1 Max-Planck-Institute for Developmental Biology, Spemannstrasse 35, 72076
Tübingen, Germany
2 MRC Centre for Developmental Neurobiology, King's College London, New Hunt's
House, Guy's Hospital Campus, London SE1 1UL, UK
* Author for correspondence (e-mail: uwe.drescher{at}kcl.ac.uk)
Accepted 13 January 2003
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SUMMARY |
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We find as expected on the basis of chick experiments that, when immobilised in the stripe assay, ephrin-A5 has a repellent effect such that retinal axons avoid ephrin-A5-Fc-containing lanes. Also, retinal axons react with repulsive turning or growth cone collapse when confronted with ephrin-A5-Fc bound to beads. However, when added in soluble form to the medium, ephrin-A5 induces growth cone collapse, comparable to data from chick.
The analysis of growth cone behaviour in a gradient of soluble ephrin-A5 in the `turning assay' revealed a substratum-dependent reaction of Xenopus retinal axons. On fibronectin, we observed a repulsive response, with the turning of growth cones away from higher concentrations of ephrin-A5. On laminin, retinal axons turned towards higher concentrations, indicating an attractive effect. In both cases the turning response occurred at a high background level of growth cone collapse. In sum, our data indicate that ephrin-As are able to guide axons in immobilised bound form as well as in the form of soluble molecules. To what degree this type of guidance is relevant for the in vivo situation remains to be shown.
Key words: Axon guidance, Retinotectal projection, Ephrin-A5, EphA receptor, Xenopus, Growth turning assay, Growth cone collapse
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INTRODUCTION |
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The role of the Eph family in the establishment of neuronal connections has
been studied in a number of different model systems, in particular the
formation of the retinotectal projection. It is now believed that this
topographically organised projection is set up to a large extent on the basis
of complementary gradients of Eph receptors and ephrins in both the retina and
the tectum, involving repulsive but also attractive interactions between Eph
receptors and ephrins (reviewed by
Knöll and Drescher, 2002;
Wilkinson, 2000
). In ephrin-A
mutant mice, mapping across the anteroposterior axis is affected
(Feldheim et al., 1998
;
Feldheim et al., 2000
;
Frisen et al., 1998
), while
recent data from in vivo analyses showed an important role of EphB family
members in mapping across the mediolateral axis
(Hindges et al., 2002
;
Mann et al., 2002
). Functional
investigations of topographically expressed transcription factors and
signalling molecules in the retina also support the idea of a prominent role
of the Eph family in retinotectal mapping
(Koshiba-Takeuchi et al.,
2000
; Mui et al.,
2002
; Sakuta et al.,
2001
; Schulte and Cepko,
2000
; Schulte et al.,
1999
).
The way in which the Eph family exerts its function has just started to be
uncovered. A characteristic of the Eph family is their capacity for
bi-directional signalling such that both Eph receptors and ephrins function as
both receptors and ligands (Klein,
2001; Knöll and Drescher,
2002
). In vivo, ephrins have to be membrane bound to fulfil either
the receptor or ligand role. As ligands, the membrane attachment is believed
to lead to a clustering of the ephrins, which is a necessary requirement for
Eph receptor activation, since soluble clustered ephrins (for example as Fc
chimeric molecules, as described in the studies here), but not soluble
monomeric ephrins, activate Eph receptors
(Davis et al., 1994
). Also, the
function of ephrins as receptors requires membrane anchorage for signalling
into the cytosol, which for GPI-anchored ephrin-As most likely requires, in
addition, a transmembrane co-receptor (Davy
et al., 1999
; Davy and
Robbins, 2000
; Huai and
Drescher, 2001
; Knöll et
al., 2001
).
We were interested in the question of whether the substratum-bound ephrins
possess the ability to guide axons, i.e. they have the capability to change
the growth direction of an axon without inducing a full growth cone collapse.
Most other molecules so far associated with axon guidance are secreted
molecules such as netrins, semaphorins and slits
(Müller, 1999). They are
thought to function in vivo by forming gradients through a localised
secretion, and to guide axons by a chemoattractive or chemorepulsive guidance
mechanism (Tessier-Lavigne and Goodman,
1996
). So far only these secreted molecules were active in the
`growth cone turning assay', which represents an assay mimicking some aspects
of the in vivo situation (Campbell et al.,
2001
; de la Torre et al.,
1997
; Hopker et al.,
1999
; Ming et al.,
1997
; Ming et al.,
1999
; Song and Poo,
2001
; Song et al.,
1998
; Song and Poo,
1999
). In this turning assay, a gradient of a particular molecule
is generated through a pulsed release from the tip of a micropipette. The
growth of axons within such a gradient is then analysed using time lapse
microscopy.
