In vivo Imaging of Retrogradely Transported Synaptic Vesicle Proteins in Caenorhabditis elegans Neurons

Article (PDF Available)inTraffic 12(1):89-101 · October 2010with25 Reads
DOI: 10.1111/j.1600-0854.2010.01127.x · Source: PubMed
Axonal transport is an essential process that carries cargoes in the anterograde direction to the synapse and in the retrograde direction back to the cell body. We have developed a novel in vivo method to exclusively mark and dynamically track retrogradely moving compartments carrying specific endogenous synaptic vesicle proteins in the Caenorhabditis elegans model. Our method is based on the uptake of a fluorescently labeled anti-green fluorescent protein (GFP) antibody delivered in an animal expressing the synaptic vesicle protein synaptobrevin-1::GFP in neurons. We show that this method largely labels retrogradely moving compartments. Very little labeling is observed upon blocking vesicle exocytosis or if the synapse is physically separated from the cell body. The extent of labeling is also dependent on the dyenin-dynactin complex. These data support the interpretation that the labeling of synaptobrevin-1::GFP largely occurs after vesicle fusion and the major labeling likely takes place at the synapse. Further, we observe that the retrograde compartment carrying synaptobrevin contains synaptotagmin but lacks the endosomal marker RAB-5. This labeling method is very general and can be readily adapted to any transmembrane protein on synaptic vesicles with a GFP tag inside the vesicle and can also be extended to other model systems.
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© 2010 John Wiley & Sons A/S
In vivo
Imaging of Retrogradely Transported Synaptic
Vesicle Proteins in
Caenorhabditis elegans
Kausalya Murthy
, Jaffar M. Bhat
and Sandhya
P. Koushika
Neurobiology, NCBS-TIFR, Bellary Road, Bangalore
560065, India
*Corresponding author: Sandhya P. Koushika,
These authors contributed equally to this work.
Axonal transport is an essential process that carries
cargoes in the anterograde direction to the synapse and
in the retrograde direction back to the cell body. We have
developed a novel
in vivo
method to exclusively mark and
dynamically track retrogradely moving compartments
carrying specific endogenous synaptic vesicle proteins
in the
Caenorhabditis elegans
model. Our method is
based on the uptake of a fluorescently labeled anti-green
fluorescent protein (GFP) antibody delivered in an animal
expressing the synaptic vesicle protein synaptobrevin-
1::GFP in neurons. We show that this method largely
labels retrogradely moving compartments. Very little
labeling is observed upon blocking vesicle exocytosis
or if the synapse is physically separated from the cell
body. The extent of labeling is also dependent on
the dyenindynactin complex. These data support the
interpretation that the labeling of synaptobrevin-1::GFP
largely occurs after vesicle fusion and the major labeling
likely takes place at the synapse. Further, we observe
that the retrograde compartment carrying synaptobrevin
contains synaptotagmin but lacks the endosomal marker
RAB-5. This labeling method is very general and can
be readily adapted to any transmembrane protein on
synaptic vesicles with a GFP tag inside the vesicle and
can also be extended to other model systems.
Key words: antibody, dyenindynactin mutants, ret-
rograde axonal transport, synaptic vesicle proteins,
Received 3 March 2010, revised and accepted for
publication 1 October 2010, uncorrected manuscript
published online 4 October 2010, published online 29
October 2010
Retrograde transport is the process in which cargo is
transported back to the cell body from the synapse.
The major retrograde motor in axons is thought of
as the dyneindynactin complex (1). The importance
of retrograde transport is shown by neuronal and neurolog-
ical phenotypes observed in animals that have mutations
in the dyneindynactin complex components (2). Retro-
grade transport plays very important roles in modification
of the outcome or progress of neurodegenerative diseases
such as amyotrophic laterla scleosis (ALS) (3).
Several cargoes are transported retrogradely, e.g. mito-
chondria, synaptic vesicle proteins, neurotrophin recep-
tors, viruses and exogenously applied toxins such as
tetanus toxins (48). As most cargoes exhibit saltatory
bidirectional movements and dynamic imaging of trans-
port is usually feasible only in the shorter time scale
of minutes, it is not always easy to determine which
endogenously expressed proteins are retrogradely trans-
ported from the synapse to the cell body. For example,
a few synaptic vesicle proteins have been inferred to be
retrogradely transported by their observed mislocalization
in mutants of the retrograde motor dynein (e.g. the trans-
membrane proteins synaptobrevin and synaptotagmin in
Caenorhabditis elegans
) or by their distal accumulation in
a nerve crush assay (e.g. synapsin, synaptotagmin, RAB-3
in vertebrates) (911). An
in vivo
method to specifically
tag retrograde cargo will offer a direct means to study
mechanisms of retrograde transport, to separate retro-
gradely moving cargo pools from anterogradely moving
pools and to study processes that depend on retrograde
transport such as axonal injury signaling (12).
Methods that specifically assay retrogradely transported
compartments use exogenously applied probes such as
radiolabeled nerve growth factor (NGF) (13), fragments
of tetanus toxin tagged with fluorophore (14), ligands
to activate Trk receptors (15) or viruses (7,8). However,
these methods have mostly been used in cell culture.
Moreover, they do not provide simple means to
specifically label a given protein to allow dynamic tracking
in vivo
. Currently, the most widely used tracking method is
to tag the protein of interest with green fluorescent protein
(GFP). However, using this technique alone it is difficult to
systematically and exclusively follow events that begin at
the synapse. A ntibodies can provide t he desired specificity
and offer an attractive means to label the desired protein
and thus the compartments in which it travels. Antibody-
based labeling techniques have been tried to study
retrograde transport (16,17), but many of these attempts
predated the development of canonical protein tags such
as hemagglutinin (HA) or GFP. Specific a ntibodies against
a non-fluorescent tag have been used recently for static
tracking, while studying receptor recycling at postsynaptic
sites in cell culture (18,19). But no general methods have
been developed to dynamically track retrograde transport
of any desired protein
in vivo
We have developed a tool to exclusively visualize the ret-
rograde transport of GFP-tagged synaptic vesicle proteins
in neurons
in vivo.
Our approach is based on inject-
ing Alexa Fluor
594-conjugated immunoglobulin G (IgG)
anti-GFP antibodies into transgenic
C. elegans
lines that
stably express GFP tagged to the synaptic vesicle protein 89
Murthy et al.
