# 2007 The Authors

Journal compilation # 2007 Blackwell Publishing Ltd

doi: 10.1111/j.1600-0854.2007.00636.xTraffic 2007; 8: 1503–1520Blackwell Munksgaard

A Functional Dynein–Microtubule Network Is Requiredfor NGF Signaling Through the Rap1/MAPK Pathway

Chengbiao Wu1,*, Alfredo Ramirez1,

Bianxiao Cui2, Jianqing Ding1,

Jean-Dominique M. Delcroix1,

Janice S. Valletta1, Jia-Jia Liu1, Yanmin Yang1,

Steven Chu2 and William C. Mobley1

1Department of Neurology and Neurological Sciences,Stanford University School of Medicine, Stanford,CA 94305, USA2Lawrence Berkeley National Laboratory, andDepartments of Physics and Molecular andCellular Biology, University of California,Berkeley, CA 94720, USA*Corresponding author: Chengbiao Wu,cbwu@stanford.edu

Rap1 transduces nerve growth factor (NGF)/tyrosine

receptor kinase A (TrkA) signaling in early endosomes,

leading to sustained activation of the p44/p42 mitogen-

activated protein kinases (MAPK1/2). However, themech-

anisms by which NGF, TrkA and Rap1 are trafficked to

early endosomes are poorly defined. We investigated

trafficking and signaling of NGF, TrkA and Rap1 in PC12

cells and in cultured rat dorsal root ganglion (DRG)

neurons. Herein, we show a role for both microtubule-

and dynein-based transport in NGF signaling through

MAPK1/2. NGF treatment resulted in trafficking of NGF,

TrkA and Rap1 to early endosomes in the perinuclear

region of PC12 cells where sustained activation of

MAPK1/2 was observed. Disruption of microtubules with

nocodazole in PC12 cells had no effect on the activation of

TrkA and Ras. However, it disrupted intracellular traffick-

ing of TrkA and Rap1. Moreover, NGF-induced activation

of Rap1 and sustained activation of MAPK1/2 were mark-

edly suppressed. Inhibition of dynein activity through

overexpression of dynamitin (p50) blocked trafficking of

Rap1 and the sustained phase of MAPK1/2 activation in

PC12 cells. Remarkably, even in the continued presence of

NGF, mature DRG neurons that overexpressed p50

became atrophic and most (>80%) developing DRG neu-

rons died. Dynein- and microtubule-based transport is

thus necessary for TrkA signaling to Rap1 and MAPK1/2.

Key words: axon, DRG, dynein, endosome, MAPK, micro-

tubule, NGF, Rap1, retrograde transport, TrkA

Received 23 May 2007, revised and accepted for publica-

tion 7 August 2007, uncorrected manuscript published

online 9 August 2007, published online 6 September 2007

A compelling body of evidence points to the importance

of retrogradely transported neurotrophin (NT) signals for

neuronal survival and function (1–12). The mechanism(s)

underlying retrograde transport of NT signaling has been

explored (1,6,8,10,12). Under the ‘signaling endosome’

hypothesis, nerve growth factor (NGF) binds to and

activates its receptor tyrosine receptor kinase A (TrkA) at

the axon (AX) terminal, and the NGF–TrkA complex is

internalized through clathrin-mediated and -independent

pathways (13–20). The signaling endosome thus formed

is retrogradedly transported to the cell body (CB) (21).

Despite much support for the hypothesis (21–23), import-

ant questions remain as to how retrograde signals are

trafficked (11,19,24). Indeed, some studies suggested that

retrograde axonal transport of neither the NGF–TrkA

complex nor the phosphorylated form of TrkA, pTrkA, is

required to convey a survival signal to the CB (25,26).

Recent studies point to an important role for dynein-based

transport of NT signals. Trk receptor appears to be asso-

ciated with members of the cytoplasmic dynein family

during retrograde transport. Both the 14 and the 74 kD

chains of dynein could be coprecipitated with Trk in brain

lysates, and both subunits accumulated together with Trk

distal to a ligature site in the rat sciatic nerve (27). Proximity

of TrkA to dynein has been shown by both confocal

imaging (28) and immunoelectron microscopy (29). In

addition, dynein-based transport was shown to be required

for retrograde trafficking of activated Trks and thus played

a critical role in promoting survival of NT-dependent DRG

neurons (30). A role for intact microtubules in transporting

NGF signals in sciatic nerve was also recently shown (28).

Taken together with other reports (31,32), a strong case

can be made for the importance of dynein and intact

microtubules for retrograde transport of NT signals.

Beyond a role for moving the signal within AXs, we

envision the possibility that trafficking events also serve

to regulate the nature of the signals generated, an issue

addressed herein.

NGF induces prolonged activation of the mitogen-activated

protein kinases (MAPK1/2) signaling pathway. While Ras

mediates the transient phase of MAPK1/2 activation,

Rap1, a small guanosine triphosphatase, contributes to

the sustained activation of theMAPK1/2 signaling pathway

(33), which is necessary for NGF-induced differentiation

in PC12 cells (34). Following NGF treatment, Rap1 was

shown to be concentrated and activated in the perinuclear

region (35), and activated Rap1 was found in a long-lasting

complex with both activated TrkA and activated MAPK1/2

(pMAPK1/2) in an early-endosome-enriched fraction (36).

We used PC12 cells and DRG neurons to pursue further

the mechanism of NGF trafficking and signaling. We found

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Figure 1: Legend on next page.

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Wu et al.

that following NGF treatment, both NGF–TrkA complexes

and Rap1 moved to the perinuclear region of PC12 cells

where they were present in early endosomes in which

persistent activation of MAPK1/2 was also registered. For

both TrkA and Rap1, trafficking to the perinuclear region

was prevented by the disruption of microtubules with

nocodazole. Notably, nocodazole suppressed the activa-

tion of Rap1 and the ensuing persistent activation of

MAPK1/2 without interfering with TrkA or Ras activation.

Disrupting the dynein–dynactin complex by overexpresss-

ing p50, dynamitin (37), also blocked trafficking of Rap1 to

the perinuclear region and inhibited activation of MAPK1/2.

Using a lentiviral system, we showed that overexpressing

p50 inhibited retrograde transport of NGF and activated

TrkA (pTrkA) and NGF-induced activation of MAPK1/2 in

DRG neurons. Even for neurons whose CBs were bathed

in NGF, the consequence of p50 overexpression was the

death of immature neurons and atrophy of mature neur-

ons. Our findings suggest that dynein and microtubules

play an essential role in mediating not just the retrograde

trafficking of activated TrkA but also activation of Rap1

signaling to MAPK1/2 in support of neuronal differentiation

and survival.

Results

Microtubule depolymerization prevents NGF-induced

translocation of Rap1 to the perinuclear region

Microtubules, comprising a- and b-tubulin, form an elab-

orate network that extends throughout the cytoplasm in

virtually all eukaryotic cells. One of the most important

functions of the microtubule is to regulate the location of

membrane-bound organelles. We reasoned that disruption

of the microtubule network might alter NGF-induced

trafficking events. First, we established that NGF treat-

ment resulted in accumulation of NGF, TrkA, Rap1 and

activated MAPK1/2 in early endosomes in the perinuclear

region in PC12 cells (Figures S1 and S2). We then

examined the microtubule network in PC12 cells using

confocal microscopy. Cells, grown on glass coverslips,

were serum starved overnight prior to treatment. The cells

were pretreated with either the nocodazole vehicle

(�nocodazole) or 5 mg/mL nocodazole (þnocodazole) for

2 h at 378C. Cells were then treated with either the NGF

vehicle or 50 ng/mL NGF (þNGF) for 30 min. The samples

were prepared for indirect immunofluorescence and ana-

lyzed using confocal microscopy. Rap1-positive structures

(magenta) and the microtubule network (green) were

visualized and analyzed. (Please note that red color in all

two-color images is presented as magenta and colocaliza-

tion with green signal will be therefore denoted as white

color hereafter.) In cells pretreated with the vehicle only

(�nocodazole), microtubules were distributed throughout

the cell and the Rap1-positive puncta were seen dispersed

throughout the cytoplasm (Figure 1A–C). Treatment with

NGF for 30 min had little or no effect on the distribution of

a-tubulin (Figure 1A versus 1D). However, NGF treatment

resulted in trafficking of Rap1 to the perinuclear region

(Figure 1E versus 1B and Figure 1F versus 1C). The

increase of Rap1 signal in the perinuclear region in NGF-

treated cells was quantified in arbitrary unit (a.u.) of

fluorescence intensity, and the results are presented in

Figure 1M.

