T H E J O U R N A L O F C E L L B I O L O G Y
© The Rockefeller University Press $30.00
The Journal of Cell Biology, Vol. 180, No. 4, February 25, 2008 827–842
Correspondence to B. Winckler: BWinckler@virginia.edu
P. Kujala ’ s present address is the Dutch Cancer Institute, 1066 CX, Amsterdam,
Abbreviations used in this paper: A/D PI, axon-dendrite polarity index; AS-
NEEP21, antisense NEEP21; DIV, day in vitro; EE, early endosome; LE, late
endosome; MVB, multivesicular body; NEEP21, neuron-enriched endosomal
protein of 21 kD; NgCAM, neuron-glia cell adhesion molecule; RE, recycling
endosome; Tf, transferrin; TGN, trans-Golgi network; Ti-VAMP, toxin-insensitive
vesicle-associated membrane protein.
The online version of this paper contains supplemental material.
Many of the diverse functions of neurons are regulated by endo-
cytosis ( Cremona and De Camilli, 1997 ; Alberts and Galli,
2003 ; for review see Garrido et al., 2003 ; Sudhof, 2004 ; for
review see Winckler, 2004 ; Deinhardt and Schiavo, 2005 ; for re-
view see Kennedy and Ehlers, 2006 ; Lai and Jan, 2006 ). Neurons
elaborate a much more complex and diversifi ed endosomal sys-
tem than nonpolarized cells ( Sachse et al., 2002 ). For instance,
somatodendritic endosomes play crucial roles in synaptic func-
tion (for review see Kennedy and Ehlers, 2006 ). Distinct axonal
endosomes are responsible for retrograde signaling from the
nerve terminal ( Susalka and Pfi ster, 2000 ; Segal, 2003 ; Howe
and Mobley, 2004 ; Deinhardt and Schiavo, 2005 ), transport of
degradative cargo, and local recycling at axonal growth cones
( Kamiguchi and Lemmon, 2000 ; Alberts and Galli, 2003 ) and
synapses ( Slepnev and De Camilli, 2000 ; Schweizer and Ryan,
2006 ). The overwhelming majority of endosomal cargos that
travel via fast axonal transport in the axon do so in a retrograde
direction ( Parton et al., 1992 ). Their fi nal destinations can be
degradative compartments in the soma, nondegradative “ signal-
ing endosomes, ” or transneuronal traffi c to another neuron.
However, long-range anterograde traffi c of endosomes along
axons is not usually observed but would be predicted to occur
for cargos that transcytose from the dendrites to axons ( von
Bartheld, 2004 ; for review see Winckler, 2004 ).
Currently, the organization and regulation of these diverse
endosomal routes is incompletely understood. Different com-
partments in the endosomal system can be distinguished by the
content of their markers as well as by their morphology. Fig. 1 D
depicts the basic organization of endosomes ( Maxfi eld and
McGraw, 2004 ). Three distinct pathways are indicated: the deg-
radative pathway ( Fig. 1 D , red arrows), the somatodendritic re-
cycling pathway (green arrows), and the transcytotic pathway
(blue arrows). In epithelial cells, recycling takes place from
the early endosome (EE) or the recycling endosome (RE).
By analogy, local recycling from somatodendritic EEs back to
adhesion molecule L1/neuron-glia cell adhesion mole-
cule (NgCAM) depends on endocytosis (Wisco, D., E.D.
Anderson, M.C. Chang, C. Norden, T. Boiko, H. Folsch,
and B. Winckler. 2003. J. Cell Biol. 162:1317 – 1328).
Two endocytosis-dependent pathways to the axon have
been proposed: transcytosis and selective retrieval/
retention. We show here that axonal accumulation of
L1/NgCAM occurs via nondegradative somatodendritic
endosomes and subsequent anterograde axonal transport,
orrect targeting of proteins to axons and den-
drites is crucial for neuronal function. We showed
previously that axonal accumulation of the cell
which is consistent with transcytosis. Additionally, we
identify the neuronal-specifi c endosomal protein NEEP21
(neuron-enriched endosomal protein of 21 kD) as a reg-
ulator of L1/NgCAM sorting in somatodendritic endo-
somes. Down-regulation of NEEP21 leads to missorting
of L1/NgCAM to the somatodendritic surface as well as
to lysosomes. Importantly, the axonal accumulation of
endogenous L1 in young neurons is also sensitive to
NEEP21 depletion. We propose that small endosomal
carriers derived from somatodendritic recycling endosomes
can serve to redistribute a distinct set of membrane pro-
teins from dendrites to axons.
The somatodendritic endosomal regulator NEEP21
facilitates axonal targeting of L1/NgCAM
Chan Choo Yap , 1 Dolora Wisco , 1 Pekka Kujala , 3,4 Zofi a M. Lasiecka , 1 Johanna T. Cannon , 1 Michael C. Chang , 1
Harald Hirling , 2 Judith Klumperman , 3,4 and Bettina Winckler 1
1 Department of Neuroscience, University of Virginia Medical School, Charlottesville, VA 22908
2 Brain Mind Institute, Ecole Polytechnique Federale de Lausanne, CH-1015 Lausanne, Switzerland
3 Department of Cell Biology and 4 Institute for Biomembranes and Center for Biomedical Genetics, University Medical Center, University of Utrecht, 3584CX Utrecht, Netherlands
JCB • VOLUME 180 • NUMBER 4 • 2008 828
to the axonal surface ( Fig. 1 D, blue). The selective retention/
retrieval model, in contrast, predicts that NgCAM is primarily
degraded in lysosomes after endocytosis, does not enter axons in
endosomes, and does not recycle to the axonal surface ( Fig. 1 D ,
red). These two pathway models can therefore be distinguished
experimentally. In this work, we show that NgCAM accumulates
on the axon by transcytosis rather than by selective retrieval/
retention. We also show that NEEP21 function is required for
effi cient accumulation of NgCAM on the axon.
Detectability of endocytosed NgCAM in
somatodendritic endosomes depends on
Because it remains controversial whether or not NgCAM is
found in somatodendritic endosomes ( Sampo et al., 2003 ; Wisco
et al., 2003 ), we fi rst tested what variables might affect detection
of endocytosed NgCAM in somatodendritic endosomes. Neither
the transfection method nor the age of the culture showed any
differences in the detectability of somatodendritic NgCAM endo-
cytosis (Fig. S1 A, available at http://www.jcb.org/cgi/content/
full/jcb.200707143/DC1). Detergent conditions, however, proved
critical for reliable detection of endocytosed NgCAM (Fig. S1,
B – E). The discrepancy between the fi ndings of Sampo et al.
(2003) and our own ( Wisco et al., 2003 ) therefore likely refl ects
differences in permeabilization conditions. Similarly to NgCAM,
human L1-myc (Fig. S1 F) and rat L1-myc (not depicted) were
internalized into somatodendritic endosomes. Cross linking of
NgCAM by divalent anti-NgCAM IgGs is not necessary for the
detectability of NgCAM in somatodendritic endosomes because
somatodendritic endocytosis can also be observed with compa-
rable effi ciency (i.e., 70 – 80% of cells) when anti-NgCAM Fab
fragments are used for the endocytosis assay (Fig. S1 G).
Endocytosed NgCAM colocalizes with
somatodendritic EEs and partially localizes
The selective retrieval/retention model predicts that endocytosed
NgCAM would be primarily degraded ( Fig.1 D , red), whereas
the transcytotic model predicts that NgCAM would largely by-
pass the late endosome (LE)/lys ( Fig. 1 D , blue). We, therefore,
asked whether endocytosed NgCAM in the somatodendritic do-
main initially colocalized with a marker for EEs (EEA1), and
whether or not it ultimately accumulated with a marker for LE/lys
(lgp120; see Materials and Methods for details on quantifi cation).
After incubating NgCAM-expressing cells for 20 min with anti-
NgCAM antibodies (t = 0), 55% of all labeled endosomes con-
tained both endocytosed NgCAM and EEA1 ( Fig. 1 C , yellow,
orange, and light green segments on the left), 23% of all labeled
endosomes contained only EEA1 ( Fig. 1 A, red; and Fig. 1 C ,
left, red). If only EEA1-positive endosomes were analyzed, 71%
of them contained some endocytosed NgCAM ( “ high ” plus
“ low ” colocalization categories). We currently do not know if the
high and low colocalization endosomes represent functionally
distinct endosomal populations but they might refl ect progressive
sorting of NgCAM out of EEA1-positive endosomes.
the somatodendritic domain might also occur in neurons ( Fig. 1 D ,
green arrow). Likewise, REs are capable of sorting cargos to
distinct plasma membrane domains in polarized epithelial cells.
In neurons, it is likely that the RE is also capable of polarized
sorting of cargos toward multiple destinations, such as axons
( Fig. 1 D , blue arrows) or dendrites (green arrows; Schmidt and
Haucke, 2007 ).
Multiple proteins identifi ed as regulators of endosomal
traffi c in nonneuronal cells are also important in neuronal endo-
somes ( Alberts et al., 2003 ; Park et al., 2004 ; Brown et al.,
2005 ; Deinhardt et al., 2006 ). Additionally, neuronal-specifi c
endosomal proteins might carry out neuronal-specifi c functions.
One such neuronal endosomal protein is the neuron-enriched
endosomal protein of 21 kD (NEEP21). NEEP21 was identifi ed as
an interacting protein of the endosomal syntaxin 13 and localizes
to rab4-positive, EEA1-negative EEs in PC12 cells ( Steiner et al,
2002 ). NEEP21 plays a crucial role in regulating the traffi cking
of multiple receptors in neurons ( Steiner et al., 2002, 2005 ;
Debaigt et al., 2004 ; Alberi et al., 2005 ), including the synaptic
traffi cking of AMPA receptors during synaptic plasticity ( Alberi
et al., 2005 ; Steiner et al., 2005 ; Kulangara et al., 2007 ).
Several groups have investigated the mechanisms under-
lying axonal targeting of the neuron-glia cell adhesion molecule
(NgCAM), the chick homologue of L1 ( Kamiguchi and Lemmon,
1998 ; Burack et al., 2000 ; Sampo et al., 2003 ; Wisco et al.,
2003 ; Chang et al., 2006 ; Boiko et al., 2007 ). Based in part on
the undetectability of NgCAM in somatodendritic endosomes,
one model proposed that NgCAM was sorted to the axon by se-
lective axonal fusion independent of endocytosis ( Sampo et al.,
2003 ). In contrast, our own work did detect somatodendritic
endocytosis of NgCAM ( Wisco et al., 2003 ). In addition,
inhibition of endocytosis greatly impaired axonal accumulation
of NgCAM ( Wisco et al., 2003 ). NgCAM therefore uses an
endocytosis-dependent pathway to the axon. Whether a “ selective
fusion ” pathway is operative for NgCAM in certain cell types or
at certain developmental stages is still an open question.
