Volume 23 May 1, 2012
MBoC | ARTICLE
Axonal and dendritic synaptotagmin isoforms
revealed by a pHluorin-syt functional screen
Camin Deana,b,*, F. Mark Dunninga, Huisheng Liua, Ewa Bomba-Warczaka, Henrik Martensc,
Vinita Bharatb, Saheeb Ahmedb, and Edwin R. Chapmana
aDepartment of Neuroscience, Howard Hughes Medical Institute, University of Wisconsin, Madison, WI 53706;
bEuropean Neuroscience Institute, 37077 Göttingen, Germany; cSynaptic Systems GmbH, 37079 Göttingen, Germany
ABSTRACT The synaptotagmins (syts) are a family of molecules that regulate membrane fu-
sion. There are 17 mammalian syt isoforms, most of which are expressed in the brain. How-
ever, little is known regarding the subcellular location and function of the majority of these
syts in neurons, largely due to a lack of isoform-specific antibodies. Here we generated
pHluorin-syt constructs harboring a luminal domain pH sensor, which reports localization, pH
of organelles to which syts are targeted, and the kinetics and sites of exocytosis and endocy-
tosis. Of interest, only syt-1 and 2 are targeted to synaptic vesicles, whereas other isoforms
selectively recycle in dendrites (syt-3 and 11), axons (syt-5, 7, 10, and 17), or both axons and
dendrites (syt-4, 6, 9, and 12), where they undergo exocytosis and endocytosis with distinc-
tive kinetics. Hence most syt isoforms localize to distinct secretory organelles in both axons
and dendrites and may regulate neuropeptide/neurotrophin release to modulate neuronal
Members of the synaptotagmin (syt) protein family are likely to regu-
late a variety of membrane-trafficking events in cells. Syts contain a
luminal tail, a transmembrane domain, and two cytoplasmic C2 do-
mains (Perin et al., 1991; Sutton et al., 1995). Calcium-sensing by the
C2 domains induces binding of some isoforms to lipids and soluble
N-ethylmaleimide–sensitive factor attachment protein receptor pro-
teins (Brose et al., 1992; Chapman et al., 1995; Schiavo et al., 1997;
Bhalla et al., 2008), which promotes fusion and exocytosis of vesicle
cargo (Chapman, 2008). The best-characterized isoform is syt-1,
which is present on synaptic vesicles and is essential for fast synaptic
transmission (DiAntonio and Schwarz, 1994; Geppert et al., 1994;
Littleton et al., 1994). However, less is known concerning additional
mammalian syt isoforms in neurons. All 17 isoforms have been de-
tected in brain at the level of mRNA, except for syt-8, 14, and 15
(Lein et al., 2007; Mittelsteadt et al., 2009). Of importance, syt mRNAs
are expressed in hippocampal neurons, except for syt-2, which is
functionally redundant with syt-1 (Stevens and Sullivan, 2003), and
expressed in distinct brain areas (Ullrich et al., 1994; Marqueze et al.,
1995). Thus hippocampal neurons provide a valid model system for
study of the localization and function of these syt isoforms.
Whereas syt-1 is present on synaptic vesicles, syt-4 was recently
localized to neurotrophin-containing dense-core vesicles in hip-
pocampal neurons, where it negatively regulates BDNF (brain-
derived neurotrophic factor) release (Dean et al., 2009). Synaptic
vesicles (harboring syt-1) and dense-core vesicles (harboring syt-4)
have distinct recycling characteristics in hippocampal neurons, as
revealed by pHluorin reporters. pHluorins fused to a luminal do-
main of synaptic vesicles exhibit a fast depolarization–induced in-
crease in fluorescence, corresponding to exocytosis, followed by a
slower decay to baseline fluorescence within 60–90 s, indicating
endocytosis and reacidification of vesicles (Sankaranarayanan and
Ryan, 2000; Granseth et al., 2006). In contrast, pHluorin–syt-4 ex-
hibits fluorescence changes in response to depolarization in both
axons and dendrites, and axonal events have slower rise and decay
kinetics than do synaptic vesicle pHluorins. In addition, pHluorin–
syt-4 fluorescence often remains elevated for several minutes fol-
lowing stimulation (Dean et al., 2009).
University of North Carolina
Received: Aug 18, 2011
Revised: Feb 17, 2012
Accepted: Feb 29, 2012
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E11-08-0707) on March 7, 2012.
*Present address: European Neuroscience Institute, 37077 Göttingen, Germany.
Address correspondence to: Edwin R. Chapman (email@example.com).
Abbreviations used: BDNF, brain-derived neurotrophic factor; BSA, bovine serum
albumin; DIV, days in vitro; EPSC, excitatory postsynaptic current; GFP, green fluo-
rescent protein; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; ROI,
region of interest.
© 2012 Dean et al. This article is distributed by The American Society for Cell Biol-
ogy under license from the author(s). Two months after publication it is available
to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society of Cell Biology.
1716 | C. Dean et al. Molecular Biology of the Cell
in axons in response to depolarization and then decreased, but with
slower kinetics than for syp-pHluorin (Figure 1B). In addition, unlike
syp-pHluorin responses, pHluorin–syt-4 axonal events were not
always coincident with the onset of depolarization. Moreover,
pHluorin-syt-4 vesicles also underwent exocytosis in dendrites
(Figure 1C), where they exhibited either a small, fast rise and decay
(Figure 1D) or a large, fast increase in fluorescence, which remained
elevated in the presence of depolarizing solution (Dean et al., 2009;
Figure 1E). These data confirm that synaptic vesicle– and dense-
core vesicle–localized proteins have spatially and temporally distinct
Of all the syt isoforms tested, only two—syt-1 and syt-2—exhib-
ited pHluorin responses indicative of localization to synaptic vesi-
cles. pHluorin–syt-1 increased in fluorescence immediately follow-
ing depolarization and then decayed back to baseline with kinetics
nearly identical to that of syp-pHluorin (Figure 2A). pHluorin–syt-2
also exhibited recycling characteristics indicating localization to syn-
aptic vesicles (Figure 2B), as expected, since syt-2 has been shown
to be functionally redundant with syt-1 (Stevens and Sullivan, 2003),
but is localized to distinct brain areas where syt-1 is absent (Ullrich
et al., 1994; Marqueze et al., 1995).
