Neuron, Vol. 42, 51–62, April 8, 2004, Copyright 2004 by Cell Press

Lipid Rafts Mediate Chemotropic Guidanceof Nerve Growth Cones
Carmine Guirland,1 Shingo Suzuki,2Masami Kojima,2 Bai Lu,3 and James Q. Zheng1,*
1
Department of Neuroscience and Cell Biology
University of Medicine and Dentistry of New Jersey
Robert Wood Johnson Medical School
Piscataway, New Jersey 08854
2
Division for Life Science Technology
National Institute of Advanced Industrial Scienceand Technology
Ikeda, Osaka 563-8577
Japan
3
Section on Synapse Development & Plasticity
National Institute of Child Health& Human Development
National Institutes of Health
Bethesda, Maryland 20892

Summary
Axon guidance requires signal transduction of extracellular cues through the plasma membrane fordirectional motility. Here we present evidence thatcholesterol- and sphingolipid-enriched membrane microdomains (lipid rafts) mediate specific guidance responses of nerve growth cones. Disruption of lipid
rafts by various approaches targeting cholesterol organgliosides selectively abolished growth cone attraction and repulsion in BDNF and netrin-1 gradients,
respectively, without affecting glutamate-induced attraction. Interestingly, local raft disruption on one sideof the growth cone in bath BDNF or netrin-1 produced
opposite turning responses to that induced by thegradients. Raft manipulation also blocked Semaphorin
3A-induced growth cone repulsion, inhibition, andcollapse. Finally, guidance responses appeared to
involve raft-dependent activation of p42/p44 MAPKand ligand-induced receptor recruitment to lipid rafts.
Together with the observation of asymmetric receptor-raft associations at the growth cone in guidance
gradients, our findings indicate that localized signaling
through membrane rafts plays a role in mediating guidance actions of extracellular cues on developing axons.
Introduction
During development, axonal growth is regulated andguided by extracellular cues that elicit permissive/
attractive and inhibitory/repulsive effects on the directional motility of axonal growth cones (Tessier-Lavigneand Goodman, 1996). In adult nervous systems, inhibitory/repulsive molecules present a major obstacle fornerve regeneration after injury and diseases (McKerracher and Winton, 2002). Thus, understanding signaltransduction mechanisms underlying specific actions ofthese extracellular cues on nerve growth and guidance*Correspondence: james.zheng@umdnj.edu

will not only enhance our understanding of brain development but also provide potential strategies to combat growth inhibition of regenerating nerve fibers. For
many guidance cues, the formation of ligand-receptor
complexes on the plasma membrane represents thefirst step in signal transduction, followed by additional
events such as receptor oligomerization and complex
formation with coreceptors and/or other membraneassociated components (Huber et al., 2003). For example, it was recently shown that cross-talk between different guidance receptors specifically enables a particularresponse while silencing another (Stein and TessierLavigne, 2001). The fact that these important eventsoccur at or within the plasma membrane suggests thatthe membrane lipid environment is crucial for signaltransduction of these extracellular cues. Whether or nota specific lipid environment of the plasma membraneplays a role in guidance signaling, however, remains
unknown. While the membrane components involved inreceptor-signaling complexes are currently being identified, how these receptors and other components interact on the membrane to generate specific signaling cascades for distinct axonal responses remains elusive.
Recent studies indicate that the plasma membrane
contains dynamic microdomains that are enriched withcholesterol and glycosphingolipids, resistant to cold detergent extraction, and of lower buoyant density thanbulk plasma membrane (Brown and London, 1998; Simons and Toomre, 2000). Known collectively as lipid
rafts, these membrane domains are also enriched withcertain membrane proteins such as caveolins, src-family
kinases, and glycosylphosphatidylinositol (GPI)-anchoredproteins (Brown and London, 1998; Simons and Toomre,
2000). Although only a limited number of transmembraneproteins are known to associate with lipid rafts, they areof significance as many are receptors for extracellularsignals, including those involved in axon elongation andguidance (Tsui-Pierchala et al., 2002). It is believed that
lipid rafts provide suitable microenvironments to enable
selective protein-protein interactions as well as local
initiation of signal transduction (Simons and Toomre,
2000; Tsui-Pierchala et al., 2002). An increasing numberof recent studies indicate that lipid rafts play importantroles in many cellular responses to extracellular signals,
including growth factor effects and immune responses(Brown and London, 1998; Simons and Toomre, 2000).
So far, only a few guidance molecules and receptors
have been examined and shown to associate with lipid
rafts, and there is no direct evidence indicating a majorrole for lipid rafts in axon guidance (Tsui-Pierchala etal., 2002). In this study, we examined the role of lipid
rafts in chemotropic guidance of nerve growth cones bydiffusible gradients of brain-derived neurotrophic factor(BDNF), netrin-1, Semaphorin 3A (Sema3A), and glutamate. Using an in vitro functional assay for guidance
responses combined with complementary approachesof raft manipulation, we show that distinct guidance
responses of nerve growth cones to BDNF, netrin-1, andSema3A, but not glutamate, depend on the integrity oflipid rafts. We further provide data that local signalingNeuron52

Figure 1. Lipid Raft Disruption by Cholesterol Manipulation Selectively Abolishes
BDNF-Induced Growth Cone Attraction(A) DIC (left) and fluorescence (right) imagesof cultured Xenopus growth cones labeledwith filipin for visualizing membrane cholesterol, without (⫺) and with (⫹) MCD treatment(5 mM, 30 min). Scale bar equals 10 ␮m.
(B) Growth cone turning in BDNF gradients
without (top pair) or with (bottom pair) 5 mM
MCD in bath saline. Digits depict the timeafter the onset of the BDNF gradient. P indicates the BDNF pipette.
(C) Superimposed traces of the trajectory ofneurite extension during the 30 min turning
assay of 15 representative cells for each condition. The origin is the center of the growthcone at the onset of the gradient; the original
direction of growth cone extension was vertical. Arrows indicate the direction of the gradient.
(D) Growth cone attraction induced by glutamate gradients without (top) and with (bottom) 5 mM MCD in the bath.
(E) Cumulative histograms showing the distribution of the turning angles for each condition. Each point represents the percentage of growth cones with final turning angles equal to orless than the values indicated on the abscissa. Top: turning induced by BDNF gradients; bottom: turning induced by glutamate gradients.
(F) Average turning angles (top) and net extension (bottom) of different groups of all the growth cones examined. Numbers indicate the total
number of growth cones examined for each condition.
Scale bars in (B)–(D) equal 20 ␮m. Abbreviations: CTRL, control medium; FLP, filipin; GLU, glutamate. Treatments were added to the bath
20–30 min prior to either fixation (A) or turning assays (C–F). MCD and FLP were used at 5 mM and 0.1 ␮g/ml, respectively. Asterisks indicate
significance when compared to the control (p ⬍ 0.01, Mann-Whitney test).