Pretreatment with 5 mM nocodazole at 378C for 2 h

resulted in a very different outcome. There was complete

disintegration of the microtubule network in approxi-

mately 95% of cells (Figure 1;þnocodazole). The staining

pattern for a-tubulin changed from the complex network

seen in normal cells (Figure 1A,D) to a pattern that was

diffused and evenly distributed (Figure 1G,J). The NGF

vehicle caused no obvious change in the pattern for Rap1

immunostaining in cells pretreated with nocodazole, that

is the Rap1-positive structures remained dispersed (Fig-

ure 1H versus 1B). However, Rap1 was no longer present

in the perinuclear region in response to NGF treatment

(Figure 1K versus 1E). Indeed, the pattern of Rap1 immu-

nostaining showed little, if any, difference from that seen

in cells treated with the NGF vehicle alone (Figure 1K

versus 1H and Figure 1L versus 1I). These studies sug-

gested that an intact microtubule network is required for

trafficking of Rap1-containing organelles to the perinuclear

region.

Disruption of the microtubule network blocks

translocation of TrkA to the perinuclear region

To examine whether TrkA trafficking was impaired when

microtubules were disrupted, PC12 cells were pretreated

with either 5 mM nocodazole (þnocodazole) or its vehicle

Figure 1: Nocodazole pretreatment blocked NGF-induced trafficking of Rap1 to the perinuclear region. PC12 cells were prepared

as described in Materials and Methods. Following pretreatment with either the nocodazole vehicle alone (�nocodazole) (A–F) or 5 mM

nocodazole (þnocodazole) (G–L) for 2 h, cells were treatedwith either NGF (50 ng/mL) (þNGF) (D–F, J–L) or the NGF vehicle (�NGF) (A–C,

G–I). Indirect immunofluorescence was carried out using confocal microscopy. Tubulin (green) and Rap1 (magenta) were stained with

a mouse antibody to a-tubulin and a rabbit antibody to Rap1, respectively. An Alexa 488 goat anti-mouse IgG conjugate was used to

visualize the microtubules, and an Alexa 568 goat anti-rabbit IgG conjugate was used to detect Rap1. Colocalization of antigens is denoted

by the white signal. Twenty-five cells were examined in each condition, and a typical representative image was shown. The size of the

collection box for all panels is 30 � 30 mm. In (M), the average line fluorescence intensity of Rap1 staining in 16 control cells and 12 NGF-

treated cells was measured. Because cells were different in size, we chose to measure the line fluorescence intensity of 10 mm distance

(2.5 mm from the nuclear edge toward the center of nucleus and 7.5 mm from the nuclear edge toward the cell periphery). The results

show that NGF treatment resulted inapproximately 30% increase in average fluorescence intensity (a.u.) in the perinuclear region

compared with that in the control groups. Standard errors are also presented.

Traffic 2007; 8: 1503–1520 1505

Dynein-/Microtubule-Mediated Trafficking of TrkA for Rap1 Signaling

(�nocodazole) for 2 h. Cells were then treated for 30 min

with either 50 ng/mL NGF (þNGF) or NGF vehicle (�NGF)

(Figure 2). The samples were prepared for confocal

microscopy to analyze the distribution of TrkA (Figure 2).

A corresponding transmitted light imagewas also captured

and superimposed to show the outline of the cell (Fig-

ure 2). In the absence of nocodazole (�nocodazole),

treatment with NGF resulted in the expected increase in

TrkA immunostaining in the perinuclear region (Figure 2B

versus 2A). However, in cells pretreated with nocodazole,

TrkA-positive puncta remained dispersed following NGF

treatment (Figure 2D versus 2C).

To confirm that TrkA trafficking was altered in cells

pretreated with nocodazole, we examined the distribution

of TrkA using subcellular fractionation on OptiPrep step

gradients of 5:10:15:20:25% (36). Serum-starved PC12

cells were pretreated with either 5 mM nocodazole (Fig-

ure 3B) or its vehicle (Figure 3A) prior to treatment with

either NGF (þNGF) or the NGF vehicle (�NGF). Cells were

then rinsed and prepared for subcellular fractionation.

Membrane fractions were collected from the four inter-

faces, and proteins were precipitated, washed and air-

dried. The samples were analyzed using SDS–PAGE/

immunoblotting. As shown previously, plasma mem-

brane proteins such as the receptor for epidermal growth

factor and Ras were concentrated in the light fractions

(fractions 1 and 2), while the early endosomal markers

Rab5 and EEA1 were enriched in the heavy fraction

(fraction 4) (36).

In cells that were pretreated with the nocodazole vehicle

(Figure 3A), NGF treatment did not alter the fractionation

of EEA1, Rab5B, Rap1 or MAPK1/2; all were enriched in

fraction 4 (Figure 3A). Without NGF treatment, TrkA (*)

was mostly detected in fraction 2, with a small amount in

fraction 1 (Figure 3A; �NGF). Following NGF treatment,

TrkA (*) was predominantly seen in fraction 4 (Figure 3A;

þNGF). The phosphorylated form of TrkA (pY490), pTrkA

(indicated by arrow), was detected in the same fraction, as

was pMAPK1/2 (Figure 3A; þNGF). The pattern for the

74 kD dynein intermediate chain (DIC74) was also exam-

ined. It was present in fractions 1 and 4 in the control

sample (Figure 3A; �NGF). NGF treatment caused the

disappearance of DIC74 from fraction 1; instead, it was

now more concentrated in fraction 4 (Figure 3A; þNGF).

These findings are consistent with the earlier results

showing that a significant amount of TrkA is present in

a light buoyant-density fraction prior to NGF treatment (38)

and that NGF treatment for 30 min resulted in the move-

ment of activated TrkA together with pMAPK1/2 to the

heavier, early-endosome-containing fraction (20,36).

The fractionation patterns for Rab5B, Rap1 and MAPK1/2

were not affected by pretreatment with nocodazole (Fig-

ure 3B versus 3A). This treatment did alter the fractionation

Figure 2: Nocodazole pretreatment dis-

rupted NGF-induced intracellular traffick-

ing of TrkA. Following pretreatment with

either the nocodazole vehicle alone

(�nocodazole) (A, B) or 5 mM nocodazole

(þnocodazole) (C, D) for 2 h, PC12 cells were

treated with either NGF (50 ng/mL) (þNGF) (B,

D) or the NGF vehicle (�NGF) (A, C). Cells were

then rinsed, fixed with 100% methanol and

processed for indirect immunofluorescence

using confocal microscopy as described in

Materials and Methods. TrkA (red) was stained

with a mouse antibody to Trk (B3) and with an

Alexa 568 goat anti-mouse IgG conjugate.

A corresponding image from the transmitted

light detector was also captured and super-

imposed onto the fluorescence image of TrkA

to illustrate the outline of the cell. Twenty-five

cells were examined in each condition, and

a typical representative image was shown. The

size of the collection box for all panels is

30 � 30 mm.

1506 Traffic 2007; 8: 1503–1520

Wu et al.

pattern for DIC74 and TrkA following NGF treatment. In

cells treated with nocodazole but not with NGF, DIC74was

present in fraction 3 and more concentrated in fraction 4

(Figure 3B; �NGF). Most TrkA (*) was detected in fraction

2, with a small amount in fraction 1 (Figure 3B; �NGF).

However, when nocodazole-pretreated cells were treated

with NGF, DIC74 was captured in fraction 1 as well as in

fraction 3 (Figure 3B; þNGF). Most TrkA (*) as well as

most pTrkA (indicated by arrow) were now present in

fraction 3 but not in fraction 4 (Figure 3B; þNGF). It is

noteworthy that activation of TrkA (arrows in Figure 3A,B)

was not affected by nocodazole, suggesting that NGF-

induced TrkA activation at the plasma membrane or initial

internalization of activated TrkA was not hindered. Further-

more, nocodazole pretreatment markedly inhibited NGF-

induced activation of MAPK1/2 (i.e. pMAPK1/2) in fraction

4 (Figure 3B versus 3A). Thus, depolymerization of micro-

tubules alters the trafficking of activated TrkA and impairs

its downstream signaling.

We also treated PC12 cells with biotinylated NGF (BtNGF)

and isolated fractions 1 and 4. Lysates of these fractions

were then incubated with streptavidin–agarose beads to

capture BtNGF and its binding proteins. The protein

complex was analyzed by SDS–PAGE and immunoblotting

with pTrkA antibody (pY490). Lysates of fraction 4 from

BtNGF-treated cells, but not from vehicle-treated cells,

contained pTrkA bound to BtNGF (Figure 3C). As a control,

pTrkA was not recovered from fraction 1 of either BtNGF-

or vehicle-treated cells (Figure 3C). We conclude that

NGF–TrkA complexes are present in early endosomes.