The targeting of several other axonal proteins is also endo-
cytosis dependent ( Garrido et al., 2001 ; Sampo et al., 2003 ;
Leterrier et al., 2006 ; Xu et al., 2006 ). Two endocytosis-dependent
pathways to the axon have been proposed: transcytosis ( Wisco
et al., 2003 ) and selective retrieval/retention ( Fache et al., 2004 ).
In the transcytotic model, the protein is sorted in the trans-Golgi
network (TGN) to the somatodendritic domain. After somato-
dendritic exocytosis, the protein is endocytosed into somato-
dendritic endosomes and traffi cked to the axon from there. In the
selective retrieval/retention model, no sorting occurs in the TGN
but proteins are inserted uniformly in both somatodendritic and
axonal domains. Preferential accumulation of the protein on
the axonal surface then occurs by selectively removing it from
the somatodendritic surface by endocytosis and degrading it.
The axonal population, however, is selectively retained on the
axo nal plasma membrane (for reviews see Garrido et al., 2003 ;
Winckler, 2004 ). The transcytotic model makes several specifi c
predictions: endocytosed NgCAM enters EEs in the somato-
dendritic domain, sorts away from somatodendritically recycling
transferrin (Tf), is not primarily degraded in lysosomes, travels
anterogradely up the axon in endosomes, and ultimately recycles
829 ENDOSOMAL TRAFFICKING OF N G CAM TO THE AXON • YAP ET AL.
yellow; and Fig. 1 C , right, yellow), whereas 32% contained
only endocytosed NgCAM ( Fig. 1, B and C , right, red). Of the
somatic endosomes containing endocytosed NgCAM at long
chase times, 47% also contained various amounts of lgp120.
Because ? 50% of endocytosed NgCAM leaves the somato-
dendritic endosomes in 90 min (see Fig. 3 B ), we estimate that ? 25%
of endocytosed NgCAM ultimately accumulates in lysosomes.
Because all proteins turn over to some degree at all times, some
NgCAM is expected to localize to lysosomes. We note that
complete quantifi cation of NgCAM localization to lysosomes is
not possible because some NgCAM and bound antibody might
have been already degraded.
Somatodendritic endosomes contain
endocytosed NgCAM by immuno-EM
Next, we sought to identify the somatic compartments contain-
ing NgCAM on the ultrastructural level. NgCAM at steady-state
was found prominently in multivesicular bodies (MVBs; Fig. 2 A ,
asterisks) in the soma. Small profi les ( Fig. 2 A , arrowheads)
were also labeled. The exact identity of these compartments is
not known but they might correspond to transport carriers or
tubular portions of EEs.
To visualize endocytosed NgCAM, neurons were incu-
bated simultaneously with an anti-NgCAM antibody and the
fl uid-phase marker gold-BSA and then processed for immuno-
EM ( Oorschot et al., 2002 ). Gold-BSA is ultimately transported
to lysosomes and does not accumulate in REs. We therefore de-
termined if endocytosed NgCAM could accumulate in compart-
ments that exclude gold-BSA. Endocytosed NgCAM, detected
by 15-nm gold-coupled protein A, was most often found in
MVBs together with 3-nm gold-BSA ( Fig. 2, B – D ). Some of
these NgCAM-positive MVBs contained no or very little gold-
BSA ( Fig. 2, C and D ). 15 nm gold was also occasionally asso-
ciated with tubules devoid of gold-BSA, which is reminiscent
of recycling endosomal compartments ( Fig. 2 E ; Peden et al.,
2004 ). NgCAM is therefore capable of entering nondegradative
endosomal compartments including REs, but is also found in
presumptive predegradative compartments.
Endocytosed NgCAM traverses
somatodendritic endosomes and recycles
preferentially to the axonal plasma
The fate of endocytosed NgCAM was followed using a pulse-
chase approach with an acid strip. We incubated NgCAM-
expressing neurons with an anti-NgCAM antibody for 20 min
(load) and then removed all remaining surface-bound antibody
using an acid strip step. Cells were then returned to the incuba-
tor for various amounts of time (chase). After loading (t = 0),
endocytosed NgCAM could be detected prominently in somato-
dendritic endosomes ( Fig. 3 A ) and was also seen, though less
brightly, in endosomes along axons ( Fig. 3 A , arrows). After
90 min of chase, the soma fl uorescence was decreased ( Fig. 3 B ),
whereas axonal endosomes were still bright ( Fig. 3 B , arrows).
Quantifi cation of the soma-associated fl uorescence showed that
the half-time for depleting the soma signal was ? 90 min ( Fig. 3 C ,
open diamonds). Tf, however, disappeared from the soma with
Endocytosed NgCAM, which had been chased for 1 h (t =
60) to allow accumulation in a terminal compartment, colocalized
partially with lgp120 (21% of all labeled endosomes; Fig. 1 B,
Figure 1. Colocalization of endocytosed NgCAM with markers for early
and late endosomes. (A) NgCAM-expressing neurons were incubated for
20 min with anti-NgCAM antibodies, which were detected after permea-
bilization with an Alexa 488 – goat anti – mouse antibody, whereas EEs
were detected with a polyclonal anti-EEA1 antibody (red). Yellow arrow-
heads indicate examples of colocalizing puncta. (B) NgCAM-expressing
neurons were incubated with anti-NgCAM antibody for 30 min at 16 ° C
and washed, and internalized NgCAM antibody was chased at 37 ° C
for 1 h. Surface NgCAM was detected before permeabilization with a
cy5 – goat anti – mouse secondary antibody (blue). After permeabilization,
endocytosed NgCAM antibodies were detected with Alexa 568 – goat
anti – mouse antibody (red) and LEs/lysosomes were detected with a poly-
clonal anti-lgp120 antibody (green). A single confocal section is shown.
The boxed regions shown in A and B are magnifi ed sections of dendrites
for easier comparison of colabeling. (C) Quantifi cation of colocalization
of EEA1 and NgCAM endocytosed for 20 min and of lgp120 and Ng-
CAM endocytosed and chased for 60 min. For each puncta, the ratio of
intensities of endocytosed NgCAM to either EEA1 (left) or lgp120 (right)
was determined as described in Materials and methods. For EEA1 co-
localization, 560 endosomes were scored. For lgp120 colocalization, 497
endosomes were scored. (D) Basic organization of endosomes, adapted
from fi broblasts. Three distinct pathways are indicated by the arrows:
degradative cargo follows the red arrows, somatodendritically recycling
cargo follows the green arrows, and transcytosing cargo follows the blue
arrows. Cargo enters via several pathways in small carriers vesicles (ECV)
that fuse with existing EEs. EEs contain a vacuolar portion as well as tubu-
lar extensions. The tubular extensions accumulate recycling cargoes and
bud off to transport recycling cargos back to the plasma membrane either
directly or via the RE. Endosomal carrier vesicles (ECVs) can be spheri-
cal or elongated and serve as transport carriers between compartments.
The vacuolar portions of the EE accumulate internal vesicles and mature
into MVBs. MVBs are transport carriers that carry cargo to LE/lysosomes
(LE/lys) for degradation (red). Some MVBs might not be predegradative
but be capable of recycling. Cargo destined for either axons or dendrites
are sorted in the RE and transported to their respective fi nal destination
JCB • VOLUME 180 • NUMBER 4 • 2008 830
expressing neurons for 1 h in a live-imaging chamber. Cells were
then washed and images were taken every 2 or 3 s for about
1 min. Endocytosed NgCAM was easily detected in somato-
dendritic endosomes in live cells ( Fig. 4, A and A ? ). The extent
of endosome motility was displayed by merging three video
frames into one RGB image such that the fi rst frame appeared
red, the second green, and the third blue ( Fig. 4, A and A ? ).
Interestingly, the majority of the bright endosomes in the soma
were stationary during the imaging duration ( Fig. 4, A and A ? ;
and Video 1, available at http://www.jcb.org/cgi/content/full/jcb
.200707143/DC1). A subset of NgCAM-containing endosomes
underwent movements ( Fig. 4, A and A ? , arrowheads). The label-
ing intensity of the endosomes usually correlated with size,
such that the large stationary endosomes were brightly labeled
but the small moving carriers were faint. We frequently ob-
served short, wiggling excursions that did not result in long-
distance translocation. We also observed longer-range transport
events of small tubules ( Fig. 4 B , arrows) or small circular com-
partments ( Fig. 4 C , arrows). Movements were often inter-
mittent with pauses of many seconds interspersed with periods
of movement. Frequently, a small transport carrier initially ap-
peared to emerge from, and ultimately disappear into, a larger
stationary endosome. 91% of NgCAM-containing endosomes
in the soma and dendrites appeared to be round or irregularly
shaped, whereas 9% appeared to be elongated ( n = 232; Fig. 4 E ).
The elongated carriers frequently appeared to be of varying
width and intensity along their lengths (see Sonnichsen et al.,
2000 ). 22% of all observed NgCAM endosomes underwent
movement during the 1-min imaging periods. Of all the moving
compartments, 84% appeared small (estimated diameter of
? 0.2 μ m) and round, whereas 14% appeared elongated. Large
(estimated diameter, > 1 μ m) and medium-sized (estimated
diameter, ? 0.7 μ m) round compartments rarely moved ( Fig. 4 E ,
white and black bars). NgCAM therefore accumulates in stationary
a half-time of ? 25 min ( Fig. 3 C , circles). To estimate the abun-
dance of endocytosed NgCAM within dendritic and axonal endo-
somes, we quantifi ed the intensity of individual endosomes in
axons and dendrites. Endosomes correspond to the peaks in
intensity line scans ( Fig. 3 D ) taken along dendrites and axons
after the loading step (t = 0 ? ) and at t = 90 ? . At t = 0, dendritic
endosomes were on average about twofold brighter than axonal
endosomes ( Fig.3 D, top; axon/dendrite intensity ratio = 0.58).
At t = 90 ? , axonal endosomes were on average 40% brighter
than dendritic endosomes ( Fig. 3 D bottom; axon/dendrite intensity
ratio = 1.39). Endocytosed NgCAM, therefore, progressively
disappeared from somatodendritic endosomes and accumulated
in axonal endosomes.
To see if internalized NgCAM ever reappeared on the
cell surface, we performed a load/acid strip/chase experiment as
described in the previous paragraph but incubated the cells with
Alexa 647 – coupled secondary anti – mouse antibodies before
permeabilization to stain the recycled surface pool (see Materials
and methods). Initially, no surface labeling could be detected
( Fig. 3 A ? ), but surface reappearance was easily detected at t = 90 ?