Figure 1: Antibody against GFP specifically labels synaptobrevin::GFP in neurons. A) Schematic of the retrograde labeling assay.
Neurons expressing SNB-1::GFP (green dotted line) on vesicles with GFP tag within the vesicle, when exocytosed, is exposed to
extracellular environment. The exposed SNB-1::GFP stably binds to injected Alexa Fluor 594 GFP antibody; the bound antibody is
endocytosed resulting in retrograde labeling in the same neuron (red dotted line). B) Schematic of
animals showing the pattern
of expression of SNB-1::GFP in anterior and posterior neurons (ALM and PLM). ALM neurons make synapses in the nerve ring (arrow)
and PLM makes synapses in the ventral cord (arrow). An arrow points to the ‘synaptic branch’ from the main PLM neuronal process
that enters the ventral cord to form synapses. C) Retrograde labeling of neurons 1 h after antibody injection in
animals expressing
SNB-1::GFP in all neurons and in the PLM neurons of
that express SNB-1::GFP (in green, top panel), SNB-1::GFP-Ab (in red, middle
panel) and merge (in lower panel). White box points to a neuronal region near the cell body where SNB-1::GFP-Ab signal is quantified in
subsequent figures. D) Immunostaining of anti-GFP antibody that has undergone endocytosis in
(left) that expresses soluble GFP
in touch receptor neuronal processes and motor neuron synapses of
(right) that expresses UNC-46::GFP (a transmembrane
presynaptic vesicle protein) in GABA neurons stained for GFP and SNB-1::GFP in mechanoreceptor neurons (
). E) Representative
images of
worms expressing GFP::RAB-3 and soluble GFP but do not show any signal in red channel. F) Coelomocyte
labeling 1 h after Ab injection as a control for working antibody.
points to non-specific autofluorescence from the pseudocoelom.
Arrowhead points to neuronal process. In all images, scale bar is 10 μm except in the
panel (D), scale bar is 5 μm.
synaptobrevin-1 (SNB-1) in neurons (20). The injected
anti-GFP antibody binds specifically to SNB-1::GFP that
exposes GFP to the extracellular milieu upon vesicle exo-
cytosis (Figure 1A). The SNB-1::GFP-Ab is subsequently
endocytosed within a vesicle. The brightness and stability
of the fluorescent antibody facilitates long-term imaging of
retrograde transport of s pecific transmembrane proteins.
Our technique gives a powerful
in vivo
labeling tool that
can be used to visualize and track GFP-tagged SNB-1, the
transmembrane synaptic vesicle protein UNC-46 or simi-
larly tagged transmembrane proteins that have been endo-
cytosed from the plasma membrane, likely at the synapse.
The only condition necessary to exploit this approach is
that the GFP domain of the tagged protein must be inside
the vesicle so that the GFP domain is exposed to the
extracellular space after the vesicle undergoes exocyto-
sis (Figure 1A). Furthermore, this method can be used
to characterize the retrogradely moving compartment,
as we show that the retrograde compartment carrying
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Specific Labeling of Retrogradely Transported Proteins
SNB-1 also contains SNT-1 but not RAB-5. Potentially, t his
general technique can also be used in other transparent
or translucent model organisms.
Results and Discussion
A fluorescently marked antibody against GFP
specifically labels synaptic vesicle proteins
in vivo
One-day adult
C. elegans
worms of the
strain that stably express SNB-1::GFP pan-neurally (20)
were injected in the pseudocoelom with a 1:10 dilution
of Alexa Fluor 594 IgG antibody against GFP (Ab). This
transgenic strain rescues a null allele of
and is
localized and trafficked like the endogenous protein (20).
After an incubation period of 1 h post-injection at 22
not only could we visualize the vesicles marked with
SNB-1::GFP in the green channel as expected, but also
SNB-1::GFP marked with Alexa Fluor 594 anti-GFP IgG
antibody (referred to henceforth as SNB-1::GFP-Ab) in
the red channel (Figure 1C, left). SNB-1::GFP-Ab could be
detected only in regions of the neuron that show the pres-
ence of SNB-1::GFP. This included synapse-rich regions of
the ventral cord, dorsal cord, nerve ring and neuronal pro-
cesses. No detectable signal was observed in the dendritic
processes in the head region (data not shown). Simi-
worms that stably express SNB-1::GFP only
in six mechanoreceptor or touch receptor neurons (20)
(Figure 1C, right; Figure S1D), when injected with Ab
and incubated f or 1 h, showed varying intensities of
SNB-1::GFP-Ab exclusively in these neurons. The inten-
sity of labeling varied from low to high with most animals
showing labeling between medium and high and fewer
animals showing low labeling and the intensity of labeling
changed over time (Figure S1B). As described earlier (21),
the scavenger cell coelomocytes were also labeled in each
experiment and their labeling remained nearly invariant
over time (Figures 1F and S1A). This labeling was used
as an indication of whether the assay worked, i.e. we
found that if coelomocytes were not labeled, no signal
was observed in neurons either and the animals and the
Ab dilution were discarded.
To rule out non-specific binding followed by endocytosis
of Alexa 594 IgG Ab, two control experiments were per-
formed. We injected and imaged
(22) transgenic
worms (
= 9), which express the peripheral membrane
protein GFP::RAB-3 on the surface of the synaptic v esi-
cle (23). Upon synaptic vesicle exocytosis, the GFP on
RAB-3 remains intracellular and will not be exposed to
the anti-GFP Ab in the extracellular space. Hence after
injection of the Ab, as expected, GFP::RAB-3 could be
visualized in the touch receptor neurons in the green
channel but no corresponding signal could be visualized in
the Cy3 channel (red), showing that the Ab binds specif-
ically only to the GFP that is exposed to the extracellular
space by exocytosis (Figures 1E and S1B). Similar results
were seen in injected
worms (
= 10) that express
soluble GFP intracellularly in the mechanoreceptor neuron
(Figures 1E and S1B) (24). In both these strains, coelo-
mocytes showed significant labeling (Figure 1F). Further,
the number of coelomocytes labeled increased with
time unlike in SNB-1::GFP animals injected with the
labeled antibody (Figure S1A). This is consistent with
more available Ab for coelomocyte uptake in soluble
GFP and GFP::RAB-3-expressing strains compared with
SNB-1::GFP-expressing strains. In SNB-1::GFP-expressing
strains c oelomocyte labeling stays constant, probably
because touch receptor neurons compete with the scav-
enger cells for uptake.