We found that three other syts—syt-5, 7, and 17—were also ex-
clusively recycled in axons (Figure 3). However, the pHluorin charac-
teristics of these syts were not synaptic vesicle like. Syt-10 was the
only syt (in addition to syt-8; unpublished data) that did not exhibit a
fluorescence change in response to depolarization (Figure 3A) in ei-
ther the absence or presence of bafilomycin to block the vacuolar
ATPase (Figure 3A, inset). The majority of pHluorin–syt-10 was local-
ized to the plasma membrane in nondepolarizing conditions in hip-
pocampal neurons. Syt-10 is most highly expressed in the olfactory
bulb, where it was reported to regulate insulin-like growth factor 1
secretion (Cao et al., 2011). In the hippocampus syt-10 is specifically
up-regulated in a subset of neurons following seizure activity (Babity
et al., 1997). Thus pHluorin–syt-10 may be “inactive” in normal condi-
tions in the majority of hippocampal neurons and specifically invoked
to recycle in subsets of neurons under conditions of high activity.
pHluorin–syt-5 was also visible on the plasma membrane, prior
to depolarization, exclusively in axons (Figure 3B), and this isoform
exhibited increases in fluorescence at select sites, coincident with
depolarization. These fluorescence responses were large in com-
parison with the majority of syt isoforms, with a slow rise time (Sup-
plemental Figure S3, A and B) reminiscent of the dense-core vesicle
recycling characteristics previously observed for pHluorin–syt-4 in
axons. pHluorin–syt-7 also exhibited fluorescence increases exclu-
sively in axons (Figure 3C), which were of smaller magnitude than for
any other axonal syt isoform (Supplemental Figure S3A) and re-
mained elevated following depolarization for at least several min-
utes. These observations suggest that syt-7 is present on internal
vesicles that undergo exocytosis, as reported in nonneuronal cells
(Wang et al., 2005), and that it is not exclusively localized to the
plasma membrane as proposed previously (Sugita et al., 2001).
Syt-7 was also previously localized to lysosomes in nonneuronal cells
(Reddy et al., 2001; Roy et al., 2004). Thus it is possible that the
pHluorin–syt-7 response following depolarization corresponds to
lysosomal fusion (Arantes and Andrews, 2006).
pHluorin–syt-17 was also localized predominantly to axons, but
this isoform had characteristics distinct from those of all other ax-
onally localized isoforms. Diffusely distributed plasma membrane
fluorescence was not observed, but rapidly trafficking vesicles were
clearly visible in nondepolarizing conditions (Figure 3D and Supple-
mental Movie S1). Syt-17 is predicted to be membrane associated
but to lack a transmembrane domain (Craxton, 2010). Thus the
These unique recycling characteristics enable a distinction of
syts localized to these and other organelles. Here we performed a
pHluorin “screen” of syt isoforms in hippocampal neurons to deter-
mine 1) whether they recycle in response to depolarization,
2) whether they undergo exo/endocytosis in axons, dendrites, or
both, and 3) whether their recycling characteristics suggest localiza-
tion to synaptic vesicles or to other vesicle subtypes. Syts-1–7, 9–12,
and 17 were tested. Of interest, we found that five syts (syt-1, 2, 5,
7, and 17) underwent exocytosis exclusively in axons, but only syt-1
and 2 exhibited recycling characteristics indicative of localization to
synaptic vesicles, whereas syt-5, 7, and 17 displayed unique recy-
cling kinetics suggesting localization to distinct vesicle subtypes.
Two syts (syt-3 and 11) recycled exclusively in dendrites, and four
(syt-4, 6, 9, and 12) recycled in both axons and dendrites. Our re-
sults suggest that the syts have diverged to regulate activity-depen-
dent fusion of a wide variety of distinct vesicle subtypes and may
therefore modulate synaptic transmission indirectly via regulation of
release of neuropeptides or neurotrophins.
To assess the membrane-recycling properties of the syts, we fused a
pH-sensitive green fluorescent protein (GFP) variant, pHluorin, to
the luminal domain of each isoform with a preprolactin leader se-
quence and a linker to promote efficient targeting (Fernandez-
Alfonso et al., 2006). Inside the lumen of acidic vesicles, pHluorin
fluorescence is quenched. When these vesicles undergo exocytosis,
their luminal domains are exposed to the more basic extracellular
solution, and they become fluorescent. Subsequent endocytosis re-
sults in vesicle reacidification and a corresponding decay in fluores-
cence. Thus recycling events can be detected as sites of transient
increases in fluorescence.
To determine whether recycling occurred in axons or dendrites
of hippocampal neurons, we examined transfected cells in which
these processes could clearly be identified using prior low-magnifi-
cation imaging, in which axons extend longer processes than do
dendrites (Supplemental Figure S1). In cases in which the two types
of processes were difficult to distinguish morphologically, retrospec-
tive immunostaining for GFP to mark pHluorin-syt and MAP-2 to
mark dendrites was used. Of interest, in these immunostaining ex-
periments, most syts were detected in both axonal and dendritic
compartments (Supplemental Figure S2); only pHluorin-syt time-
lapse imaging experiments during depolarization revealed recycling
of specific syts in distinct compartments.
We first compared the recycling characteristics of synaptophysin
(syp)-pHluorin, which is exclusively localized to synaptic vesicles
(Granseth et al., 2006), and of pHluorin–syt-4, which is present on
neurotrophin-containing dense-core vesicles (Dean et al., 2009). Both
of these fusion proteins have been validated to localize identically to
their endogenous counterparts (Granseth et al., 2006; Dean et al.,
2009). Hippocampal neurons were transfected with these fusion con-
structs, and transfected cells, which could be detected by faint fluo-
rescence prior to stimulation, were imaged during depolarization with
45 mM KCl, which has been shown to efficiently induce exocytosis of
both synaptic vesicles and neurotrophin-containing dense-core vesi-
cles (Hartmann et al., 2001; Kolarow et al., 2007; Dean et al., 2009).
Depolarization caused a rapid fluorescence increase at presyn-
aptic boutons in axons of syp-pHluorin–transfected neurons, with no
significant fluorescence or change in fluorescence detected in den-
drites (Figure 1A). This increase in fluorescence in axons decayed
(corresponding to endocytosis and reacidification) within 90 s, as
previously shown for this reporter of synaptic vesicle recycling
(Granseth et al., 2006). pHluorin–syt-4 fluorescence also increased
Volume 23 May 1, 2012 Axonal and dendritic synaptotagmin isoforms | 1717
Two syt isoforms exhibited pHluorin responses upon depolariza-
tion exclusively in dendrites: syt-3 and syt-11. Syt-3 was the only
isoform to undergo endocytosis, instead of exocytosis, in response
to depolarization. In resting conditions syt-3 was present in a punc-
tate pattern resembling postsynaptic sites in both proximal (Figure
4A) and distal (Figure 4B) dendrites. Following depolarization, the
fluorescence of these puncta decayed exponentially (Figure 4C).