( Fig. 3 B ? and C , triangles and broken line). To determine to
which surface endocytosed NgCAM recycled, we determined the
axon-dendrite polarity index (A/D PI; see Materials and methods)
for recycling NgCAM. At t = 40 ? , surface NgCAM staining
already was threefold higher on the axonal surface. By t = 90 ? ,
it was 4.7-fold higher. By 2.5 h of chase, the recycling A/D PI
was 5.5, the same as the steady-state A/D PI for NgCAM. NgCAM
therefore recycled with at least a threefold bias toward the axon.
Endocytosed NgCAM accumulates in
stationary somatodendritic endosomes and
is transported in small endosomal carriers
We next imaged traffi cking of endosomal NgCAM by feeding
anti-NgCAM antibodies coupled to Alexa 488 to NgCAM-
Figure 2. Ultrastructural identifi cation of
NgCAM-containing somatic endosomes. (A)
Hippocampal neurons were infected with
AdNgCAM for 24 h and subsequently fi xed.
NgCAM was detected with anti-NgCAM anti-
bodies and 15 nm gold – protein A. NgCAM is
found on the plasma membrane, in numerous
MVBs (asterisks), and in small profi les of un-
known identity (arrowheads). (B – E) NgCAM-
expressing neurons were fed with anti-NgCAM
antibodies and 3 nm gold-BSA for 15 – 60 min
and then washed and fi xed. Endocytosed Ng-
CAM (15 nm gold) is found in MVBs together
with varying amounts of 3 nm gold BSA. The
labeled MVB in B contains many 3-nm gold
particles (appearing as dark, grainy material)
as well as large 15-nm gold particles (arrows).
A larger magnifi cation of the boxed region is
shown in the inset below to highlight the two
different sizes of particles. The MVB in C has
few 3-nm gold particles, whereas the one in D
has virtually none. (E) Occasionally, NgCAM is
found in tubules that are devoid of cointernalized
BSA-gold and likely represent RE. Bars, 150 nm.
831 ENDOSOMAL TRAFFICKING OF N G CAM TO THE AXON • YAP ET AL.
Figure 3. Endocytosed NgCAM leaves somatic endosomes and recycles preferentially to the axonal plasma membrane. (A) After 20 min of anti-NgCAM
antibody loading (t = 0), intracellular endocytosed NgCAM can be visualized after acid stripping of surface-bound antibodies in somatodendritic but
also axonal (arrows) endosomes. (B) After 90 min of chase at 37 ° C (t = 90 ? ), somatodendritic endosomes are less prominently labeled, whereas axonal
endosomes are still clearly detected (arrows). (A ? and B ? ) Surface reappearance of endocytosed NgCAM in the same cells was assayed with an Alexa
647 – goat anti – mouse antibody after acid stripping and chase. No surface labeling was detectable at t = 0 (A ? ), demonstrating the effi ciency of the acid
strip. After 90 min of chase, axonal surface labeling was apparent (B ? ), demonstrating recycling of NgCAM to the plasma membrane. (C) The disappear-
ance of somatic endosomal NgCAM was plotted as percentage of fl uorescence remaining after acid stripping over time (open diamonds). Recycling of Tf
from somatic endosomes was plotted as well (circles). Surface reappearance of endocytosed NgCAM was plotted as percentage of the chase end point
of 2.5 h (triangles and broken line). The mean of four independent experiments is shown. SEM is indicated for each time point. (D) Representative intensity
line scans are shown for dendrites at t = 0 (top left), axons at t = 0 (top right), dendrites at t = 90 min (bottom left), and axons at t = 90 min (bottom right).
Brightly staining endosomes correspond to the peaks on the trace. The ratio of axon/dendrite average puncta intensity for t = 0 and t = 90 min is indicated
to the right of the traces. n = 4 independent experiments.
JCB • VOLUME 180 • NUMBER 4 • 2008 832
the mobility of NgCAM endosomes in the axon (see Materials
and methods). Again, most of the bright round endosomes con-
taining NgCAM were not motile ( Fig. 4, D1 – D3 , white puncta).
Because the moving endosomal carriers are small and faint, imag-
ing of axonal motile endosomes was technically challenging and
extensive quantifi cation proved not to be feasible. Nonetheless,
we could observe clear examples of moving transport carriers
containing endocytosed NgCAM ( Fig. 4 D and Video 2, avail-
able at http://www.jcb.org/cgi/ content/full/jcb.200707143/DC1)
somatodendritic endosomes but is transported intermittently in
small transport carriers.
Endocytosed NgCAM travels anterogradely
Retrograde transport of endosomes has been well described ( Overly
and Hollenbeck, 1996 ; Segal, 2003 ; Deinhardt and Schiavo,
2005 ). Whether or not endosomal cargo travels anterogradely in
axons has not been established. We therefore sought to determine
Figure 4. Live imaging of NgCAM-containing endosomes. (A – C) Neurons (DIV10) transfected with NgCAM were allowed to endocytose Alexa 488 – anti-
NgCAM antibodies in live imaging chambers for 60 min before washing and imaging at an acquisition speed of 0.5 Hz. Somatodendritic endosomes
can be easily visualized (A and A ? ). See Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200707143/DC1). (A and A ? ) Three frames
of video were merged so that frame 1 (0 s) is red, frame 2 (2 s) is green, and frame 3 (6 s) is blue. Nonmoving endosomes therefore appear white.
Moving endosomes appear colored (arrowheads). (B and C) Single frames of endocytosed NgCAM, 2 s apart, are shown to illustrate examples of a moving
NgCAM-containing tubule (B, arrow), a nonmoving tubule (B, arrowhead), or a moving NgCAM-containing round carrier (C, arrow). The trajectory of the
carrier in C is depicted on the graph. The speeds of observed movements ranged from 0.1 to 1.4 μ m/s, averaging 0.5 μ m/s. (D) Axonal endosomes were
imaged live after acid stripping. A lower magnifi cation image of the labeled cell is shown. Axons and dendrites are indicated with arrows and arrowheads,
respectively. (D1 – D3) Anterograde and retrograde movements of small NgCAM-containing endosomal carriers in the axon are observed (arrows). Three
examples are shown in D1 – D3, where frame 1 is red, frame 2 is green, and frame 3 is blue. Frames are 10 s apart as indicated (see Video 2). Nonmoving
endosomes appear white. Anterograde movement is toward the top. Fig. S2 shows a single channel display. (E) Quantifi cation of the behavior of NgCAM-
containing somatodendritic endosomes. Three size classes are distinguishable: large round (apparent diameter, 1 – 1.3 μ m), medium round (apparent
diameter, 0.6 – 0.8 μ m), small round (0.2 – 0.3 μ m), and elongated. NgCAM was mostly transported in small round endosomal carriers. n = 232.
833 ENDOSOMAL TRAFFICKING OF N G CAM TO THE AXON • YAP ET AL.
motile endosomes (Vidoes 3 – 6, available at http://www.jcb.org/
cgi/content/full/jcb.200707143/DC1). Of all labeled endosomes
( n = 365), 17% underwent movements in 50 s of imaging.
Tf-containing compartments frequently wiggled about randomly,
but only a few examples of rapid directed movement of Tf-
containing compartments could be observed ( Fig. 5 B , red arrow;
and Fig. 5 D , right). Many of the compartments containing only
Tf were large and pleiomorphic. Shape changes in these large
structures did not permit quantifi cation of individual compart-
ments. In contrast, we frequently saw clear examples of mobile
small spherical and tubular elements. These motile compartments
tended to be largely devoid of Tf, with 70% of mobile compart-
ments containing only NgCAM ( Fig. 5 D , middle; and Video 3).
Small spherical endosomes showed the most motility ( Fig. 5 B ,
green arrow; Fig. 5 D , middle; and Video 4).
In addition, we observed red and green puncta that moved
as a unit ( Fig. 5 C and Video 5), which is reminiscent of the
traffi c light patterns observed in fi xed samples ( Fig. 5 A , right).
These images might represent single membrane-bound elon-
gated compartments in which Tf and NgCAM are laterally seg-
regated. Such lateral segregation of cargos in REs is also found
in other cell types, including MDCK cells ( Thompson et al.,
2007 ) and macrophages ( Manderson et al., 2007 ). To visualize
the laterally segregated cargos, a fl uorescent lipid (DiIC18-DS)
was allowed to coendocytose. This assay enabled the observa-
tion of two differently labeled cargos encompassed in single
DiI-labeled structures ( Manderson et al., 2007 ). Even though
this technique still relies on light microscopy, observation of
such structures is consistent with the existence of single com-
partments with laterally segregated components. We adapted
this assay, feeding Tf and anti-NgCAM antibody as well as DiI.
Surprisingly, DiI labeled the NgCAM-containing endosomes
much more reliably than it labeled Tf-containing endosomes
(Fig. S3 A, available at http://www.jcb.org/cgi/content/full/
jcb.200707143/DC1), which suggests that the lipid composi-
tion of NgCAM and Tf endosomes is not identical and that DiI
partitions preferentially into a subset of endomembranes in
neurons. We occasionally observed endosomes labeled by all
three tracers (two to fi ve per cell out of hundreds). Among the
triple-labeled profi les, endosomes with laterally offset NgCAM
and Tf staining seemingly encompassed by a single DiI-labeled
profi le were common (Fig. S3, B – D). Endocytosed NgCAM
therefore was found together with Tf in stationary REs in the
soma but it segregated away from Tf and moved over long dis-
tances in small spherical or tubular endosomal carriers largely
devoid of Tf.
Axonal polarization of NgCAM is sensitive
to interference with NEEP21 but not
membrane protein (Ti-VAMP)
In PC12 cells, two proteins affect the endosomal recycling of
L1, the toxin-insensitive VAMP Ti-VAMP/VAMP7 ( Martinez-
Arca et al., 2001 ) and NEEP21 ( Steiner et al., 2002 ). Ti-VAMP
is found in both somatodendritic and axonal endosomes,
whereas NEEP21 is found only in somatodendritic endosomes.
We used two function-interfering constructs, Ti-VAMP-DN
in both the anterograde and retrograde direction ( Fig. 4, D1 – D3 ,
arrows; and Video 2) with speeds ranging from 0.3 to 1.8 μ m/s.
Slightly more than half of the movements occurred in the antero-
grade direction. Therefore, small NgCAM-containing endosomal
carriers travel up the axon by anterograde axonal transport,
whereas large stationary endosomes in the axon accumulate
endocytosed NgCAM. We do not currently know whether the
retrogradely transported NgCAM is degraded in lysosomes or
can recycle to the plasma membrane.