Detection of SNB-1::GFP-Ab but not GFP::RAB-3 nor solu-
ble GFP suggests that the GFP attached to SNB-1 is likely
to be exposed to the extracellular surface, where it is
recognized by the Ab. This labeling could occur at synap-
tic regions as SNB-1::GFP is exposed to the extracellular
milieu upon synaptic vesicle fusion. The labeling a ll along
the neuron and the cell body could be a consequence of
retrograde axonal transport.
We used our images to estimate how many SNB-1::GFP
punctae also contain SNB-1::GFP-Ab and observed that
more than 90% of the large green SNB-1::GFP vesicles
were also labeled with SNB-1::GFP-Ab (150/158,
= 9
worms) (Figure S1D). These data suggest that many of the
bright SNB-1::GFP vesicles may be in retrogradely moving
compartments, while anterogradely moving SNB-1::GFP
may be in smaller compartments and thus more difficult to
clearly detect as distinct bright punctae. Consistent with
this, we see significant diffuse’ SNB-1::GFP signal in t he
neuronal processes between the bright punctae, which
contain both SNB-1::GFP and SNB-1::GFP-Ab (Figure 1D,
Figure 3A
, data not shown).
To test if this method could be used on other synaptic vesi-
cle proteins tagged with GFP on their lumenal domain, we
injected an anti-GFP Ab in the UNC-46::GFP-expressing
(25). UNC-46 encodes a transmembrane
protein present on synaptic vesicles and is required to traf-
fic the vesicular γ-Aminobutyric acid (GABA) transporter to
synaptic vesicles (25). We injected an anti-GFP Ab uncon-
jugated to fluorophore, allowed time for endocytosis, fixed
and stained with a secondary Ab (refer to
Materials and
). We see specific labeling of a UNC-46::GFP-
carrying compartment in motor neurons after allowing for
anti-GFP Ab endocytosis even for 1015 min. At this time-
point, UNC-46::GFP-Ab is present at synapses (Figure 1D)
and a small amount of this signal is also visible in the neu-
ronal process near the cell body and in the cell body, sug-
gesting that the UNC-46::GFP-Ab compartment may also
be retrogradely transported. Identical data were obtained
when living
animals were visualized after being
injected with Alexa 594-conjugated anti-GFP antibodies
(Figure S1D). The fluorophore-unconjugated anti-GFP Ab
likewise specifically labels SNB-1::GFP in touch receptor
neurons (Figure 1D). This labeling looked nearly identical
to animals injected with Alexa 594-conjugated anti-GFP
Ab and visualized in living animals (Figure 1C,D). Further,
2011; 12: 89101 91
Murthy et al.
worms that express soluble GFP in neurons do not show
any endocytosed anti-GFP Ab signal while still showing
soluble GFP signal, which is not abolished by the fixation
procedures (Figure 1D).
Taken together, we have developed a method to label
specific lumenally tagged synaptic vesicle proteins
in vivo
The labeling depends on the exposure of the GFP tag to
the extracellular milieu allowing the antibody access to the
proteins as they cycle through the plasma membrane.
Retrograde labeling kinetics in mechanoreceptor
If the labeling of SNB-1::GFP takes place predominantly
at synaptic regions, one would expect it to label synaptic
regions more quickly, followed by an increase in other
parts of the neuronal process if the labeled SNB-1::GFP
undergoes retrograde transport. To determine the labeling
kinetics, we quantitated the labeling intensity of SNB-
1::GFP-Ab in the anterior lateral microtubule cells (ALM)
and posterior lateral microtubule cells (PLM) (Figure 1B) in
different regions of these neurons and at different time-
points. Each posterior mechanoreceptor neuron (PLM)
gives out a single synaptic branch (Figure 1B), which ends
in approximately 11 en passant synapses closely clustered
in about an 815-μm region in the ventral cord (20,26)
(Figure 2C). Similarly, the anterior cell makes several
synapses restricted to a small region in the nerve ring. The
close spacing of the synapses below the optical resolution
of a light microscope precludes our ability to track
movement of labeled vesicles between synapses, unlike
such observations made by Darcy et al. (27) in cultured
vertebrate neurons. The regions quantitated in the
synaptic branch (Figure 2, box) and the neuronal process
near t he cell body are both spatially several millimeters
away from the synaptic c lusters. Thus, the labeling
quantitated along the neuronal process is likely to arise
only from retrograde transport of labeled compartments.
To make appropriate quantitative comparisons, all data
were normalized to the synaptic signal observed at the
15-min time-point with SNB-1::GFP. We collected images
of SNB-1::GFP and SNB-1::GFP-Ab at 15 min, 30 min,
45 min, 1 h, 3 h, 6 h, 9 h and 12 h post-injection. Some
representative images of the PLM neuron at the neuronal
process near the cell body (indicated in figures and graphs
as cell body), at the mechanoreceptor neuron branch and
at the synaptic region are shown in Figure 2AC, respec-
tively. Imaging ALM neuron synapses proved challenging
because the synapses are distributed over different opti-
cal planes unlike in the PLM neurons. Further, the branch
geometry is not easily visualized and there is very high
autofluorescence in the region near the ALM cell body
as it is in the middle of the animal. Region-specific quan-
tification of the data in PLM neurons (
= 8–12 worms)
reveals that both at the synapse and in the neuronal pro-
cess near the cell body, the intensity initially increases
and then decreases with time. But the rise near the cell
body is slower and to a lower peak level compared to the
rise at the synapse. The subsequent decrease also occurs
later near the cell body than at the synapse (Figure 2D).
The decrease in labeling at synaptic regions in both ALM
and PLM neurons at later time-points could arise because
of decreased uptake (endocytosis) of the antibody along
with retrograde transport of SNB-1::GFP-Ab away from the
synapse. After 912 h almost no SNB-1::GFP-Ab signal is
observed in both ALM and PLM neurons.