through lipid rafts underlies growth cone responses tothese guidance cues. Finally, we demonstrate a guidance cue-induced recruitment of specific receptors tolipid rafts and asymmetric receptor-raft association on
the growth cone surface in guidance gradients. Thesefindings thus indicate a functional role for lipid rafts inmediating localized signal transduction during growthcone guidance.
Results
Disruption of Lipid Rafts Selectively Blocks
Growth Cone Attraction by BDNF
Embryonic Xenopus neurons cultured on laminin wereused in the turning assay to examine chemotropic responses of growth cones to a number of guidance cues
(Guirland et al., 2003). We manipulated the integrity oflipid rafts using methyl-␤-cyclodextrin (MCD), a watersoluble cyclic oligomer that extracts cholesterol fromthe plasma membrane (Simons and Toomre, 2000). Incubation of Xenopus neurons with a relatively low concentration of MCD (5 mM) for 30 min largely depleted membrane cholesterol without significantly affecting the
growth cone motility and morphology, as examined bylight microscopy and fluorescent staining of membranecholesterol with filipin, a polyene antibiotic that binds
to membrane cholesterol (Figure 1A; Robinson and Karnovsky, 1980). We first investigated the effects of cholesterol extraction by MCD on BDNF-induced growthcone attraction using the standard in vitro turning assay(Ming et al., 1997; Song et al., 1997). We found thata gradient of BDNF (50 ␮g/ml in the ejection pipette,
ⵑ50 ng/ml reaching the growth cone) induced marked

attraction of Xenopus growth cones within 30 min of
the gradient application, whereas acute MCD treatment
abolished the attractive response (Figure 1B). Composite traces of the trajectories of 15 randomly selected
growth cones from each group further confirm the abolition of BDNF-induced attraction by MCD treatment (Figure 1C). While the majority of growth cones exposed to
the BDNF gradient extended toward the BDNF pipette,
MCD-treated growth cones did not exhibit any directional preference in the BDNF gradient (Figure 1C). The
elimination of BDNF-induced attraction by MCD raft disruption is best depicted by the cumulative distributionsof turning angles (Figure 1E): growth cones exposed
only to BDNF gradients displayed a distribution shiftedto the positive territory, while MCD-treated growthcones exhibited a distribution overlapped with that of
the control group. The effects of MCD raft disruption on
BDNF guidance are summarized by the average turning
angles (Figure 1F) of all growth cones. While the BDNF
gradient induced significant attraction relative to thecontrol group (average turning angles ⫾ SEM, 13.2⬚ ⫾3.7⬚ and 0.6⬚ ⫾ 4.4⬚, respectively; p ⬍ 0.01, MannWhitney test), no preferential turning was observed for
growth cones treated with MCD (average turning angles,
0.9⬚ ⫾ 3.1⬚; p ⬎ 0.5, Mann-Whitney test). To assurethat the blockade of BDNF-induced attraction is not an
artifact of membrane cholesterol depletion by MCD, wealso disrupted lipid rafts using filipin, which is structurally different from MCD and does not deplete cholesterol
from the membrane (Simons and Toomre, 2000). Consistently, BDNF-induced growth cone attraction was alsoabolished by bath application of 0.1 ␮g/ml filipin (Figures
1C, 1E, and 1F). Statistical comparison of turning angles
among the control, MCD-treated, and filipin-treatedAxon Guidance Depends on Lipid Rafts for Signaling
53

groups shows no difference (p ⬎ 0.5, Mann-Whitneytest), further confirming the effective elimination of
the BDNF attractive effects by either MCD or filipin
treatment.
To determine that MCD or filipin abrogation of BDNFinduced attraction is not due to their nonspecific actions
on growth cone motility, we examined growth cone turning induced by glutamate gradients since it is mediatedby Ca2⫹ signals through NMDA channels (Zheng et al.,1996), which are not associated with lipid rafts (Suzuki
et al., 2001; Wu et al., 1997). Consistently, a glutamate
gradient (50 mM in pipette, ⵑ50 ␮M reaching the growthcone) induced marked attraction of Xenopus growthcones on laminin. Unlike BDNF, however, glutamateinduced attraction was not affected by cholesterol extraction using 5 mM MCD as evidenced by composite
traces (Figure 1D), cumulative distributions (Figure 1E),
and average turning angles (Figure 1F). Both groups
exposed to glutamate gradients (⫺MCD and ⫹MCD)
exhibited similar average turning angles (15.0⬚ ⫾ 4.4⬚
and 13.5⬚ ⫾ 4.9⬚, respectively; p ⬎ 0.5, Mann-Whitneytest), which are significantly different from that of thecontrol (p ⬍ 0.01). These results provide direct evidence
that lipid raft disruption by cholesterol manipulation did
not impair the growth cone’s ability to advance, sense,
and respond to guidance gradients per se, but selectively blocked BDNF-induced attraction. Taken together,our data indicate that the integrity of lipid rafts is required for chemoattractive effects of BDNF gradients.
Growth Cone Guidance by Netrin-1 Depends
on Lipid Rafts
Netrin-1 is a well-established guidance cue that attracts
as well as repels nerve growth cones in vivo (TessierLavigne and Goodman, 1996), so we examined the raftinvolvement in netrin-1 guidance in vitro. Under thesame culture and assay conditions as above, netrin-1
gradients (5 ␮g/ml in pipette, ⵑ5 ng/ml at the growthcone) significantly repelled Xenopus growth cones onlaminin (Figure 2A), which is consistent with previous
reports that young Xenopus spinal neurons were repelled by netrin-1 (Buck and Zheng, 2002; Ming et al.,2001) and laminin favored netrin-1 repulsion for Xenopus
retinal neurons (Hopker et al., 1999). The cumulative
distribution of turning angles shows that a larger percentage of growth cones exhibit negative turning angles(Figure 2C), producing an average of ⫺18.0⬚ ⫾ 5.4⬚ (Figure 2D; p ⬍ 0.01, compared to the control group shownin Figure 1). However, bath application of 5 mM MCD
completely abolished the repulsion (Figure 2B), resulting
in an angle distribution centering at zero (Figure 2C) andan average of 2.3⬚ ⫾ 5.0⬚ (Figure 2D; p ⬎ 0.5, comparedto the control group). We also found that bath application of 0.1 ␮g/ml filipin blocked the growth cone repulsion induced by netrin-1 gradients (Figures 2C and 2D).
The net neurite extension was not significantly affectedby either MCD or filipin treatment (p ⬎ 0.5). Based onthese findings, we conclude that the integrity of lipid
rafts is also crucial for netrin-1 signaling in growth cones.
Together with the inhibition of BDNF-induced attraction
by lipid raft disruption, our data indicate that cholesterol-enriched membrane domains, i.e., lipid rafts, play
an important role in both attractive and repulsive guid-Figure 2. Growth Cone Turning Induced by Netrin-1 Depends on
Lipid Raft Integrity(A and B) Representative images showing Xenopus growth conesat the onset and the end of 30 min exposure to a netrin-1 gradient(5 ␮g/ml in pipette) without (A) and with (B) 5 mM MCD in bath.
Dashed lines represent the corresponding positions of the growthcone at the onset of the turning assay. Scale bars equal 20 ␮m.
(C) The cumulative histogram depicts the responses to netrin-1 gradients without or with lipid raft disruption by 5 mM MCD or 0.1 ␮g/
ml filipin.
(D and E) Average turning angles (D) or net extension (E) for eachcondition are depicted by bar graphs, and the total numbers ofgrowth cones examined for each condition are indicated in (D).
Asterisks indicate significance over the control (p ⬍ 0.01, MannWhitney test).