To show if pMAPK1/2 activated by NGF in fraction 4 were

accessible to its downstream signaling molecules, we

carried out an in vitro kinase phosphorylation assay using

recombinant Elk-1, a substrate immediate downstream to

pMAPK1/2 in NGF signaling cascade. Fraction 4 from cells

treated with NGF containing pMAPK1/2 was capable of

Figure 3: Nocodazole pretreatment disrupted the intracellu-

lar trafficking of TrkA as analyzed using a step gradient of

OptiPrep. PC12 cells were starved and pretreated with either

vehicle (A, control) or 5 mM nocodazole (B) for 2 h and were

subsequently treated with NGF (þNGF, 50 ng/mL) or the NGF

vehicle (�NGF) for 30 min. Cells were rinsed and homogenized.

The homogenates were centrifuged to remove nuclei. The result-

ing supernatants were fractionated by centrifugation using the

step-density gradient system as described in Materials and

Methods. Four membrane fractions (1, 5:10%; 2, 10:15%; 3,

15:20% and 4, 20:25%) were collected from the indicated

interface, and proteins from each fraction were precipitated in

7% trichloroacetic acid and washed in acetone. The pellets were

then dried and boiled in SDS–PAGE loading buffer. The proteins

were analyzed using SDS–PAGE and immunoblotting. The blots

were independently probed with the indicated antibodies. In

a separate experiment, following treatment with either the

NGF vehicle (�) or 50 ng/mL BtNGF (þ) for 30 min, cells were

fractionated as in (A). Fractions 1 and 4 from the vehicle- and

BtNGF-treated samples were lysed and precipitated with

streptavidin–agarose beads as described inMaterials andMethods.

The precipitates were washed, separated on SDS–PAGE and

blotted with a rabbit antibody to pTrkA (C). In (D), membrane

fractions (fraction 4) were collected from both NGF-treated (þNGF)

and vehicle-treated (�NGF) samples and were reconstituted into an

in vitro Elk-1 phosphorylation assay system. Phospho-Elk-1 and Elk-

1 were detected using specific antibodies. All blots were visualized

using SuperSignal, and the results shown are representative of

at least three independent experiments.

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Dynein-/Microtubule-Mediated Trafficking of TrkA for Rap1 Signaling

phosphorylating Elk-1 (Figure 3D; þ), while fraction 4 from

cells that were treated with NGF vehicle did not contain

pMAPK1/2 and was incapable of phosphorylating Elk-1

(Figure 3D;�). Therefore, a population of early endosomes

is capable of transmitting the NGF signal.

Disruption of the microtubule network suppresses

Rap1 and MAPK1/2 signaling

To pursue further the idea that aberrant trafficking of TrkA

and Rap1 would compromise activation of the MAPK1/2

signaling pathway, we analyzed the effects of nocodazole

pretreatment on the activation of Ras and Rap1. We first

confirmed that TrkA was activated to a level similar to that

in control samples when microtubules were disrupted.

Serum-starved PC12 cells were pretreated with either

vehicle (�nocodazole) or 5 mM nocodazole (þnocodazole)

prior to treatment with NGF or the NGF vehicle (Figure 4).

Cells were rinsed, lysed and centrifuged. The supernatant

was immunoprecipitated with a specific antibody to

Trk (MCTrk). The immunoprecipitate was analyzed using

SDS–PAGE/immunoblotting. As shown in Figure 4A, no-

codazole pretreatment did not significantly alter the mag-

nitude or the duration of TrkA activation induced by NGF.

Samples were also prepared to assay the active forms (i.e.

GTP-bound form) of Ras and Rap1 using an established

protocol (36). As shown in Figure 4B, nocodazole pre-

treatment had little effect on GTP loading of Ras. In

contrast, nocodazole significantly suppressed the activa-

tion of Rap1 (Figure 4B). Consistent with this result, there

was also suppression of NGF-induced sustained activation

of MAPK1/2. While the early phase of MAPK1/2 activation

was unaffected by nocodazole, the activation of MAPK1/2

seen at 30 min of NGF treatment was markedly reduced

(Figure 4B). The levels for total MAPK1/2 were not

affected by nocodazole. We also examined activation of

the phosphatidylinositide 3-kinase (PI3K)/Akt pathway fol-

lowing NGF treatment. Nocodazole pretreatment caused

partial suppression of this pathway, as evidenced by

a decrease in the level of phosphorylated Akt (pAkt,

Ser473) but not total Akt (Figure 4B). Thus, disrupting the

microtubule network suppressed NGF-induced activation

of Rap1 signaling, the sustained phase of MAPK1/2

activation and activation of PI3K/Akt.

The overexpression of p50 blocks NGF-induced

retrograde transport of Rap1 and abolishes the

sustained phase of MAPK1/2 activation

DIC74 was found to be associated with the Trk receptor in

mouse brain lysates (27). We confirmed the association of

DIC74 with TrkA in PC12 cells by immunoprecipitation

(data not shown). Thus, cytoplasmic dyneins might play

a role in intracellular trafficking of Trk (30). To examine

what role dynein plays in retrograde trafficking of TrkA and

Rap1 signaling, we inhibited the activity of the dynein–

dynactin complex by overexpressing the 50 kD subunit of

dynactin and dynamitin (p50) (37,39). The overexpression

of p50 causes dissociation of the microtubule- and cargo-

binding subunits of the dynein–dynactin complex, thus

serving to disrupt dynein-based transport (39,40). In a trans-

genic mouse model, p50 overexpression led to marked

inhibition of retrograde axonal transport (41).

We reasoned that p50 overexpression in PC12 cells might

block NGF-induced retrograde trafficking of TrkA and Rap1

and, as a result, inhibit the sustained activation ofMAPK1/2.

To test this idea, we transiently transfected PC12 cells

with a pcDNA3 construct that contained the full-length

human p50 complementary DNA (cDNA) sequence. We

used immunostaining to examine the effects of p50 over-

expression. Forty-eight hours posttransfection, PC12 cells

were serum starved for 16 h prior to receiving treatment

Figure 4: Nocodazole pretreatment selectively inhibited

Rap1 activation and the sustained phase of MAPK1/2 acti-

vation induced by NGF. PC12 cells were starved and treated

with either the vehicle (�nocodazole) or 5 mM nocodazole

(þnocodazole) for 2 h prior to treatment with NGF (50 ng/mL) or

NGF vehicle (0 min) for the indicated time intervals. In (A), TrkA

was immunoprecipitated from all samples with a mouse antibody

to Trk (MCTrk) and was analyzed using SDS–PAGE/immunoblot-

ting. TrkA was detected using B3, and pTrkA was revealed using

a rabbit antibody to pTrkA (pY490). In (B), the level of RasGTP and

Rap1GTP was analyzed as described in Materials and Methods. In

addition, cells were washed and then lysed in RadioImmuno-

Precipitation Assay (RIPA) buffer. Ten micrograms of proteins

from the resulting supernatant of each sample was separated on

SDS–PAGE and analyzed by immunoblotting with a mouse anti-

body to the phosphorylated MAPK1/2. The blot was stripped and

reprobed for the total amount of MAPK1/2 to show equal loading

of protein samples. Samples from the lysates were also immuno-

blotted with a rabbit antibody to phosphorylated Akt (Ser473) and

reprobed with a rabbit antibody to total Akt. The results shown are

representative of at least three independent experiments.

1508 Traffic 2007; 8: 1503–1520

Wu et al.

with either the NGF vehicle (�NGF) or the NGF (þNGF,

50 ng/mL for 30 min) (Figure 5). Cells were then pro-

cessed for immunostaining using a mouse monoclonal

antibody to p50 and a rabbit polyclonal antibody to Rap1.

