eventually these domains coalesce, yielding a featureless, bright
image (see Fig. 4b).
The adsorption of proteins to fluid interfaces reflects the amphipathic na-
ture of the polypeptide chain. However, in contrast to simple amphiphilic
molecules, the mechanism of adsorption is determined not only by the in-
trinsic gradient of chemical potential but also by a complex interrelation
between entropic (conformational), hydrophobic, electrostatic, and van der
Waals interactions. The surface of the annexin C-terminal core is mostly hy-
drophilic. In consequence, adsorption to the fluid interface implies conforma-
tional changes that expose hydrophobic segments to the non polar medium.
Several studies by neutron reflectivity on the adsorption to the air/water in-
terface of rigid28,29 and non rigid30 globular proteins have revealed that most
of the conformational changes associated with adsorption tend to conserve
secondary structure. This is achieved by the promotion of specific forms of
11
aggregation or assembly that are consistent with the tertiary structure in so-
lution.28,29 A recent fluorescence microscopy study of lysozyme,31 consistent
with this model, presents a similar phase behavior as the one reported here
for annexin A1. Our fluorescence microscopy results confirm the formation
of a protein condensed phase accompanying the surface tension relaxation,
suggesting that a similar behavior can be expected for annexin A1.
Annexin A1 interaction with DPPC Biphasic Monolayers. To
study the interaction between annexin A1 and the DPPC monolayer, fluores-
cence microscopy was conducted while the monolayer was held at stationary
values of surface pressure, as shown in Fig. 5. Constant surface pressure
experiments, as opposed to constant area, present the protein with a phos-
pholipid monolayer that has a stationary phase distribution. Because the LC
phase of DPPC is metastable,26 a monolayer held at fixed specific area expe-
riences a surface pressure relaxation and accompanied partial dissolution of
LC domains. The extent and specific evolution of this relaxation process will
depend on the specific compression/expansion history.23 In consequence, to
achieve the desired nearly constant phase distribution, the magnitude of the
surface pressure has to be maintained stationary by continuously adjusting
the trough area.
Figure 5 shows the evolution of the surface pressure and trough area
throughout a complete experiment. Region I corresponds to the initial com-
pression to a specific area in the LE-LC biphasic region after which the trough
barriers are controlled so as to keep a constant surface pressure. The experi-
12
ments were performed at a LC area fraction of 33±1%. Region II corresponds
to a transient stabilization period. The asterisk marks the time of injection
of annexin A1. The first indication of annexin A1 at the air/buffer interface
occurs about 800 s after injection. During the period identified as Region
III, the presence of annexin A1 at the air/buffer interface is observed in iso-
lated locations. These small domains have no measurable impact on the
trough area. Only after a uniform distribution of small protein domains at
the LE/LC boundaries exists is a monotonic trough area increase observed
(region IV in Fig 5). This monotonic increase in the trough area, with a
corresponding constant surface pressure remains, can only be explained as a
displacement of the phospholipid by the adsorbed protein due to the pene-
tration of annexin A1 into the monolayer.
Figure 6 shows a sequence of micrographs obtained with the Texas Red
filter corresponding to the interaction process of the protein with the phos-
pholipid monolayer at pH 6.0 containing EGTA in the subphase. The initial
nucleation and uniform distribution of protein domains at the LE/LC bound-
aries is shown in Figs. 6a and 6b, respectively. The subsequent growth by co-
alescence of the initial domains consists of a wetting of the LE/LC boundary.
Only after complete coverage of the LE/LC boundaries are protein domains
observed in the LE phase and underneath the LC phase (see Figs. 6c and 6d).
The protein domains at the boundaries grow towards the LE phase keeping a
circular interface with it. A micrograph (Fig. 7a) taken with the wide-band
filter during this stage, revealing the protein domains in light gray (red in
the visual observation), confirms the growth of the protein domains and the
13
wetting of LE/LC boundary.
During the initial stages of adsorption, there are no apparent changes in
shape or size of the LC domains, suggesting that annexin A1 has displaced the
LE phospholipid phase without compressing it to form new LC phase. Hence,
the observed increase in trough area. The next step of the interaction process
is the coalescence of protein domains located on different LE/LC boundaries.