Taken together, injecting a fluorophore-tagged anti-GFP
antibody into the pseudocoelom specifically labels SNB-1::
GFP. Further, the labeling kinetics support the hypothe-
sis that this Ab is labeling vesicles at synapses and that
at least a portion of them may contribute to the slower
increase in signal seen in the neuronal process near the cell
body. However, we cannot exclude that labeling occurs
directly in the cell body as has been reported for trafficking
of synaptobrevin in cell culture (28). Nor can we assess
with this method how much of the signal arises from
the known plasma membrane pools of SNB-1 that escape
endocytosis (29). However, our approach still provides a
useful tagging method and the experiments in the ensuing
sections will support the validity of our method to study
retrograde transport of SNB-1.
Time-lapse imaging of movement of SNB-1::GFP-Ab
in neurons
We hypothesized from t he observed patterns in the sig-
nal of SNB-1::GFP-Ab that majority of the labeling of
SNB-1::GFP was likely occurring at synapses and specif-
ically it is SNB-1::GFP-carrying retrograde compartments
that are labeled in the touch receptor neurons. We tested
this by time-lapse imaging of
strains that were
injected with the Ab at the 1 h post-injection time-point.
Regions of the ALM neuron proximal to the cell body were
imaged. Not only could we track many retrograde events
along with very few anterograde events, imaging could be
performed for approximately a minute without significant
photobleaching (Movie S1, cell body on the right). This
allowed us to quantitatively analyze the transport proper-
ties of carriers with SNB-1::GFP-Ab, such as velocity and
flux, and compare them with the transport properties of
SNB-1::GFP carriers (Figure 2E,F). We found that more
than 95% of the SNB-1::GFP-Ab particles move in the ret-
rograde direction, while only 3545% of the SNB-1::GFP
particles move in the retrograde direction. We also find
that the velocity of retrogradely moving SNB-1::GFP-Ab
is very similar to retrogradely moving SNB-1::GFP
(Figure 2F). Further, we estimate that only about 2030%
of the retrogradely moving SNB-1::GFP are labeled with
SNB-1::GFP-Ab. This may arise from inefficient labeling
of retrograde compartments after endocytosis, but could
also arise from anterogradely moving SNB-1::GFP show-
ing reversals to move retrogradely in the neuronal process.
It is also possible that retrogradely moving cargoes move
infrequently with long pauses, thus at any given imag-
ing time most SNB-1::GFP-Ab are stationary (Figure 2E,
arrow). Consistent with this possibility, we observe a large
number of stationary collections of SNB-1::GFP-Ab in the
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Specific Labeling of Retrogradely Transported Proteins
Figure 2: Labeling kinetics and dynamic imaging of SNB-1::GFP-Ab in touch receptor neurons. Images of retrograde labeling
in different regions: (A) neuronal process near the cell body, (B) neuronal process near the branch and (C) synaptic regions of PLM
neurons in
that express SNB-1::GFP (top panel) with SNB-1::GFP-Ab (lower panel). Boxed region of the branch was quantified
in each image (B; SNB-1::GFP-Ab panel). D) Quantitative representation of SNB-1::GFP-Ab labeling in different regions of the PLM and
ALM neurons normalized to synaptic SNB-1::GFP signal plotted as a semilog plot, where incubation = hours after anti-GFP injection
represented on the
-axis as log of time to the base 2. Data are represented as mean ± SEM,
= 8 12. E and F) Dynamic imaging
of SNB-1::GFP-Ab and SNB-1::GFP. E) Kymographs showing displacement (
-axis) of SNB-1::GFP (
= 15) and SNB-1::GFP-Ab (
= 6)
vesicles over time (
-axis) of the same neuron, acquired by live imaging in green and red channels of Ab-injected
neurons. White
arrowhead points to a SNB-1::GFP-Ab particle that may be switching direction of movement from anterograde to retrograde (reversal).
Arrow points to stationary SNB-1::GFP-Ab particle (F). Average velocity and flux of SNB-1::GFP and SNB-1::GFP-Ab calculated from
kymographs generated from time-lapse movies.
points to non-specific autofluorescence from the pseudocoelom and arrowhead
points to the neuronal process in A and B. Box in B points to region of the synaptic branch quantified in Figures 2, 3 and 5. Data are
represented as mean ± SEM. Scale bar = 10 μm.
2011; 12: 89101 93
Murthy et al.
neuronal process, whose intensity increases with time.
The few anterogradely moving SNB-1::GFP-Ab vesicles
could arise from short reversals of retrogradely moving
SNB-1::GFP-Ab carriers (Figure 2E, arrowhead), although
yet again we cannot exclude the possibility that these
vesicles are labeled at the cell body or along the neuronal
process. Nevertheless, these data show that SNB-1::GFP-
Ab is largely present only in retrogradely moving carriers
that likely a re labeled at the synapse.
Anterograde transport and physical connection
to the synapse are necessary for labeling
To further test that the observed SNB-1::GFP-Ab signal
largely arises from labeling at synapses, we tested labeling
under two conditions. We used a mutant with a strong
block in anterograde transport and we physically severed
the PLM neuronal process such that the cell body lost
connection with the synapse.
The kinesin-3 motor UNC-104 is the major carrier for
presynaptic vesicles in neurons (30). In strong hypomor-
phic mutants of
, the majority of the SNB-1 is
stuck in the cell body and in the proximal neuronal
process near the cell body (Figure 3A) and nearly no
SNB-1::GFP is seen at synapses (20). Injecting Ab into
unc-104(e1265); jsIs37
= 8), we see a s trong reduction
of labeling in the PLM neuronal process near the cell
body compared to
(Figure 3A,A
). This reduction
is presumably because of the inability of SNB-1::GFP to
be transported to the synapse. This therefore results in
less exposure of SNB-1::GFP to the extracellular environ-
ment because of reduced numbers of vesicles available
for release and consequent reduction in labeling with
the Ab and therefore fewer retrogradely labeled carriers
being transported back to the cell body. This showed that
robust anterograde transport of SNB-1::GFP is required for
SNB-1::GFP-Ab signal in the neuronal process near the cell
To further test the necessity of the synapse, the neuronal
process of the
PLM neuron was severed by laser
ablation (31), physically disconnecting the synapse from
the main PLM process. These worms, after a recovery
period of 1218 h, were injected with the anti-GFP Ab,
incubated for 1 h and imaged. In these worms, drastic
reduction in labeling was observed in the proximal neu-
ronal process connected to the cell body. However, the
SNB-1::GFP present in the distal cut fragment attached
to the synapse is able to label with the anti-GFP Ab
(Figure 3B). The distal labeling is brightest at the synapse
and at the synaptic branch but reduces along the neu-
ronal process. The reduced labeling in the distal neuronal
process may arise from ‘Wallerian-like’ degenerative pro-
cesses that the neuronal process can undergo when sep-
arated from the cell body. This result not only shows that
a physical connection to the synapse is needed for robust
labeling ( Figure 3B) but also further suggests that trans-
port of the Ab from the synapse must occur in a retrograde
manner along the process to robustly label the neuronal
process near the cell body. The corresponding uncut PLM
in the same animal that has undergone axotomy shows
strong labeling near the cell body (Figure 3B, uncut).