The response of growth cones to gradients of soluble molecules might be
fundamentally different from their response to membrane-bound ephrins, which
involves a local cell-cell contact between the responsive growth cone and the
ephrin-expressing cell, and which is, as such, classified as a guidance
mechanism via contact repulsion or attraction
(Tessier-Lavigne and Goodman,
1996). Such an interaction might for example involve reciprocal
signalling between these cells. Also the clustering of ligands and receptors
and/or their lateral mobility within the membrane might play an important
role. These features might be exclusive for membrane-bound molecules.
So far ephrin-As have been investigated in the `stripe assay' with retinal
ganglion cell (RGC) axons, where they force a striped outgrowth of retinal
axons, and in the `collapse assay', where they induce a full growth cone
collapse (Davenport et al.,
1999; Drescher et al.,
1995
; Jurney et al.,
2002
; Menzel et al.,
2001
; Monschau et al.,
1997
; Shamah et al.,
2001
; Wahl et al.,
2000
). However, these results do not address the question of
whether ephrins have the ability to induce turning of growth cones, which is
believed to be the consequence of a local or partial growth
cone collapse (e.g. Fan and Raper,
1995
; Zhou et al.,
2002
). It is conceivable that ephrin-As for some, as yet unknown,
reasons might only be able to induce a general growth cone collapse (thus
acting in vivo as a stop signal), which would suffice to explain the striped
outgrowth in the stripe assay.
Here we show for Xenopus retinal ganglion cell growth cones that
ephrin-A5 has the ability to induce a specific collapse reaction in the
collapse assay and a striped outgrowth in the stripe assay. These results are
comparable with those using chick retinal axons
(Drescher et al., 1995;
Monschau et al., 1997
). Also,
ephrin-A5-Fc is able to induce the turning of Xenopus retinal growth
cones when applied as a soluble clustered molecule and as an immobilised
molecule linked to beads. The turning response is substratum-dependent, a
feature which has also been shown recently for netrins
(Höpker et al., 1999
).
This turning response occurs under special conditions, that is at higher
concentrations and/or steeper slopes of the gradient. Under these conditions a
subpopulation of about 20% reacted with turning, while the rest of the growth
cones showed a collapse reaction. Thus it has been shown for the first time
that ephrin-A5 is in principle able to guide retinal axons in vitro.
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MATERIALS AND METHODS |
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Staining of retinal growth cones in culture with alkaline phosphatase
fusion proteins
Retinae of stage 28 Xenopus embryos were explanted on matrigel and
incubated at room temperature for 48 hours in culture medium containing 0.4%
methylcellulose. Following roughly the method described by Cheng and Flanagan
(Cheng and Flanagan, 1994) for
chick retinae, cultures were washed in PBS and incubated for 2 hours either
with ephrin-A5-alkaline phosphatase fusion protein (ephrin-A5-AP, 5 nM) or, as
control, 5 nM AP alone. Then the cultures were washed six times with HBHA (0.5
mg/ml BSA; 0.1% NaN3, 20 mM Hepes pH 7.0 in Hank's A solution),
fixed in 60% acetone, 3% formaldehyde and washed twice with HBS (20 mM Hepes
pH 7.0, 150 mM NaCl). Endogenous phosphatases were inactivated at 65°C for
15 minutes. After washing with AP buffer (0.1 M Tris-HCl pH 9.5, 0.1 M NaCl, 5
mM MgCl2), the colour reaction was performed in 4.5 µl
nitro-blue-tetrazolium (50 mg/ml; NBT) (Promega, Madison, WI, USA), 3.5 µl
5-bromo-4-chloro-3-indoyl phosphate (BCIP) (50 mg/ml; Promega, Madison, WI,
USA) per ml AP buffer. One hour later the staining reaction was stopped by
washing with PBS.
