Release of large synaptic DCV proteins is triggered by Ca 2+ -independent Rugose-localized complexin phosphorylation

Neuronal dense-core vesicles (DCVs) contain neuropeptides and much larger proteins such as the 70 kDa protease TPA, which contributes to long-term potentiation and synaptic growth. Rather than using full collapse exocytosis that is common in endocrine cells, DCVs at a native intact synapse, the Drosophila neuromuscular junction, release their contents via fusion pores formed by kiss and run exocytosis. Here fluorogen activating protein (FAP) imaging reveals the limited permeability range of synaptic DCV fusion pores and then shows that this constraint is circumvented by cAMP-induced extra fusions with dilating pores that result in DCV emptying. These Ca 2+ independent full fusions require PKA-R2, a PKA phosphorylation site on the fusion clamp protein complexin and the acute presynaptic function of Rugose/Neurobeachin, a PKA-R2 anchor implicated in learning and autism. Therefore, localized Ca 2+ -independent signaling triggers the opening of dilating fusion pores to release large DCV cargo proteins that cannot pass through fusion pores that normally dominate Ca 2+ -dependent synaptic protein release.


INTRODUCTION
Neuronal dense-core vesicles (DCVs) contain a wide variety of secretory protein cargos including neuropeptides (with molecular weights of 0.6-30 kDa in Drosophila) and proteases that function in synaptic plasticity and growth (e.g., 70 kDa tissue plasminogen activator) (Huang et al., 1996;Baranes et al., 1998). Early studies of secretion from endocrine cells emphasized Ca 2+ -induced DCV emptying by full collapse exocytosis that follows fusion pore dilation. However, studies of spontaneous and activity-evoked release by DCVs in the intact Drosophila neuromuscular junction (NMJ) with two different imaging approaches failed to detect DCV emptying and instead produced data consistent with release via fusion pores formed by kiss and run exocytosis (Wong et al., 2015;Bulgari et al., 2019). Partial release by kiss and run exocytosis of presynaptic DCVs is conducive with the cell biology of neurons: because these organelles are replaced by axonal transport that can take days, kiss and run exocytosis may prevent rapid depletion of stores at distal sites of release that are slow to refill. However, it is not known whether presynaptic fusion pores allow for release of large DCV cargos.
Imaging permeation through DCV fusion pores is difficult in native synapses that contain DCVs and small synaptic vesicles (SSVs) because fluid phase fluorophores used with endocrine cells (e.g., Takahashi et al., 2002) cannot distinguish between the two vesicle types and cutting-edge superresolution microscopy methods that resolve fusion pore dilation rely on in vitro preparations (e.g., Anantharam et al., 2011).
Likewise, electrical and electrochemical fusion pore measurements (Sharma and Lindau, 2018) are not well suited to typical native cotransmitting synaptic boutons.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 4 However, synaptic DCV fusion pores were detected recently using membraneimpermeant malachite green (MG)-based fluorogens and a fluorogen activating protein (FAP) targeted to the DCV lumen so that, upon opening of a fusion pore, fluorogens pass through the fusion pore into the DCV to bind the FAP with picomolar affinity and generate far-red fluorescence (Bulgari et al., 2019). Because previously used fluorogens varied in shape and constituent moieties, and their hydrodynamic sizes were not characterized (Bulgari et al., 2019), the permeability of presynaptic fusion pores was not studied systematically. Nevertheless, an experimental approach for studying synaptic fusion pore permeability was established.
Here a series of single chain PEGylated membrane impermeant MG fluorogens and a FAP inserted into the neuropeptide Drosophila insulin-like peptide 2 (i.e., Dilp2-FAP, Bulgari et al., 2019) are used at the Drosophila NMJ to show that synaptic fusion pores that open spontaneously and in response to activity are not permeable to large DCV cargos. Then the conundrum of how such proteins can be efficiently released from presynaptic DCVs is resolved by establishing that anchored, activated PKA acts via complexin phosphorylation to open dilating fusion pores that results in DCV emptying.