The PLM neuronal process begins to show robust regen-
eration and process outgrowth 6 h after axotomy (32).
Further, immature neurons are shown to undergo vesi-
cle exocytosis along the axon (33). Thus, the regrowing
axotomized PLM nerve in
animals could share char-
acteristics with an immature neuron and the observed
low level of labeling could arise from SNB-1::GFP cycling
through the plasma membrane close to the growth cone,
along the neuronal process or at the cell body.
Taken together, these data show that anterograde trans-
port t o the synapse and physical connection to synapses
are necessary for robust labeling with Ab, thus it is
likely t hat majority of the labeling occurs at or near
Both exocytosis and endocytosis are necessary
for robust labeling of retrograde compartments
To determine if exocytosis and endocytosis are necessary
steps in the labeling of the retrogradely moving compart-
ment containing SNB-1::GFP, we determined the labeling
intensity in two mutants. We used
, a hypo-
morphic allele of syntaxin essential for synaptic vesicle
fusion, and
, a null mutant in a clathrin adaptor
protein AP180 with a demonstrated role in endocytosis
of SNB-1
in vivo
(3436). PLM neurons in
mutants showed highly reduced labeling in the
neuronal process (Figure 3C). Moreover, the little label-
ing that does occur initially is nearly absent after 6 h
(Figure 3C,E). This reduced labeling is consistent with
the predicted role for syntaxin in vesicle fusion, thereby
less SNB::1-GFP is exposed to the extracellular milieu
for labeling with the Ab. Further, the SNB-1::GFP-Ab
label in the AP180 endocytosis mutant
similar to SNB-1::GFP in these same cells (Figure 3C).
That is, the SNB-1::GFP-Ab signal looks like it is dis-
tributed on the plasma membrane (36), which looks more
spread and a lot thicker, presumably because most of the
label is restricted to the cell surface. Consistent with the
endocytic block in
animals, we observe that the
SNB-1::GFP-Ab in the cell body is largely restricted at or
near the plasma membrane instead of intracellularly, as
observed in wild type
animals (Figure 3D). In addi-
tion, compared to SNB-1::GFP-Ab labeling in wild type,
the SNB-1::GFP-Ab signal in most
animals is
very low (Figure 3E). These results show that both endo-
cytosis and exocytosis are important steps for labeling
SNB-1::GFP with the anti-GFP Ab.
Reduced labeling is observed in retrograde transport
Data thus far indicate that SNB-1::GFP is labeled with the
anti-GFP Ab largely at or near the synapses and that the
labeling is dependent on anterograde transport, exocytosis
2011; 12: 89101
Specific Labeling of Retrogradely Transported Proteins
Figure 3: Anterograde transport to synapses and physical connection to synapses are necessary for SNB-1::GFP labeling.
A) Labeling in
1 h after injection of anti-GFP Ab. Strong reduction of labeling with SNB-1::GFP-Ab in
unc-104(e1265); jsIs37.
Boxed region in (A) is shown in higher magnification in (A
). B) Strong reduction of labeling with SNB-1::GFP-Ab
near the cell body in axotomized PLM
worms (right) compared to non-axotomized
(left). Labeling in distal axotomized
remnant is seen upto the severed point.
indicates background non-specific fluorescence from the pseudocoelom that is usually
greatly increased upon axotomy. Arrowhead points to the neuronal process. C) Synaptic vesicle exocytosis mutant
in the
t-SNARE syntaxin (middle panel) showed reduced SNB-1::GFP-Ab labeling compared to
. Qualitatively, the labeling in endocytosis
in the
gene (lowest panel) near the cell body and synapse was more spread out and thicker when compared
, consistent with significant labeling on the plasma membrane even at the cell body. D) PLM cell body showing surface staining
of SNB-1::GFP-Ab (2) compared to SNB-1::GFP (1) in
animals 1 h after Ab injection. E) Quantification of labeling in
shows reduced labeling with time and nearly constant intensity of labeling
. Data are represented as a semilog
plot, where incubation time = hours after anti-GFP injection represented as log to the base 2. Scale bar = 10 μm.
2011; 12: 89101 95
Murthy et al.
and endocytosis of SNB-1::GFP. Moreover, labeling near
the cell body should arise from the retrograde transport of
SNB-1::GFP-Ab in a dyneindynactin-dependent process.
To verify this latter inference directly, we tested if labeling
is sensitive to reduction in retrograde transport. For this,
we assayed the extent of labeling in mutants of the
dyneindynactin complex with known roles in SNB-1 ret-
rograde transport. We used two hypomorphic alleles,
, a mutation in the dynein heavy chain gene, and
, a mutation in the p150 glued component of
the dynactin complex. In these mutants, SNB-1::GFP and
synaptotagmin accumulate in the tip of the mechanore-
ceptor neuron (9). In worms injected with antibody, this
tip accumulation of SNB-1::GFP is also labeled with SNB-
1::GFP-Ab and as expected this labeling increases with
time in both PLM and ALM neurons (Figure 4B).