Collapse assay
The collapse assay was performed essentially as described previously
(Raper and Kapfhammer, 1990;
Cox et al., 1990
). Retinal
explants of Xenopus were grown overnight on laminin (20 µg/ml)
coated cover slips. The growth cones were investigated using an inverted phase
contrast microscope. By using a computer-controlled scanning stage, up to 15
growth cones could be observed simultaneously in time lapse. Pictures were
taken under automatic control every 3-5 minutes, starting 30 minutes before
and finishing 60 minutes after the addition of proteins. A growth cone was
considered as collapsed when the motile structures, i.e. lamellipodia and
filopodia, disappeared and the growth cone stopped growing or even retracted
its whole axon shaft.
In vivo, ephrin-A5 is membrane anchored. Here we have used a soluble
version of ephrin-A5, in which the GPI-anchor was replaced by the Fc part of a
human IgG antibody [named ephrin-A5-Fc
(Ciossek et al., 1998)]. As a
control protein in these collapse assays, Fc alone was added, which is known
to have no or very little collapse-inducing activity
(Ciossek et al., 1998
).
Modified stripe assay
The technique for the modified stripe assay has been described previously
(Vielmetter et al., 1990;
Hornberger et al., 1999
). 660
µl of a solution of 25 cm2 nitrocellulose dissolved in 12 ml
methanol was dropped in a 6 cm Petri dish and evaporated overnight. The
silicon matrix containing channels of 90 µm width was pressed on the
nitrocellulose-coated Petri dish and the protein solution for the first set of
lanes was injected with a Hamilton syringe using 100 µl of a solution
containing 8 µg/ml ephrin-A5-Fc (or Fc for control experiments) clustered
with 80 µg/ml of an anti-human Fc antibody conjugated with FITC (Sigma, St.
Louis, MO). After 30 minutes incubation in 5% CO2 at 37°C, the
silicon matrix was carefully removed, the stripes were washed once with Hank's
A solution and then the solution for the second set of lanes was applied (80
µl of a solution containing 3 µg/ml Fc clustered with 30 µg/ml
non-labelled anti-human Fc-antibody). After 30 minutes incubation, the stripes
were washed twice with Hank's A solution and covered with laminin (20
µg/ml) for 1-2 hours. Finally the stripes were washed again and covered
with medium until explantation of the Xenopus retinae. Owing to the
clustering of ephrin-A5-Fc with an FITC-conjugated Fc-antibody, alternating
lanes can be distinguished using fluorescence microscopy. After 2 days in
culture, explants were fixed in 4% paraformaldehyde and analysed.
Growth cone turning assay using Ephrin-A5 coated beads
Following the protocol of Kuhn et al.
(Kuhn et al., 1995),
carboxylated latex beads (4.5 µm in diameter) were coated with Protein-A.
Remaining active groups were subsequently blocked with BSA. These beads were
incubated with ephrin-A5-Fc (clustered with an FITC-conjugated antibody)
overnight at 4°C, centrifuged and washed with PBS. To verify the binding
of ephrin-A5-Fc, the beads were examined under blue light with 450-490 nm
excitation wavelength. Successfully labelled beads showed green
fluorescence.
Using a laser tweezers device (600 mW diode laser with a wavelength of 1200 nm, P.A.L.M.), beads were placed in front of Xenopus retinal ganglion growth cones. Beads without any or only protein A coating were used in control experiments. Pictures were taken every 30 seconds. The growth cones were observed for about 1.5 hours in each experiment. Subsequently, the turning angle relative to the direction of the last 10 µm segment of the axon shaft at the beginning of the experiment and the growth cone collapse rate were determined.
Growth cone turning in an ephrin-A5 gradient
Gradients of diffusible substances were established as described previously
(Lohof et al., 1992;
Zheng et al., 1994
). In brief,
a stable gradient was produced by ejecting the substance of interest out of a
capillary having a tip opening of 1 µm. Pulses were created by applying a
pressure of 3 psi for 10 mseconds at 2 Hz. The growth cones were positioned at
a distance of 100 µm or in the case of ephrin-A5-Fc gradients at a
distance of 60 µm from the micropipette tip with an angle of
45° relative to the initial direction of the axon shaft. After
establishment of the gradient, pictures were taken every 5 minutes up to the
end of the experiment for about 1 hour. Concentration of ephrin-A5-Fc in the
capillary was 20 µg/ml. In controls, pure medium without guidance molecules
was loaded into the capillary.
