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 sequence 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 results in vesicle reacidification and a corresponding decay in fluorescence. 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-magnification 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, retrospective immunostaining for GFP to mark pHluorin-syt and MAP-2 to mark dendrites was used. Of interest, in these immunostaining experiments, 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 constructs, and transfected cells, which could be detected by faint fluorescence 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 vesicles (Hartmann et al., 2001
; Kolarow et al., 2007
; Dean et al., 2009
Depolarization caused a rapid fluorescence increase at presynaptic boutons in axons of syp-pHluorin–transfected neurons, with no significant fluorescence or change in fluorescence detected in dendrites (). 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 in axons in response to depolarization and then decreased, but with slower kinetics than for syp-pHluorin (). 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 (), where they exhibited either a small, fast rise and decay () or a large, fast increase in fluorescence, which remained elevated in the presence of depolarizing solution (Dean et al., 2009
; ). These data confirm that synaptic vesicle– and dense-core vesicle–localized proteins have spatially and temporally distinct pHluorin responses.
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 (more ...)
Of all the syt isoforms tested, only two—syt-1 and syt-2—exhibited pHluorin responses indicative of localization to synaptic vesicles. pHluorin–syt-1 increased in fluorescence immediately following depolarization and then decayed back to baseline with kinetics nearly identical to that of syp-pHluorin (). pHluorin–syt-2 also exhibited recycling characteristics indicating localization to synaptic vesicles (), 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
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. (more ...)
We found that three other syts—syt-5, 7, and 17—were also exclusively recycled in axons (). However, the pHluorin characteristics 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 () in either the absence or presence of bafilomycin to block the vacuolar ATPase (, inset). The majority of pHluorin–syt-10 was localized to the plasma membrane in nondepolarizing conditions in hippocampal 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 conditions in the majority of hippocampal neurons and specifically invoked to recycle in subsets of neurons under conditions of high activity.
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 (more ...)
pHluorin–syt-5 was also visible on the plasma membrane, prior to depolarization, exclusively in axons (), and this isoform exhibited increases in fluorescence at select sites, coincident with depolarization. These fluorescence responses were large in comparison with the majority of syt isoforms, with a slow rise time (Supplemental 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 exclusively in axons (), which were of smaller magnitude than for any other axonal syt isoform (Supplemental Figure S3A) and remained elevated following depolarization for at least several minutes. 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 axonally localized isoforms. Diffusely distributed plasma membrane fluorescence was not observed, but rapidly trafficking vesicles were clearly visible in nondepolarizing conditions ( and Supplemental Movie S1). Syt-17 is predicted to be membrane associated but to lack a transmembrane domain (Craxton, 2010
). Thus the 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
). pHluorin–syt-17 fluorescence did increase at isolated puncta in response to depolarization, suggesting that syt-17 is an integral vesicle protein, 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 ().
Two syt isoforms exhibited pHluorin responses upon depolarization 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 punctate pattern resembling postsynaptic sites in both proximal () and distal () dendrites. Following depolarization, the fluorescence of these puncta decayed exponentially (). This fluorescence decay corresponded to endocytosis, since perfusion with NH4Cl to neutralize all internal acidic compartments resulted in recovery of fluorescence at these sites (Supplemental Figure S4), and the depolarization-induced fluorescence decay was abolished in the presence of bafilomycin to block reacidification (, inset). Syt-11–containing vesicles also recycled exclusively in dendrites but with very different kinetics compared with syt-3. Syt-11 vesicles were often visible prior to depolarization and exhibited small increases in fluorescence ( and Supplemental Figure S3A), which decayed back to baseline within 60–90 s.
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. (more ...)
Three syt isoforms in addition to syt-4 exhibited pHluorin responses upon depolarization, in both axons and dendrites. Syt-6 was predominantly axonal (), with a relatively large proportion of pHluorin–syt-6 on the plasma membrane under resting conditions. 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 present in a punctate pattern in dendrites (), 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).
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 (more ...)
Although syt-9 mRNA is not present at high levels in hippocampus (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 () and dendrites (), and increased in fluorescence in both compartments in response to depolarization (). We note that syt-9 was recently postulated to localize to synaptic vesicles, where it was reported to rescue fast synaptic transmission in syt-1 knockouts in cortical neurons (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 () and failed to rescue fast synaptic transmission in hippocampal neurons from syt-1–knockout mice (). This raises the possibility that this isoform may regulate dense-core vesicle release in the hippocampus.
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 (more ...)
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 (more ...)
Syt-12 also exhibited responses in both axons and dendrites. In axons pHluorin–syt-12 responded to depolarization with fluorescence increases, which remained elevated in depolarizing conditions (). In dendrites (), puncta were often visible prior to stimulation; they increased in fluorescence and then decayed back to baseline within 2 min, with kinetics exhibiting little variation between different puncta.
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. (more ...)
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 extracellular surface of the plasma membrane, whereas addition of NH4
Cl 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 depolarization and then successively perfused pH 5.5 and NH4
Cl solution and measured the fluorescence at these sites (); the percentage of surface versus internal pHluorin was plotted for each syt (, upper graph). Of interest, axonally recycling syts had higher amounts of surface expression compared with dendritic syts or to syts that recycled in both compartments. 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 fluorescence and thus are likely not exclusively plasma membrane localized, 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 stimulation with high-potassium solution, which could promote a larger amount of surface expression of syt-1.
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 (more ...)
In addition, we assessed the amount of syt present in nonacidified 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 fluorescence levels in nonacidic compartments than axonal syts localized to other vesicle subtypes (, lower graph). This analysis relies on comparison of fluorescence intensities between different syts, which might reflect differences in expression levels. To address this, we calculated the average total fluorescence () in the presence of NH4Cl, which should reflect the total expression level, for each isoform. Total fluorescence did not correlate with nonacidified 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 ().
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 () or with synaptophysin to mark synaptic sites (). We found that syt-1 and syt-3 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 intermediate 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 synaptophysin, 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 varying degrees of localization to dendrites in addition to axons.
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 (more ...)
In summary, using pHluorin-tagged syt isoform reporters, we assayed the site and kinetics of recycling in response to depolarization of vesicles harboring each isoform to determine whether they recycle in axons and/or dendrites and to discern whether they are targeted to synaptic vesicles. We found that subsets of syts are differentially targeted to distinct vesicle subtypes in axons, dendrites, or both compartments (). 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 localized 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.
Summary of localization of syt isoforms based on pHluorin-syt characteristics.