ance of nerve growth cones by BDNF and netrin-1, respectively.
Further Evaluation of Raft Dependence of GrowthCones’ Responses to BDNF and Netrin-1
The above findings were all based on cholesterol manipulation for lipid raft disruption. Cholesterol extraction
by MCD was shown to partially release certain protein
components from the plasma membrane (Ilangumaran
and Hoessli, 1998), which could contribute to the blockade of BDNF and netrin-1 guidance reported above.
Although our immunostaining found no detectablechange in the level of either TrkB or DCC receptors at
the growth cone after MCD cholesterol extraction (datanot shown), we performed additional experiments toaddress this issue. We first performed a “rescue” experiment in which Xenopus neurons were depleted of cholesterol by a 30 min MCD treatment followed by readdition of a low concentration of cholesterol (cholesterol/
MCD complex) and then turning assays. We found that
readdition of 20 ␮M cholesterol (balanced by 160 ␮M

Neuron54

Figure 3. Growth Cone Responses to BDNF or Netrin-1 Selectively
Depend on Membrane Lipid Environment(A) Cumulative histograms depict the turning responses of Xenopus
growth cones in BDNF, netrin-1, or glutamate gradients under the“rescue” condition (top) or exogenous GM1 application (bottom). The“rescue” experiments involved 30 min cholesterol extraction by 5
mM MCD followed by addition of 20 ␮M cholesterol (with 160 ␮M
MCD). Res-ctrl: growth cones exposed to 20 ␮M cholesterol (with160 ␮M MCD) without prior extraction. For GM1 experiments, 20 ␮M
GM1 was added to the bath medium 30 min before the turning assays.
(B) Average turning angle and net extension of each condition are
depicted by the bar graph, and the numbers denote sample sizefor each experiment. Asterisks indicate significance over the control
group (p ⬍ 0.01, Mann-Whitney test). MDC (monodansylcadaverine)
was used at 50 ␮M.

MCD) immediately restored BDNF-induced attraction(Figure 3). The cumulative distribution of turning angles
of “rescued” growth cones essentially overlaps with thatof untreated growth cones in the BDNF gradient, bothshowing marked attraction (Figure 3A). The averageturning angle of cholesterol-rescued growth cones to
BDNF gradients is 11.1⬚ ⫾ 4.7⬚, which is significantly
different from the control group (p ⬍ 0.05) but similar
to the growth cones exposed only to BDNF gradients(p ⬎ 0.5). Similarly, we found that netrin-1-induced repulsion was also restored in the similar rescue experiments(Figures 3A and 3B). As an important control, 20 ␮Mcholesterol (balanced by 160 ␮M MCD) itself did notaffect normal turning responses to BDNF and netrin-1,
respectively (marked as “res-ctrl,” Figure 3B), further
supporting the specificity of the rescue experiments.
These results thus indicate that cholesterol extraction
blocked the guidance effects of BDNF and netrin-1 bymainly affecting the integrity of lipid microenvironments,
rather than by stripping away guidance receptors andother protein components from the plasma membrane.
While the above results clearly indicate the involvement of membrane cholesterol in growth cone guidance,
the importance of lipid raft integrity is further addressed
through a second set of complementary experiments
by targeting a different lipid constituent of rafts, thegangliosides. It was previously shown that applicationof exogenous ganglioside GM1 affects the integrity andstability of lipid rafts such that raft-associated proteinsare excluded from raft microdomains (Simons et al.,1999). When we added 20 ␮M GM1 to the bath medium
prior to the turning assays, BDNF-induced attraction
and netrin-1-induced repulsion were all abolished (Figure 3). Statistical analysis shows clearly that exogenous
GM1 blocked turning responses of Xenopus growthcones to BDNF and netrin-1 gradients, respectively (p ⬎0.5 comparing to the control; p ⬍ 0.01 comparing torespective BDNF and netrin-1 groups without raft manipulation). The selectivity of exogenous GM1 on blocking
guidance responses to BDNF and netrin-1 was confirmed by the ineffectiveness of GM1 to block glutamateinduced growth cone attraction (Figure 3). These results
therefore indicate that BDNF and netrin-1 guidance
effects depend on lipid rafts. Finally, we have excluded
the involvement of clathrin-mediated endocytosis inraft-dependent chemotropic guidance by BDNF andnetrin-1. We found that monodansylcadaverine (MDC,
50 ␮M), a potent inhibitor of clathrin-mediated endocytosis, did not affect BDNF-induced attraction and netrin1-induced repulsion (Figure 3B). Taken together, resultsfrom these complementary studies strongly support thenotion that lipid rafts play an essential role in mediating
growth cone guidance by BDNF and netrin-1.
Although MCD or filipin treatment abolished BDNFinduced attraction, the growth-promoting effect of
BDNF appeared to be largely unaffected. MCD- or filipintreated growth cones in BDNF gradients still extended
more than those exposed to control medium (Figure 1F).
On average, growth cones exposed to BDNF gradientswith or without cholesterol manipulation extended
ⵑ150% of that of the growth cones in control. However,
the growth-promoting effects appeared to be abolishedby exogenous GM1 (Figure 3B), implicating a difference
between cholesterol extraction and GM1 addition on lipid
rafts. To further verify this observation, we performeda growth assay in which randomly selected, isolated
neurons were first monitored for 1 hr to determine thenet extension of each growth cone. These cells werethen exposed to 50 ng/ml BDNF with or without 5 mM
MCD or 20 ␮M GM1 for another hour to assess the extension of the same growth cones. For each growth cone,
the net extension of the second hour was normalizedagainst that of the first hour to yield the growth ratio (inpercentage). Consistently, BDNF exposure significantly
increased the neurite extension to 149% ⫾ 21% (n ⫽16) of that in the control hour. Similar enhancement by
BDNF was also observed for MCD-treated growth cones(158% ⫾ 22%, n ⫽ 14), but not GM1-treated cells (97% ⫾13%; n ⫽ 10). Growth cones exposed to the control
medium or medium containing MCD or GM1 without
BDNF exhibited no effects on neurite extension (106% ⫾12%, n ⫽ 15; 93% ⫾ 14%, n ⫽ 15; and 111% ⫾ 11%,Axon Guidance Depends on Lipid Rafts for Signaling55

bath, focal MCD application resulted in marked repulsion, contrasting with the attraction induced by a BDNF
gradient (Figures 4A and 4B). Conversely, when 5 ng/ml
netrin-1 was present in the bath, focal MCD application
resulted in a significant attractive turning response (Figures 4A and 4B), which is opposite to the repulsion
induced by netrin-1 gradients. No difference in neurite
extension was observed for all three groups (Figure 4C),
further indicating that local MCD application did notaffect the growth cone’s normal motility. These results
support the notion that asymmetric receptor activation
and/or local signaling through lipid rafts underlie growthcone turning responses to guidance gradients.