This results in the formation of a continuous protein network with the LC
domains as nodes (Fig. 6e and Figs. 7b and 7c). As a consequence of this
process, the LE regions are fully confined, and the protein domains appear to
occupy most of the LE area. The formation of this network and the nucleation
and growth of protein domains in the LE phase region occur independently.
The completion of the interdomain protein-network is followed by a loss of
curvature and an overall change in shape of the LC domains. It is worth
noting that this shape transition coincides with the formation of the protein
network and not with the complete wetting of the LC domains.
In the final stage of adsorption, the fluorescence signal originating from
the annexin A1 essentially fills the viewing field (Fig. 6f). However, compar-
ison with Fig. 7d confirms that LE phase is still present in the monolayer.
Additionally, the fraction of LC domains remains unchanged within the ex-
perimental uncertainty at the end of the protein adsorption process. These
facts suggest that the features revealed by fluorescent microscopy during lat-
est stages of adsorption do not reflect a process that occurs entirely at the
air/buffer interface but immediately underneath. Also, contributing to the
14
features in the Texas Red images is the larger fluorescence intensity of Texas
Red. This tends to amplify the size of the protein domains, such as the ones
observed in Fig. 6. This effect was verified by contrasting these images with
those taken with the wide-band filters on the same areas.
A pH of 6.0 for the aqueous subphase was selected based on previously re-
ported results19 indicating that annexin A1 shows it highest thermodynamic
stability between pH 5.0 and 6.0. To investigate a potential dependence of
the protein surface activity on pH, experiments were also performed at pH
7.4 with a similar LC area fraction. Taking the rate of change in trough area
as a qualitative measure of kinetics, the comparison of Fig. 8 with Fig. 5 re-
veals a similar penetration kinetics at pH 6.0 and pH 7.4, once full coverage
of LC domains occurs. Fluorescence microscopy revealed mostly similar mi-
crostructural features and overall interaction processes between annexin A1
and the DPPC monolayer for pH 7.4 as observed in the pH 6.0 experiments
(see Figs. 9 and 10).
Comparison of Fig. 8 with Fig. 5 reveals that the length of time spent in
region III, the initial adsorption of the protein into the monolayer, differs by
approximately 3000 s, with it being longer for the pH 7.4 system. However,
consistent with the pH 6.0 systems, the non-zero rate of change of area (region
IV) for pH 7.4 occurs when a uniform distribution of small protein domains
covers the LE/LC boundaries (see Figs. 9a and b and Figs. 10a and 10b).
At this point, the rate of trough area expansion is very similar at both values
of pH. It is also noticeable that the time interval between the first protein
15
adsorption events and the full coverage of LE/LC domain boundaries in all
of these experiments is within the same time scale (between 1500 and 2000 s)
as the onset of surface pressure increase for annexin A1 at the bare air-water
interface.
For both systems at pH 6.0 and pH 7.4, the microstructural organization
consists of a network of protein domains. Also, at the late stages, the LC do-
mains undergo a shape transition to long, skinny domains (see Fig. 10d). The
only real difference is the magnitude and extent of the domains. For pH 7.4,
the growth by coalescence of protein domains at the LE/LC boundaries does
not progress to a full extent before the experiment is terminated. Similarly,
in contrast to the observations at pH 6.0, the inter-domain network formed is
not uniformly extended (Figs 9d and 10d). Therefore, the LE regions are not
confined by the protein network (Fig. 9c). Additionally, as a consequence of
the protein domain formation, it appears that the distribution of LC domains
becomes clustered or at least less uniform.
Given that the chosen values of pH are on opposite sides of the protein
calculated isoelectric point, these results suggest that there is not a strong
pH dependence for the protein-monolayer interaction. For both pH values,
the initial and final trough areas are approximately equal. Therefore, the
observed difference in protein coverage in the late stages may reflect the
amount of protein aggregated below the interface and not be related to the
monolayer-protein interaction. Moreover, differences in LC domains size and
morphology were observed for monolayers spread over the two buffer solutions
16
in the absence of protein. Therefore, the possibility that the observed dif-
ferences in overall microstructural organization between the two systems are
due to constitutional differences introduced by the different buffer solutions
and intrinsic to the phospholipid monolayer themselves can not be discarded.
Further experiments are needed to completely understand the late-time differ-
ence in network coverage. Experiments were conducted substituting EGTA
in the subphase with CaCl2 at both pH 6.0 and pH 7.4 (results not shown).