Endocytosed NgCAM transiently colocalizes
with recycling Tf
Tf is a marker for the somatodendritic recycling pathway ( Fig. 1 D,
green; Hemar et al., 1997 ). The transcytotic model predicts
that NgCAM initially internalizes into the same EE as Tf but
ultimately sorts away from Tf into distinct endosomes and is
sorted to axons from there. We incubated FITC-Tf – loaded cells
with anti-NgCAM antibody at 16 ° C for 30 min to allow entry
into the EE ( Schmid and Smythe, 1991 ). Cells were then washed
and incubated at 37 ° C in the continued presence of FITC-Tf for
chase times of 5 – 120 min. At 5 min of NgCAM chase, NgCAM
and Tf showed numerous clear examples of precise overlap
(unpublished data), suggesting that NgCAM initially entered the
Tf-containing EE, which is consistent with the colocalization of
EEA1 and endocytosed NgCAM ( Fig. 1 A ). At 15 min of chase,
we already observed many puncta containing only endocytosed
NgCAM ( Fig. 5 A , left, red). Because Tf was loaded to a steady
state, these puncta correspond to a compartment in which Tf
does not signifi cantly accumulate. Precise colocalization of
Tf and endocytosed NgCAM at 15 min of chase could also be
observed ( Fig. 5 A , arrowheads). After chase times of 60 min
( Fig. 5 A , right), Tf- and NgCAM-positive regions frequently
appeared closely apposed but not precisely overlapping. In these
cases, the red and green channels were laterally offset, appear-
ing as multicolored “ traffi c lights ” ( Fig. 5 A , right, arrowheads),
which suggests that Tf and NgCAM largely occupied either
distinct compartments or distinct domains of the same com-
partment (see also Thompson et al., 2007 ). This pattern of
localization suggested that NgCAM and Tf entered the same
EEA1-positive EE and subsequently sorted away from one an-
other in a somatodendritic endosomal compartment, presum-
ably an RE.
Dual live imaging of endocytosed NgCAM
We next imaged NgCAM-expressing cells loaded with Texas
red – Tf and Alexa 488 – coupled anti-NgCAM antibodies to see
if Tf and NgCAM were transported together in endosomal
carriers (see Materials and methods). The Tf-positive compart-
ments correspond to REs because they still contained Tf after
> 20 min of chase. We observed somatodendritic endosomes
containing both ( Fig. 5, B and C , yellow), or either one of the
cargos ( Fig. 5, B and C, green and red). 42% of endosomes con-
tained both NgCAM and Tf, 30% contained only NgCAM, and
28% contained only Tf ( n = 365). Fig. 5 B demonstrates some
of the classes of behaviors we observed: arrowheads designate
some of the stationary endosomes, whereas arrows designate
JCB • VOLUME 180 • NUMBER 4 • 2008 834
endosomes containing both NEEP21 and endocytosed NgCAM
fell to 48% ( Fig. 6 C , right). The NEEP21 compartment, therefore,
behaves kinetically as an intermediate on the NgCAM pathway.
Interestingly, the rate of clearance of endocytosed NgCAM from
NEEP21 endosomes is slow, indicating that NgCAM has a long
residence time in this compartment. Substantial colocalization
with NEEP21 was also observed for endo cytosed human and rat
myc-L1 (not depicted). In contrast, NEEP21 ( Fig. 6 D , green)
showed only poor overlap with EEA1-positive EEs (red) in cul-
tured hippocampal neurons ( Fig. 6 D ), as is the case in PC12
cells ( Steiner et al., 2002 ).
Down-regulation of NEEP21 causes
missorting of NgCAM in endosomes
Increased missorting to the somatodendritic domain could arise
by several mechanisms. NgCAM might be expressed at higher
( Martinez-Arca et al., 2001 ) and antisense NEEP21-GFP (AS-
NEEP21; see Materials and methods for details; Steiner et al.,
2002 ). GFP was cotransfected with NgCAM as a control.
Coexpression of Ti-VAMP-DN did not signifi cantly affect A/D PI
for NgCAM ( Fig. 6 A , left), even though it profoundly inhibited
axon outgrowth if introduced at day in vitro (DIV) 2 (not de-
picted; Martinez-Arca et al., 2001 ). Coexpression of AS-NEEP21,
however, led to missorting of NgCAM to dendrites, resulting in
a statistically signifi cant reduction of NgCAM A/D PI ( Fig. 6 A ,
right). NEEP21 is therefore a regulator of NgCAM transport in
Endocytosed NgCAM (red) substantially and precisely
overlapped with endogenous NEEP21 ( Fig. 6 B , green) in so-
matodendritic endosomes (66% of all labeled endosomes; Fig. 6 C ,
left). Of NEEP21-containing endosomes, 86% contained endo-
cytosed NgCAM at t = 0. At 1 h, the percentage of all labeled
Figure 5. Dual live imaging of internalized
Tf and NgCAM. (A) Neurons were transfected
with NgCAM and incubated with anti-NgCAM
antibodies (red) and FITC-Tf (green) at 16 ° C,
washed and chased at 37 ° C in the continued
presence of Tf for 15 (A, left) or 60 min (A,
right), and then fi xed. Surface NgCAM (blue)
was detected with a cy5 – anti – mouse secondary
antibody before permeabilization. Yellow arrow-
heads indicate overlapping puncta. At 60 min
of chase, the overlap is frequently not precise,
resulting in a traffi c light appearance. Single
confocal sections are shown. The traffi c light
pattern is most abundant at late chase times.
(B and C) Neurons (DIV12) expressing NgCAM
were loaded with Texas red – Tf and Alexa
488 – anti-NgCAM antibodies for 1 h and then
washed and imaged live every 3 s. Tf (red) and
endo cytosed NgCAM (green) can be easily de-
tected in somatodendritic endosomes in live neu-
rons. See Video 3 (available at http://www.jcb
(B) Examples of endosome behavior is shown
in four consecutive frames 3 s apart: stationary
endosomes containing only NgCAM (green),
only Tf (red), or both NgCAM and Tf (yellow)
are indicated with arrowheads. Motile endo-
somal carriers are indicated by arrows (red, Tf
only; green, NgCAM only). The four compart-
ments marked with symbols were traced over
time and their trajectories plotted in the adja-
cent graph. For the moving compartments, the
arrow points at the fi rst frame of the sequence.
A proximal dendrite is shown. See Video 4.
(C) Consecutive frames of a traffi c light endo-
some are shown. The endosome appeared
initially yellow but in subsequent frames ap-
pears as one slightly elongated carrier (or two
tethered carriers) in which Tf (red) is laterally
segregated away from endocytosed NgCAM
(green). The fi eld shown is from the soma of
the cell. See Video 5. (D) Labeled endosomal
puncta were categorized as Tf only, NgCAM
only, or both NgCAM and Tf. All labeled
structures in each panel were subdivided by
shape (round or elongated) and size (large,
medium, or small) and by motility (stationary
or moving). Structures containing both Tf and
NgCAM rarely moved, whereas structures
containing Tf only moved occasionally. Struc-
tures containing NgCAM only were the most
motile. n = 365 endosomes.
835 ENDOSOMAL TRAFFICKING OF N G CAM TO THE AXON • YAP ET AL.
NgCAM missorting occurs in the EE
We next asked whether the kinetics of endosomal traffi cking
of NgCAM were changed in AS-NEEP21 – expressing cells.
We predicted that endocytic uptake of NgCAM would be increased
because higher levels of NgCAM are available for endocytosis
on the somatodendritic surface at steady state in AS-NEEP21 –
expressing cells. We do in fact see more than twofold higher
levels of endocytosed NgCAM in the soma at t = 0 in AS-NEEP21
compared with GFP controls ( Fig. 7 B ). For several other recep-
tors, expression of AS-NEEP21 caused increased retention of
the receptors in endosomes. We therefore compared the extent
of NgCAM clearance from somatic endosomes at t = 40 min.
Signal levels at t = 40 ? were normalized to signal levels at t = 0
for each set because the initial signal levels were much higher
for AS-NEEP21 to begin with ( Fig. 7 C ). We found a modest
increase in NgCAM retention in AS-NEEP21 – expressing cells
(P = 0.055 by Mann Whitney U test).
In epithelial cells, cargo can leave the EE in three direc-
tions: direct local recycling to the plasma membrane, transport
levels in AS-NEEP21 – expressing cells, leading to saturation of
the sorting machinery. We therefore compared the total mean sur-
face intensity of NgCAM in cells expressing GFP as control or
AS-NEEP21. No statistically signifi cant differences in total sur-
face NgCAM were observed (surface intensity in GFP = 5208 ±
832 arbitrary units; AS-NEEP21 = 5952 ± 534 arbitrary units).
Because NEEP21 is found in the TGN as well as in endosomes
( Steiner et al., 2002 ), either one could be the site of missorting.
We therefore determined the A/D PI at steady state of a mutant
NgCAM containing a point mutation in the somatodendritic
sorting signal, NgCAM Y33A. This NgCAM mutant travels to
the axon on a direct pathway from the TGN, bypassing somato-
dendritic endosomes ( Wisco et al., 2003 ). The steady-state A/D PI
of NgCAM Y33A was not reduced by coexpressing AS-NEEP21
( Fig. 7 A ). Additionally, we determined the A/D PI of endosomally
recycling NgCAM (using the same assays as for Fig. 3 ). The re-
cycling A/D PI of NgCAM was also signifi cantly diminished
compared with control cells ( Fig. 7 A , right). AS-NEEP21 there-
fore causes missorting of NgCAM during endosomal recycling.
Figure 6. Endocytosed NgCAM traverses the NEEP21-positive EE. (A) Neurons were cotransfected with NgCAM and either dominant-negative Ti-VAMP
(left) or anti-sense NEEP21 (right). GFP was expressed as a control. 18 h after transfection, surface NgCAM was detected with immunostaining.
Coexpression of AS-NEEP21 but not Ti-VAMP-DN led to a signifi cant decrease in the A/D PI (see Materials and methods). Error bars indicate SEM; **, statis-
tical signifi cance from GFP controls at P < 0.001. n = 4 independent experiments, scoring 20 – 25 cells per experiment for each condition. (B) NgCAM-expressing
neurons were allowed to endocytose anti-NgCAM antibodies for 20 min before fi xation. Endocytosed NgCAM was detected with a red secondary anti-
body, whereas endogenous NEEP21 was detected with a rabbit anti-NEEP21 antibody (green). Precise overlap of NgCAM and NEEP21 is observed
(colocalization appears yellow). Single channels as well as overlaid channels are shown for the boxed area. A single confocal section is shown. Arrow-
heads indicate dendrites. (C) Extent of overlap was scored for cells loaded with anti-NgCAM antibodies for 20 min without a chase (left; t = 0) and after
a 1-h chase (right; t = 60). Extent of overlap was binned into strong colocalization, NEEP only, and NgCAM only. NgCAM showed high colocalization
(black) at t = 0, which diminished with time (right), whereas the no colocalization categories NEEP only and NgCAM only increased with time. (D) The EE
populations enriched in EEA1 (red) or in NEEP21 (green) show poor colocalization. Single channel panels are shown on the left.