This fluorescence decay corresponded to endocytosis, since perfu-
sion with NH4Cl to neutralize all internal acidic compartments
resulted in recovery of fluorescence at these sites (Supplemental
pHluorin fused to syt-17 may be cytoplasmic, resulting in strong
fluorescence. Alternatively, these vesicles may have a higher pH
than synaptic vesicles and could correspond to nonacidified dense-
core vesicles or signaling endosomes (Cosker et al., 2008). pHluo-
rin–syt-17 fluorescence did increase at isolated puncta in response
to depolarization, suggesting that syt-17 is an integral vesicle pro-
tein, but at far fewer sites than other syt isoforms. These pHluorin
responses were characterized by fast, large increases in fluorescence
(Supplemental Figure S3), which remained elevated in the presence
of depolarizing solution (Figure 3D).
FIGURE 1: Synaptic vesicle–pHluorins and dense-core vesicle–pHluorins have distinct fluorescence characteristics.
(A) Axonal regions of a synaptophysin-pHluorin–transfected neuron before and after depolarization (left). Sample
regions that change fluorescence during depolarization are indicated with hatched white circles. Middle and right,
sample and average fluorescence, respectively, during depolarization. Arrow indicates addition of 45 mM KCl to
depolarize neurons. (B) Axon of a pHluorin-syt-4–transfected neuron before, during, and after depolarization, with sites
of fluorescence change indicated (left). Sample (middle) and average (right) fluorescence traces of puncta before, during,
and after depolarization. (C) Dendrites of a pHluorin-syt-4 transfected–neuron before, during, and after depolarization.
(D) Sample (left) and average (right) traces of small, fast dendritic events. (E) Sample (left) and average (right) traces of
large, slow dendritic events (n = 27–57 ROIs from three cultures; error bars indicate SEM). Scale bars, 5 μm.
02550 75 100125 150
syt 4 axons
syt 4 dendrites
0 s 50 s 120 s
0 s 40 s 120 s
1718 | C. Dean et al. Molecular Biology of the Cell
sibility that this isoform may regulate dense-core vesicle release in
Syt-12 also exhibited responses in both axons and dendrites. In
axons pHluorin–syt-12 responded to depolarization with fluores-
cence increases, which remained elevated in depolarizing condi-
tions (Figure 8A). In dendrites (Figure 8B), puncta were often visible
prior to stimulation; they increased in fluorescence and then de-
cayed back to baseline within 2 min, with kinetics exhibiting little
variation between different puncta.
We also compared the plasma membrane versus intracellular
(vesicular) distribution of each syt isoform. Bath application of
pH 5.5 solution acts to quench pHluorin fluorescence on the ex-
tracellular surface of the plasma membrane, whereas addition of
NH4Cl acts to dequench internal pHluorins if they are localized in
acidic compartments. These treatments can therefore be used to
assess the relative amounts of surface versus internal pHluorin in
neurons expressing different pHluorin-tagged syt isoforms. We
selected sites that had undergone exo/endocytosis following de-
polarization and then successively perfused pH 5.5 and NH4Cl
solution and measured the fluorescence at these sites (Figure
9A); the percentage of surface versus internal pHluorin was plot-
ted for each syt (Figure 9B, upper graph). Of interest, axonally
recycling syts had higher amounts of surface expression com-
pared with dendritic syts or to syts that recycled in both compart-
ments. These findings indicate that, surprisingly, the recycling of
syts in specific subcellular compartments can be predicted by
the ratio of surface-to-internal protein levels. As mentioned, both
syt-7 and syt-3 exhibited a significant amount of internal fluores-
cence and thus are likely not exclusively plasma membrane local-
ized, as previously suggested (Sudhof, 2002). We note that the
surface fraction of syt-1 ranges between ∼25 and 33% following
field stimulation (Fernandez-Alfonso et al., 2006; Wienisch and
Klingauf, 2006). The slightly higher surface fraction detected
in the present study (43.7 ± 15.3%) may be a result of prior
Figure S4), and the depolarization-induced fluorescence decay was
abolished in the presence of bafilomycin to block reacidification
(Figure 4C, inset). Syt-11–containing vesicles also recycled exclu-
sively in dendrites but with very different kinetics compared with syt-
3. Syt-11 vesicles were often visible prior to depolarization and ex-
hibited small increases in fluorescence (Figure 4D and Supplemental
Figure S3A), which decayed back to baseline within 60–90 s.
Three syt isoforms in addition to syt-4 exhibited pHluorin re-
sponses upon depolarization, in both axons and dendrites. Syt-6
was predominantly axonal (Figure 5A), with a relatively large propor-
tion of pHluorin–syt-6 on the plasma membrane under resting con-
ditions. Large increases in pHluorin–syt-6 fluorescence in axons
were observed in response to depolarization (Supplemental Figure
S3A), the majority of which remained elevated for at least 2–3 min
following addition of high-potassium solution. Syt-6 was also pres-
ent in a punctate pattern in dendrites (Figure 5B), and subsets of
these puncta initially decreased and then increased in fluorescence
following depolarization but with a magnitude approximately one-
fourth the size of axonal events (Supplemental Figure S3A).
Although syt-9 mRNA is not present at high levels in hippocam-
pus (Mittelsteadt et al., 2009), our Western blot analysis of wild-type
and syt-9–knockout mice indicates that syt-9 protein is present in
this brain region (Supplemental Figure S5). Syt-9 exhibited several
pHluorin characteristics that were similar to those of syt-4; vesicles
were visible prior to stimulation, trafficked rapidly in both axons
(Figure 6A) and dendrites (Figure 6B), and increased in fluorescence
in both compartments in response to depolarization (Figure 6, A
and B). We note that syt-9 was recently postulated to localize to
synaptic vesicles, where it was reported to rescue fast synaptic trans-
mission in syt-1 knockouts in cortical neurons (Xu et al., 2007). How-
ever, we found that the same construct used in that study was poorly
localized to synaptic sites in comparison to syt-1 (Figure 7, A and B)
and failed to rescue fast synaptic transmission in hippocampal neu-
rons from syt-1–knockout mice (Figure 7, C–H). This raises the pos-
FIGURE 2: Syt-1 and syt-2 are the only isoforms that exhibit synaptic vesicle–like pHluorin responses. (A) Axonal regions
of a pHluorin-syt-1–transfected neuron (left), with sample (middle) and average (right) fluorescence traces during
depolarization. (B) Axonal regions of a pHluorin-syt-2–transfected neuron (left), sample (middle), and average (right)
fluorescence traces during depolarization (n = 27–30 ROIs from three cultures; error bars indicate SEM). Scale bars, 5 μm.