To better illustrate the perinuclear region, we also co-

stained with Hoechst 33258 to visualize the nucleus by

epifluorescence (Figure 5A/b,d,f,h). In untransfected cells

(Figure 5A/a,b,e,f), NGF induced accumulation of Rap1

Figure 5: The overexpression of p50 inhibited NGF-induced translocation of Rap1 and pMAPK1/2 signals to the perinuclear

region. PC12 cells were transiently transfected with pcDNA3 plasmids that contained the full-length human dynamitin cDNA sequence for

48 h as described inMaterials and Methods. Cells were then reseeded on glass coverslips and starved. Cells were treated with either the

NGF vehicle (�NGF) or NGF (þNGF, 50 ng/mL for 30 min). In both (A) and (B), p50 (green) was immunostained with a specific mouse

antibody (1/500 dilution) and detected with a secondary goat anti-mouse IgG–Alexa 488 conjugate. Rap1 (magenta) in (A) was stained with

a specific rabbit antibody and visualizedwith a secondary goat anti-rabbit IgG–Alexa 568 conjugate. The sample in (A) (b, d, f and h) was also

co-stained with Hoechst 33258 (blue) to mark the nucleus. The resulting samples were captured and analyzed using a Nikon E800

epifluorescence microscope. Similarly, the pMAPK1/2 signals (magenta) in (B) were stained with a specific rabbit antibody to pMAPK1/2

and visualized with a secondary goat anti-rabbit IgG–Alexa 568 conjugate. The p50 (green) was stained as in (A). The resulting samples

were analyzed using a confocal microscope (B). Typically, 25 cells were examined in each condition, and a typical representative image is

shown. The size of the collection box is 60 � 60 mm for (A) and 30 � 30 mm for (B).

Traffic 2007; 8: 1503–1520 1509

Dynein-/Microtubule-Mediated Trafficking of TrkA for Rap1 Signaling

signal in the perinuclear region. In cells that overexpressed

p50 (Figure 5A/c,d,g,h), NGF treatment failed to cause con-

centration of Rap1 in the perinuclear region (Figure 5A/b–f

versus 5A/d–h). The effect was specific for p50 because

overexpression of enhanced green fluorescent protein

(EGFP) did not affect NGF-induced trafficking of Rap1

(data not shown).

We next investigated the sustained phase of MAPK1/2

activation in cells that transiently overexpressed p50.

Without NGF treatment (�NGF; Figure 5B), the baseline

level for pMAPK1/2 signal in transfected (Figure 5B/c) cells

was similar to that in nontransfected (Figure 5B/a) cells

(Figure 5B/d versus 5B/b). Treatment of nontransfected

(Figure 5B/e) cells with NGF for 30 min showed a marked

increase in pMAPK1/2 in the perinuclear region (Figure 5B/f

versus 5B/b). Similarly, a marked increase in pMAPK1/2

was also seen in cells that were transfected with a plasmid

vector expressing EGFP, pEGFP (data not shown). In

contrast, NGF treatment failed to elicit this response when

p50 was overexpressed (Figure 5B/g,h versus 5B/e,f).

Thus, inhibition of cytoplasmic dynein blocked NGF-

induced retrograde transport of Rap1 and abolished the

sustained phase of MAPK1/2 activation.

The overexpression of p50 blocks retrograde

transport of the NGF–pTrkA complex in

mature DRG neurons in culture

We next examined the trafficking of NGF signals in

cultured neurons. Rat embryonic DRGs were cultured on

collagen-coated cover glasses for 21 days (DIV-21) as

described in Materials and Methods. At this point, the

cultures contain large, mature neurons with well-devel-

oped AXs. At least 94% of neurons were judged to be NGF

responsive as evidenced by immunopositivity for TrkA

(data not shown). Of note, these neurons no longer require

NGF for survival (42). Following NGF deprivation for 24 h,

cells were treated with either 100 ng/mL NGF (Figure 6

B,D,F) or NGF vehicle (Figure 6A,C,E) and were prepared

for immunostaining. NGF treatment resulted in redistribu-

tion of both Rap1 and TrkA to the perinuclear region, where

they significantly colocalized (Figure 6A versus 6B). Con-

sistent with our results for PC12 cells, NGF induced

activation of MAPK1/2 and the signal for pMAPK1/2 over-

lapped with that for Rap1 in the perinuclear region (Fig-

ure 6D). Finally, we confirmed colocalization of Rap1 with

EEA1 in the perinuclear region following NGF treatment

(Figure 6F versus 6E). Based on these observations, we

conclude that NGF treatment of DRGs resulted in an

increase of these signaling proteins within the perinuclear

region as expected.

To show whether or not p50 overexpression blocks

retrograde axonal transport of NGF, we used Campenot

chamber cultures (CAMP10; Figure 7A), in which distal

axons (DAXs) were separated from their CBs and proximal

axons (PAXs) (9,25). DRGs (DIV-21) were transduced with

either a control virus or lenti-p50 or were not transduced,

as described in Materials and Methods. The lentiviral

system is highly efficient and results in sustained trans-

duction of nondividing cells, such as DRG neurons (43).

Following NGF withdrawal together with serum starvation

for 24 h, BtNGF (100 ng/mL) was added to the DAX

chamber. We elected to carry out most observations at

60 min because at this time-point there was robust trans-

port of BtNGF to CBs. Samples were rinsed, fixed and

incubated with a Streptavidin–Alexa 488 conjugate to

detect BtNGF in neuron CBs and PAXs. The fluorescence

signal for BtNGF/Streptavidin–Alexa 488 was measured

and quantified as described in Materials and Methods.

The results showed that p50 overexpression markedly

decreased the amount of BtNGF present in the CB in

comparison with the no-virus control (35 � 60 a.u. versus

210 � 40 a.u.). This represents a decrease of approxi-

mately 83% (p < 0.01; t-test) in retrogradely transported

BtNGF when p50 was overexpressed (Figure 7B). Trans-

duction with a control virus had no significant effect on

transport as judged in relation to nontransduced neurons

(210 � 40 a.u. versus 244 � 57 a.u.; p > 0.05).

To show that retrograde transport of BtNGF/pTrkA was

also blocked by p50 overexpression, we cultured DRGs

(DIV-21) in CAMP15 chambers (Figure 7A). Cells were

transduced with either lenti-EGFP or lenti-p50 or were

not transduced, as described previously. Following NGF

deprivation and serum starvation, BtNGF (100 ng/mL) was

added to the DAX chamber for 60 min. Samples from the

CB were rinsed, collected and lysed. BtNGF in lysates was

precipitated with a streptavidin–agarose conjugate. The

precipitates were washed and analyzed by SDS–PAGE/

Western blotting using a rabbit-specific antibody to mouse

NGF. BtNGFwas detected in the CB chamber in the control

virus sample (Figure 7C). In addition, pTrkA (pY490) was

present in the precipitate, as assessed by reprobing with

a rabbit-specific antibody to pTrkA (pY490) (Figure 7C).

Transduction with lenti-EGFP resulted in relatively small

(approximately 20%) decrease in pTrkA under this condi-

tion (Figure 7C). However, in cells transduced with lenti-

p50, the amount of BtNGF in the CBwasmarkedly reduced

and there was little pTrkA (Figure 7C). To ensure that NGF

activated TrkA in the AXs of these cells, we treated the

DAX chamber with NGF (100 ng/mL) for 15 min and

collected the axonal lysate for Western blotting. The level

of pTrkA induced by NGF in lenti-p50 DAXs was approxi-

mately 75% of controls (Figure 7D). These results show

that p50 overexpression blocks retrograde transport of the

NGF–pTrkA complex in DRG neurons.

Overexpression of p50 in dividing cells causes disintegra-

tion of the Golgi complex (39,44). Because DRG neurons

are postmitotic, we expected that the Golgi would remain

intact. To confirm this, we stained DRG neurons with a

mouse antibody (1/200) that specifically recognizes the

38 kD trans Golgi network protein TGN38. The flag-tagged

p50 was visualized using a rabbit antibody to the flag tag

1510 Traffic 2007; 8: 1503–1520

Wu et al.

(1/100). Alexa 568 goat anti-mouse immunoglobulin G

(IgG) and Alexa 488 goat anti-rabbit IgG conjugates were

used to visualize the respective primary antibodies. We

compared the pattern of TGN38 staining in cells trans-

duced with lenti-p50 with that in cells not transduced

(Figure 7E). There was no apparent change in the

appearance of the overall structures as marked by

TGN38 antibody following viral transduction. Moreover,

there was no apparent loss of DRG neurons during these

experiments.

Although these experiments showed that DRG neuron

survival was not affected by p50 overexpression, we

noticed an apparent decrease in the size of neuronal

somas even when neurons were bathed in NGF. Cellular

images were captured and quantified. In the continuing

Figure 6: NGF-induced trafficking

of Rap1, TrkA and pMAPK1/2 to

the perinuclear region in DRGs

(DIV-21). DRGs (DIV-21) were cul-

tured and starved as described in

Materials and Methods. Cells were

treated with either NGF vehicle

(�NGF; A, C and E) or 100 ng/mL

NGF (þNGF; B, D and F) for 60 min.

Cells were rinsed, fixed and prepared

for immunostaining with indicated

antibodies. Alexa 568 goat anti-rabbit

IgG conjugates were used to visualize

Rap1 (magenta). Alexa 488 goat anti-

mouse IgG conjugates were used to

reveal Trk, pMAPK1/2 and EEA1 (all in

green). Representatives of confocal

microscopic images from at least

three independent experiments were

shown. The size of the collection box

is 50 � 50 mm.