No substantial or systematic differences were found in either microstructural
features or overall kinetics.
In contrast to the canonical behavior of annexins in the presence of calcium
ions and anionic phospholipids, the results presented so far suggest that the
annexin A1/DPPC monolayer interaction is predominantly non-electrostatic.
The adsorption of annexin A1 to the phospholipid monolayer is likely to be
accompanied by a change in protein conformation, in a manner consistent
with the adsorption behavior of the protein at the bare air/buffer interface.
In both cases, specific domains in the protein chain are more likely to be
attracted to the lipid interface through hydrophobic interactions. The for-
mation of a condensed phase in discrete domains by the adsorbed protein
suggests an aggregation processes regulated by this change in conformation
at the surface. The presence of the LE/LC domain boundaries appears to
modulate both the nucleation and the growth of the protein domains, as in-
dicated by the occurrence of wetting and networking. One consequence of
this modulated growth could be differences in protein chain packing between
the domains at the boundary and those that nucleate in the interior of LE
17
phase. This would explain why at first coalescence occurs only between do-
mains nucleated at the boundaries, since it is the process that leads to the
network formation.
The difference in protein domain morphology between the present case
and the adsorption to the bare aqueous interface suggest that the protein
domains are insoluble in the LE phase, since the formation of circular domain
boundaries minimizes the contact between the protein and the lipid LE phase.
This idea is reinforced by the fact that, even though the LE regions are being
compressed due to the penetration of the protein into the monolayer, no
secondary nucleation of LC domains is observed in the interior of the LE
phase. Any additional contribution to the state of stress arising from the
fine compressibility of the LE phase is being relaxed by an increase in the
trough area. Immiscibility between the protein domains and the LE phase
could also explain why those protein domains nucleated in the interior of LE
phase seem to participate in the coalescence process only at a late stage when
their surface coverage is high and/or the network of domains nucleated at the
LE/LC boundaries is sufficiently thick.
Annexin A1 Interaction with Monophasic Monolayers. To further
confirm the previous assessment, the specific interaction of annexin A1 with
each phospholipid monolayer phase was studied through constant pressure
experiments conducted above the onset of the LE-LC to LC transition at 9.0
mN/m, and below the onset of the LE to LE-LC at 3.5 mN/m. These results
confirm the preference for protein adsorption at domain boundaries (the line
18
activity of annexin A1) and the insolubility of annexin A1 in the LE phase.
At 9.0 mN/m, the monolayer consists of fully grown LC domains with
flattened boundaries, which are in contact with all their neighbors. In other
words, the LC monophasic region is characterized by a granular texture.
As indicated before, achieving a steady state starting from a monophasic LC
state, involves some relaxation. In this case, the relaxation process introduces
small isolated domains of LE phase as shown in Figure 13a. Notice, however,
that this microstructure is not the same as the one for monolayers in the
biphasic region. After injection of annexin A1, an image with the Texas
red filter shows that these boundaries are fully decorated with small protein
domains (see Fig. 12a) after 1000 s. As in the case of the biphasic monolayer,
the protein domains at the LC/LE boundaries grow by coalescence forming a
continuous interphase among the LC domains (see Fig. 12b). Subsequently,
the LC domains become completely isolated from each other and the protein
layer thickens. At the same time, the LC domains are elongated until both
lipid and protein form a striped pattern (see Fig. 12c,d). Penetration kinetics
are substantially slower than for the biphasic experiments (see Fig. 11). The
striped microstructural pattern was maintained with minimum change until
the experiments were stopped. Similar experiments performed with labeled
protein but without fluorescent label in the monolayer (results not shown)
produced a consistent behavior for the adsorption and domain formation of
the protein.
These results confirm the preferential adsorption at monolayer domain
19
boundaries and the line active character of annexin A1. The lack of extended
regions of LE phase confirms that the line activity is a distinct characteristic
of the quasi two-dimensional protein domains. The complete alteration of
the DPPC monolayer microstructure can only be achieved through a change
of the electric dipole field distribution over and across the amphiphilic mono-
layer. This suggests again specificity in conformation of the protein domains
either at a mesoscopic level or at the level of chain conformation.