0 s 40 s 120 s
0 s 40 s 120 s
syt 1 axons
syt 2 axons
Volume 23 May 1, 2012 Axonal and dendritic synaptotagmin isoforms | 1719
for each isoform. Total fluorescence did not correlate with nonacidi-
fied internal fluorescence, suggesting that expression levels do not
affect the relative amount of internal versus external syt. In addition,
expression levels did not influence the recycling of syt isoforms in
axons versus dendrites (Figure 9C).
As mentioned, in many cases syt isoform–specific antibodies are
not yet available, and only antibodies against syt-1, 4, and 9 (in the
current study) have been validated using knockouts. However, we
tested the localization of a subset of isoforms using anti-syt-1, 3, 4,
5, 9, and 12 antibodies in hippocampal cultures colabeled with
MAP2 to mark dendrites (Figure 10A) or with synaptophysin
to mark synaptic sites (Figure 10B). We found that syt-1 and syt-3
stimulation with high-potassium solution, which could promote a
larger amount of surface expression of syt-1.
In addition, we assessed the amount of syt present in nonacidi-
fied internal compartments (fluorescence above background in the
presence of pH 5.5 solution) for each isoform and found that syts
with synaptic vesicle–like recycling kinetics exhibited higher fluores-
cence levels in nonacidic compartments than axonal syts localized
to other vesicle subtypes (Figure 9B, lower graph). This analysis re-
lies on comparison of fluorescence intensities between different
syts, which might reflect differences in expression levels. To address
this, we calculated the average total fluorescence (Figure 9C) in the
presence of NH4Cl, which should reflect the total expression level,
FIGURE 3: Syt-5, 7, and 17 undergo exocytosis exclusively in axons, with distinct kinetics. (A) pHluorin-syt-10–
transfected neurons before and after depolarization (left). Sample (middle) and average (right) fluorescence traces
during depolarization. Inset shows average fluorescence traces during depolarization in the presence of bafilomycin to
block reacidification of vesicles. (B) pHluorin–syt-5 in axons before and after depolarization, and sample and average
fluorescence traces. (C) pHluorin–syt-7 in axons before and after depolarization, with sample and average fluorescence
traces. (D) pHluorin–syt-17 vesicles in axons before and after depolarization, with sample and average traces of
fluorescence at exocytotic sites (n = 30–60 ROIs from three cultures; error bars indicate SEM). Scale bars, 5 μm.
syt 5 axons
0 s 70 s
syt 7 axons
syt 17 axons
syt 10 axons
0 s 70 s
0 s 70 s
025 50 75100
1720 | C. Dean et al. Molecular Biology of the Cell
ized to distinct secretory organelles in both axons and dendrites and
thus have diverged to regulate exocytosis of a wide variety of vesicle
subtypes in neurons.
The best-characterized synaptotagmin isoform is syt-1, which
is present on synaptic vesicles and is essential for fast synaptic
exhibited the greatest colocalization with synaptophysin at synapses,
syt-9 and syt-12 showed an intermediate value, and syt-4 and syt-5
exhibited the least colocalization. In terms of localization to axons or
dendrites, syt-3 and syt-4 exhibited the highest colocalization with
MAP2 signal in dendrites, syt-5, syt-9, and syt-12 showed intermedi-
ate colocalization, and syt-1 overlapped the least with MAP2. These
findings are largely in agreement with the pHluorin-syt results, in
which syt-1 is present on synaptic vesicles marked with synapto-
physin, syt-3 is predominantly localized to dendrites, and syt-4, 5, 9,
and 12 are present at some but not all synaptic sites and have vary-
ing degrees of localization to dendrites in addition to axons.
In summary, using pHluorin-tagged syt isoform reporters, we as-
sayed the site and kinetics of recycling in response to depolarization
of vesicles harboring each isoform to determine whether they recy-
cle in axons and/or dendrites and to discern whether they are tar-
geted to synaptic vesicles. We found that subsets of syts are differ-
entially targeted to distinct vesicle subtypes in axons, dendrites,
or both compartments (Table 1). Surprisingly, only two isoforms—
syt-1 and syt-2—had pHluorin fluorescence responses characteristic
of synaptic vesicles. The majority of syt isoforms appear to be local-
FIGURE 4: Syt-3 and 11 recycle in dendrites, where they exhibit unique pHluorin responses upon depolarization.
(A) Cell body and proximal dendrites of pHluorin-syt-3–transfected neuron before and after depolarization. (B) Distal
dendrites of the same cell. Fluorescent puncta are visible prior to depolarization, which decrease in fluorescence upon
depolarization. (C) Representative (left) and average (right) traces of puncta fluorescence decay during depolarization.
Inset shows average fluorescence traces during depolarization in the presence of bafilomycin to block reacidification.
(D) Dendrite of a pHluorin-syt-11–transfected neuron prior to, during, and following depolarization (left). Representative
(middle) and average (right) fluorescence traces during depolarization (n = 30 ROIs from three cultures; error bars
indicate SEM). Scale bars, 5 μm.
0255075 100125 150
syt 3 dendrites
syt 11 dendrites
0 s 60 s
AxonsDendritesBothNo responseNot in brain
SV, synaptic vesicle.
TABLE 1: Summary of localization of syt isoforms based on pHluorin-
Volume 23 May 1, 2012 Axonal and dendritic synaptotagmin isoforms | 1721
biochemical properties; different isoforms have distinct calcium
sensitivities and kinetics of interactions with phospholipids (Bhalla
et al., 2005; Hui et al., 2005). In addition, the distribution of syt
isoform mRNAs has been well described (Mittelsteadt et al., 2009).
However, information regarding the subcellular localization and
function of syt proteins has lagged behind, largely due to the in-
herent difficulties in generating isoform-specific antibodies.
transmission (DiAntonio and Schwarz, 1994; Geppert et al., 1994;
Littleton et al., 1994). The subsequent discoveries of additional
isoforms led to speculation that different syts may regulate distinct
aspects of synaptic vesicle function or be localized to distinct or-
ganelles to affect various intracellular trafficking events (Schiavo
et al., 1998; Marqueze et al., 2000; Sudhof, 2002; Craxton, 2004).