Figure 4. Turning Responses Induced by Local Lipid Raft Disruptionin Uniform Guidance Cues
A pipette containing 400 mM MCD (P ) was placed 50 ␮m from thegrowth cone for focal lipid raft disruption (5 ms pulse duration). The
growth cones were exposed to bath medium (B ) containing either
BDNF (50 ng/ml) or netrin-1 (5 ng/ml).
(A) The cumulative histogram depicts the responses of growth cones
exposed to local MCD with bath medium containing control or BDNF
or netrin-1.
(B and C) Average turning angles (B) or net extension (C) for eachcondition are depicted by bar graphs, and numbers in (B) indicate
the total number of growth cones examined for each condition.
Asterisks indicate significance when compared to the control (p ⬍0.01, Mann-Whitney test).

n ⫽ 15, respectively). Statistical analysis using the Student’s t test confirms that BDNF significantly increases
the extension of Xenopus growth cones regardless of
MCD treatment (p ⬍ 0.05). However, such enhancementof extension was eliminated by exogenous GM1 addition(p ⬎ 0.5 comparing to the control groups). These results
thus suggest that different aspects of lipid rafts (cholesterol-sensitive and GM1-sensitive) may mediate these
distinct effects of BDNF on the growth cone.
Local Signaling through Lipid Rafts Mediates
Growth Cone Turning
Chemotropic turning of growth cones in diffusible gradients likely involves asymmetric activation of membrane
receptors and localization of intracellular signaling(Hong et al., 2000; Zheng, 2000; Zheng et al., 1994),
which may depend on membrane rafts. We thus reasoned that uniform application of these guidance cuesin the bath followed by local disruption of lipid rafts
on one side of the growth cone would also create an
asymmetry in surface receptor activation and downstream signaling across the growth cone. Importantly,
however, such asymmetry would be opposite to thatinduced by the gradient of the same guidance molecule,thus leading to an opposite turning response. We tested
this hypothesis by using a focal application method(Buck and Zheng, 2002) to largely restrict MCD to one
side of the growth cone. Focal MCD application(400 mM in pipette, ⵑ1 mM reaching the growth cone)in the absence of any guidance molecules in the bathdid not affect the direction or the extension rate (Figure4). However, when 50 ng/ml BDNF was present in the

Lipid Rafts also Mediate Semaphorin 3A Effects
on Growth Cones
We also examined turning responses of Xenopus neurons to another well-known guidance cue: Semaphorin
3A/collapsin-1 (Sema3A) (Kolodkin et al., 1993). We
found that Sema3A gradients exhibited marked inhibition on the extension of Xenopus growth cones during
turning assays. The majority of growth cones that did
advance in Sema3A gradients exhibited marked repulsion, whereas growth cones exposed to pipette application of the vehicle control solution showed no directional
preference (Figure 5A). The average turning angles fromthese two groups highlight the significant repulsion induced by Sema3A (Figure 5B; average angles: ⫺18.8⬚ ⫾4.1⬚ and 1.1⬚ ⫾ 4.3⬚ for the Sema3A and control groups,
respectively; p ⬍ 0.01). Bath application of 5 mM MCD
effectively blocked Sema3A-induced repulsion (Figure
5A), resulting in an average turning angle of 6.0⬚ ⫾ 3.9⬚
(Figure 5B; p ⬎ 0.1 when compared to the control vehicle, Mann-Whitney test). Since only growth cones that
extended during the 30 min assay were scored, theneurite extension from our turning assays (Figure 5C)
did not reflect Sema3A inhibition on growth cone motility. We therefore performed the growth assay describedabove to evaluate the role of lipid rafts in growth inhibition by Sema3A. We found that bath application of
Sema3A, not the vehicle, significantly inhibited growthcone extension, but such inhibition was largely removed by MCD treatment (Figure 5D). Since Collapsin-1/
Sema3A was initially identified for its ability to collapse
growth cones, we next examined if lipid rafts are also
involved in Sema3A-induced growth cone collapse. Because embryonic Xenopus spinal neurons in culture do
not exhibit drastic growth cone collapse in response to
Sema3A, we used growth cones from basilar pontine
explants in culture (Rabacchi et al., 1999) for collapse
assays. Consistently, bath application of Sema3A resulted in marked collapse of pontine growth cones asevidenced by the loss of actin-rich lamellipodia and filopodia (Figure 5E). About 80% of the growth cones exhibited collapsed morphology after Sema3A exposure,compared to about 20% in the group exposed to thevehicle (p ⬍ 0.01, Mann-Whitney test). Disruption of lipid
rafts by 1 ␮g/ml filipin (added 25 min prior to the Sema3A
exposure), however, diminished growth cone collapse
induced by Sema3A, resulting in a similar collapse percentage to that of the control (Figure 5E; p ⬎ 0.1, MannWhitney test). We could not use MCD for raft disruption
on pontine growth cones, as they were susceptible toNeuron56

duced a large increase in p42/p44 phosphorylation,
whereas no change in the total p42/p44 level was observed using a different antibody that recognizes total
p42/p44 (Figures 6A and 6B). Treatment of Xenopus
neurons with 5 mM MCD or 20 ␮M GM1 largely diminishedthe guidance cue-induced increase in p42/p44 phosphorylation without affecting the total p42/p44 level at
the growth cone (Figures 6A and 6B). We also observed
an asymmetric increase of p42/p44 phosphorylation
across the growth cone subjected to the BDNF gradient,suggesting its role in growth cone turning (Figure 6C).
We thus used a specific inhibitor PD98059 to block p42/
p44 activation and examined the requirement of p42/
p44 activation in the turning response. Consistent withprevious findings (Campbell and Holt, 2003; Forcet
et al., 2002; Ming et al., 2002), application of 10 ␮M
PD98059 (30 min before the turning assay) largely
blocked the turning responses to gradients of BDNF,
netrin-1, and Sema3A, but did not affect glutamateinduced attraction (Figure 6D). These parallel results
point to the involvement of raft-dependent p42/p44 activation in guidance responses of growth cones to BDNF,
netrin-1, and Sema3A.