At 3.5 mN/m, the protein penetrates the LE monolayer forming circular
domains (see Figs. 15a,b and Fig. 16a), and ultimately, an emulsion-like pat-
tern forms between the annexin A1 and the LE phase. The initial protein
domains nucleate uniformly in regions of about 5 µm and grow by coales-
cence (see Fig. 15c and Figs. 15b,c ). No apparent condensation of the LE
phase to LC phase was observed. At the end of the experiment individual
protein domains were on the order of 70-80 µm in diameter (see Fig. 15d
and Fig. 15d). At that time, these large domains collapse onto each other
to form larger extended regions. The formation of such large domains and
extended regions is consistent with the behavior observed for the adsorption
to the bare aqueous interface.
These observations are consistent with the results obtained with the bipha-
sic monolayers, and confirm the immiscibility of the protein domains in the
phospholipid LE phase. No domain networking was observed on the LE phase
in the monophasic experiment, confirming that the growth of protein domains
in the biphasic monolayer system is modulated by the presence of the LE/LC
20
boundaries. In the same way, it can be asserted that nucleation of protein
domains in the interior of the LE phase is an independent state from the
preferential adsorption to the LE/LC boundaries. (This will be discussed in
more detail in the next section in the context of the model by Netz et al.18)
Additionally, as was observed in the biphasic monolayer systems, the protein
domains that nucleate in the LE phase reach a critical size before starting
coalescence. This behavior is consistent with a specific pattern of aggregation
for the adsorbed protein at the scale of tertiary structure.
Discussion
We have presented fluorescence microscopy results on the interaction of an-
nexin A1 with DPPC monolayers as a function of the lipid monolayer phase
state. The central features are that annexin A1 preferentially adsorbs to LC-
LE domain boundaries, and that it ultimately induces a shape change of the
LC domains. Both of these results indicate that the annexin A1 is line active,
relative to LC-LE domains. The adsorption in the presence of LC-LE domains
results in the formation of a protein network, something that does not occur
for adsorption in the absence of the monolayer or in the LE phase. This
suggests that two different adsorbed states exist in the monolayer. Finally,
some protein fluorescent signal was also observed at the location of the LC
domains. Small differences in focal length indicated that these small protein
domains were not at the surface. Also, the late-time protein images suggest
the existence of protein aggregates below the air-water interface. These find-
21
ings suggest that it is necessary to consider the possibility that the complexity
of the observed adsorption process and surface behavior could imply that the
protein domains are only partially at the surface, and that the phenomena
of conformational change and aggregation have a multilayer character.
Preferential adsorption to the LE/LC domain boundaries of phospholipid
monolayers has been reported for other proteins that present interfacial activ-
ity, such as concanavalin A,25 bacterial surface layer proteins,32 fibronectin,33
and surfactant protein A.34 Netz, Andelman and Orland18 have developed a
Flory-Huggins type mean-field theory that is able to account for this phe-
nomenon. According to this model, the preferential adsorption of a protein
to LE/LC domain boundaries is an entropic effect due to the constitutional
differences between the adsorbed protein phase and the phospholipid mono-
layer. The model predicts a reduction of the line tension associated with the
LE/LC boundary due to the protein adsorption. This is consistent with the
observed wetting of the LC phase by the annexin A1 domains located at the
LE/LC. The change in shape of the LC domains can also be explained in
this context. The full coverage of the LE/LC boundary by coalesced protein
domains could screen the dipole-dipole interaction between neighboring LC
domains. At the same time, a reduction of the line tension allows the LC do-
main morphology to be dominated by the repulsive dipole-dipole interaction
within the domains. It has been predicted35,36 that under these circumstances
the LC domains would assume elongated shapes, as was observed during the
last stages of the annexin A1/ DPPC monolayer interaction process. The
screening of the LC interdomain dipolar interaction by the adsorbed protein
22
could also explain the clustering of LC domains observed at pH 7.4.
The theory by Netz et al.18 also accounts for the observed nucleation of
new protein domains in the LE phase, as an event that could occur due to
a change in the protein chemical potential at the surface. This is consistent
with the reported observation that the adsorption in the interior of the LE
phase occurs after the protein domains have completely covered the LE/LC
boundaries. Consequently, nucleation of new protein domains in the interior
of the LE phase could be attributed to a critical increase on protein surface
concentration. The model of Netz et al. is based only on pairwise interac-
tions between the system components, which justifies the assumption that
hydrophobic interactions are dominant in the annexin A1/DPPC monolayer
system.