The syt isoforms have been well characterized in terms of their
FIGURE 5: Syt-6 undergoes recycling in both axons and dendrites. (A) pHluorin–syt-6 in axons before and after
depolarization (left). Representative (middle) and average (right) traces of fluorescence during depolarization.
(B) pHluorin–syt-6 in dendrites (left), and sample and average fluorescence responses to depolarization (n = 27–30 ROIs
from three cultures; error bars indicate SEM). Scale bars, 5 μm.
syt 6 axons
syt 6 dendrites
0 25 5075
0 2550 75100
FIGURE 6: Syt-9–harboring vesicles recycle in both axons and dendrites. (A) pHluorin–syt-9 vesicles in axons before and
after depolarization (left). Note that some puncta are visible prior to depolarization; these puncta traffic rapidly in
anterograde and retrograde directions. (B) pHluorin–syt-9 in dendrites before and after depolarization (left), with
sample and average fluorescence traces in response to depolarization (n = 30 ROIs from three cultures; error bars
indicate SEM). Scale bars, 5 μm.
syt 9 axons
syt 9 dendrites
1722 | C. Dean et al. Molecular Biology of the Cell
A number of fusion events play impor-
tant roles in either mediating or modulating
synaptic transmission, including the exocy-
tosis of synaptic vesicles (Heuser and Reese,
1973), recycling of adhesion molecules (Tai
et al., 2007), ion channels (Misonou and
Trimmer, 2004) and postsynaptic receptors
(Shi et al., 1999; Linnarsson et al., 2000; Park
et al., 2004), and release of neuropeptides
(Baraban and Tallent, 2004) and neurotro-
phins (Figurov et al., 1996). Specific synap-
totagmins with distinct biochemical proper-
ties could differentially regulate these
exocytotic and endocytotic events in re-
sponse to calcium. Syt-3, for example, un-
dergoes endocytosis exclusively in dendrites
in response to depolarization, with kinetics
similar to that of pHluorin-tagged postsyn-
aptic receptors retrieved from the plasma
membrane in response to N-methyl-d-aspar-
tic acid treatment (Lin and Huganir, 2007;
Gong and De Camilli, 2008). These pHluo-
rin-tagged postsynaptic receptors initially
endocytose and then exhibit a gradual re-
covery to the plasma membrane surface
over the course of ∼10 min. Syt-3 may there-
fore act to down-regulate surface expres-
sion of postsynaptic receptors to modulate
Syt-6, which was also present in a punc-
tate pattern in dendrites, initially decreased
and then increased in fluorescence follow-
ing depolarization. This could correspond to
the initial internalization of a receptor in re-
sponse to stimulation, followed by reinser-
tion into the plasma membrane.
In addition, a number of neurotrophins
and neuropeptides are required for specific
aspects of synaptic function (Baraban and
Tallent, 2004; Figurov et al., 1996). The de-
polarization-dependent exocytosis of BDNF,
which is essential for the plasticity of neu-
ronal circuits in the hippocampus (Korte
et al., 1995), has recently been found to oc-
cur through multiple modes (Dean et al.,
2009; Matsuda et al., 2009) regulated by
syt-4 (Dean et al., 2009). Both the amount and duration of BDNF
release affect synaptic function differentially (Rutherford et al., 1998);
precise regulation of the release kinetics of various neuropeptides
and neurotrophins via distinct syt isoforms may therefore contribute
to the plasticity of circuits.
We found that pHluorin–syt-9 has characteristics similar to those
of pHluorin–syt-4, where vesicles are visible prior to stimulation, traf-
fic rapidly, and undergo recycling in both axons and dendrites. How-
ever, pHluorin–syt-9 vesicles fuse coincident with depolarization in
axons, whereas there was often a delay prior to fusion of syt-4 vesi-
cles. In addition, pHluorin–syt-9 dendritic fluorescence responses
remained elevated following stimulation, whereas fast pHluorin–
syt-4 dendritic responses decayed to baseline within 20 s. These ob-
servations are consistent with the ability of syt-9 to promote fusion
(Bhalla et al., 2008), possibly of neurotrophin- or neuropeptide-con-
taining vesicles, whereas syt-4 inhibits their fusion (Dean et al., 2009).
Our findings from a “screen” of pHluorin-syt dynamics in neurons
shed new light on the localization and function of the syt isoforms
expressed in brain. This screen revealed differential localization of
syts to distinct vesicle subtypes in axons versus dendrites, in which
specific isoforms recycle only in axons, only in dendrites, or in both
compartments. In addition, we found that the majority of syt isoforms
are localized to vesicles with recycling characteristics different from
those of synaptic vesicles. Five syts (syt-1, 2, 5, 7, and 17) undergo
exocytosis exclusively in axons, but only syt-1 and 2 exhibited pHluo-
rin responses indicating a localization to synaptic vesicles, whereas
syt-5, 7, and 17 each displayed unique recycling kinetics, suggesting
differential localization to distinct vesicle subtypes. Two syts (syt-3
and 11) recycled exclusively in dendrites, with distinct kinetics, and
four syts (syt-4, 6, 9, and 12) were observed to undergo exo/endocy-
tosis in both axons and dendrites. Different syt isoforms may have
thus evolved to regulate fusion of distinct vesicle subtypes.
FIGURE 7: Syt-9 fails to rescue fast synaptic transmission in syt-1–knockout (KO) hippocampal
neurons. (A) Hippocampal neurons infected with myc–syt-9 or myc–syt-1 lentiviral constructs
were immunostained with anti-myc and anti-synapsin antibodies to mark synaptic vesicles. Scale
bar, 10 μm. (B) Syt-1 colocalizes with the synaptic vesicle marker synapsin (0.48 ± 0.03; 850
puncta from four samples), whereas significantly less syt-9 colocalizes with synaptic vesicles
(0.17 ± 0.02; 780 puncta from four samples), p < 0.001. (C) Evoked EPSCs from neurons in
dissociated cultures were recorded as previously reported (Liu et al., 2009). Syt-1 expression
rescued fast synaptic transmission (arrow) in syt-1 KOs, but syt-9 failed to rescue. (D) Statistical
analysis of EPSC amplitude (in pA; syt-1 KO, 150 ± 25; syt-1, 866 ± 179; syt-9, 170 ± 33) and
charge (E) calculated over 1 s (in nC; syt-1KO, 22 ± 6; syt-1, 44 ± 9; syt-9, 25 ± 6). The number of
neurons in each group (n) is indicated. (F) Normalized cumulative charge transfer further
indicates that expression of syt-9 does not alter release kinetics in syt-1–knockout neurons.