Figure 5. Lipid Rafts Mediate Sema3A-Induced Repulsion, Growth
Inhibition, and Growth Cone Collapse(A) The cumulative histogram shows the distribution of turning
angles for growth cones exposed to control vehicle or Sema3A
gradients with or without MCD treatment.
(B and C) The average turning angles (B) and net extension (C) for
each group are presented in the bar graphs. Numbers in (B) indicate
the total number of growth cones examined for each condition.
(D) Growth inhibition by bath application of Sema3A as analyzed by
the growth assay.
(E) Sema3A-induced collapse of pontine growth cones. Pontine explant cultures were exposed to control vehicle or Sema3A withor without filipin treatment. The fluorescent images illustrate therepresentative growth cone actin cytoskeleton resulting from eachcondition. The scale bar equals 10 ␮m.

damage during MCD exposure. Taken together, theseresults indicate that lipid rafts mediate the inhibitory
actions of the guidance molecule Sema3A in both Xenopus spinal neurons and mammalian CNS neurons.
Raft-Dependent Activation of p42/p44 MAPKin Guidance Responses
Our findings that lipid rafts mediate growth cone responses suggest a role for lipid rafts in signal transduction of guidance molecules. We therefore examined theraft dependence of p42/p44 MAPK activation by thesecues (Campbell and Holt, 2003; Forcet et al., 2002; Ming
et al., 2002). Using a specific antibody against phosphop42/p44 and quantitative fluorescence imaging, wefound that a brief exposure of Xenopus growth cones
to BDNF, netrin-1, and Sema3A, but not glutamate, in-

Association of Guidance Receptors
with Lipid Rafts
The above findings make a strong case that lipid rafts
are functionally involved in growth cone guidance andsuggest that receptors for BDNF, netrin-1, and Sema3A,
but not glutamate, are associated with lipid rafts anddependent on them for functioning. To test this possibility, we examined whether the BDNF receptor TrkB andthe netrin-1 receptor DCC are localized in lipid rafts
using standard membrane fractionation and Western
blot methods (Kawabuchi et al., 2000). Due to the limited
number of spinal neurons in Xenopus cultures, we used
relatively pure cortical cultures from embryonic rats forthe membrane fractionation experiments (Ma et al.,
2003; Suzuki et al., 2001). We detected a small but prominent portion of DCC and TrkB in the lipid raft fraction
as indicated by the lipid raft marker caveolin-2 (Cav-2;
Figure 7A, lane 1; Simons and Toomre, 2000). Interestingly, a brief exposure of cortical neurons to BDNF
(200 ng/ml, 30 min) before fractionation resulted in a
noticeable increase in the amount of TrkB in the twomost buoyant fractions, including the lipid raft fraction(Figure 7A). Netrin-1 exposure (200 ng/ml, 30 min) also
increased the amount of DCC in the lipid raft fraction,although the increase was relatively small. Moreover,netrin-1 exposure increased the levels of DCC in fractions two through four (Figure 7A, lanes 2–4), suggestinga netrin-1-dependent translocation of DCC toward
buoyant partitions. Collectively, these results indicatethat guidance cues induced further association of their
receptors with lipid rafts (Ma et al., 2003; Paratcha et al.,2001). Since this biochemical assay relies on membrane
fractions from whole-cell lysates, it lacks the sensitivity
to detect receptor-raft association at the growth cone.
We thus used a more sensitive and well-establishedfluorescence copatching assay to examine raft association of specific guidance receptors on the surface of
Xenopus growth cones (Harder et al., 1998; Simons andToomre, 2000). This copatching method is based on

Axon Guidance Depends on Lipid Rafts for Signaling
57

Figure 6. Raft-Dependent Activation of p42/
p44 MAPK in Growth Cone Guidance(A) Representative fluorescence images of
Xenopus growth cones stained with a specificantibody against phospho-p42/p44. The growthcones were exposed to either control medium(Ctrl), netrin-1 (N-1), or Sema3A (S3A), without(⫺) or with (⫹) MCD treatment. Scale bar
equals 10 ␮m.
(B) Levels of phospho-p42/p44 and total p42/
p44 in Xenopus growth cones induced by different guidance cues, with or without lipid raft
disruption by bath application of MCD or GM1.
Numbers denote the growth cones examinedin each condition. Asterisks indicate statistical significance (p ⬍ 0.01, Student’s t test).
(C) Local application of BDNF induced asymmetric activation of p42/p44. The top pairshows a Xenopus growth cone exposed to
the BDNF gradient (arrows) for 5 min, fixed,
and immunostained for total p42/p44 (T) andphospho-p42/p44 (P). For visualization, theintensity of the fluorescence images is codedby 8-level pseudocolors (see the color bar).
The bottom pair shows a control growth cone
without BDNF application.
(D) Inhibition of p42/p44 by PD98059 blocked
turning responses. The average turning
angles and neurite lengths are displayed as
bar graphs. Numerals indicate the number ofgrowth cones examined in each group.

the physical attraction among raft components whereby
membrane proteins associated with lipid rafts can becrosslinked by antibodies to form patches that colocalize with raft patches formed by crosslinking GM1 gangliosides using cholera toxin B subunit (CTxB) (Harder etal., 1998). The raft association of a particular receptor
can then be quantified by the percentage of receptor
patches colocalizing with CTxB patches. We examined
four receptors using this copatching method: NMDA
receptor subunit 2B (NR2B), TrkB, DCC, and neuropilin-1(NPN-1). We found that most patches of NR2B receptors
did not colocalize with CTxB patches (Figures 7B and
7C), indicating the lack of raft association of NR2B (Suzuki et al., 2001; Wu et al., 1997). We therefore considered the percentage of NR2B patches colocalized with
CTxB patches (ⵑ10%) as the baseline of the copatching
method for significant raft association in our culture
system. Consistent with the Western blot data (Figure
7A), only a small percentage of DCC, TrkB, and NPN-1
were associated with lipid rafts before ligand exposure(Figure 7C). However, a brief exposure to specific guidance cues (5 min) induced marked increase in the colocalization percentage of TrkB, DCC, and NPN-1 withraft patches (Figure 7C). In contrast, NMDA receptor
patches did not exhibit any changes in colocalization
with lipid rafts. These results illustrate that BDNF,
netrin-1, and Sema3A, but not glutamate, increased theassociation of their respective receptors with lipid rafts
and suggest the existence of ligand-induced receptor
recruitment to lipid microdomains (Hering et al., 2003;
Ma et al., 2003).