JCB • VOLUME 180 • NUMBER 4 • 2008 836
cells ( Fig. 7 F , aqua), whereas endocytosed NgCAM was addi-
tionally found in axonal endosomes ( Fig. 7 F , red; arrows indi-
Polarity of endogenous L1 is affected by
down-regulation of NEEP21
We then asked if the axonal polarity of endogenous L1 was also
dependent on NEEP21 function. We introduced the antisense
NEEP21 plasmid by electroporation into dissociated hippo-
campal neurons before plating and then assayed the distribution
of surface L1 at DIV3. Endogenous L1 was highly enriched on
the axonal surface ( Fig. 8 A , arrows) in GFP-expressing control
cells ( Fig. 8, A and A ? , red) and little L1 was detected on the
somatodendritic surface (arrowheads). When AS-NEEP21-GFP
was expressed, L1 was readily detected on the axon ( Fig. 8 B ,
arrows) but also on soma and dendrites ( Fig. 8, B and B ? , red,
arrowheads). The A/D PI of endogenous L1 was signifi cantly
to the RE, or transport to the LE/lysosome (see Fig. 1 D ). We next
asked if the retention of endocytosed NgCAM in somatic endo-
somes ( Fig. 7 C ) might be, at least in part, caused by terminal
missorting to LE/lysosomes. In control cells ( Fig. 7 D , top),
little overlap between NgCAM (red) and the lysosomal lgp120
(blue or aqua in insets) was observed (overlap appears white in
the insets). AS-NEEP21 – coexpressing cells, however, showed
increased colocalization of endocytosed NgCAM with lgp120
( Fig. 7 D , bottom; and Fig. 7 E ). NEEP21 function therefore
biases the exit route from the EE such that NgCAM is prefer-
entially transported to the RE rather than recycled to the somato-
dendritic surface or traffi cked to lysosomes.
Lastly, we asked if Tf was missorted to the axon in AS-
NEEP21 – expressing cells. Such missorting would suggest that
the somatodendritic EE could serve as a direct exit station toward
the axon. No such missorting was observed and Tf remained
somatodendritically restricted in antisense NEEP21 – expressing
Figure 7. Down-regulation of NEEP21 leads
to missorting of endosomal NgCAM to the
somatodendritic surface and to lysosomes.
(A) The A/D PI was determined for NgCAMY33A
at steady state (left) and for NgCAM for endo-
somal recycling to the plasma membrane (t =
2.5 h; right) in cells coexpressing GFP as controls
(black) or AS-NEEP21 (gray). Error bars indi-
cate SEM; **, statistical signifi cance from GFP
controls at P < 0.001. n = 4 independent ex-
periments scoring 12 – 17 cells per experiment
for each condition. (B) The extent of anti-
NgCAM antibody loading at t = 0 into somatic
endosomes was quantifi ed in control cells (black)
or AS-NEEP-GFP coexpressing neurons (gray).
Values were normalized to GFP-expressing
control cells (Ab loading). (C) The presence of
endocytosed NgCAM in somatic endosomes
(soma retention) was quantitated at t = 0 and
t = 40 min for cells coexpressing either GFP
as control (black) or antisense NEEP21-GFP
(AS-NEEP21; gray). Values were normalized
to the t = 0 levels for each condition. n = 4
independent experiments, scoring 12 – 17 cells
per experiment for each condition. Error bars
indicate SEM. *, statistical signifi cance from
controls at P < 0.01; #, statistical signifi cance
from controls at P = 0.055 (Mann Whitney U
test). (D) The extent of colocalization between
the lysosomal lgp120 (blue) and endocytosed
NgCAM (90-min chase; red) was visualized
for cells coexpressing NgCAM and GFP (top) or
NgCAM and AS-NEEP21-GFP (bottom). Single
channel and merged images of the soma region
are shown separately on the right. In these
panels, lgp120 is displayed in aqua. Overlap
with the red channel appears white. (E) Quan-
tifi cation of the number of colocalizing puncta
from the experiment in D. n = 3 independent
experiments scoring 12 – 17 cells per experi-
ment and condition. Error bars indicate SEM;
**, statistical signifi cance from GFP controls
at P < 0.001. (F) Localization of internalized
Tf (aqua) and endocytosed NgCAM (red;
20-min load at t = 0) in cells coexpressing
NgCAM and AS-NEEP21-GFP. The right panel
shows the single channel for Tf. Although
endocytosed NgCAM can be detected in
endosomes along axons (red; left, arrows),
no missorting of Tf (aqua) to the axon (arrows)
837 ENDOSOMAL TRAFFICKING OF N G CAM TO THE AXON • YAP ET AL.
endogenous L1. We observed extensive labeling of endosomes,
both in axons and in the somatodendritic domain ( Fig. 8, D1
and D2 ).
Live imaging of NEEP21-GFP and
Lastly, we determined if the NEEP21 compartment corre-
sponded to the stationary NgCAM-containing endosomes or
whether NEEP21 was also found in moving transport carriers
together with NgCAM ( Fig. 9 and Videos 6 and 7, available
Colocalization of endocytosed NgCAM and NEEP21-GFP was
striking and precise ( Fig. 9, A and C , yellow). These yellow com-
partments containing both NgCAM and NEEP21 were largely
stationary ( Fig. 9 A , asterisk), with only 2.5% of them moving
( Fig. 9 C ; n = 443). Unexpectedly, NEEP21-containing compart-
ments did show frequent movements ( Fig. 9 A , arrowheads) but
the moving NEEP21 compartments rarely contained NgCAM.
If only moving compartments were considered, 66% of them
contained only NEEP21 ( Fig. 9 D ). Compartments containing
NgCAM but not NEEP21 also moved (20.5%; Fig. 9 A , white
arrows; and Fig. 9 C ). The observed movements were episodic,
frequently punctuated by pauses of many seconds ( Fig. 9 B ).
reduced in antisense NEEP21 – expressing cells ( Fig. 8 C ).
NEEP21 therefore promoted axonal polarization of endogenous
L1 in young cultures. Because of two technical constraints,
we were unable to assess the effects of AS-NEEP21 on endog-
enous L1 in mature cultures. (1) Endogenous L1 accumulates
stably to high levels on the axonal surface. Introducing anti-
sense NEEP21 at DIV8/9 (as we do for exogenously expressed
NgCAM) made it impossible to detect the fate of the newly
synthesized pool of L1 because such a large amount of endog-
enous L1 was already accumulated stably on the axonal surface
at the time of transfection. (2) Mature cultures of DIV9 – 11 have
a very dense network of criss-crossing axons. Trying to assess
the polarity of endogenous L1 proved not to be possible be-
cause individual axons cannot be unambiguously traced to the
cell body. Furthermore, axons commonly grow along dendrites,
giving the erroneous impression that MAP2-positive processes
The observation that endogenous L1 is sensitive to
NEEP21 depletion suggested that a signifi cant fraction of en do g -
enous L1 also travels via somatodendritic endosomes to the
axon. We therefore tested if endogenous L1 can be endocytosed
in the somatodendritic domain by performing antibody uptake
experiments in DIV3 neurons with an antibody against the
Figure 8. Endogenous L1 mislocalizes to
the somatodendritic domain when NEEP21 is
down-regulated. (A and B) Dissociated neu-
rons were electroporated with GFP as control
(A and A ? ) or AS-NEEP21-GFP (B and B ? ) and
surface L1 was detected with a polyclonal anti-
L1 antibody (red) at DIV3. In GFP-expressing
control cells (A and A ? ), endogenous L1 is
highly polarized to the axon (arrows) and only
weakly found on dendrites (arrowheads). In
AS-NEEP21-GFP – expressing cells (B and B ? ),
endogenous L1 is additionally detected on the
soma and dendrites. (C) The A/D PI was de-
termined for endogenous surface L1 for control
cells expressing GFP or AS-NEEP21-GFP. n =
50 cells for GFP; n = 63 cells for AS-NEEP21-
GFP from two independent experiments. Error
bars indicate SEM. **, statistical signifi cance
at P < 0.0001. (D) DIV3 neurons were incu-
bated with polyclonal anti-L1 antibody for
20 min and then fi xed and stained with second-
ary antibody after permeabilization. Labeled
endosomes can be seen in axons, somata, and
dendrites. Two examples are shown.
JCB • VOLUME 180 • NUMBER 4 • 2008 838
round carriers without Tf. Once NgCAM endosomal carriers
enter the axon, many are transported by fast axonal transport.
Large stationary endosomes along the axon accumulate endo-
cytosed NgCAM and might provide intermediary stopover points
into which motile carriers fuse and from which new motile car-
riers are generated. We propose that NEEP21 is required for
faithful sorting of NgCAM to axons by facilitating traffi cking
from somatodendritic EEs to REs rather than local somato-
dendritic recycling or degradation. We conclude that functioning
somatodendritic endosomes are required for axonal localization
Live imaging of NgCAM-containing
We developed live imaging approaches to visualize the endo-
somal traffi cking of NgCAM in cultured primary neurons. Traf-
fi cking of all NgCAM pools was shown previously by imaging
NgCAM-GFP ( Burack et al., 2000 ). To monitor endosomal but
not post-TGN traffi c, we labeled specifi cally endosomal pools of
NgCAM by incubation with Alexa-labeled anti-NgCAM antibodies.
In agreement with fi xed samples, endocytosed NgCAM was found
We conclude that NgCAM accumulates in stationary NEEP21
endosomes and is transported in NEEP21-negative transport
carriers. NEEP21-positive carriers are frequently observed but
it is not known which cargo might be cotransported.
NgCAM accumulates on the axonal plasma
membrane via transcytosis, not via
In this work, we tested two endocytosis-dependent models for
axonal accumulation and show that NgCAM accumulates on the
axon via transcytosis rather than via selective retrieval/retention.