(G) Representative EPSC traces in response to high-frequency stimulation (30 action potentials
at 20 Hz). No fast component of release was observed in syt-9–expressing syt-1–knockout
neurons, whereas syt-1 fully rescued fast release in knockout neurons. (H) The peak amplitude of
each EPSC was normalized to the first EPSC and plotted vs. time to assess short-term plasticity.
There is no statistically significant difference between syt-9–rescue and syt-1–knockout neurons.
All data shown represent mean ± SEM. *p < 0.05, ***p < 0.001.
n 5 . 0
n 4 . 0
syt 1 KO
syt 1 KO
syt 1 KO
1 t y
1 t y
9 t y
1 t y
1 t y
9 t y
syt 1 KO
g r a
n , s
1. .00.80.6 0.4
Nor. Cumul. Charge (100%)
1 t y
9 t y
Volume 23 May 1, 2012 Axonal and dendritic synaptotagmin isoforms | 1723
content and were estimated to be present at five copies per vesicle
in total. These syts may be localized to other vesicle subtypes, per-
haps in greater abundance, where they could indirectly affect synap-
tic transmission, as in the case of syt-4 (Dean et al., 2009).
PHluorin-syt-7–harboring vesicles underwent exocytosis exclu-
sively in axons but also with kinetics distinct from that of synaptic
vesicles. Syt-7 was previously localized to lysosomes in nonneuronal
cells, where it regulates calcium-triggered exocytosis and plasma
membrane repair (Reddy et al., 2001; Roy et al., 2004), and to
dense-core vesicles in PC12 cells (Wang et al., 2005). However, a
role of syt-7 in affecting neuronal function in brain has not been
described; syt-7 knockouts were reported to exhibit unaltered neu-
rotransmitter release and short-term plasticity (Maximov et al.,
For several syts (including syt-4, 9, 11, 12, and 17) highly mobile
fluorescent (nonacidifed) vesicles were visible in nondepolarizing
conditions and remained fluorescent in the presence of pH 5.5 solu-
tion (to quench surface fluorescence), suggesting localization to
nonacidified internal compartments. These compartments could
correspond to recycling or signaling endosomes, which can range in
pH from 5.5 to 6.5 (Demaurex, 2002; Ibanez, 2007; Cosker et al.,
The fraction of each syt in the plasma membrane also differs, and,
of interest, the localization of isoforms to axons versus dendrites can
be predicted based on how much is in the plasma membrane. In ad-
dition, the fraction of each syt in acidic versus nonacidic internal or-
ganelles differs, further supporting the idea that different syts are lo-
calized to distinct organelles. Finally, we found that each syt isoform
exhibits different kinetics of pHluorin fluorescence response to depo-
larization. Surprisingly, only two isoforms—syt-1 and 2—were local-
ized to synaptic vesicles based on their pHluorin recycling kinetics,
whereas many additional syt isoforms exhibited dense-core
vesicle–like pHluorin responses. These syts may be localized to dis-
tinct neuropeptide or neurotrophin vesicles to regulate their release.
We cannot exclude the possibility that the additional syt isoforms
Syt-4 is one of four syt isoforms, in addition to syt-8, 11, and 12, that
do not appear to sense Ca2+ and thus may act to inhibit fusion in
neurons (Bhalla et al., 2008), thereby conferring exocytosis with ad-
ditional regulation. The pHluorin-syt-9 results in combination with
the inability of syt-9 to substitute for syt-1 on synaptic vesicles to
rescue fast transmission in syt-1 knockouts (Figure 7) raises the pos-
sibility that this isoform may regulate dense-core vesicle release. We
note that syt-9 was recently postulated to localize to synaptic vesi-
cles, in which it was reported to rescue fast synaptic transmission in
syt-1 knockouts (Xu et al., 2007). However, we found that the same
construct used in that study was poorly localized to synaptic sites in
comparison to syt-1 (Figure 7, A and B) and failed to rescue fast syn-
aptic transmission in syt-1 knockouts (Figure 7, C–H). A possible ex-
planation for this discrepancy is that syt-9 may be localized to distinct
vesicle subtypes in hippocampus compared with the striatum, since
Xu et al. focused on inhibitory postsynaptic currents recorded from
striatal neurons and our study tested the effects of syt-9 on excitatory
postsynaptic currents (EPSCs) from hippocampal neurons. Given the
finding that syt-9 is present in hippocampus and syt-1 knockouts lack
fast synchronous synaptic transmission in hippocampus (Geppert
et al., 1994; Liu et al., 2009), we can exclude syt-9 as a fast synchro-
nous calcium sensor for synaptic transmission in hippocampus.
The observation that pHluorin–syt-12 vesicle recycling occurs in
both axons and dendrites with kinetics distinct from those of synap-
tophysin-pHluorin and pHluorin-syt-1 also contrasts with a recent
study in which syt-12 was localized to synaptic vesicles, where it in-
creased spontaneous neurotransmitter release without affecting
evoked release (Maximov et al., 2007). It is possible, however, that
syt-12 is localized, perhaps to a greater extent, to other vesicle sub-
types. Mass spectrometry of purified synaptic vesicles identified syt-
1, 2, 9, 12, and 17 on synaptic vesicles (Takamori et al., 2006), but
syt-9, 12, and 17 were detected at significantly lower levels than
syt-1. Syt-1 comprises 6.9% of the total synaptic vesicle protein at
15 copies per vesicle, whereas the remaining syt-9, 12, and 17 com-
bined made up only 2.3% of the total synaptic vesicle protein
FIGURE 8: pHluorin-syt–12 harboring vesicles recycle in both axons and dendrites. (A) Axons of a pHluorin-syt-12–
transfected neuron before and after depolarization (left), and sample and average fluorescence traces of pHluorin-syt-12
responses. (B) Dendritic regions of pHluorin-syt-12–transfected neurons before and 50 and 120 s after depolarization
(left). Representative (middle) and average (right) fluorescence traces in response to depolarization (n = 30 ROIs from
three cultures; error bars indicate SEM). Scale bars, 5 μm.
0 s 50 s
0 s 50 s 120 s
syt 12 axons
syt 12 dendrites
1724 | C. Dean et al. Molecular Biology of the Cell
regulated by the syts. The localization of specific syts to distinct
axonal and dendritic vesicle subtypes suggests that they may reg-
ulate a broader range of exocytotic events than previously
MATERIALS AND METHODS
Hippocampal neuron cultures
Rat hippocampi were isolated from E18-19 rats as described previ-
ously (Banker and Cowan, 1977). Hippocampi were treated with
trypsin for 20 min at 37°C, triturated to dissociate cells, plated at
25,000–50,000 cells/cm2 on polylysine-coated coverslips (Carolina
Biological Supply, Burlington, NC), and cultured in Neurobasal sup-
plemented with 2% B-27 and 2 mM Glutamax (Life Technologies,
Invitrogen, Carlsbad, CA).