Traffic 2007; 8: 1503–1520 1511

Dynein-/Microtubule-Mediated Trafficking of TrkA for Rap1 Signaling

presence of NGF, p50-overproducing cells were smaller,

with a mean profile of 685 � 200 mm2 (n ¼ 50) compared

with 943 � 193 mm2 (n ¼ 50) for cells transducedwith the

control virus (i.e. a decrease to approximately 73% of the

control, p < 0.01). We conclude that it is possible that p50

overexpression led to a change in cell function, resulting in

cellular atrophy. An alternative explanation is that NGF

signaling from the surface of neuron CBs was inadequate

to sustain cellular differentiation in the absence of the

ability to traffic endosomes.

Overexpression of p50 blocks NGF-induced activation

of MAPK1/2 and results in atrophy in mature DRG

neurons in culture

To examine if p50 overexpression suppressed NGF sig-

naling in mature neurons, DRG neurons (DIV-21) were

Figure 7: Ectopic expression of p50-blocked retrograde transport of NGF–pTrkA signaling in rat DRGs (DIV-21) in Campenot

chambers. A) Diagrams of two types of Campenot nerve cell growth chamber (CAMP10 and CAMP15). In (B), 20 000 cells from

dissociated rat E15-16 DRGs were plated in the inner chamber of CAMP10. Cells were maintained for 21 days and then transduced with

the indicated lentivirus. Seven days posttransduction, both the inner and the outer chambers were washed extensively and starved.

Samples were rinsed again, and 50 ng/mL BtNGF was added to the outer chamber, followed by incubation at 378C for 1 h. BtNGF was

detected using a Streptavidin–Alexa 488 conjugate, and the amount of BtNGF in a.u. that was transported to the CBs and part of the PAXs

from the DAX was quantified as described inMaterials andMethods. In (C), DRGs (DIV-21) were cultured in CAMP15 and transduced as in

(B). Following NGF deprivation, 100 ng/mL BtNGF was added to the axonal chamber, and the samples were incubated at 378C for 1 h.

Samples were rinsed, and the CBs were lysed, collected and cleared by centrifugation. The resulting supernatant was incubated with

streptavidin–agarose conjugate. The precipitates were analyzed using SDS–PAGE/immunoblotting to detect BtNGF and pTrkA as

indicated. In (D), NGF (100 ng/mL) (þ) or NGF vehicle (�) was added to the DAX chamber for 15 min, and the AXs were collected for SDS–

PAGE/immunoblotting analysis with the pTrkA antibody. Representative blots from at least three independent experiments were shown.

In (E), DRG neurons (DIV-21) were transduced with either a control virus or lenti-p50 overnight, and the cultures were continued for an

additional 7 days. Cells were extensively washed and prepared for immunostaining with a mouse antibody to TGN38 and a rabbit antibody

to flag for visualizing p50. Alexa 568 goat anti-mouse IgG conjugates were used to visualize TGN38 (magenta). Alexa 488 goat anti-rabbit

IgG conjugates were used to reveal dynamitin (green). Representatives of confocal microscopic images from at least three independent

experiments were shown. The size of the collection box is 50 � 50 mm.

1512 Traffic 2007; 8: 1503–1520

Wu et al.

cultured on coated cover glasses and transduced with

either a control virus or lenti-p50, as described previously.

Cells were then starved and treated with either NGF or the

NGF vehicle and processed for immunostaining for

pMAPK1/2. As expected, NGF treatment of neurons trans-

duced with a control virus induced a marked increase in

pMAPK1/2 in both CBs and AXs (Figure 8B versus 8A). In

cultures transduced with lenti-p50, approximately 90% of

the neurons overexpressed p50 in both the CB and the

AXs. In these cells, NGF failed to elicit activation of

Figure 8: Overexpression of p50 in mature rat DRG neurons inhibited NGF-induced activation of MAPK1/2. DRGs (DIV-21) were

prepared and transduced as described in Figure 7. Prior to processing for immunostaining and immunoblotting, cells were starved, washed

and treatedwith either the NGF vehicle (�NGF) or NGF (þNGF, 100 ng/mL for 60 min). In both (A) and (B), p50 (green) was immunostained

with a specific mouse antibody and detected with a secondary goat anti-mouse IgG–Alexa 488 conjugate. pMAPK1/2 (magenta) was

stained. The samples were captured and analyzed using a Nikon E800 epifluorescence microscope (A and B). Twenty-five cells were

examined in each condition, and a typical representative image is shown. The scale bar in the CB and AX is 20 mm. In (C), lysates from cells

that were treated with either the NGF vehicle (�NGF) or NGF (þNGF, 100 ng/mL for 60 min) were analyzed using SDS–PAGE/

immunoblotting. The blots were probed with a rabbit antibody to either pMAPK1/2 or the phosphorylated form of TrkA (pY490) or to pAkt.

Blots that fell within the linear range of exposures were scanned and measured using Scion Image Beta 4.0.2 (Scion Corporation). The

relative activity in a.u. was obtained by subtracting the background value in the vehicle-treated sample from that of the NGF-treated

sample. The results shown are representative of at least three independent experiments. In (D), images of 100 cells from each sample

were analyzed, and the cell size was measured using Scion Image Beta 4.0.2 as described inMaterials and Methods. The size distribution

was shown.

Traffic 2007; 8: 1503–1520 1513

Dynein-/Microtubule-Mediated Trafficking of TrkA for Rap1 Signaling

MAPK1/2 in either the CB or the AX (Figure 8B ver-

sus 8A).

To examine the effect of p50 overexpression on NGF

signaling, we harvested cell lysates from cultures treated

with either the vehicle or the NGF (100 ng/mL) for 60 min.

Samples were analyzed using SDS–PAGE, followed by

immunoblotting with an antibody to pTrkA (pY490). As

shown in Figure 8C, there was a small (approximately

17%) and statistically insignificant reduction (p > 0.5) in

the level of pTrkA induced by NGF in p50-overproducing

cells compared with cells transduced with a control virus.

Thus, consistent with previous results (Figure 7D), p50

overexpression did not appear to significantly affect TrkA

activation. Next, we measured the level of pMAPK1/2.

While a robust response was evident in cells that were

transduced with the control virus, activation of MAPK1/2

by NGF was virtually completely inhibited in neurons that

overexpressed p50 (Figure 8C). NGF-mediated activation

of the PI3K/Akt was also suppressed, albeit to a lesser

extent (Figure 8C). Therefore, as for PC12 cells, over-

expression of p50 in DRG neurons blocked or severely

inhibited the sustained phase of MAPK1/2 activation

induced by NGF while having little effect on the activation

of TrkA.

To address more thoroughly what role dynein-based trans-

port has on NGF signaling, we examined the neuronal

soma size, a well-recognized phenotype regulated by NGF

signaling inmature DRG neurons (45) and in NGF-dependent

basal forebrain cholinergic neurons (46,47). Rat DRG

neurons cultured for DIV-21 on cover glasses were trans-

duced with either a control virus or lenti-p50 in the

continuous presence of NGF. Seven days later, cells were

imaged using confocal microscopy. In control samples, cell

size varied from 200 to 1800 mm2, with a mean value of

1010 � 28 mm2. In cells that overproduced p50, the mean

value was significantly decreased to 651 � 28 mm2

(p < 0.01) (Figure 8D). Overexpression of p50 thus re-

sulted in severe atrophy (to approximately 65% of control

value), despite the fact that NGF was continuously present

in the medium bathing the surface of CBs and their AXs. If

compromised NGF signaling and not some other conse-

quence of p50 overexpression was responsible, the same

effect should be shown with simple removal of NGF from

the bathing medium. This is what was observed. The NGF

withdrawal from mature DRG neurons (DIV-21) for 5 days

resulted in a decrease in somal size to approximately 70%

of the NGF-treated control. We conclude that interrupting

dynein function through p50 overexpression markedly

inhibited the NGF signaling events needed to maintain

the somal size of mature DRG neurons.

Overexpression of p50 in developing DRG neurons

caused cell death even in the presence of NGF

Because the survival of developing DRGs is critically

dependent on the availability of NGF (42), we asked

whether or not overexpression of p50 in developing DRG

neurons would result in cell death in the presence of NGF.