Based on previous kinetic experiments, we estimate that ? 80%
of NgCAM travels via transcytosis ( Wisco et al., 2003 ). We pro-
pose the following model of the transcytotic pathway and the
endosomal compartments involved ( Fig. 9 E ): NgCAM is taken
up into somatodendritic EEA1-positive EEs. NgCAM traverses
NEEP21-positive endosomes, from where it is preferentially
trafficked to REs. Subsequently, endosomal NgCAM sorts
away from Tf in the RE and is transported in tubules and small
Figure 9. Dynamic behavior of NEEP21-GFP and endocytosed NgCAM in endosomes. (A and B) Live imaging of endocytosed NgCAM (red) and NEEP21-
GFP (green). A portion of a proximal dendrite is shown (A). Images were captured every 2 s as indicated above each panel. Frames not shown displayed
no movements. The trajectories for all twenty frames (0 – 38 s) are displayed in B for the three compartments marked by arrowheads, arrows, and asterisks
in A. The starting point at 0 s is indicated. The NEEP21-containing and the NgCAM-containing compartments (marked with arrows/arrowheads) show
movements, whereas the compartment containing both NEEP21 and endocytosed NgCAM (marked with an asterisk) does not move. See Video 6 (avail-
able at http://www.jcb.org/cgi/content/full/jcb.200707143/DC1). (C) Quantifi cation of all scored endosomes ( n = 443) in the 1-min imaging period.
(D) Quantifi cation of moving endosomes ( n = 79; colors as defi ned in C). (E) Model of endosomal compartments involved in NgCAM transcytosis. Tf and
NgCAM (aquamarine arrows) enter EEA1-positive EEs (orange) in the somatodendritic domain as well as NEEP21-positive EEs (purple), from where they
are transported to the somatodendritic RE. Tf (green arrows) but not NgCAM (blue arrows) recycles to the somatodendritic surface from the EE and the RE.
NgCAM (blue), however, sorts away from Tf into putative “ transcytotic REs ” (red) and travels anterogradely up the axon in small carriers. Large stationary
endosomes along the axon (blue) accumulate endocytosed NgCAM and might provide intermediary stopover points into which motile carriers fuse and
from which new motile carriers are generated. When NEEP21 is down-regulated by antisense, NgCAM is missorted, presumably in the NEEP21 endo-
some, toward the somatodendritic surface as well as to lysosomes (gray arrows; LE/lys). Tf, however, is not missorted to the axon.
839 ENDOSOMAL TRAFFICKING OF N G CAM TO THE AXON • YAP ET AL.
NEEP21 function in the transcytotic
NEEP21 was initially identifi ed as an interacting partner of the
endosomal syntaxin13 ( Steiner et al., 2002 ). NEEP21 plays cru-
cial roles in regulating recycling of multiple cargos ( Steiner
et al., 2002, 2005 ; Debaigt et al., 2004 ; Alberi et al., 2005 ). Endo-
cytosed NgCAM colocalizes precisely with NEEP21 in cultured
hippocampal neurons and exits with slow kinetics. The NEEP21
endosomes might therefore serve as a “ warehouse ” not only for
synaptic AMPA receptors but also for transcytosing NgCAM.
Furthermore, down-regulation of NEEP21 affects sorting of
NgCAM. The NEEP21 compartment therefore is functionally
on the pathway of endosomal NgCAM traffi cking toward the
axon. Importantly, endogenous L1 is also affected by down-
regulation of NEEP21, which argues that transcytosis is a rele-
vant pathway for L1 in young neurons. Interestingly, NEEP21
and syntaxin13 are highly expressed during development but
down-regulated in adulthood ( Hirling et al., 2000 ; Steiner et al.,
2002 ). Therefore, the traffi cking pathways of receptors might
be developmentally regulated. Our work provides new insights
into the dynamics and organization of neuronal endosomes and
indicates the importance of neuronal endosomes in the polar-
ized membrane traffi c to axons.
Materials and methods
Cell culture and reagents
Primary cultures of hippocampal neurons were grown as described previ-
ously ( Wisco et al., 2003 ) and cultured for 8 – 16 d. The 8D9 anti-NgCAM
hybridoma was obtained from the National Institutes of Health Hybridoma
Bank. Tissue culture supernatants were concentrated over a T-gel column
(Thermo Fisher Scientifi c). Purifi ed IgGs were coupled to Alexa-488 accord-
ing to manufacturer ’ s instructions (Invitrogen). 8D9 Fab fragments were pre-
pared using the Fab preparation kit according to manufacturer ’ s instructions
(Thermo Fisher Scientifi c). Rabbit anti-EEA1 antibody was obtained from
Chemicon and Texas red – rat Tf was obtained from Jackson ImmunoResearch
Laboratories. Polyclonal antibodies raised against NEEP21 and plasmid
AS-NEEP2-GFP were previously described by Steiner et al. (2002) . Anti-lgp120
rabbit antiserum “ Mingus ” was a gift from I. Mellman (Yale University, New
Haven, CT). Anti-L1 rabbit serum was provided by V. Lemmon (University of
Miami, Miami, FL). Secondary reagents were obtained from Invitrogen ex-
cept anti – mouse Fab-specifi c FITC, which was obtained from Sigma-Aldrich.
Human L1-myc was provided by D. Benson (Mount Sinai School of Medi-
cine, New York, NY), and rat L1-myc was provided by D. Felsenfeld (Mount
Sinai School of Medicine). Ti-VAMP reagents were provided by T. Galli
(Institut National de la Sant é et de la Recherche M é dicale, Paris, France).
NgCAM cDNA was subcloned into pCB6 with the Bluescript multiple clon-
ing site containing a nonenhanced cytomegalovirus promoter. NgCAM
Y33A has been described previously ( Wisco et al., 2003 ).
Transfections and infections
Neuronal cultures at DIV9 – 12 were transfected using Lipofectamine 2000
with 1 μ g DNA and 3 μ l Lipofectamine 2000 (Invitrogen) for 60 – 90 min,
washed, and incubated for 16 – 20 h. To reduce the number of over-
expressing cells, the NgCAM plasmid was mixed with an empty plasmid
containing no insert. For some experiments, an NgCAM adenovirus was
used as described previously ( Wisco et al., 2003 ).
Down-regulation of NEEP21 and Ti-VAMP
To test if NEEP21 was functionally important for traffi cking of NgCAM to
the axon, we used the same anti-sense strategy that has previously been
used successfully ( Steiner et al., 2002 ). For these experiments, neurons
were transfected simultaneously with antisense NEEP21-GFP and NgCAM
plasmids; control cells were transfected with GFP and NgCAM plasmids.
In order to quantify the extent of downregulation by AS-NEEP21 expression,
the endogenous NEEP21 staining in the soma of individual cells ( n = 23 for
each of the AS-NEEP21 – transfected cells or untransfected control cells) was
determined using Image J. NEEP21 showed a mean reduction of ? 50%
prominently in somatodendritic endosomes. Most of the labeled
endosomes were nonmobile, but small mobile transport carriers
(either spherical or tubular) could be visualized in the soma
Endosomal transport in axons is thought to occur in a retro-
grade direction toward either somatic lysosomes or signaling
endosomes ( Parton et al., 1992 ). Anterograde fast axonal trans-
port of endosomes is not usually observed. We show here directly
by live imaging that NgCAM is transported anterogradely in
endosomes in axons. The axonal NgCAM endosomes are remi-
niscent of compartments revealed by live imaging of syntaxin13-
GFP in cultured neurons ( Prekeris et al., 1999 ). Fast axonal
anterograde transport of endosomes might thus serve to re-
distribute a distinct set of membrane proteins from the dendrite
to axons. Other cargos (such as the type 1 cannabinoid receptor;
Leterrier et al., 2006 ) might require some of the same compart-
ments as NgCAM.
Identity of somatodendritic compartments
Endocytosed NgCAM is initially but transiently found with
EEA1 and Tf in EEs. It is subsequently found in compartments
that are closely apposed to Tf-containing REs. We suggest,
based on confocal images of fi xed cells and on dual live imag-
ing, that NgCAM progressively sorts away from Tf into mobile
tubules and small spherical carriers. These NgCAM-containing
carriers might fuse with intermediate compartments before en-
tering the axon. It remains to be determined if the REs found at
and within dendritic spines ( Park et al., 2006 ) are accessible
to recycling NgCAM as well or constitute a discrete subset.
By immuno-EM, we observed a preponderance of endocytosed
NgCAM in MVBs in the soma. Although many of these MVBs
are likely to be predegradative and degradative compartments,
it remains to be determined if NgCAM-containing MVBs are
intermediates on the transcytotic pathway.
In MDCK cells, all transcytosing cargo traverses the
RE ( Sheff et al., 1999 ). Our data similarly suggest that trans-
cytosis to the axon does not occur directly from the EE but
from the RE. It additionally suggests that traffi cking from REs
toward either the axon or the somatodendritic surface is medi-
ated by distinct signals in the cargo proteins. Whether or not
sequences in the cytoplasmic tail of NgCAM mediate sort-
ing to the axon from somatodendritic endosomes remains to
Our studies provide insights into the dynamics and orga-
nization of neuronal endosomes by using dual live imaging of
two differentially sorted recycling cargos. In cultured neurons,
no singular, perinuclear RE was observed. ( de Marco et al.,
2006 ; Thompson et al., 2007 ). Rather, NgCAM-containing
endosomes were dispersed throughout the soma at all times of
chase. Similar to endosomes in nonneuronal cells ( Mukherjee
and Maxfi eld, 2000 ), lipid composition appears to be mosaic in
neuronal endosomes as well, such that NgCAM endosomes ac-
cumulate, whereas Tf endosomes exclude DiI. Our observations
are consistent with lateral segregation of Tf and NgCAM in sin-
gle endosomes but only ultrastructural studies ultimately have
the resolution to prove this point.
JCB • VOLUME 180 • NUMBER 4 • 2008 840
labeling. For visualizing endocytosed L1, endocytosis assay with acid strip
Image quantifi cation
The same adjustments were performed on all images equally for each set of
quantifi cations. t tests were performed using Statistics software 13 (SPSS).
Endocytosis pulse/chase experiments ( Fig. 2 ). Total soma fl uo-
rescence was determined using Image J after background subtraction.
The soma was outlined using the freehand tracing function and total fl uo-
rescence was measured in the outlined region of interest. Measurements
were taken for 15 – 20 cells per experiment and condition.
Endosome intensity ( Fig. 2 D ). A one-pixel-wide line was traced
along axons and dendrites in Image J and plotted as an intensity profi le.
The maximal intensity of the peaks in the trace, which correspond to Ng-
CAM-containing endosomes, were measured and averaged from 10 axons
and 10 dendrites each for four experiments.
A/D PI ( Figs. 2 and 9 ). Axon and dendrite fl uorescence intensities
were measured along a one-pixel-wide line in Image J after background
subtraction and normalized to length. A/D PI was obtained by dividing the
mean axon intensity by the mean dendrite intensity.