One immediate implication of the ligand-induced receptor-raft association is the generation of localized
signaling that underlies directional responses of nerve
growth cones in guidance gradients. A guidance gradient likely elicits more receptor-raft association on thenear side of the growth cone where it is exposed to ahigher concentration of the cue, thus creating an asymmetry for downstream signaling. To test this possibility,
we used an in situ cold detergent extraction method toisolate membrane rafts and associated receptors on thegrowth cone. By double labeling raft marker GM1 and thespecific receptor, we examined the spatial distributionof receptor-raft association on the growth cone. We
selected BDNF for this study since our Western blot andcopatching data showed a strong increase in TrkB-raftassociation after BDNF exposure. The growth cone was
exposed to 3 min BDNF gradient and rapidly detergentextracted and fixed by a prechilled solution containing
Triton X-100 and fixative. The TrkB receptors were then
immunostained and lipid rafts were labeled by staining
GM1 with FITC-CTxB. Without cold detergent extraction,
both GM1 and TrkB staining appeared to be homogenous
across the growth cone surface (Figure 8A). Growthcones subjected to the cold detergent extraction displayed punctate staining of TrkB and GM1 and, mostimportantly, almost all the TrkB puncta overlapped with
GM1 staining, indicating that these are detergent-resistant raft-associated TrkB receptors (hereafter referred
to as TrkB-raft). For growth cones not exposed to the
BDNF gradient, the TrkB-raft distribution appeared tobe symmetric across the growth cone surface (Figure

Neuron58

Figure 7. Association of Guidance Receptors with Lipid Rafts(A) Immunoblots of membrane fractions fromrat cortical neurons were probed with antibodies against TrkB or DCC. Lane numbers
indicate the membrane fractions where 1 and
5 are the relative top to bottom fractions, respectively. Lane 1 represents the lipid raft
fraction as indicated by the raft marker caveolin-2 (Cav-2), and lane 5 represents thenonraft fraction as indicated by transferrin receptors (TfR).
(B) Fluorescent copatching in Xenopus neurons. Representative fluorescent images
show Xenopus growth cones with lipid raft
patches induced by FITC-cholera toxin B
subunit (CTxB, green) and receptor patches
induced by antibodies against NR2B receptors (top) or DCC receptors (bottom). The receptor patches were further labeled by secondary antibody conjugated with Cy3 (red).
Arrows indicate colocalized patches that appear yellow/orange. Scale bar equals 10 ␮m.
(C) Quantification of raft association of four
different receptors by the copatching method
on Xenopus neurons with or without ligand
application. Asterisks indicate statistical significance when comparing between thegroups without and with ligand exposure (p ⬍0.01, Student’s t test).

8B). However, 3 min exposure of growth cones to the
BDNF gradient resulted in a significant increase of TrkBraft labeling on the near side of the growth cone relative
to the far side (Figure 8C). Quantitative analysis of TrkBraft staining on two sides of the growth cone revealed a
2:1 difference in TrkB-raft distribution (Figure 8D). Theseobservations provide evidence that asymmetric receptor-raft association is induced by the guidance gradientand suggests a role for receptor-raft association in localized signaling in the growth cone during guidance responses.
Discussion
Increasing evidence suggests that cells utilize dynamic
membrane microdomains (lipid rafts) to provide an ordered lipid microenvironment for specific molecular interactions between membrane receptors and distinct
intracellular signaling components (Brown and London,
1998; Paratcha and Ibanez, 2002; Simons and Toomre,
2000; Tsui-Pierchala et al., 2002). Here we present evidence that lipid rafts selectively mediate growth coneguidance by BDNF, netrin-1, and Sema3A. This conclusion is based on results from the functional guidance
assays coupled with lipid raft manipulation as well asbiochemical and imaging studies on receptor-raft association. Several lines of evidence further support the
selectivity of our raft manipulations in blocking guidance
responses. First, acute cholesterol manipulation by two
different molecules, MCD and filipin, did not drasticallyaffect the growth cone’s morphology or motility on thelaminin substrate. These results also imply that growthcone motility on laminin (through ␤1 integrin) does notdepend on lipid rafts (Nakai and Kamiguchi, 2002). Second, the consistent turning responses that resulted

shortly after cholesterol rescue argue against the possibility that cholesterol extraction depleted membrane receptors and other proteins to affect guidance. Third,
focal MCD application (in the absence of guidance cues)
did not affect growth cone extension, further indicatingthat MCD does not influence general aspects of steering.
Fourth, the abolishment of guidance responses by exogenous GM1 implicates the lipid microenvironment, notcholesterol itself, in mediating guidance responses. Finally, the fact that Xenopus growth cones subjected tolipid raft disruption still responded attractively to glutamate gradients provides, arguably, the best evidence
that lipid rafts selectively mediate guidance actions of
BDNF, netrin-1, and Sema3A.
Our examination of p42/p44 MAPK activation by
BDNF, netrin-1, and Sema3A supports the notion that
lipid rafts are involved in signal transduction. We foundthat either MCD or GM1 attenuated p42/p44 phosphorylation induced by these molecules. In particular, the increase in p42/p44 phosphorylation induced by netrin-1
and Sema3A was completely blocked by MCD or GM1.
BDNF-induced p42/p44 phosphorylation, on the other
hand, was only reduced by MCD cholesterol extraction
but effectively abolished by GM1 addition. While the exact
mechanism underlying the difference between MCD and
GM1 on inhibiting p42/p44 phosphorylation elicited by
BDNF is not clear, these findings are consistent with
MCD and GM1 effects on BDNF promotion of neurite
extension. Further elucidation of the precise signaling
pathways underlying these two BDNF effects would provide important insights toward the differential dependence on lipid rafts. Nevertheless, our results, together
with the blockade of turning responses by MAPK inhibitors, suggest that raft-dependent p42/p44 activation underlies chemotropic actions of BDNF, netrin-1, andSema3A.

Axon Guidance Depends on Lipid Rafts for Signaling
59

Figure 8. Asymmetric TrkB Receptor-Raft Association on the
Growth Cone Surface Induced by BDNF Gradients(A) A Xenopus growth cone fixed and double stained for surface
GM1 by FITC-CTxB and TrkB receptors by immunostaining. The DIC
image of the growth cone is shown on the right. Scale bar equals
10 ␮m.
(B) A Xenopus growth cone subjected to in situ cold detergent
extraction and fixation. GM1 staining by FITC-CTxB and TrkB byimmunostaining exhibited a punctate pattern. For quantification,
each fluorescent image was set with a threshold (50% of the maximum intensity of the growth cone), pseudocolored (green for GM1
and red for TrkB), and overlaid on the DIC image. The yellow color
indicates the overlapped area between GM1 and TrkB.
(C) A Xenopus growth cone subjected to the BDNF gradient (arrows)for 3 min before in situ cold detergent extraction/fixation andstaining.
(D) Quantification of overlapped GM1 and TrkB on the two sides of
the growth cone without (control) and with local BDNF exposure.
The ratios are derived from the TrkB-raft colocalized areas on thetwo sides of the growth cone, each normalized against its respective
GM1 staining area.