These ideas can be connected to the three different penetration kinetics
reported by Rosengarth et al.13 for annexin A1 phospholipid monolayer sys-
tems: first-order kinetics for the system containing both calcium ions and
DPPS; second-order kinetics for the systems containing DPPS in the absence
of calcium ions; and a slower second-order kinetics for the DPPC monolayer
system. It can be speculated that the occurrence of the first-order kinetics
characterizes unambiguously the canonical electrostatic interaction between
annexins and anionic phospholipids. On the other hand, the mixed mono-
layer DPPC-DPPS tested in that study has been reported to present DPPS-
rich domains in the absence of chelator agents,17,37 it is then possible that
the second-order kinetics corresponds to the kind of complex interfacial phe-
23
nomena described here, whereby protein aggregation and line activity play a
dominant role. The reported differences in kinetics between the DPPC and
the monolayers containing DPPS could be attributed to the different nature
of the domains formed in these systems.
There is good evidence that the phase behavior reported here for annexin
A1 is directly linked to interactions involving the N-terminal domain. No
penetration into DPPC monolayers was observed by Rosengarth et al.13 for
a proteolytic fragment of annexin A1 lacking the amphipathic N-terminal
domain and for annexin A5 which lacks an N-terminal domain. Further ev-
idence for the role of the N-terminal domain comes from considering the
association to membranes of annexin A12 and annexin A5 in the absence of
calcium. Under acidic conditions (pH below 5.0) these annexins appear to
refold and insert into bilayers, yielding a transmembrane configuration.38–40
This phenomenon has been shown to depend on hydrophobic interactions be-
tween the protein and zwitterionic components of the model membranes.39,40
It is highly sensitive to the protonation state of the C-terminal core. This
is in contrast to the results reported here. The fact that the interaction of
annexin A1 with zwitterionic phospholipid monolayers presents the same mi-
crostructural features at neutral and acidic pH allows us to speculate that
this behavior is not related to the C-terminal core conformation but rather, in
accordance with the results of Rosengarth et al.,13 related to the amphipathic
nature of the N-terminal domain.
24
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28
30 40 50 60 70 80 90 100
Area/lipid (Å2)
0
10
20
30
40
50
Π (
mN
/m)
Figure 1: Surface pressure vs. specific area isotherm for DPPC on MES/NaOH buffer at 20 C.
29
c d
a b
Figure 2: Fluorescence micrographs for DPPC at 20 C:(a) Π = 4.7 mN/m on MES/NaOH buffer; (b) Π = 9.1
mN/m on MES Na/OH buffer. Imaging performed with a wide-band blue excitation filter and a long-pass
green emission filter, sensitive to both NBD-PC and Texas Red (herein identified as Wide-Band filter).
30
0 2000 4000 6000 8000time (s)
0
5
10
15
Π (
mN
/m)
c
a b
Figure 3: Surface Pressure evolution after the injection of annexin A1 in MES/NaOH buffer to a final
concentration of 24nM. Points a and b correspond to the fluorescence microscopy images shown in Fig. 4
31
e
c
a
b
Figure 4: Fluorescence micrographs for the adsorption of annexin A1to the Air-MES/NaOH buffer interface.
Imaging performed with a narrow band-filter selective for Texas Red (herein identified as Texas Red filter).
Times after injection: (a) 1120 s; (b) 1330 s; (c) 2800 s
32
0 1000 2000 3000 4000 5000time (s)
0
20
40
60
80
100
Are
a (c
m2
)
0
5
10
15
20
Π (
mN
/m)
I
IIIII
IV
* ba c ef
Figure 5: Surface Pressure and Trough Area evolution for the system DPPC monolayer - annexin A1 at pH
6.0. The asterisk indicates the time of injection of annexin A1 to a final concentration of 24 nM. Points a to
f correspond to the fluorescence microscopy images shown in Fig. 6. See text for explanation of the regions
labeled I-IV.
33
c d
ef
a b
Figure 6: Fluorescence micrographs for the system DPPC monolayer - annexin A1 at pH 6.0 and with the
phospholipid monolayer in a biphasic state (LC area fraction is 33 % ) . Imaging performed with the Texas
Red filter (see Fig. ?? and text for more details on the filter sets). The scale bar is 30 µm. Times after
protein injection are: (a) 1346 s; (b) 1604 s; (c) 1731 s; (d) 1621 s; (e) 1917 s; (f) 2203 s.