Colocalization of endocytosed NgCAM and markers of endosomal
compartments ( Figs. 5 and 8 ). When quantifying the extent of colocalization,
we found that the relative intensities of the two markers under study often
differed over a wide range, from roughly equal intensities to 10 – 20-fold
higher intensity of one marker compared with the other. To refl ect this
observation, colocalization was scored as being either “ none ” ( > 25-fold
higher intensity of one marker compared with the other), “ high ” (intensity
ratios of the two markers between 1 and 5), or “ low ” (intensity ratios of the
two markers between 5 and 25). Black levels of images were adjusted to
drop out nonspecifi c background using Photoshop (Adobe). The cursor
was centered on all identifi able endosomal puncta and the red and green
levels were recorded. The ratio of red/green was then determined and
binned into fi ve bins such that the ratio of intensities for the two channels
was in the range of 1 – 5 (high colocalization), 5 – 25 (low colocalization), and
> 25 (no colocalization). In Fig. 8 , high colocalization and low colocalization
bins were combined into a single bin because the low colocalization bins
were small and did not change with chase time.
Colocalization with lysosomes ( Fig. 9 ). The number of puncta con-
taining strong signals of both lgp120 and endocytosed NgCAM was
counted in the soma region of 12 – 17 cells per experiment and condition.
Online supplemental material
Fig. S1 shows detectability of endocytosed NgCAM in somato dendritic
endosomes. Fig. S2 shows a single-frame display of Fig. 4 D . Fig. S3
shows that the lipophilic dye DiIC18(5)DS preferentially labels NgCAM-
containing endosomes. Video 1 shows the dynamic behavior of NgCAM-
containing endosomes in the soma of a DIV10 cultured hippocampal neuron.
Video 2 shows the dynamics of NgCAM-containing endo somes in the
axon of a DIV10 cultured hippocampal neuron. Video 3 shows dual live
imaging of two endosomal cargos in proximal dendrites. Video 4 is
a high-contrast, cropped movie showing long distance movement of a
small NgCAM-positive endosomal carrier in a dendrite. Video 5 is a high-
contrast, cropped movie showing coordinated movement of a small endo-
somal carrier containing laterally segregated cargos in the soma. Video 6
shows dual live imaging of NEEP21-GFP and endocytosed NgCAM in a
proximal dendrite. Video 7 shows a second example of dual live imaging
of NEEP21-GFP and endocytosed NgCAM in a proximal dendrite. Online
supplemental materials is available at available at http://www.jcb.org/
Crucial reagents were generously provided by Drs. Deanna Benson and Dan
Felsenfeld, Thierry Galli, Ira Mellman, and Vance Lemmon. Special thanks go
to Drs. George Bloom and Ed Perez-Reyes for help and access to their live-
imaging rigs. We thank Drs. George Bloom, Kevin Pfi ster, and Frank Solomon
for comments on the manuscript. We thank the members of the Winckler Labo-
ratory Julie Lim and Chad Lane for technical help with the experiments and
Max Vakulenko for insightful discussions.
This work was supported by the National Institutes of Health/National
Institute of Neurological Disorders and Stroke (grant 1RO1NS 045969-06 to
B. Winckler), a Basil O ’ Connor grant from the March of Dimes Foundation
(FY01-517 to B. Winckler), and the Swiss National Science Foundation (grant
3100A0-111935/1 to H. Hirling). P. Kujala was supported by grant AL-
W8PJ/00-31 from the Research Council for Earth and Life Sciences (Nederlandse
Organisatie voor Wetenschappelijk Onderzoek-Aard en Levens Wetenschappen)
to J. Klumperman.
due to antisense expression. For Ti-VAMP, a dominant-negative construct
containing the longin domain of Ti-VAMP was used. This construct leads to
inhibition of neurite outgrowth ( Martinez-Arca et al., 2001 ) and delay of
L1 recycling from PC12 cells ( Alberts et al., 2003 ). Ti-VAMP longin, in our
hands, also had a profound inhibitory effect on axon outgrowth of hippo-
campal neurons, showing that it was active as a dominant-negative.
Microscopy and live imaging
For live imaging, cells were grown on large round coverslips (Bellco Bio-
technology) or glass-bottom dishes (MatTek). Cells were transfected with
NgCAM plasmid 18 h before imaging and incubated with Alexa 488 –
coupled 8D9 anti-NgCAM antibody for 20 – 60 min, washed, and mounted
in a live imaging chamber on a heated stage under 5% CO 2 . To visualize
axonal NgCAM-containing endosomes, cells were acid stripped (see next
paragraph) and washed before imaging. Live imaging was performed on
a microscope (Axiovert; Carl Zeiss, Inc.) using a 63 × Plan Apo lens (Carl
Zeiss, Inc.) and a camera (Orca ER; Hamamatsu). Openlab software
was used for image capture at exposures of 80 – 150 ms with 2 × binning.
For some experiments, imaging was performed on an IX81 microscope
(Olympus) using a 100 × oil immersion objective and captured with 100 ms
exposures and 1 × binning. For dual imaging of endocytosed NgCAM
and NEEP21-GFP, cells were transfected with NgCAM and NEEP21-GFP
plasmids and uptake was performed with an Alexa 568 – antiNgCAM anti-
body. Only cells expressing low to moderate amounts of NEEP21-GFP
were chosen for imaging to minimize overexpression artifacts. Confocal
imaging was performed on a laser scanning microscope (LSM 510; Carl
Zeiss, Inc.) with a 63 × 1.4 differential interference contrast microscopy oil
Plan Apochromat objective or on a UV confocal laser scanning microscope
(TCS-SP1; Leica) equipped with a four-channel spectrophotometer scan
head and four lasers (350 nm Ar-UV, 488 nm argon, 568 nm krypton, and
633 nm HeNe). A 63 × 1.4 NA Plan Apo objective lens was used.
Endocytosis assay with acid strip
Neurons expressing NgCAM for 18 h were incubated with 8D9 anti-Ng-
CAM antibodies for 20 min at 37 ° C and washed several times; all anti-
body remaining on the surface was then stripped by treatment with MEM,
pH 2, for 2 min ( Fourgeaud et al., 2003 ), washed extensively, and re-
turned to the incubator for various amounts of times before fi xation in 2%
paraformaldehyde/3% sucrose/PBS, pH 7.4. Recycled 8D9 antibody was
detected before permeabilization with Alexa 647 or Cy5 secondary goat
anti – mouse antibody. Because all remaining primary antibodies had been
removed from the surface by acid stripping before the chase period, any
surface-associated Alexa 647 fl uorescence at later chase times was caused
by the reappearance of previously internalized anti-NgCAM primary anti-
bodies. Internalized NgCAM antibody was detected by applying Alexa-
568 goat anti – mouse antibody after permeabilization. Permeabilization
was achieved either with 0.05% saponin or 0.2% Triton X-100 for 10 min
at room temperature as described previously ( Wisco et al., 2003 ). The up-
take of lipophilic dye (DiIC18-DS; Invitrogen) in neurons was conducted
as described by Manderson et al. (2007) .
Immunogold EM of ultrathin cryosections was performed according to the
fl at-embedding technique described previously ( Oorschot et al., 2002 ).
Cells were fi xed in 2% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M
PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 2 mM
MgCl 2 , pH 6.9) for 2 h at room temperature after an endocytosis assay.
After rinsing fi rst in PBS with 0.02% M glycine and then in PBS with 0.1%
BSA, the fi xed cell monolayer was directly embedded in a thin layer of
12% gelatin phosphate buffer, which was prewarmed to 37 ° C. The gelatin
was solidifi ed at 4 ° C and subjected to a prolonged infi ltration with 2.3 M
sucrose for 48 h at 4 ° C on a rocker. Coverslips were gently removed from
the gelatin layer now containing some cells transferred from the coverslip.
Approximately 1 mm 3 blocks of selected areas with cells were cut off from
the gelatin layer, mounted on aluminum pins in a way that the gelatin-
embedded cells were orientated upwards, and then frozen in liquid nitrogen.
Ultrathin cryosections parallel to the gelatin-embedded cell monolayer
were cut at ? 120 ° C in a cryo ultramicrotome (Diatome) and picked up in
a 1:1 mixture of 2.3 M sucrose and 2% methylcellulose and immuno-
labeled according to the protein A – gold method.
AS-NEEP21 expression in young neurons
Nucleofection of freshly dissociated neurons was performed using a Nucleo-
fector device (Amaxa Biosystems) according to the manufacturer ’ s instructions.
At DIV3 after nucleofection, neurons were surface-labeled live with anti – rat
L1 and fi xed with 2% paraformaldehyde, followed by secondary antibody
841 ENDOSOMAL TRAFFICKING OF N G CAM TO THE AXON • YAP ET AL.
protein 1 regulates surface expression of glutamate receptors. J. Biol.
Chem. 282 : 2395 – 2404 .
Lai , H.C. , and L.Y. Jan . 2006 . The distribution and targeting of neuronal voltage-
gated ion channels. Nat. Rev. Neurosci. 7 : 548 – 562 .
Leterrier , C. , J. Laine , M. Darmon , H. Boudin , J. Rossier , and Z. Lenkei . 2006 .
Constitutive activation drives compartment-selective endocytosis and axonal
targeting of type 1 cannabinoid receptors. J. Neurosci. 26 : 3141 – 3153 .
Manderson , A.P. , J.G. Kay , L.A. Hammond , D.L. Brown , and J.L. Stow . 2007 .
Subcompartments of the macrophage recycling endosome direct the dif-
ferential secretion of IL-6 and TNF ? . J. Cell Biol. 178 : 57 – 69 .
Martinez-Arca , S. , S. Coco , G. Mainguy , U. Schenk , P. Alberts , P. Bouille , M.
Mezzina , A. Prochiantz , M. Matteoli , D. Louvard , and T. Galli . 2001 .
A common exocytotic mechanism mediates axonal and dendritic outgrowth.
J. Neurosci. 21 : 3830 .
Maxfi eld , F.R. , and T.E. McGraw . 2004 . Endocytic recycling. Nat. Rev. Mol. Cell
Biol. 5 : 121 – 132 .
Mukherjee , S. , and F.R. Maxfi eld . 2000 . Role of membrane organization and
membrane domains in endocytic lipid traffi cking. Traffi c . 1 : 203 – 211 .
Oorschot , V. , H. de Wit , W.G. Annaert , and J. Klumperman . 2002 . A novel fl at-
embedding method to prepare ultrathin cryosections from cultured cells
in their in situ orientation. J. Histochem. Cytochem. 50 : 1067 – 1080 .
Overly , C.C. , and P.J. Hollenbeck . 1996 . Dynamic organization of endo-
cytic pathways in axons of cultured sympathetic neurons. J. Neurosci.