All procedures involving animals were performed in accordance
with the guidelines of the National Institutes of Health, as approved
may be localized to subsets of synaptic vesicles with pHluorin fluores-
cence kinetics distinct from that of vesicles containing synaptophysin,
syt-1, and syt-2. However, this seems unlikely, given that synaptic
vesicle populations lacking synaptophysin have not been reported
and that individual synaptic vesicles in a mixed population all contain
synaptophysin at similar copy numbers (Mutch et al., 2011).
Analysis of chimeras composed of different domains of syt iso-
forms should make it possible to determine the structural elements
that underlie differential targeting of this family of proteins to
distinct organelles. Moreover, the finding that some isoforms are
targeted to organelles that recycle in axons or in dendrites or are
present in both classes of neurites opens the door to study aspects
of polarized transport in neurons. Future studies will focus on how
this polarity is achieved. In addition, a future challenge is to deter-
mine which cargoes are contained within these novel syt isoform–
harboring vesicles and how the release of these cargoes might be
FIGURE 9: Surface vs. internal pHluorin-syt localization. (A) Fluorescence of the indicated pHluorin-syt isoforms during
perfusion of pH 5.5 solution to quench surface fluorescence and NH4Cl solution to alkalinize all internal acidic
compartments and dequench internal pHluorin fluorescence. (B) Quantitation of percentage of surface vs. internal
pHluorin-syt (top graph) and of nonacidified internal fluorescence compared between each isoform (expressed as
percentage of total fluorescence). Isoforms are grouped in order of recycling exclusively in axons, in both axons and
dendrites, or exclusively to dendrites. (C) Quantitation of total fluorescence for each syt isoform (total fluorescence
above background in the presence of NH4Cl).
syt 1 axons
syt 2 axons
syt 3 dendrites
syt 5 axons
syt 6 axons
syt 7 axons
syt 9 axons/
syt 10 axons
syt 11 dendrites
syt 12 axons/
syt 17 axons
(% total fluorescence)
total fluorescence (au)
Volume 23 May 1, 2012 Axonal and dendritic synaptotagmin isoforms | 1725
Seville, Seville, Spain). ImageJ software
(National Institutes of Health, Bethesda,
MD) was used to analyze the colocalization
of myc-tagged syt-1 and syt-9 with anti-syn-
apsin (Synaptic Systems).
acquisition, quantitation, and statistical
Cells were fixed with 4% paraformaldehyde,
permeabilized with 0.1–0.2% Triton X-100
and 10% goat serum or 1% bovine serum al-
bumin (BSA), blocked with 0.1% Triton X-100,
10% goat serum, or 10% BSA, and immunos-
tained at room temp for 1–2 h or overnight at
4°C. Images were acquired on an Olympus
FV1000 upright confocal microscope (Olym-
pus, Center Valley, PA) with a 60×, 1.10 nu-
merical aperture water immersion lens and a
Zeiss Axiovert epifluorescence microscope
with a 100× oil objective (Carl Zeiss, Jena,
Germany). For quantitation of percentage
colocalization, images were acquired with
identical laser and gain settings and imported
into MetaMorph (Molecular Devices, Sunny-
vale, CA). Channels were thresholded sepa-
rately to include all recognizable puncta. Per-
centage colocalization was determined as
percentage of the total thresholded area of
one channel (syt signal) that overlapped with
the thresholded area of the corresponding
channel (synaptophysin or MAP-2 signal).
Transfection of hippocampal neurons
Neurons growing on 12-mm coverslips in
24-well plates were transfected with Lipo-
fectamine (Invitrogen) or calcium phosphate. For Lipofectamine
transfection (at 5–6 d in vitro [DIV]), medium was removed, saved,
and replaced with 500 μl of fresh medium. One microliter of Lipo-
fectamine 2000 in 50 μl of Neurobasal medium and 0.5 μg of DNA
in 50 μl of Neurobasal medium were incubated separately for 5 min
and then mixed and incubated for 30 min at room temperature. This
mixture was added to the well of neurons, incubated for 4 h at 37°C
and 5% CO2, and then removed and replaced with half saved me-
dium and half fresh medium.
Neurons were transfected using calcium phosphate at 3–6 DIV as
described previously (Dresbach et al., 2003). Prior to transfection,
medium was removed, saved, and replaced with 500 μl of 37°C
Optimem (Life Technologies, Carlsbad, CA) and incubated for
30–60 min. A 105-μl amount of transfection buffer (274 mM NaCl,
10 mM KCl, 1.4 mM Na2HPO4, 15 mM glucose, 42 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH
7.06) was added dropwise to a solution containing 7 μg of DNA and
250 mM CaCl2, with gentle vortexing. This mixture was incubated
for 20 min at room temperature, 30 μl was added per well, and the
neurons were incubated for 60–90 min. This medium was then re-
moved, cells were washed three times in 37°C Neurobasal medium,
and saved medium was added back to the transfected cells.
For time-lapse experiments, neurons were transferred to a live-
imaging chamber (Warner Instruments, Hamden, CT) containing
by the Animal Care and Use Committee of the University of Wiscon-
Antibodies and mammalian expression constructs
Antibodies used were as follows: polyclonal syt-1 (cat. #105102),
syt-3 raised against amino acids 86–169 of human syt-3, syt-4 (cat.
#105043), syt-5 raised against amino acids 74–147, syt-9 (cat.
#105053), synaptophysin (Synaptic Systems, Göttingen, Germany),
syt-12, GFP (Abcam, Cambridge, MA), MAP2 (Millipore, Billerica,
MA; Synaptic Systems), and Alexa 405, Alexa 488, Alexa 546, and
Alexa 647 secondaries (Invitrogen). pHluorin-syt-1 was provided by
T. Ryan (Weill Medical College of Cornell University, New York, NY).
Additional pHluorin-syt constructs were generated by replacing the
syt-1 in pHluorin-syt-1 by PCR-amplified syts. Syt-2, 5–8, 10, and 11
were provided by M. Fukuda (Tohoku University, Sendai, Japan).