To further confirm our observations, SNB-1::GFP-Ab inten-
sity was quantified in the PLM neuronal process near the
cell body, in the neuronal branch region, at the synapse and
at the neuronal end of
dhc-1(js319); jsIs37
1(or404ts); jsIs37
. We observed that the increase in label-
ing at synaptic regions is greatest in
and significantly
lower in both retrograde transport mutants (Figures 5B
and S1C). This suggests that the balance of endocyto-
sis and exocytosis of vesicles in these mutants may be
altered. Further, upon Ab labeling in
and in
, the neuronal ends in the PLM neurons
label more than in wild type
. In all three genotypes,
this labeling increases with time before decreasing even-
tually (Figure 5C). This eventual decrease is much slower
in both the mutants t han in wild type
, consistent
with reduced retrograde transport (Figure 5A,C,D). Further
Figure 4: Labeling in wild type and dyneindynactin complex mutants. Examples of images of SNB-1::GFP and SNB-1::GFP-Ab
in (A) the neuronal process near the cell body of ALM (right) and PLM (left) of
dhc-1(js319); jsIs37
(dynein heavy chain) and
dnc-1(or404ts); jsIs37
(p150 glued) mutants. Both ALM and PLM (B) neuronal ends and (C) synapses show increased SNB-1::GFP-Ab
intensity with time.
points to autofluorescence from the pseudocoelom. Box in (B) shows the characteristic ‘tip’ accumulation of
SNB-1::GFP observed in dynein mutants. Scale bar: (A, B) 5 μmand(C)10μm.
2011; 12: 89101
Specific Labeling of Retrogradely Transported Proteins
Figure 5: Quantification of SNB-1::GFP-Ab labeling and
transport in retrograde transport mutants. Unsaturated
images were collected of both the SNB-1::GFP (green) and
SNB-1::GFP-Ab (red) and quantified. All SNB-1::GFP-Ab signal
(obtained from various regions) was normalized with synaptic
SNB-1::GFP signal of the corresponding genotype at 15 min.
Labeling was quantified in the (A) neuronal process near the
cell body, (B) synaptic region, (C) neuronal end and neuronal
(D) process near the synaptic branch in PLM neurons in
1); jsIs37
(dynein heavy chain) and
dnc-1(or404ts); jsIs37
glued dynactin) mutants. All data are represented as mean ±
SEM on a semilog plot (
= 810), where incubation = hours
after anti-GFP injection represented as log to the base 2. E)
Average retrograde flux and (F) retrograde velocity of SNB-1::GFP
and SNB-1::GFP-Ab calculated from kymographs (Figure S1E)
generated from time-lapse movies of
js319(dhc-1); jsIs37
dnc-1(or404ts); jsIs37
. Data are represented as mean ± SEM.
corroboration is provided by the reduction in labeling
intensity in
compared to
wild type
in the neuronal process region near the
cell body (Figure 5A). Such a reduction in the
compared to
is also visible near
the PLM neuronal branch, suggesting that fewer SNB-
1::GFP-Ab likely exit t he synaptic region. Broadly similar
trends are observed in the ALM (data not shown).
As another test to confirm the dependence of SNB-1::GFP-
Ab on the dyneindynactin complex, we dynamically
imaged transport of these labeled compartments in the
PLM neuronal processes of t he hypomorphic mutant
animals. As one might predict, t he retrograde flux (number
of moving compartments per second) of moving SNB-1::
GFP-Ab-labeled compartments obtained from kymographs
(Figure S1E) of time-lapse movies was reduced consid-
erably in
= 8) and
= 8)
when compared with wild type
= 7) (Figure 5E).
The retrograde flux of SNB-1::GFP likewise was also
reduced. The velocity of the retrogradely moving com-
partments carrying SNB-1::GFP-Ab was largely unaffected
in the dyneindynactin mutants compared to velocity of
SNB-1::GFP-Ab in wild type animals (Figure 5F). This may
arise because the dyenindynactin alleles are only partial
loss-of-function mutants with relatively modest effects
on animal behavior and viability and likely on axonal
transport (9).
Taken together, these data imply that the labeling in the
neuronal process near the cell body and at the mechanore-
ceptor neuron tip is dependent on the activity of the
dyneindynactin complex and thus this method is useful in
specifically labeling retrogradely transported SNB-1::GFP
Characterization of the SNB-1::GFP-Ab-labeled
To characterize the SNB-1::GFP-Ab-labeled retrogradely
moving compartments, we assessed colocalization with
other proteins. We used a selection of synaptic vesi-
cle markers, endosome markers and molecular motors
to characterize the retrograde SNB-1::GFP-Ab-carrying
compartments 520 min after anti-GFP Ab injection.
We observe that nearly all SNB-1::GFP-Ab com-
partments also contain the synaptic vesicle protein
synaptotagmin-1 (SNT-1) (Figure 6A,
= 15 animals) (37).
Of the 452 puncta counted, 435 retrograde compart-
ments contain both SNB-1::GFP-Ab and SNT-1; 13/452
(3%) puncta contain only SNT-1 but do not label with
SNB-1::GFP-Ab; 4/452 (<1%) SNB-1::GFP-Ab puncta do
not label with SNT-1. The SNT-1 puncta that do not
label with SNB-1::GFP-Ab may represent vesicles moving
anterogradely and therefore lacking SNB-1::GFP-Ab. Alter-
nately, these vesicles may lack SNB-1::GFP and therefore
do not label with anti-GFP Ab. To test whether there are
compartments that contain SNT-1 but not SNB-1, we car-
ried out a double-labeling experiment using a nti-SNT-1
and anti-GFP antibodies (not injected) that potentially
recognize all SNB-1::GFP (Figure 6G); 114/126 puncta con-
tain both SNT-1 and SNB-1::GFP (
= 9 animals), 7/126
(5%) puncta contain only SNT-1 and 5/126 (4%) puncta
contain only SNB-1::GFP. These data suggest that there
may be a few compartments that contain SNT-1 that either
lack or have very low levels of SNB-1. Such compartments
may account for SNT-1 puncta that lack SNB-1::GFP-Ab.
However, we cannot rule out that the lack of co-staining
with SNT-1 and SNB-1::GFP could arise from inefficient
labeling with either primary or secondary antibodies.
Another synaptic vesicle protein RAB-3 shows poor colo-
calization where only 4/15 animals examined show some
colocalization with the SNB-1::GFP-Ab compartments in
very small parts of their neurons (Figure 6D) (38). The
2011; 12: 89101 97
Murthy et al.
small amount of colocalization could be a consequence of
RAB-3 dissociating from the vesicle surface and diffusing
in the very narrow neuronal process to give an appea-
rance of colocalizing with a subset of SNB-1::GFP-Ab
compartments. These data are consistent with prior
observations that the retrograde dyneindynactin com-
plex motor transports a compartment containing SNB-1
and SNT-1 in
C. elegans
neurons but does not transport
endogenous RAB-3 (9), although total pools of SNB-
1, SNT-1 and RAB-3 do all colocalize with each other
consistent with all of them being on synaptic vesicle
proteins (35,38).