16 : 6056 – 6064 .
Park , M. , E.C. Penick , J.G. Edwards , J.A. Kauer , and M.D. Ehlers . 2004 .
Recycling endosomes supply AMPA receptors for LTP. Science .
305 : 1972 – 1975 .
Park , M. , J.M. Salgado , L. Ostroff , T.D. Helton , C.G. Robinson , K.M. Harris ,
and M.D. Ehlers . 2006 . Plasticity-induced growth of dendritic spines by
exocytic traffi cking from recycling endosomes. Neuron . 52 : 817 – 830 .
Parton , R.G. , K. Simons , and C.G. Dotti . 1992 . Axonal and dendritic endocytic
pathways in cultured neurons. J. Cell Biol. 119 : 123 – 137 .
Peden , A.A. , V. Oorschot , B.A. Hesser , C.D. Austin , R.H. Scheller , and J.
Klumperman . 2004 . Localization of the AP-3 adaptor complex defi nes a
novel endosomal exit site for lysosomal membrane proteins. J. Cell Biol.
164 : 1065 – 1076 .
Prekeris , R. , D.L. Foletti , and R.H. Scheller . 1999 . Dynamics of tubulo-
vesicular recycling endosomes in hippocampal neurons. J. Neurosci.
19 : 10324 – 10337 .
Sachse , M. , G. Ramm , G. Strous , and J. Klumperman . 2002 . Endosomes: multi-
purpose designs for integrating housekeeping and specialized tasks.
Histochem. Cell Biol. 117 : 91 – 104 .
Sampo , B. , S. Kaech , S. Kunz , and G. Banker . 2003 . Two distinct mechanisms
target membrane proteins to the axonal surface. Neuron . 37 : 611 – 624 .
Schmid , S.L. , and E. Smythe . 1991 . Stage-specifi c assays for coated pit forma-
tion and coated vesicle budding in vitro. J. Cell Biol. 114 : 869 – 880 .
Schmidt , M.R. , and V. Haucke . 2007 . Recycling endosomes in neuronal mem-
brane traffi c. Biol. Cell . 99 : 333 – 342 .
Schweizer , F.E. , and T.A. Ryan . 2006 . The synaptic vesicle: cycle of exocytosis
and endocytosis. Curr. Opin. Neurobiol. 16 : 298 – 304 .
Segal , R.A. 2003 . Selectivity in neurotrophin signaling: theme and variations.
Annu. Rev. Neurosci. 26 : 299 – 330 .
Sheff , D.R. , E.A. Daro , M. Hull , and I. Mellman . 1999 . The receptor recycling
pathway contains two distinct populations of early endosomes with dif-
ferent sorting functions. J. Cell Biol. 145 : 123 – 139 .
Slepnev , V.I. , and P. De Camilli . 2000 . Accessory factors in clathrin-dependent
synaptic vesicle endocytosis. Nat. Rev. Neurosci. 1 : 161 – 172 .
Sonnichsen , B. , S. De Renzis , E. Nielsen , J. Rietdorf , and M. Zerial . 2000 .
Distinct membrane domains on endosomes in the recycling pathway vi-
sualized by multicolor imaging of Rab4, Rab5, and Rab11. J. Cell Biol.
149 : 901 – 914 .
Steiner , P. , J.C. Sarria , L. Glauser , S. Magnin , S. Catsicas , and H. Hirling . 2002 .
Modulation of receptor cycling by neuron-enriched endosomal protein of
21 kD. J. Cell Biol. 157 : 1197 – 1209 .
Steiner , P. , S. Alberi , K. Kulangara , A. Yersin , J.C. Sarria , E. Regulier , S. Kasas ,
G. Dietler , D. Muller , S. Catsicas , and H. Hirling . 2005 . Interactions be-
tween NEEP21, GRIP1 and GluR2 regulate sorting and recycling of the
glutamate receptor subunit GluR2. EMBO J. 24 : 2873 – 2884 .
Sudhof , T.C. 2004 . The synaptic vesicle cycle. Annu. Rev. Neurosci. 27 : 509 – 547 .
Susalka , S.J. , and K.K. Pfi ster . 2000 . Cytoplasmic dynein subunit heterogeneity:
implications for axonal transport. J. Neurocytol. 29 : 819 – 829 .
Thompson , A. , R. Nessler , D. Wisco , E. Anderson , B. Winckler , and D. Sheff .
2007 . Recycling endosomes of polarized epithelial cells actively sort
apical and basolateral cargos into separate subdomains. Mol. Biol. Cell .
18 : 2687 – 2697 .
Submitted: 20 July 2007
Accepted: 28 January 2008
Alberi , S. , B. Boda , P. Steiner , I. Nikonenko , H. Hirling , and D. Muller . 2005 .
The endosomal protein NEEP21 regulates AMPA receptor-mediated syn-
aptic transmission and plasticity in the hippocampus. Mol. Cell. Neurosci.
29 : 313 – 319 .
Alberts , P. , and T. Galli . 2003 . The cell outgrowth secretory endosome (COSE):
a specialized compartment involved in neuronal morphogenesis. Biol. Cell .
95 : 419 – 424 .
Alberts , P. , R. Rudge , I. Hinners , A. Muzerelle , S. Martinez-Arca , T. Irinopoulou ,
V. Marthiens , S. Tooze , F. Rathjen , P. Gaspar , and T. Galli . 2003 . Cross
talk between tetanus neurotoxin-insensitive vesicle-associated membrane
protein-mediated transport and L1-mediated adhesion. Mol. Biol. Cell .
14 : 4207 – 4220 .
Boiko , T. , M. Vakulenko , H. Ewers , C.C. Yap , C. Norden , and B. Winckler . 2007 .
Ankyrin-dependent and -independent mechanisms orchestrate axonal
compartmentalization of L1 family members neurofascin and L1/neuron-
glia cell adhesion molecule. J. Neurosci. 27 : 590 – 603 .
Brown , T.C. , I.C. Tran , D.S. Backos , and J.A. Esteban . 2005 . NMDA receptor-
dependent activation of the small GTPase Rab5 drives the removal of
synaptic AMPA receptors during hippocampal LTD. Neuron . 45 : 81 – 94 .
Burack , M.A. , M.A. Silverman , and G. Banker . 2000 . The role of selective trans-
port in neuronal protein sorting. Neuron . 26 : 465 – 472 .
Chang , M.C. , D. Wisco , H. Ewers , C. Norden , and B. Winckler . 2006 . Inhibition
of sphingolipid synthesis affects kinetics but not fi delity of L1/NgCAM
transport along direct but not transcytotic axonal pathways. Mol. Cell.
Neurosci. 31 : 525 – 538 .
Cremona , O. , and P. De Camilli . 1997 . Synaptic vesicle endocytosis. Curr. Opin.
Neurobiol. 7 : 323 – 330 .
de Marco , M.C. , R. Puertollano , J.A. Martinez-Menarguez , and M.A. Alonso .
2006 . Dynamics of MAL2 during glycosylphosphatidylinositol-anchored
protein transcytotic transport to the apical surface of hepatoma HepG2
cells. Traffi c . 7 : 61 – 73 .
Debaigt , C. , H. Hirling , P. Steiner , J.P. Vincent , and J. Mazella . 2004 . Crucial
role of neuron-enriched endosomal protein of 21 kDa in sorting between
degradation and recycling of internalized G-protein-coupled receptors.
J. Biol. Chem. 279 : 35687 – 35691 .
Deinhardt , K. , and G. Schiavo . 2005 . Endocytosis and retrograde axonal traffi c
in motor neurons. Biochem. Soc. Symp. 72 : 139 – 150 .
Deinhardt , K. , S. Salinas , C. Verastegui , R. Watson , D. Worth , S. Hanrahan , C.
Bucci , and G. Schiavo . 2006 . Rab5 and Rab7 control endocytic sorting
along the axonal retrograde transport pathway. Neuron . 52 : 293 – 305 .
Fache , M.P. , A. Moussif , F. Fernandes , P. Giraud , J.J. Garrido , and B. Dargent .
2004 . Endocytotic elimination and domain-selective tethering constitute
a potential mechanism of protein segregation at the axonal initial seg-
ment. J. Cell Biol. 166 : 571 – 578 .
Fourgeaud , L. , A.S. Bessis , F. Rossignol , J.P. Pin , J.C. Olivo-Marin , and A. Hemar .
2003 . The metabotropic glutamate receptor mGluR5 is endocytosed by a
clathrin-independent pathway. J. Biol. Chem. 278 : 12222 – 12230 .
Garrido , J.J. , F. Fernandes , P. Giraud , I. Mouret , E. Pasqualini , M.P. Fache , F.
Jullien , and B. Dargent . 2001 . Identifi cation of an axonal determinant in
the C-terminus of the sodium channel Na(v)1.2. EMBO J. 20 : 5950 – 5961 .
Garrido , J.J. , F. Fernandes , A. Moussif , M.P. Fache , P. Giraud , and B. Dargent .
2003 . Dynamic compartmentalization of the voltage-gated sodium chan-
nels in axons. Biol. Cell . 95 : 437 – 445 .
Hemar , A. , J.C. Olivo , E. Williamson , R. Saffrich , and C.G. Dotti . 1997 .
Dendroaxonal transcytosis of transferrin in cultured hippocampal and
sympathetic neurons. J. Neurosci. 17 : 9026 – 9034 .
Hirling , H. , P. Steiner , C. Chaperon , R. Marsault , R. Regazzi , and S. Catsicas .
2000 . Syntaxin 13 is a developmentally regulated SNARE involved in neu-
rite outgrowth and endosomal traffi cking. Eur. J. Neurosci. 12 : 1913 – 1923 .
Howe , C.L. , and W.C. Mobley . 2004 . Signaling endosome hypothesis: A cellular
mechanism for long distance communication. J. Neurobiol. 58 : 207 – 216 .
Kamiguchi , H. , and V. Lemmon . 1998 . A neuronal form of the cell adhesion
molecule L1 contains a tyrosine-based signal required for sorting to the
axonal growth cone. J. Neurosci. 18 : 3749 – 3756 .
Kamiguchi , H. , and V. Lemmon . 2000 . Recycling of the cell adhesion molecule
L1 in axonal growth cones. J. Neurosci. 20 : 3676 – 3686 .
Kennedy , M.J. , and M.D. Ehlers . 2006 . Organelles and traffi cking machinery for
postsynaptic plasticity. Annu. Rev. Neurosci. 29 : 325 – 362 .
Kulangara , K. , M. Kropf , L. Glauser , S. Magnin , S. Alberi , A. Yersin , and
H. Hirling . 2007 . Phosphorylation of glutamate receptor interacting