Syt-3 was provided by S. Seino (Kobe University, Kobe, Japan). Syt-4
was provided by B. Hilbush (Roche Research Center, Nutley, NJ).
Syt-9 was provided by M. Birnbaum (University of Pennsylvania,
Philadelphia, PA). Syt-17 was provided by M. Craxton (MRC Labora-
tory, Cambridge, United Kingdom). A myc-tagged syt-9 lentiviral
construct based on the pFUGW vector with the human polyubiq-
uitin promoter was provided by T. Sudhof (Stanford University, Palo
Alto, CA). Myc–syt-9 and myc–syt-1 were constructed by tagging
syt-9 or syt-1 with a myc epitope and signal sequence at the N-ter-
minus and subcloning into a double synapsin promoter lentiviral
expression plasmid (provided by F. Gomez-Scholl, University of
FIGURE 10: Localization of endogenous syts in hippocampal neurons. (A) Coimmunostaining of
hippocampal neurons with anti-syt-1, 3, 4, 5, 9, and 12 (green) and anti-MAP2 (red) to mark
dendrites. Percentage overlap of syt signal with MAP2 signal was as follows: 13 ± 2 (syt-1), 47 ±
16 (syt-3), 43 ± 2 (syt-4), 39 ± 8 (syt-5), 23 ± 4 (syt-9), and 24 ± 9 (syt-12). (B) Immunostains of
anti-syt-1, 3, 4, 5, 9, and 12 with anti-synaptophysin to mark synaptic sites. Percentage overlap
of syt signal with synaptophysin signal was as follows: 90 ± 4 (syt-1), 78 ± 1 (syt-3), 13 ± 3 (syt-4),
12 ± 1 (syt-5), 26 ± 5 (syt-9), and 54 ± 10 (syt-12). Scale bars, 10 μm.
syt / MAP2
syt-12 syt-9 syt-5 syt-4 syt-3 syt-1
syt isoform synaptophysin merge
syt-12 syt-9 syt-5 syt-4 syt-3 syt-1
1726 | C. Dean et al. Molecular Biology of the Cell
bathing saline solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2,
2 mM MgCl2, 5.5 mM glucose, 20 mM HEPES, pH 7.3). Transfected
cells (identified by faint GFP fluorescence in nondepolarizing condi-
tions) in which axons and dendrites were clearly discernible by
morpho logy were selected. Images were acquired at 1-s intervals
and 500-ms exposure times, with 484/20-nm excitation and 517/20-
nm emission filters, on a Nikon TE300 inverted microscope (Nikon,
Melville, NY) with a Roper Scientific Photometrics Cascade IIB EM-
CCD camera (Photometrics, Tucson, AZ) and Lambda DG-4 fast-
switching light source interfaced with MetaMorph software. A base-
line of 10 images was collected before addition of high-potassium
buffer (100 mM NaCl, 45 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 5.5
mM glucose, 20 mM HEPES, pH 7.3) to depolarize neurons. Axonal
or dendritic regions were selected in MetaMorph, and the fluores-
cence intensity was plotted versus time. Puncta that did not exhibit
any lateral movement during image acquisition and that exhibited a
change in fluorescence in response to KCl were chosen for analysis;
in the case of syt-10, which did not respond to depolarization, arbi-
trary regions of interest (ROIs) were chosen for comparison. Uni-
form, round, 1.5-μm-diameter ROIs were selected. For measure-
ment of the surface versus internal amount of each syt isoform at
exo/endocytotic sites, neurons were first depolarized to confirm
that recycling events could be detected within the field of view. Sub-
sequent sequential perfusion of pH 5.5, pH 7.3, and NH4Cl solu-
tions were then performed to determine surface and internal pHluo-
rin at these same regions, using an MPS-2 multichannel perfusion
system (World Precision Instruments, Sarasota, FL). For inhibition of
the vesicular H+ ATPase, neurons were incubated with 1 μM bafilo-
mycin for 2 min (Calbiochem, La Jolla, CA) prior to imaging
Whole-cell patch-clamp recordings were made from dissociated syt-
1–knockout hippocampal cultures (Geppert et al., 1994). All record-
ings were done 12–15 d after neurons were plated on coverslips.
The pipette solution consisted of 130 mM K-gluconate, 1 mM ethyl-
ene glycol tetraacetic acid, 5 mM Na-phosphocreatine, 2 mM
Mg-ATP, 0.3 mM Na-GTP, 10 mM HEPES, and 5 mM QX-314, pH 7.3
(290 mOsm). Neurons were continuously perfused with extracellular
solution consisting of 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10
mM glucose, 10 mM HEPES, pH 7.3 (300 mOsm), 50 μM d-2-amino-
5-phosphonopentanoate, 0.1 mM picrotoxin, and 5 mM Ca2+. All
drugs were from Sigma-Aldrich (St. Louis, MO). The stimulating bi-
polar electrode was filled with extracellular solution. Neurons were
voltage clamped at −70 mV with an EPC-10/2 amplifier (HEKA Elec-
tronics, Lambrecht/Pfalz, Germany). Only cells with series resistances
of <15 MΩ, with 70–80% of this resistance compensated, were ana-
lyzed. Currents were acquired using PATCHMASTER software
(HEKA), filtered at 2.9 kHz, and digitized at 10 kHz. Data were ana-
lyzed using Clampfit (Molecular Devices, Sunnyvale, CA), IGOR
(WaveMetrics, Portland, OR), and ImageJ software. All experiments
were carried out at room temperature.
For Western blots of syt-9, brain regions were isolated from adult
syt-9–knockout mice and wild-type littermates (Jackson Labora-
tory, Bar Harbor, ME; deposited by T. Sudhof) and homogenized
in 320 mM sucrose and 4 mM HEPES, pH 7.4, using a Teflon
glass homogenizer. Homogenates were centrifuged for 10 min at
1000 × g to pellet nuclei and insoluble debris. Supernatants were
collected, and the protein concentration was determined accord-
ing to a modified Lowry Peterson method that includes solubili-
We thank Sam Kwon for independent verification of pHluorin-syt
results. This work was supported by National Institutes of Health
Grant MH 61876 to E.R.C. and by Epilepsy Foundation, National
Institutes of Health National Research Service Award, and Sofja
Kovalevskaja Alexander von Humboldt and European Research
Council grants to C.D. E.R.C is an investigator of the Howard Hughes
zation and precipitation of proteins by trichloroacetic acid
(Peterson, 1977). Equal amounts of protein were loaded per lane
for comparison of brain regions between wild-type and syt-9–
knockout mice, resolved by SDS–PAGE, and analyzed by
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