To further characterize the SNB-1::GFP-Ab compartments,
we double-stained with an antibody to the specific
anterograde synaptic vesicle motor UNC-104/kinesin-
3 (30). No colocalization was observed between SNB-
1::GFP-Ab and UNC-104/kinesin-3 (Figure 6C,
= 14 ani-
mals). Further, very few SNB-1::GFP-Ab compartments
colocalize (4/17 animals show some colocalization) with
another anterograde motor UNC-116/kinesin-1 motor
(Figure 6B) (39). These data are consistent with the expec-
tation that retrogradely moving compartments are unlikely
to contain anterograde motors known to transport presy-
naptic vesicle proteins. We also assessed the presence of
an early endosomal marker RAB-5 (40) and an active zone
marker UNC-10/RIM (41). Neither of these proteins are
present in the SNB-1::GFP-Ab compartments (Figure 6E,
= 13, 15 animals, respectively).
Figure 6: Characterization of SNB-1::GFP-Ab-labeled retrograde compartments. Images of worms expressing SNB-1::GFP either
pan-neurally (
) or in touch receptor neurons (
) injected with either fluorophore-unconjugated mouse anti-GFP Ab or rabbit
anti-GFP Ab to label the retrogradely transported compartments, fixed and incubated with antibodies to (A) synaptotagmin SNT-1 show
a lot of colocalization. Co-staining with (B) UNC-116/kinesin-1 shows occasional colocalization; (C) with UNC-104/kinesin-3 shows n o
colocalization; (D) with RAB-3 shows no colocalization; (E) with RAB-5 shows no colocalization and (F) with UNC-10/RIM shows no
colocalization. G) Colocalization between endogenous SNT-1 and SNB-1::GFP. SNB-1::GFP is detected by a mouse anti-GFP antibody
(uninjected). Most of the SNT-1 compartments also contain SNB-1::GFP. In all double-label experiments, the fluorophore conjugated to
the secondary antibody is listed in parentheses. Scale bar: 5 μm.
2011; 12: 89101
Specific Labeling of Retrogradely Transported Proteins
Taken together, these data suggest that the compartment
that carries SNB-1 away from t he synapse contains
synaptotagmin but lacks many other markers, such as
other presynaptic vesicle markers RAB-3 and UNC-104,
the early endosomal marker RAB-5 or the anterograde
kinesin motor UNC-116/kinesin-1. These data suggest
that the SNB-1 that exits t he synapse is present in a
postendocytic compartment that contains a subset of
synaptic vesicle proteins. This compartment may differ
from both a synaptic vesicle and an early endosome,
although it could retain the ability to undergo exocy-
tosis if recruited to an active zone containing UNC-10
and RAB-3. Our work suggests the presence of spe-
cific retrogradely moving compartments carrying vary-
ing subsets of synaptic vesicle proteins away from the
We have developed a simple method to specifically label
and track retrogradely transported transmembrane pro-
teins based on the uptake of a fluorescent tag upon exo-
cytosis. As proof of principle, we have shown t his method
to label SNB-1::GFP after vesicle fusion at synapses so that
the protein can subsequently be tracked in the neuron by
dynamic imaging and quantitative labeling of steady-state
intensities. The labeling is dependent on the number of
SNB-1::GFP-carrying vesicles that reach the synapse and
the ability of these vesicles to undergo both endocyto-
sis and exocytosis at synapses. This idea can easily be
adapted to other transmembrane vesicular proteins that
have a tag (GFP or other) within the vesicle to which a flu-
orescently labeled antibody can be targeted. Our method
has the advantage that it assays only those molecules
that have cycled through the plasma membrane, so they
can be separated from the same protein molecules that
have not undergone fusion with the plasma membrane.
Although this Ab is labeled with a fluorophore, other
tags (42) can be used to further probe the characteris-
tics of retrogradely transported compartments. Moreover,
the principles behind our method can be used to label
protein-carrying retrograde compartments in other organ-
isms such as
Materials and Methods
Nematodes were cultured according t o the study by Brenner (43). The
strains used in this study were
) (20),
) (22,23),
) (24) and mutant strains
(9) in the
background and
) (25).
Injection of antibody
Anti-GFP antibody tagged with Alexa Fluor Red 594 (Molecular Probes)
diluted in freshly autoclaved and filter-sterilized 1
× PBS buffer (1:10)
was injected into the pseudocoelom of one-day-old adult
C. elegans
After injection, worms were incubated at 22
C or various time-points and
imaged. Labeled antibodies usually stop working in this assay 2 3 months
after receipt of the Ab from Molecular Probes.
and o
strains were also injected with mouse anti-GFP antibody unconjugated
to fluorophore (Bangalore Genei) incubated at room temperature, fixed
as described below, subsequently stained with secondary anti-mouse
Alexa 568. Data obtained with the fluorophore-labeled anti-GFP antibody
and-unconjugated anti-GFP antibody were identical.
Static imaging was performed by using Axiovert Zeiss 200M inverted
microscope fitted with a Zeiss Axio Cam MRm at 63
× and 100× using
the fluorescein isothiocyanate (FITC) and Cy3.5 channels, respectively.
Unsaturated images were collected and processed in I
Laser axotomy
L4 larvae were anesthetized using 0.13% (w/v) sodium azide on
an agar pad Nd:YAG (Spitlight 600, Innolas) nanosecond single-shot laser
pulses (
λ = 355 nm) with 0.8 μJ energy used was used to cut the PLM
neuronal process with a 100
×, 1.3 numerical aperture (NA) objective on an
inverted microscope (Olympus iX71) (31). Worms were allowed to recover
for several hours (1218 h) before injection with the anti-GFP Ab.