Synaptic DCV fusion pore permeability
Fluorogens were synthesized via conjugating single chain polyethylene glycol (PEG) adducts to MG. Their hydrodynamic sizes were then determined in comparison to proteins by size exclusion fast protein liquid chromatography (FPLC) (see Methods).
The hydrodynamic properties of these fluorogens scaled with molecular weight . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Responses to 1 minute of 70 Hz stimulation led to robust labeling with the 4.3 kDa fluorogen, but were attenuated with larger fluorogens (Fig. 1A-D). For the three smaller fluorogens (4.3, 12.5 And 32.4 kDa), the incremental changes could reflect the slower diffusion of larger fluorogens and the effect of approaching the size cutoff of the fusion pore, which in this case must be 4-5 nm (i.e., ~2 times larger than in chromaffin cells; Albillos et al., 1997). However, the two largest fluorogens (53.2 and 71.1 kDa), with incrementally modest increases in hydrodynamic size (because radius is proportional to the cube root of protein molecular weight), barely produced any signals and 32.4 kDa fluorogens, time-dependent labeling was not seen with incubation of the NMJ with the 53.2 kDa fluorogen in the absence of Ca 2+ for 8 minutes (Fig. 1E,F,G).
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 6 Thus, synaptic fusion pore permeability is sufficient for the release of Drosophila neuropeptides, but excludes DCV cargos that exceed the fusion pore cutoff, which falls between 32.4 and 53.2 kDa.

Frequency of DCV emptying events
Given the above synaptic DCV fusion pore permeability, we explored whether NMJ DCVs ever empty to efficiently release large cargos, a process often referred to as full fusion (FF). For this purpose, 1 µM MG-BTau was used to detect spontaneous events that occur in the absence of extracellular Ca 2+ (Bulgari et al., 2019), thus enabling imaging of individual release sites without interference from surrounding events that are stimulated by activity. Single labeling events via kiss and run fusion pores ( Fig. 2A, K&R) were readily detected by their sustained labeling and occurred with variable kinetics (Fig. 2B, top and middle); overall, they grew over 23.7 + 2.7 seconds (n = 37) and could persist for the duration of imaging (up to 4 minutes).
However, on rare occasions labeling via the opening of a fusion pore was followed within seconds by the rapid loss of fluorescence ( Fig. 2A, FF). Again, time courses were variable (Fig. 2C); the lifetime of these events was 17.25 + 3.81 seconds with a rise time to peak fluorescence of 5.62 + 0.53 seconds (n=8). Based on the imaging field of view and the depth of field, such events could not be attributed to undocking and transport of DCVs. Rather, following MG fluorogen influx through the fusion pore and binding to the FAP, MG-FAP complexes that normally are retained (i.e., cannot exit through fusion pores) must have been released by fusion pore dilation that resulted in DCV emptying.
Interestingly, such full fusions could occur with a DCV that had already had a kiss and . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. ; https://doi.org/10.1101/2022.12.24.521856 doi: bioRxiv preprint 7 run event (Fig. 2C, bottom). Overall, DCV full fusions only occurred in 3.6% of exocytotic events at the synapse in these experiments (Fig. 3A, Con; filled bar shows kiss and run, open bar shows full fusion; frequencies expressed per bouton).