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RESULTS |
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In control experiments using 1 µg/ml Fc only, 6 of 67 (9%,
Table 1) of the growth cones
showed a growth cone collapse. Similar levels of this `non-specific'
background collapse, which is possibly caused by changes in pH or temperature,
have also been seen in other experiments
(Drescher et al., 1995;
Monschau et al., 1997
). In
contrast, application of 1 µg/ml unclustered chick ephrin-A5-Fc increased
the percentage of collapsed growth cones from 9% to 44% (70 of 158,
Table 1). A further rise in
collapse rate was achieved when the concentration was raised to 20 µg/ml
(also unclustered). Here 88% of the growth cones (23 of 26) showed collapse. 5
µg/ml of clustered ephrin-A5-Fc resulted in the collapse of 80% of the
analysed growth cones (23 of 29). These data show that Xenopus
retinal growth cones are similarly sensitive to ephrin-A5-Fc as, for example,
are chick retinal axons (Drescher et al.,
1995
; Monschau et al.,
1997
). It also shows that ephrin-A5-Fc can exert its activity as a
dimer, while other ephrins, in particular members of the ephrin-B family,
require tetrameric or higher oligomerised forms for appropriate receptor
activation (e.g. Stein et al.,
1998
).
|
Using a substratum consisting of immobilised ephrin-A5-Fc versus Fc, retinal axons strongly preferred to grow on lanes containing Fc, but not ephrin-A5-Fc stripes (Fig. 2A). In control experiments, in which both lanes contained Fc, retinal axons did not show any preference (Fig. 2B). These data indicate that substratum-bound ephrin-A5 has a repellent guidance activity on Xenopus retinal axons.
|
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Analysis of ephrin-A5 in the growth cone turning assay
The growth cone turning assay developed by M. Poo and co-workers is
regarded as a sophisticated technique to study the ability of a molecule to
guide axons in vitro (Lohof et al.,
1992; Zheng et al.,
1994
). We have applied this assay to investigate the ability of
ephrin-A5 to induce changes in growth direction of Xenopus retinal
axons not only when presented in substratum-bound form (see above), but also
as a soluble molecule in a gradient.
Exposure to gradients of soluble ephrin-A5-Fc did indeed result in a
turning of retinal growth cones. This occurred, however, only if the
micropipette was moved closer to the growth cone, such that the distance
between pipette tip and growth cone was reduced from 100 µm to 60 µm
(Lohof et al., 1992;
Zheng et al., 1994
). The
experiments were performed with unclustered ephrin-A5-Fc at a concentration of
20 µg/ml at the pipette tip (Fig.
5). Using a lower concentration of ephrin-A5-Fc (5 µg/ml) and
positioning the pipette tip at a distance of 100 µm from the growth cone
did not lead to a turning response (data not shown). By decreasing the
distance between growth cone and pipette tip, the slope of the gradient gets
steeper and the concentration of ephrin-A5-Fc at the growth cones gets higher.
We could not use higher concentrations of ephrin-A5-Fc, as these higher
concentrations cause aggregation and thus blocked the tip opening such that no
stable gradient could be formed (data not shown).
|
|
In another set of controls, we analysed Xenopus retinal growth
cones growing on laminin in BDNF and netrin 1 gradients. In a gradient of BDNF
(50 µg/ml at the pipette tip), growth cones were repulsed with a mean
turning angle of 37° (±3.6; n=13), which is in good
correlation with data obtained by Höpker et al.
(Höpker et al., 1999). In
the second set of experiments, myc-tagged netrin 1 (a gift from M.
Tessier-Lavigne) at a concentration of 5 µg/ml was applied
(Höpker et al., 1999
).
Here Xenopus growth cones were repelled with a mean turning angle of
21° (±1.3; n=16). Netrin 1 typically elicits an
attractive response (de la Torre et al.,
1997
; Ming et al.,
1997
); however, as shown recently
(Höpker et al., 1999
),
netrin 1 activity is also substratum dependent, such that the growth cone
response is converted from attraction to repulsion if axons are grown on
laminin instead of e.g. fibronectin.
In control experiments with only medium in the pipette, no turning of axons was observed (Fig. 5). The mean turning angles of all experiments are summarized in Fig. 6.
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DISCUSSION |
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Growth cone collapse versus turning
In agreement with data from other species, retinal axons from Xenopus
laevis express EphA receptors. In the stripe assay they (consequently)
avoid ephrin-A5-Fc-containing lanes and their growth cones collapse when
exposed uniformly to soluble ephrin-A5-Fc.