Dynamic imaging and analysis
worms were incubated for 1 h after injection at 22
imaged using spinning disc confocal microscope. Images were acquired
at five frames per second at a magnification of 100
× (Olympus) using an
Andor monochrome camera (iXon DU897-UVB). Simultaneous acquisition
of red and green was not possible, thus in some cases we sequentially
imaged SNB-1::GFP-Ab followed by SNB-1::GFP. Some control SNB-1::GFP
moves were taken in animals that were not injected to ensure that the Ab
injection did not alter the movement of SNB-1::GFP-carrying vesicles. All
SNB-1::GFP movement data were pooled. Particle velocity analysis was
carried out on kymographs made from the acquired movies in I
(kymograph plug-in). Flux was calculated by counting the number of
synaptic vesicles that moved in a specific direction within a 20-
μm segment
of the process. Velocity was calculated from the slopes of lines from the
Quantitation of signal
Eight bit images were obtained using 300-millisecond exposure below sat-
uration (0255 pixel intensity) levels. Quantification of SNB-1::GFP (green)
as well as the SNB-1::GFP-Ab (red) signal in different regions was per-
formed by thresholding; thereafter, average pixel intensity was obtained
by subtracting average background pixel intensities from the thresholded
signal intensity. Data represented in different regions have been normal-
ized to the intensity of SNB-1::GFP at the synapse at the 15-min time-point
of the corresponding genotype to account for day-to-day as well as animal-
to-animal variations in SNB-1::GFP, which in turn could affect labeling
by the anti-GFP antibody. Levels of SNB-1::GFP-Ab were quantitated at
15 min, 30 min, 45 min, 1 h, 3 h, 6 h, 9 h and 12 h after injection. Data
were plotted on a semilog plot, where the scale on t he
-axis uses log of
time (in hours) to the base 2.
Labeling colocalization of SNB-1::GFP-Ab with other
transgenic animals were injected with either
mouse anti-GFP antibody (undiluted) (Bangalore Genei) or rabbit anti-GFP
antibody (1:10) (Invitrogen) and fixed in 4% PFA and 50% methanol for
15 min on ice. Animals were cut near the head o r tail t o break the
cuticle barrier for entry of second primary Ab and secondary antibodies.
After fixation, animals were washed with 1
× PBS for 2 h after every
10 min to get rid of unendocytosed anti-GFP antibody. Animals were then
washed with borate buffer, permeabilized with Triton in 1
× PBS before
2011; 12: 89101 99
Murthy et al.
the second primary was added (o/n, 4
C). Under these fixation condi-
animals expressing soluble GFP in touch receptor neurons
showed occasional faint staining in the cell body and no staining along
the neuronal process. For double immunostaining, the following primary
antibodies were used: mouse anti-GFP (1:50) (Bangalore Genei); rabbit
anti-SNB-1 (1:800) (35); rabbit anti-SNT-1 (1:1000) (37); mouse anti-RAB-3
(1:5000) (38); rabbit anti-UNC-116/kinesin heavy chain (1:400) (39); rabbit
anti-UNC-10/RIM (1:2000) (41)) and rabbit anti-RAB-5A (1:100) (Santa Cruz
Biotech Inc). In double-label experiments u sing the mouse anti-GFP and any
other rabbit primary antibody, the secondaries used were anti-mouse Alexa
488 (1:400) (Molecular Probes) and anti-rabbit Alexa 568 (1:400) (Molecular
Probes). In double-label experiments using the r abbit anti-GFP with any
other mouse primary antibody, the secondaries used were anti-rabbit Alexa
(1:400) 568 or 488 (Molecular Probes) and, respectively, anti-mouse Alexa
488 or 568 (1:400) (Molecular Probes).
Double-labeled specimens were imaged on a confocal microscope using
a 100
× 1.4 NA oil immersion lens. Images obtained in the green and red
channels were merged to check for colocalization. Multiple regions and
isolated individual mechanoreceptor or sublateral neurons were imaged in
at least 917 independently stained animals.
K. M. and J. M. B. carried out experiments and helped in manuscript
preparation. S. P. K. carried out experiments and wrote the paper. We
thank Dr Krishnamurthy at CIFF (Department of Science and Technology,
Government of India Centre for Nanotechnology; No. SR/S5/NM-
36/2005) and Dr VijayRaghavan for use of the Zeiss apotome. We thank
Bikash Chowdhary for preliminary live imaging and Sucheta Kulkarni for
help with axotomy. We thank Michael Nonet for early discussions about
setting up the assay and antibodies against SNT-1, SNB-1, RIM and RAB-3.
We thank Dr Frank McNally for anti-UNC-116 and Dr Mayor for anti-RAB-
5A. J. M. B. is supported by a fellowship from CSIR, K. M. is supported by
a post-doctoral fellowship from DBT. This work was partially supported by
a grant from DST to S. P. K.
Supporting Information
Additional Supporting Information may be found in the online version of
this article:
Figure S1: Labeling of coelomocytes and neurons with anti-GFP-
Alexa-Ab. a) Number of coelomocytes labeling with the anti-GFP antibody
increases in
animals compared to
animals. Data
represented as mean
± SEM,
= 10. b) Average labeling with the anti-GFP
Ab in PLM neurons of
animals. 0, no labeling; 1,
low labeling; 2, medium labeling; 3, high labeling;
= 10. c) SNB-1::GFP
intensities in the synapse are higher in dyneindynactin mutants compared
. Uptake of Ab in
seems significantly altered while it
is relatively unaffected in
animals. Data represented as mean
± SEM,
= 810. d) Confocal images show significant colocalization
(lower panel; merge) of SNB-1::GFP-Ab- labeled compartments (middle
panel) with vesicles expressing SNB-1::GFP (top panel). Confocal images
showing significant colocalization (lower panel; merge) of UNC-46::GFP-
Ab-labeled compartments (middle panel) with vesicles expressing UNC-
46::GFP (top panel). e) Kymographs of time-lapse movies tracking
movement of SNB-1::GFP compartments (top panel) and SNB-1::GFP-
Ab-labeled compartments (lower panel) showing displacement (
over time (
-axis) acquired by live imaging in green and red channels,
respectively, of Ab-injected
= 7),
dhc-1(js319); jsIs37
= 8)and
dnc-1(or404ts); j sIs37
= 8). Scale bar (D) 5 and (E) 10 μm.
Movie S1: Movie of SNB-1::GFP-Ab in the PLM neuron captured at five
frames per second and played back speeded up four times. One can also
see the second PLM neuron slightly out of focus below the PLM neuron
that is in focus. Cell body is to the right and most SNB-1::GFP-Ab particles
move toward the cell body.
Please note: Wiley-Blackwell are not responsible for t he content or
functionality of any supporting materials supplied by the authors.
Any q ueries (other than missing material) should be directed to the
corresponding author for the article.
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2011; 12: 89101 101
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