It is of interest to note that BDNF-elicited neurite extension and chemoattraction appear to depend on lipid
rafts differently. While chemoattractive effects of BDNF
were abolished by lipid raft disruption through MCD
cholesterol extraction, the growth promotion was notaffected, suggesting that growth promotion and chemoattraction are mechanistically separate events (Guirland
et al., 2003; Markus et al., 2002; Ming et al., 1997, 1999).
On the other hand, we find that BDNF-induced growthpromotion was largely abolished by exogenous GM1 addition. BDNF is known to elicit multiple signaling cascades leading to diverse neuronal functions (Kaplan andMiller, 2000; Markus et al., 2002), thus it is conceivablethat signaling cascades underlying chemoattraction andgrowth promotion may depend on different aspects oflipid microdomains on the membrane, which can be
independently altered by cholesterol extraction and GM1
addition. Intriguingly, p42/p44 MAPK appears to be involved in both BDNF chemoattraction and growth promotion but differently affected by MCD and GM1, which
may be attributed to distinct spatiotemporal localization
and/or intensity of MAPK signals. Contrary to the BDNF
findings, we found that the growth inhibition, growthcone collapse, and growth cone repulsion induced bySema3A all depended on lipid rafts and could beblocked by MCD treatment, suggesting a common signaling pathway but with distinct spatiotemporal regulation. For example, local Collapsin-1/Sema3A induced
growth cone repulsion through local collapse of growthcone lamellipodia, while uniform Collapsin-1 caused thecollapse of the whole growth cone and growth inhibition(Fan and Raper, 1995). Recent studies find that Sema3A
signaling involves the formation of a complex consistingof neuropillin-1, plexin A, and the adhesion molecule L1(Castellani and Rougon, 2002; Pasterkamp and Kolodkin, 2003). It has been reported that L1 is associatedwith lipid rafts and plays an important role in growthcone adhesion and migration (Nakai and Kamiguchi,
2002). Our findings thus indicate that lipid rafts mediate
Sema3A signaling for distinct inhibitory actions.
How exactly are lipid rafts involved in the signal transduction of guidance cues? Several nonmutually exclusive models have been proposed (Simons and Toomre,
2000): (1) receptors can strongly associate with lipid
rafts, (2) receptors can be recruited into lipid rafts upon
ligand binding, leading to signal localization and amplification, and/or (3) rafts can be aggregated upon ligand
binding to link particular receptors with distinct raftdependent signaling complexes. Our Western blots andfluorescent copatching data indicate that receptors forthe guidance cues studied here were weakly associatedwith lipid rafts under baseline conditions, but the receptor-raft association was markedly enhanced after stimulation by their respective ligands. It is possible that association of ligand-receptor complexes with lipid rafts
enables the assembly of appropriate signaling complexes for guidance functions (Simons and Toomre,
2000; Tsui-Pierchala et al., 2002). For example, growthcone guidance by BDNF and netrin-1 involves phospholipase C (PLC) and PI-3 kinases (Ming et al., 1999), bothof which associate with lipid rafts (Inoue et al., 2002).
Many nonreceptor tyrosine kinases involved in adhesion
are also associated with lipid rafts (Simons and Toomre,
2000), and active Rho-GTPases are targeted to lipid rafts
for coupling to downstream effectors (del Pozo et al.,
2004; Michaely et al., 1999; Palazzo et al., 2004). Therefore, lipid rafts may mediate growth cone guidance by
providing a critical platform for localized coupling ofactivated receptors and/or their downstream effectors
for cytoskeletal regulation.
A gradient of guidance cues is thought to activate
surface receptors asymmetrically to generate localized
responses that steer the growth cone in a particular
direction. The local raft disruption data (see Figure 4)
provide direct evidence that localized raft-dependent
signaling is sufficient to initiate directional responses ofgrowth cones. Previous studies on chemotactic cells

Neuron60

have shown that the overall distribution of membrane
receptors for specific chemokines remains uniform during chemotaxis and localization of responses is downstream of membrane receptors (Parent and Devreotes,
1999). Exactly how is such localization generated
through uniformly distributed surface receptors? We
propose that an asymmetry in lipid raft association of theguidance receptor, without apparent changes in overall
distribution, represents the first step of signal localization during directional responses. In particular, theasymmetric translocation of guidance receptors into
lipid rafts after ligand binding could lead to local signal
amplification by concentrating signaling molecules and/or excluding unwanted modulatory components (Simons and Toomre, 2000), which might be essential for
successful sensing of extracellular gradients. Our observation of asymmetric TrkB-raft distribution on growthcones exposed to BDNF gradients provides evidence tosupport this local signaling model. Future experiments
involving high-resolution imaging such as fluorescenceresonance energy transfer (FRET) would provide detailed insights into the spatiotemporal interactions ofsignaling components across the growth cone surface
during guidance.
One of the important findings in this study is theinvolvement of lipid rafts in inhibitory and collapsing
effects of Sema3A. One can postulate that lipid rafts
may also be involved in signaling by other inhibitory
molecules. For example, p75 receptors have recently
been identified as the coreceptors for growth inhibitory
molecules including myelin-associated glycoprotein(MAG) and NOGO (Wang et al., 2002a; Wong et al., 2002)
and are known to associate with lipid rafts (Bilderback etal., 1997). Furthermore, MAG has been shown to interact
with specific lipid gangliosides (Vyas et al., 2002), and
the GPI-linked NOGO receptor may partition in lipid rafts
for its functions (Wang et al., 2002b). It is conceivablethat lipid rafts play a major role in MAG/NOGO inhibitionof axonal growth. Since growth inhibition represents amajor barrier for nerve regeneration in the adult nervous
system, our results are of particular interest as they
identify lipid rafts as a potential target for further determination of signaling components responsible for inhibition. Moreover, the finding that disruption of lipid rafts
abolishes Sema3A-induced inhibition suggests that manipulation of lipid raft integrity or individual lipid raftcomponents could be an effective approach to overcoming growth inhibition. Clearly, a deeper understanding of the specific guidance receptors, signaling components, and kinetics with which they are recruited to lipid
rafts to interact with other signaling components would
provide invaluable insights into mechanisms underlying
the precise wiring of complex neuronal connections.
Importantly, such an understanding can contribute tonovel strategies of enhancing nerve regeneration after
injury that specifically rely on manipulation of lipid rafts
and their associated components.