As the stripe assay evaluation is normally done when the striped pattern is
already established, it is not possible to determine whether the patterned
outgrowth is the result of a mechanism involving growth cone collapse or
guidance. In one scenario, it is conceivable that each time an axon hits an
ephrin-A5-containing stripe its growth cone collapses, followed by a
subsequent regrowth in a slightly different direction, followed again by
growth cone collapse, regrowth etc., leading thus to a striped outgrowth. In
the other scenario there might be a `smooth' turning of retinal axons away
from ephrin-A5-containing stripes involving no or only a partial growth cone
collapse (Fan and Raper, 1995)
(B. K. Müller, Diplomarbeit, University of Tübingen, 1988). The
former case would indicate that ephrin-A5 might simply act as a stop signal in
vivo, whereas in the latter case it would function as a true guidance molecule
steering growth cones towards their target in vivo. On a more general level,
an investigation of this question would help to understand more about the way
in which growth cones `work', meaning here how they integrate Eph receptor
signalling.
The relationship between growth cone turning and growth cone collapse has
been considered for some time now (Fan and
Raper, 1995; Walter et al.,
1990
). On the one hand, there is the more extreme view that there
are two classes of molecules, with one class only able to induce a
full growth cone collapse, while the other class is able to induce growth cone
turning. On the other hand there is the view that the same molecule can induce
either turning or collapse, depending on the way in which it is presented to
the growth cone. Growth cone turning is understood here as a consequence of a
local or partial growth cone collapse (Fan
and Raper, 1995
; Zhou et al.,
2002
). Mechanistically, in this view a local application of a
repulsive guidance molecule would lead to a reorganisation and retraction of
actin filaments and microtubules at the site of ligand-receptor activation,
while non-activated domains of the growth cone continue to advance further,
which would lead finally to a turning away from the repulsive molecules
causing the local collapse. The same guidance molecule might also induce a
general growth cone collapse if presented uniformly to the growth cone, i.e.
from all sides. There might be various parameters that determine stop versus
guidance activity, such as the length/strength of receptor activation or the
diffusion rates of activated signalling molecules inside the growth cone.
Thus, some signalling molecules might readily spread throughout the growth
cone, whereas others would be confined to the immediate vicinity of the
activated guidance receptor (e.g. Kasai
and Petersen, 1994
). Therefore, the growth cone collapse-inducing
activity of ephrin-A5, as shown here in the collapse assay and in the bead
assay, is not necessarily in disagreement with a possible function as a
guidance molecule. Interestingly, in ephrin-A5 mutant mice a transient
overshooting of retinal axons beyond their target area, the superior
colliculi, into the inferior colliculi has been observed, in addition to
topographic targeting errors within the superior colliculi, consistent with a
function of ephrin-A5 as both guidance and stop signal
(Frisen et al., 1998
).
Indeed, as we show here in the case of a focal presentation to Xenopus retinal axons, ephrin-A5-Fc in either substratum-bound (i.e. bound to beads) or soluble form induces growth cone turning, albeit on the background of a high percentage of growth cone collapse (see below). It is very possible that, by changing the concentration of ephrin-A5-Fc on the beads, or the shape of the gradient or the angle between the gradient and growth cone, the reaction of growth cones could be shifted more towards growth cone collapse or turning.
In this context, it came as a surprise to us that lower concentrations of
ephrin-A5 result in collapse only, whereas higher ephrin-A5 concentrations
induce turning (still on a high background level of collapse reaction). We
expected rather the opposite, with growth cone collapse at higher
concentrations, and turning at lower concentrations. At present we have no
clear idea of the meaning of this finding. One interpretation in
contrast to the currently prevailing hypothesis might be that growth
cone collapse and turning are two different processes, which nevertheless can
be induced concentration-dependently by the same molecule. It is also possible
that the differential steepness of the ephrin-A5-Fc gradient, which is
dependent on the distance from the micropipette tip, might be important with
respect to growth cone guidance versus collapse
(Goodhill and Baier, 1998;
Rosentreter et al., 1998
).
Nonetheless, our data from the turning assay allow the conclusion that substratum-bound and soluble ephrin-A5 have the ability to function as guidance molecules in vitro.