Embryonic rat DRGs (E15-16) were dissected, dissociated

and cultured for 7 days (DIV-7). Cultures were then trans-

duced with either a control virus or lenti-p50 and main-

tained for additional 7 days in the continuous presence of

NGF (100 ng/mL). Under these conditions, we routinely

achieved >95% transduction efficiency. Cultures were

rinsed and apoptotic neurons detected using the In Situ

Cell Death Detection Kit, TMR red (Roche). This assay

specifically labels DNA strand breaks (‘nicks’) in genomic

DNA produced during apoptosis. Apoptotic cells, whose

nuclei were fluorescently labeled with TMR red, were

visualized and counted. Transmitted light images were

simultaneously collected and superimposed onto the cor-

responding confocal images to allow for visualization of

cells. Representative images (Figure 9) show cells trans-

duced with no virus (Figure 9A), a control virus (Figure 9B)

or lenti-p50 (Figure 9C). The DRG neurons transduced with

lenti-p50 showed massive cell death (Figure 9C), whereas

relatively few apoptotic cells were observed in either of the

control cultures (Figure 9A,B). Scoring the percentage of

apoptotic cells showed a marked and highly significant

(p < 0.01) increase when neurons were transduced with

lenti-p50 (Figure 9D). Thus, even though NGF (100 ng/mL)

was continuously present throughout the culture period,

transduction with lenti-p50 resulted in the death of approx-

imately 85% of neurons. Consistent with other recent

findings (30), we conclude that dynein-based transport of

NGF signaling appear to play a critical role in promoting

survival of developing DRG neurons.

Discussion

Herein, we show that intact microtubules and functional

dynein are critical for the ability of NGF signaling to induce

sustained activation of MAPK1/2 and to support the

survival and maintenance of DRG neurons. The underly-

ing trafficking events induced by NGF treatment included

translocation of TrkA and Rap1 to the perinuclear region

where they were present in early endosomes. Import-

antly, the trafficking events supported by microtubules

and dynein were necessary not just for NGF signals

created and transported retrogradely within AXs but also

when signals arose at the level of CBs. The data are

evidence that endosomal trafficking plays an important

role in both the creation of NGF signals and their intracel-

lular transport.

Our working hypothesis posits that several distinct steps

are required for generating the TrkA/Rap1 signaling endo-

some. First, NGF binds to TrkA at the cell surface to

activate the receptor. Second, the NGF–pTrkA complex is

internalized through either clathrin-mediated or non-

clathrin-mediated pathways (18,19,48). Third, the newly

formed NGF–pTrkA signaling endosome is likely docked

onto the microtubule network through the binding of both

the intermediate and the light chains of the dynein

1514 Traffic 2007; 8: 1503–1520

Wu et al.

complex (30), thus linking it to the retrograde transport

system.

The studies reported herein provide evidence for another

step in trafficking of the NGF–pTrkA complex, leading to

‘maturation’ of the NGF–pTrkA signaling complex into one

that is now competent to persistently activate MAPK1/2.

Rap1 plays a key role in transducing and sustaining the

NGF signal from TrkA to MAPK1/2. Disruption of the

microtubule network by nocodazole or the dynein–

dynactin complex through ectopic expression of p50,

dynamitin, prevents the genesis of a fully functional

NGF–TrkA–Rap1 signaling endosome.

There is compelling evidence that the NGF–pTrkA–Rap1

signaling endosome contributes critically to sustained

activation of MAPK1/2, a pathway important for PC12 cell

differentiation (33,36). The current study provides some

key evidence that following NGF treatment, TrkA and Rap1

are trafficked to signaling endosomes in the perinuclear

region. This process likely represents an important step in

delivering the NGF signal to the nucleus (9,48,49). There

are significant potential advantages to the compartmental-

ization of TrkA signaling in endosomes. First, the signal

would be specific for NGF. Second, by concentrating the

signaling machinery used to transmit TrkA signals, the

probability of conducting signals that are robust and of high

fidelity may be increased. Third, providing the signal near

the nucleus a local source of downstream signals, such as

those propagated by activated MAPK1/2, would facilitate

effective delivery of signals through diffusion.

Similar observations were made for the c-Jun NH(2)-

terminal kinase (JNK) signaling pathway in neurons (50).

The JNK-interacting protein-1, JIP1, that serves as a scaf-

fold for the stress-activated JNK pathway was found to be

highly concentrated in the neurite tips of primary hippo-

campal neurons. On anoxic stress (oxygen and glucose

deprivation), JIP1 as well as activated JNK were trans-

located to the perinuclear region (50,51). Ablation of JIP1

prevented JNK activation and rendered neurons less

susceptible to stress-induced apoptosis. Trafficking of

signaling proteins from the cell periphery to the perinuclear

region may contribute critically to the ability to transmit

a variety of signals that modulate neuronal survival and

function.

The mechanistic basis for trafficking events involving TrkA

is the topic of recent studies (18–20). Our findings suggest

that the cytoplasmic motor dynein plays an important role

in trafficking of the NGF–pTrkA complex and in the

activation of the MAPK1/2 and PI3K signaling pathways.

It remains to be determined how the dynein–dynactin

complex interacts with TrkA and, perhaps, with elements

Figure 9: Ectopic expression of p50

in early developing cultured rat DRG

neurons (DIV-7) caused apoptosis.

DRG neurons were isolated and cul-

tured for 7 days as described. Cells

were transduced with no virus (A),

a control virus (B) or lenti-p50 (C) over-

night, and the cultures were continued

for an additional 7 days in the presence

of 100 ng/mL NGF. Seven days post-

transduction, samples were pro-

cessed, and apoptotic cells were

detected using the In Situ Cell Death

Detection Kit, TMR red. The samples

were analyzed and images captured

using a confocal microscope, and the

nuclei of apoptotic cells were specifi-

cally labeled and revealed with

magenta fluorescence. Images from

transmitted light detector were simul-

taneously collected and superimposed

onto their corresponding fluorescence

images. The size of the collection box

is 500 � 500 mm. In (D), we scored

the number of apoptotic DRG neurons

in each condition. The percentile of

apoptotic cells was shown.

Traffic 2007; 8: 1503–1520 1515

Dynein-/Microtubule-Mediated Trafficking of TrkA for Rap1 Signaling

of the NGF–pTrkA–Rap1 signaling complex. Whether or

not the activity of dynein itself is regulated by NGF

signaling is another important question. In addition, the

receptor for transforming growth factor-b (52,53) and the

activated receptor for epidermal growth factor (54) were

recently shown to undergo translocation to the perinuclear

region through early endosomes and that this process was

driven by dynein. It will be important to define whether or

not dynein contributes routinely to the trafficking of the

signals of other NTs and cytokines.

The signaling endosome hypothesis focuses on the retro-

grade transport of neurotrophic signals within AXs. We

expected to show that overexpressing p50 would severely

inhibit retrograde transport of NGF signaling from AX

terminals of DRG neurons. Unexpectedly, we discovered

that ectopic expression of p50 also inhibited NGF signaling

from the somal surface of DRG neurons, leading to the

death of immature neurons and the atrophy of mature ones.

These findings make the case that NGF signaling events,

including at least those that involve MAPK1/2 and PI3K,

need to engage the endosomal system. Indeed, our findings

are evidence that endosomal traffic not only delivers signals

but is also used to create them. It is important to point out

that the present study does not rule out the existence of

other paradigms by which NGF signal can be conveyed to

the CB. We speculate that it is advantageous to have

multiple pathways coexit to support neuronal function.

Disruption of trafficking of NGF signal can readily be

envisioned as adversely affecting neuronal function.

Indeed, an emerging body of data suggests that such

events are impaired or altered in the central nervous

system of patients suffering from neurodegenerative dis-

orders such as Alzheimer’s disease (6,55–62). It is impera-

tive to pursue further the mechanisms of retrograde

trafficking of NGF and other NT signaling and their possible

involvement in neurodegenerative disorders.

Materials and Methods

Antibodies and reagentsMouse antibodies to EEA1, dynamitin (p50), Ras, Rap1 and TGN38 were

purchased from BD Transduction Laboratories. Rabbit antibodies to Rap1

(Krev-1) and EGFP and mouse antibodies to Trk (B3 and MCTrks), DIC74

and pMAPK1/2 were purchased from Santa Cruz Biotechnology, Inc. Rabbit

antibodies to Elk-1, phospho-Elk-1, pTrkA (pY490), Akt, phosphorylated Akt

(Ser473), MAPK1/2 (Erk1/2) and the phosphorylated (i.e. activated) MAPK1/2

were purchased from Cell Signaling, Inc. Rabbit antibody to flag was

purchased from Upstate Biotechnology, Inc. A rabbit antibody to mouse

NGF was purchased from Alomone labs. Control mouse IgGs were

obtained from Chemicon. Nocodazole and n-Octyl-b-D-glucopyranoside

were purchased from Calbiochem-Novabiochem Corp. Nocodazole was

dissolved at a concentration of 5 mg/mL (1000 � stock) in dimethyl

sulfoxide (DMSO). Mouse NGF was purified as previously described (63).