34
c d
a b
Figure 7: Fluorescence micrographs for the system DPPC monolayer - annexin A1 at pH 6.0 and with the
phospholipid monolayer in a biphasic state (LC area fraction is 33 % ) . Imaging was performed with the
wide-band filter (see Fig. 2 and text for more details on filter sets). White and gray regions appeared green
and red, respectively, in the visual observation. The scale bar is 30 µm. Times after protein injection are:
(a) 1626 s; (b) 1915 s; (c) 1802 s; (d) 2441 s.
35
0 1000 2000 3000 4000 5000 6000 7000time (s)
0
20
40
60
80
100
Are
a (c
m2
)
0
5
10
15
20
Π (
mN
/m)
*a
c
d
Figure 8: Surface Pressure and Trough Area evolution for the system DPPC monolayer - annexin A1 at pH
7.4. The asterisk indicates the time of injection of annexin A1 to a final concentration of 24 nM. Points a-f
correspond to the fluorescence microscopy images shown in Fig. 9.
36
ef
c d
a b
Figure 9: Fluorescence micrographs for the system DPPC monolayer - annexin A1 at pH 7.4 and with the
phospholipid monolayer in a biphasic state (LC area fraction is 33 % ) . Imaging performed with the Texas
Red filter. The scale bar is 30 µm. Times after protein injection are: (a) 3212 s; (b) 3332 s; (c) 3625 s; (d)
4280 s.
37
c d
a b
Figure 10: Fluorescence micrographs for the system DPPC monolayer - annexin A1 at pH 7.4 and with
the phospholipid monolayer in a biphasic state (LC area fraction is 33 % ) . Imaging performed with the
wide-band filter. The scale bar is 30 µm. Times after protein injection are: (a) 3087; (b) 3653 s; (c) 3716 s;
(d) 3917.
38
0 2000 4000 6000 8000time (s)
0
20
40
60
80
100
Are
a (c
m2
)
0
5
10
15
20
25
30
35
40
Π (
mN
/m)
* ab c d
Figure 11: Surface Pressure and Trough Area evolution for the system DPPC monolayer - annexin A1 at pH
6.0. The protein was injected while the monolayer was held at 9.0 mN/m. The asterisk indicates the time
of injection to a final concentration of 24 nM. Points a-f correspond to the fluorescence microscopy images
shown in Fig. 12.
39
ef
c d
a b
Figure 12: Fluorescence micrographs for the system DPPC monolayer - annexin A1 at pH 6.0 with the
phospholipid monolayer in the LC phase. Imaging performed with the Texas Red filter. The scale bar is 30
µm.Times after protein injection are: (a) 1126; (b) 3683; (c) 4187; (d) 4733.
40
ef
c d
a b
Figure 13: Fluorescence micrographs for the system DPPC monolayer - annexin A1 at pH 6.0 with the
phospholipid monolayer in the LC phase. Imaging performed with the wide-band filter. The scale bar is 30
µm.Times after protein injection are: (a)
41
0 1000 2000 3000 4000 5000 6000time (s)
0
20
40
60
80
100
Are
a (c
m2
)
0 1000 2000 3000 4000 5000 6000time (s)
0
5
10
15
20
Π (
mN
/m)
* ab c
d
Figure 14: Surface Pressure and Trough Area evolution for the system DPPC monolayer - annexin A1 at pH
6.0. The protein was injected while the monolayer was held at 9.0 mN/m. The asterisk indicates the time
of injection to a final concentration of 24 nM. Points a-f correspond to the fluorescence microscopy images
shown in Figs. 15.
42
ef
c d
a b
Figure 15: Fluorescence micrographs for the system DPPC monolayer - annexin A1 at pH 6.0 with the
phospholipid monolayer in the LE phase. Imaging performed with the Texas Red filter. The scale bar is 30
µm. Times after protein injection are: (a)
43
ef
c d
a b
Figure 16: Fluorescence micrographs for the system DPPC monolayer - annexin A1 at pH 6.0 with the
phospholipid monolayer in the LE phase. Imaging performed with the wide-band filter. The scale bar is 30
µm. Times after protein injection are: (a)
44