ney test to analyze turning angles since they do not follow a normal
distribution. Focal application of MCD was achieved by a modified
pipette application method (Buck and Zheng, 2002). Glutamate waspurchased from Sigma. Human recombinant BDNF (rHu-met-BDNF)
was generously provided by Regeneron (Tarrytown, NY). Purified
netrin-1 proteins were a gift from Dr. Marc Tessier-Lavigne (Genetech). Sema3A was collected from the supernatant of confluent COS
cells expressing human Sema3A (Kolodkin et al., 1993) and dialyzed
into Ringer’s saline for pipette application. The vehicle control solution was obtained from the supernatant of mock-transfected COS
cells that expressed the same vector lacking the hSema3A insert.
Methyl-␤-cyclodextrin, filipin, and GM1 gangliosides were purchasedfrom Sigma and were added to the bath medium 20–30 min before
the onset of turning assay.
Pontine Explant Culture and Collapse Assay
Pontine nuclei explants were prepared as described previously(Baird et al., 1992). Collapse assays were performed on cultureswith or without filipin pretreatment (1 ␮g/ml, 25 min) by incubatingthem with Sema3A or the vehicle control solutions for 30 min. The
Sema3A stock solution (described above) was added at a 1:20 dilution, which resulted in ⵑ80% collapse of pontine growth cones(Rabacchi et al., 1999). The vehicle stock solution was also used at
the same dilution. Explants were then quickly fixed with 4% paraformaldehyde and 0.25% glutaraldehyde in a cacodylate buffer(Guirland et al., 2003) for 30 min, permeabilized with Triton X-100(0.1%), and labeled with rhodamine-phalloidin (1.5%; Molecular
Probes) for 15 min. Fluorescence imaging was performed on a Nikon
inverted microscope (TE2000) equipped with a 40⫻ Plan Fluor oilimmersion objective (numeric aperture: 1.3), a CCD camera(PXL1400, Roper Scientific), and Axon Imaging Workbench software(Axon Instruments). Images were analyzed for percentages of collapsed growth cones.
Quantitative Imaging of Phospho-p42/p44 and p42/p44
Xenopus cultures, treated with or without 5 mM MCD or 20 ␮M GM1(30 min), were exposed to glutamate (50 ␮M), BDNF (50 ng/ml),
netrin-1 (200 ng/ml), Sema3A (1:20), or control medium for 5 min.
The cells were then fixed as above for 10 min, permeabilized, andlabeled with antibodies against phospho-p42/44 or total p42/p44(Cell Signaling Technology, Boston, MA) and Cy3-conjugated secondary antibodies (Jackson ImmunoResearch). Imaging was doneusing a 60⫻/1.4 Plan Apo objective with the same settings between
the control and exposed groups. Background subtracted images
were analyzed by creating a region of interest (ROI) that was circumscribed by the growth cone. For each growth cone, the ROI intensity
was normalized to the average from the parallel control. Data foreach condition were from at least two separate batches of Xenopus
cultures. To assess asymmetric activation of p42/p44, the growthcone was subjected to 5 min BDNF pipette application (50 ␮m fromthe growth cone) and then rapidly fixed, stained, and imaged. We
double stained the total and phopho-p42/44 using a polyclonal antip42/p44 and a monoclonal anti-phospho-p42/p44 (Cell Signaling),
followed by FITC- and Cy3-conjugated secondary antibodies. Eachchannel of the staining was acquired using the same parametersand saved for further processing and analysis.

Experimental Procedures

Membrane Fractionation and Immunoblots
Membrane fractions were prepared from cultured cortical neurons(Ma et al., 2003; Suzuki et al., 2001) as described previously (Kawabuchi et al., 2000). Six membrane fractions were collected from the
top of the gradient with the second fraction containing caveolin-2(Cav-2), thus designated as the lipid raft fraction (Figure 7A, lane 1),
and the last fraction containing transferrin receptor (TfR), therefore
designated as the nonraft fraction (Figure 7A, lane 5). The six fractions were then subjected to SDS-PAGE and probed with antibodies
for TrkB receptor (BD Biosciences), DCC (Santa Cruz), or caveolin-2(BD Biosciences) using a standard immunoblot procedure (Kawabuchi et al., 2000).

Xenopus Culture, Turning Assay, and Focal Application
Xenopus cultures and turning assays are described previously (Guirland et al., 2003) except assays were done in a modified Ringer’s
solution (Ming et al., 1999). We used the nonparametric Mann-Whit-

Fluorescent Copatching of Lipid Rafts
and Membrane Receptors
We adapted the previously described patching method (Harder etal., 1998) for Xenopus neurons. Briefly, membrane rafts and recep-

Axon Guidance Depends on Lipid Rafts for Signaling
61

tors were clustered by FITC-conjugated cholera toxin B subunit(CTxB; 8 ␮g/ml; Sigma) and an appropriate primary antibody for
1 hr at 12⬚C, followed by a Cy3-labeled secondary antibody for
45 min at 12⬚C. The cells were then fixed in 4% paraformaldehyde
and 0.25% glutaraldehyde for 4 min, followed by cold MeOH for
5 min. Digital images of both green (FITC) and red (Cy3) channelsof each neuron were individually acquired, background subtracted,
and merged as separate layers using Adobe Photoshop (Adobe
Systems). We quantified the raft association on the growth cone
and adjacent neurite shafts by determining the percentage of receptor clusters colocalized with GM1 patches. Antibodies were rabbit
against NR2B (Upstate Biotechnology), chicken IgY against TrkB
extracellular domain (kindly supplied by L. Reichardt), mouse against
DCC (Oncogene), and rabbit against NPN-1 (generously provided by
Dr. David Ginty).

son, R.G., and Schwartz, M.A. (2004). Integrins regulate Rac targeting by internalization of membrane domains. Science 303,
839–842.

In Situ Cold Detergent Extraction and Fixation
Xenopus growth cones were first exposed to BDNF gradients for
3 min (100 ␮M in pipette, 50 ␮m away, 10 ms pulse duration), immediately subjected to simultaneous cold detergent extraction and fixation using an ice-cold cacodylate buffer containing triton X-100(0.05%) and paraformaldehyde (2%) on ice for 5 min. After staining
GM1 and TrkB, images of both channels were acquired and a threshold (50% of the maximum intensity of the growth cone) was set foreach image to generate two color images (red, TrkB; green, CTxB),
which were then merged and overlaid on its corresponding DIC
image. To analyze the degree of asymmetric localization of TrkB to
lipid rafts, we divided the growth cone into near and far halves withrespect to the pipette (for control cells, near was the right hand side),measured the yellow area (TrkB localized with GM1), and normalized itagainst the green area (GM1 staining by FITC-CTxB) in each half ofthe growth cone. The area ratio of the growth cone (near/far) was
simply calculated from the normalized values and an even distribution of TrkB-raft produces a ratio of 1.

Harder, T., Scheiffele, P., Verkade, P., and Simons, K. (1998). Lipid
domain structure of the plasma membrane revealed by patching of
membrane components. J. Cell Biol. 141, 929–942.

Acknowledgments
We thank Dr. Marc Tessier-Lavigne at Genetech for providing purified netrin-1 proteins, Dr. Corey Goodman at UC Berkeley for supplying Sema3A-expressing COS cells, and Dr. David Ginty at John
Hopkins Medical School for sharing the anti-neuropilin-1 antibodies.
We are also grateful to Dr. Donald Winkelemann (Department of
Pathology, UMDNJ-RWJMS) for imaging assistance and Dr. Kuo
Wu (Department of Neuroscience and Cell Biol., UMDNJ-RWJMS)
for help in immunostaining. This work is supported by a grant from
National Institutes of Health.
Received: June 18, 2003
Revised: November 18, 2003
Accepted: March 2, 2004
Published: April 7, 2004
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