The NGF was stored in 0.2% acetic acid (the NGF vehicle). The BtNGF was

produced according to a published method (64). The efficiency of biotin

labeling (>95%) was verified by retarded mobility of BtNGF versus NGF on

SDS–PAGE. The BtNGF was fully potent in activating TrkA and in inducing

neurite outgrowth as confirmed using PC12 cells (data not shown).

Horseradish peroxidase (HRP) conjugated to goat anti-rabbit or anti-mouse

IgGs was obtained from Jackson Immunoresearch Laboratories, Inc. Alexa

568 or Alexa 488 goat IgG conjugates (anti-mouse or anti-rabbit) and

Hoechst 33258 (10 mg/mL), Streptavidin–Alexa 488 were purchased from

Molecular Probes. Protein G–agarose conjugates and SuperSignal reagents

were obtained from Pierce. OptiPrep was obtained from Life Technologies.

Mouse IgGs against a-tubulin (Clone DM1A) and agarose–glutathione

conjugates and all other chemicals were purchased from Sigma. In Situ

Cell Death Detection Kit, TMR red (Cat# 2 156 792) was purchased from

Roche Applied Science. Campenot nerve cell growth chambers (CAMP10

and CAMP15) were purchased from Tyler Research Corporation.

Cell culture and transient transfectionPC12 cells were maintained in DMEM (4.5 mg/L glucose) supplemented

with 10% horse serum and 5% FBS. Cells were incubated at 378C with 5%

CO2. Cells that were 50–60% confluent and serum starved for 24 h prior to

treatments were used in all experiments. Unless indicated otherwise, all

pretreatments and treatments were carried out by adding growth factors

and other reagents to the media at 378C. The final concentrations for the

nocodazole vehicle (DMSO) and the NGF vehicle (0.2% acetic acid) were

0.1 and 0.0001%, respectively. For transient transfection, the pcDNA3

plasmids containing the full-length, flag-tagged human dynamitin cDNA

sequence (a generous gift from Dr E. Holzbaur, University of Pennsylvania)

were introduced into 70–80% confluent PC12 cells using Lipofectamine

2000 (Invitrogen). For mock transfection, pEGFP-N1 (BD Clontech) was

used as a control. In general, a transfection efficiency of 5–10% was

obtained.

Rat DRG culture and lentiviral transductionThe DRG neurons were isolated aseptically from E15-16 Sprague Dawley

rat following previously established methods (42,65). Dissociated neurons

were plated on either collagen-coated 12-well plates (25 000) or 18-mm

glass coverslips in 12-well plates (10 000/plate) in NGF (100 ng/mL) in

‘maintenance medium’ [(MM), containing sodium phosphate-free MEM

with Earle’s salts, L-glutamine, 10% heat-inactivated fetal bovine serum and

4.5 g/L D-glucose]. Selection medium (MM with antimitotic factors) was

added to the culture next day. The selection was continued for 2 days.

Cultures were then maintained in MM containing NGF (100 ng/mL) for 18

additional days (a total of 21 days), feeding every 3 days. The 21-day-old

cells in vitro are referred as DIV-21. At this time, they contain large, mature

neurons, with an average diameter of 48 mm, at least 94% of which are

immunopositive for TrkA and extend well-developed neurites that also stain

positively for TrkA (data not shown).

For lentiviral transduction, a cDNA encoding either EGFP or dynamitin was

cloned into the pLenti6/V5 expression plasmid using the ViraPower

Lentivirus Expression System (Invitrogen) following the manufacture’s

instruction. The plenti6-EGFP, pLenti6-dynamitin plasmid or the empty

vector was cotransfected together with pLP1, pLP2 and pLP/VSVG into

293FT cells. The lentivirus (lenti-EGFP or lenti-dynamitin or control virus)

was harvested from the supernatant (5000� g for 15 min). The DRG

neurons of DIV-7 or DIV-21 were transduced with the resulting lentivirus

at a multiplicity of infection of approximately 10. A control virus was

produced using the empty pLenti6/V5 vector and was also introduced into

DRGs in parallel experiments. Virus was removed from DRG culture

following an overnight incubation. The DRG neurons were switched back

to MM, and the culture was continued for an additional 7 days. Prior to

experiments, DRG neurons were extensively washed in serum-free, NGF-

free MM and were deprived of NGF for 24 h in serum-free, NGF-free MM

containing a goat antibody against mouse NGF (1:500). After washing in

MM, NGF (100 ng/mL) or equal volume of the vehicle control (0.05% acetic

acid in PBS, pH 7.0) was added for 60 min. Cells were washed and

processed for either immunostaining for confocal analysis or lysed in

1516 Traffic 2007; 8: 1503–1520

Wu et al.

RadioImmunoPrecipitation Assay (RIPA) buffer for SDS–PAGE/immuno-

blotting as described subsequently.

Ras and Rap1 activation assayEstablished methods were used to detect endogenous GTP-bound Ras and

Rap1 proteins as described previously (36). The fusion constructs between

glutathione S-transferase (GST) and either the Rap-binding domain of

RalGDS (RalGDSRBD) or the Ras-binding domain of C-Raf (C-RafRBD)

(gifts from Dr J. L. Bos, Utrecht University, The Netherlands) were over-

expressed in Escherichia coli DH5a cells. The fusion proteins were purified

and used to assay Rap1GTP and RasGTP, respectively. Briefly, an equal

number of treated or untreated PC12 cells were lysed in ice for 30 min in

fishing buffer (FB) (10% glycerol; 1% Nonidet P-40; 50 mM Tris–HCl, pH

7.5; 200 mM NaCl; 2.0 mM MgCl2; 250 mM phenylmethylsulfonyl fluoride;

2 mg/mL aprotinin; 1 mg/mL leupeptin; 10 mg/mL soybean trypsin inhibitor;

10 mM NaF and 1 mM Na3VO4). The samples were centrifuged at 16 000� g,

for 30 min at 48C. Either 5 mg RalGDSRBD/GST or 5 mg C-RafRBD/GST

proteins that were prebound to agarose–glutathione conjugates was added

to the resulting supernatants and incubated at 48C for 60–120 min with

gentle rotation. The beads were washed four times in cold FB and boiled in

SDS–PAGE sample buffer. The amounts of RasGTP and Rap1GTP were

analyzed using SDS–PAGE and immunoblotting.

Cell fractionationFractionation of PC12 cells was carried out using a published protocol (36).

Briefly, an equal number of treated or untreated PC12 cells were rinsed and

harvested by centrifugation (800� g for 5 min). Cells were resuspended

and homogenized by douncing 20 times in a Teflon-coated homogenizer in

1 mL cold homogenization buffer (HB) (250 mM sucrose; 20 mM Tricine–

NaOH, pH7.8; 1 mM ethylenediaminetetraacetic acid and 2 mM MgCl2). The

samples were centrifuged (800� g for 10 min), and the supernatant was

adjusted to 25% OptiPrep with 50% OptiPrep in HB. The resulting mixture

(2 mL in 25% OptiPrep) was placed at the bottom of an Ultra-Clear� Tube

(14 � 89 mm; Beckman Instruments, Inc.) and was overlaid successively

with 2 mL each of 20, 15, 10 and 5% OptiPrep in cold HB. The samples

were centrifuged for 16–18 h at 27 000 r.p.m. at 48C in a SW41 rotor

(Beckman Instruments, Inc.). Membrane fractions were collected from

each of the four interphases of the OptiPrep gradients. For all studies,

proteins were precipitated from the membrane fractions using 7% tri-

chloroacetic acid and washed with acetone. The pellets were air-dried,

boiled in SDS–PAGE loading buffer and analyzed using SDS–PAGE and

immunoblotting.

Elk-1 in vitro phosphorylation assay,

immunoprecipitation, SDS–PAGE and

immunoblottingBriefly, protein samples were separated on 7.5–12.5% gels and proteins

electrotransferred onto polyvinylidene fluoride (PVDF) membranes (NEN

Life Science Products, Inc.). The PVDF membranes were preblotted with

5% nonfat milk (carnation) and probed with primary antibodies, as indi-

cated, at concentrations suggested by the suppliers. The blots were

washed in Tris Buffered Saline Tween 20 (TBST) (20 mM Tris–HCl, pH

7.4; 150 mM NaCl and 0.1% Tween-20), followed by incubation with either

goat anti-mouse or anti-rabbit IgG–HRP conjugates at a dilution of 1/10 000

to 1/40 000. The blots were washed and developed with SuperSignal

(Pierce). For quantification, exposures within the linear ranges were

scanned and determined using the Scion Image program (Scion Corp.).