Substratum dependency of the turning response
The direction of growth cone turning in an ephrin-A5-Fc gradient was
dependent on the substratum on which the axons were grown. On laminin we
observed an attraction with a turning towards higher concentrations of
ephrin-A5. To confirm this turning response, we changed the substratum to
fibronectin resulting in a conversion of the turning response from attraction
on laminin to repulsion on fibronectin. This substratum dependency supports
the view that the turning of retinal axons in response to an ephrin-A5-Fc
gradient does not represent an artefact, but is a reflection of the well-known
fact that the substratum on which axons are growing plays an important role in
the interpretation of guidance molecules. A similar phenomenon has been
described, for example, for netrin 1, which results in an attraction on
fibronectin (and other substrata) and a repulsion on laminin
(Höpker et al., 1999;
Song and Poo, 2001
) (for
reviews, see Song and Poo,
1999
).
Generally, our finding strengthens the idea that extracellular matrix molecules, which make up the scaffold within which growth cones migrate in vivo, have an important influence on the way in which growth cones interpret guidance cues.
Laminin was also used as an outgrowth-promoting substratum in the stripe
assay experiments. Here, however, ephrin-A5 leads to striped outgrowth, which
is normally interpreted as a repulsive effect. While at first view these
stripe assay results are in contradiction to the data from the turning assay,
there are some aspects which might lead to a more coherent picture (see also
Holmberg and Frisen, 2002). In
the turning assay only a minor fraction of axons (20%) responded with a
turning response (either attractive or repulsive), while the majority of
growth cones (80%) collapsed upon interaction with ephrin-A5-Fc on both
laminin and fibronectin. A similar situation holds true for the stripe assay.
Here also the majority of axons are prevented from invading ephrin-A5
containing stripes. Thus, in sum, in both assays the majority of retinal axons
behaves in the same way, i.e. with avoidance of ephrin-A5.
There is a smaller subpopulation of axons showing no striped outgrowth,
meaning they are not repelled by ephrin-A5-Fc. Investigating the behaviour of
retinal axons in the stripe assay using time-lapse microscopy showed that
indeed a fraction of growth cones were not immediately repulsed on hitting the
border of ephrin-A5-containing stripes, but grew for some time within the
ephrin-A5 stripes and were displaced from these stripes only after some time,
if at all (data not shown). This suggests that the repellent response in a
fraction of retinal axons is biphasic with an initial attraction or
insensitivity towards ephrin-A5 and a second phase involving a repulsive
reaction. Given that the turning assay was performed for only one hour, it is
possible that only the first phase in the response to ephrin-A5 was observed.
It is known that turning responses can be switched from repulsion to
attraction, and vice versa, from attraction to repulsion using a large variety
of different stimuli (Song and Poo,
2001). Future experiments will be directed towards investigating
the functional meaning of this biphasic response of retinal axons.
In sum, we have shown here for the first time that ephrin-As are able to guide axons both in immobilised form and as soluble molecules. Thus, for guiding axons, the membrane attachment of ephrin-As is not necessary. The relevance of these findings for the in vivo situation remains to be shown.
A difference between in vivo and in vitro conditions is potentially the
dynamics of ligand clustering. In the in vitro experiments the level of
oligomerisation of ephrin-A5 is fixed, such that ephrin-A5 is presented, for
example, in the growth cone turning assay as a dimer. In vivo, in the context
of a cell-to-cell interaction, the oligomerisation of ephrin-As is possibly a
much more dynamic process. Here ephrin-A and EphA molecules are localised in
subdomains of the membrane called lipid rafts, which are very small entities
(<70 nm) in the non-activated state and might contain only a few proteins
(Simons and Toomre, 2001).
However, during activation, which means here the interaction of ephrin-As with
EphA receptors, rafts fuse to large (and then microscopically visible)
structures. Although the mechanism/s of lipid raft fusion are yet poorly
understood, this clustering could finally lead to large assemblies of
EphA/ephrin-A complexes, and in turn to a considerably stronger interaction
than that occurring in the in vitro experiments. In consequence the off-rate,
i.e. the separation of ligands and receptors, could be different and could
affect the guidance behaviour, for example owing to an increased duration of
the response (see also Hattori et al.,
2000
). It might also be possible that in vivo signals from the
(activated) ephrin-A-expressing cell are sent to the EphA receptor-expressing
axon and that these signals modulate the biological response. Thus it appears
that in particular the role of lipid rafts in axon guidance has to be
investigated in more detail to gain a deeper understanding of
EphA/ephrin-A-mediated signalling processes.
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ACKNOWLEDGMENTS |
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