For in vitro Elk-1 phosphorylation assay, a previously published method was

followed (28).

Indirect immunofluorescence and

confocal microscopyThe PC12 cells were grown for 24–48 h on glass coverslips coated with

matrigel (Becton Dickinson). Cells were starved, washed twice briefly

with cold PBS and fixed with ice-cold 100% methanol for 5 min at �208C.The samples were rinsed thrice with PBS at room temperature. The fixed

cells were preblocked with 1% BSA and 0.2% Triton-X-100 in PBS for

20 min at room temperature. Primary antibodies were diluted at 1/300 to

1/500 in PBS containing 1% BSA and were incubated with the fixed cells

for 1 h at room temperature. The samples were washed with PBS

containing 0.8% BSA thrice and then incubated with goat anti-rabbit– or

anti-mouse–IgG Alexa conjugates (1/600) for 1 h at room temperature.

The coverslips were washed with PBS containing 0.2% BSA thrice,

followed by a rinse with PBS and dH2O. In experiments where nuclear

staining was required (Figure S2C,D and 5A), samples were further

incubated with 2 mg/mL Hoechst 33258 (final concentration) in PBS for

1 min at room temperature, followed by a rinse with PBS and dH2O. The

coverslips were air-dried and mounted in antifade medium for observa-

tion. Images shown in Figure S2C,D and 5A were analyzed using a Nikon

E800 epifluorescence microscope and were captured using a SPOT

charge-coupled device (CCD) camera (Diagnostic Instruments). The

images were processed using SPOT 3.4.3 and Adobe Photoshop 5.0

(Adobe Systems). For confocal microscopy, the images were captured

using a Nikon Eclipse E800 microscope and a Bio-Rad Laser Scanning

System Radiance2000. Using a 60� Plan-Apo immersion objective

(NA1.4), images at the half height of the cell were collected in a

512 � 512 collection box. The images were processed using Confocal

Assistant 4.02 (Bio-Rad) and Adobe Photoshop 7.0 (Adobe Systems).

In experiments in which the periphery of cells was visualized, a bright field

image was captured and superimposed onto the fluorescence image.

Transport of BtNGF in rat DRGs using

Campenot chamberE15-16 rat DRGs were dissected and dissociated, and 20 000 cells were

placed in the middle chamber of a CAMP10 Campenot chamber (Figure 7).

After seven DIV, AXs crossed the barrier and extended into the outer

chamber. Cultures were maintained for 3 weeks in NGF-containing

medium as described previously. The DRGs were then transduced with

lentivirus with different constructs and maintained for an additional 7 days.

Cells were extensively rinsed and starved as described previously. We

routinely checked and discarded those chambers with leakage problem. For

transport assay, BtNGF (100 ng/mL) was added to the DAXs in the outer

chamber and incubated for 60 min at 378C. Cells were rinsed, fixed,

permeabilized and incubated with Streptavidin–Alexa 488 (1/1000 dilution)

for 1 h at room temperature. Samples were rinsed and mounted. The

BtNGF transported to the CB was measured as follows: images of CBs in

the middle chamber were acquired using an inverted Nikon fluorescence

microscope (TE2000U) equipped with Nikon 10� Plan objective. The

fluorescence signal was detected and captured using a CCD camera

(CoolSNAP HQ; Roper Scientific, Inc.), with a detection area of

1392 � 1040 pixels (pixel size: 6.45 � 6.45 mm2). This cooled (�308C)camera has a 12-bit dynamic range, which allows imaging very dim and very

bright objects at the same time without saturation. Binary data were

collected and saved using a home-built software package with 2 � 2

hardware binning and 0.5 seconds exposure time (Stanford University).

Subsequent data analysis was performed using Data Interactive Language

(IDL) software with scripts written in the Chu laboratory. For each fluores-

cence image, the average signal intensity within a selected region of

interest (ROI) encompassing 100 cells was measured. At least four ROIs

were selected to obtain the mean and the standard deviation. A background

value was obtained by incubating a sample with Streptavidin–Alexa 488

conjugate only. This value was subtracted from those obtained from the

samples that were treated with BtNGF. All statistical analyses were carried

out using Student’s t-test.

For detection of BtNGF that was transported retrogradely to the CB/PAXs

by Western blot, 200 000 DRGs were placed in the small chamber of

a CAMP15 apparatus (Figure 7) and maintained for 3 weeks. Following

lentiviral transduction and NGF deprivation, BtNGF (50 ng/mL) was added

into the larger chamber that harbored only the AXs and was incubated for

12 h. The CBs/PAXs were rinsed, lysed and cleared by centrifuge. Forty

microliters of streptavidin–agarose conjugate slurries was added to the

supernatant to precipitate BtNGF. The precipitates were analyzed by

SDS–PAGE/Western blotting.

Traffic 2007; 8: 1503–1520 1517

Dynein-/Microtubule-Mediated Trafficking of TrkA for Rap1 Signaling

Acknowledgments

We thank the members of the Mobley laboratory for their assistance. We

also thank Drs Francisca Bronfman and Mike Fainzilber for their invaluable

advice on the production of BtNGF. These studies are supported through

the grants fromNational Institutes of Health (NS24054, NS38869, AG16999

and NS055371), the John Douglas French Alzheimer’s Foundation, the

McGowan Charitable Trust, the Larry L Hillblom Foundation, the Adler

Foundation and the Deane Johnson Alzheimer’s Disease Fund.

Supplementary Materials

Figure S1: The BtNGFwas internalized into EEA1-positive endosomes

in PC12 cells. The PC12 cells were cultured for 24–48 h on cover glasses

coated with matrigel and were serum starved overnight. Cells were chilled

and incubated with BtNGF (50 ng/mL) for 45 min on ice. Cells were rinsed

and immediately processed for immunostaining (A, C, E and G). A parallel

sample was shifted to 378C for 30 min to allow endocytosis of BtNGF prior

to immunostaining (B, D, F and H). The BtNGF was detected with

a Streptavidin–Alexa 488 conjugate (green, A–D). The following specific

primary antibodies were used: a mouse antibody to Trk, B3 (1/200) for TrkA

(A, B, E and F); a mouse antibody to EEA1 (1/50) for EEA1 (C and D); a rabbit

antibody to Rab5B (1/200) for Rab5B (E, F, G and H) and a mouse antibody

to pMAPK1/2 (1/100) for pMAPK1/2 (G and H). Secondary antibody

conjugated to either Alexa 568 or Alexa 488 was used to visualize the

primary antibody. Colocalization of proteins is denoted by the white signal.

The size of the collection box is 30 � 30 mm.

Figure S2: The NGF-induced trafficking of Rap1, TrkA and pMAPK1/2

to the perinuclear region. The PC12 cells were cultured as described in

Figure 1. Following pretreatment with either the NGF vehicle alone (�NGF)

or the NGF (50 ng/mL) (þNGF), indirect immunofluorescence was carried

out using confocal microscopy as described in Materials and Methods.

Rap1 (A–D and G–L) was immunostained using a specific rabbit antibody

(1/250) and detected with an Alexa 568 goat anti-rabbit IgG conjugate. The

TrkA (E and F and G and H) was detected using B3 (1/200) and visualized

with either an Alexa 568 goat anti-mouse IgG conjugate (E and F) or an Alexa

488 goat anti-mouse IgG conjugate (G and H). The signals for pMAPK1/2

and EEA1 were each detected using a mouse monoclonal antibody specific

for these proteins as shown in Figure 1. The dilutions of primary antibodies

were as follows: 1/100 for pMAPK1/2 and 1/50 for EEA1. An Alexa 488 goat

anti-mouse IgG conjugate was used to visualize these antibodies. Colocal-

ization of antigens is denoted by the white signal. In A, B, E and F,

a corresponding image from the transmitted light detector was simul-

taneously captured and superimposed onto the fluorescence image of Rap1

or TrkA to illustrate the outline of the cell. In (C) and (D), Rap1 (magenta) was

co-stained with Hoechst 33258 to reveal the nucleus (blue). The resulting

samples were captured and analyzed using a Nikon E800 epifluorescence

microscope. Twenty-five cells were examined in each condition (A–L), and

a typical representative image was shown. The size of the collection box is

30 � 30 mm for (A) and (B) and E–L and 60 � 60 mm for (C) and (D).

Supplemental materials are available as part of the online article at http://

www.blackwell-synergy.com

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