cAMP evokes opening of dilating fusion pores
Because it seems unlikely that such rare full fusions wholly account for release of large DCV cargo proteins, we considered that signaling might trigger synaptic DCV full fusions. A candidate for such a trigger is cAMP, which evokes Ca 2+ independent release from DCVs at the Drosophila NMJ (Shakiryanova et al., 2011;Bulgari et al., 2018). Therefore, we tested whether cAMP in the absence of extracellular Ca 2+ affects the frequency of kiss and run and/or full fusions.
Four independent experimental approaches showed that cAMP selectively evokes presynaptic DCV full fusions. First, the membrane permeant cAMP analog 8parachlorophenyl-thio-cAMP (CPT, 1 mM) increased the frequency of full fusions without affecting kiss and run events (Fig. 3A). Second, bath application of 100 µM octopamine, an insect neuromodulator that acts via cAMP to promote the growth of NMJ arbors (Koon et al., 2011), also increased the frequency of DCV emptying with no effect on the kiss and run event frequency (Fig. 3A, Oct). Third, bath application of 100 µM forskolin (FSK), an adenylate cyclase activator, increased the full fusion frequency without altering kiss and run fusions frequency (Fig. 3B) or the rise times of responses (25.5 + 3 s (n = 23) for kiss and run; 6.0 + 1.0 (n = 11) for full fusions). Thus, frequency of one subtype of release is affected without changing kinetics of individual events.
Finally, following motor neuron specific expression of the photoactivatable adenylate . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. ; https://doi.org/10.1101/2022.12.24.521856 doi: bioRxiv preprint 8 cyclase PACα (Schröder-Lang et al, 2007), blue light activation of the adenylate cyclase increased the frequency of DCV full fusions, again with no change in kiss and run frequency (Fig. 3C). Together, these results reveal that cAMP evokes release from presynaptic DCVs by selectively increasing the frequency of full fusions without affecting ongoing kiss and run exocytosis.

Anchored PKA-R2 is required for cAMP-evoked full fusions
The effects of cAMP are mediated by two ubiquitously expressed intracellular cAMP effectors, protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) (de Rooij, 1998). To test for a role of Epac, we began with bath application of the Epac activator 8-(4-methoxyphenylthio)-2′-O-methyl-cAMP (Me, 200 µM).
However, no changes in the frequencies of emptying or kiss and run events were In contrast, presynaptic PKA containing the type 2 regulatory subunit (PKA-R2) is required for cAMP-evoked DCV full fusions. First, the PKA catalytic subunit inhibitor H89 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. This genetic presynaptic perturbation inhibited the effect of cAMP on DCV full fusions, again without affecting kiss and run exocytosis frequency (Fig. 5H). Taken together, . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. ; https://doi.org/10.1101/2022.12.24.521856 doi: bioRxiv preprint these experimental results demonstrate that cAMP activation of anchored PKA-R2 triggers Ca 2+ -independent full fusion of presynaptic DCVs.

Complexin phosphorylation is required for cAMP-evoked release from DCVs
The above results pose the question of how rugose-anchored PKA activity induces Ca 2+ -independent synaptic DCV full fusions. Recent findings directed our attention to complexin (Cpx), which is often referred to as fusion clamp because it reduces spontaneous SSV fusion. First, Drosophila complexin is phosphorylated by PKA at S126 to upregulate (or unclamp) spontaneous SSV release events (Cho et al., 2015). Second, an in vitro reconstitution sytem devoid of much of the cellular exocytosis machinery suggested the potential for facilitation of fusion pore dilation by complexin (Pierson and Shin, 2021). Hence, these observations support the hypothesis that complexin phosphorylation mediates Ca 2+ -independent PKA-induced spontaneous DCV full fusions. Therefore, we examined the effect of replacing native complexin with the unphosphorylatable S126A mutant (Cpx S126A ) using the approach described previously by Cho et al., 2015: a Cpx null mutant was rescued with neuronal expression of Cpx S126A . First, to test whether the mutant rescue was effective for DCVs, a GFP-tagged neuropeptide (ANF-GFP) (Rao et al., 2001) that reports native DCV-mediated release (e.g., Husain and Ewer, 2004) was expressed while replacing Cpx with Cpx S126A and release was measured as the loss of ANF-GFP fluorescence. In the presence of extracellular Ca 2+ , activity-evoked DCV-mediated release in Cpx S126A animals was intact: the ~20% release (Fig. 6A), followed by some recovery due to activity-dependent . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. ; https://doi.org/10.1101/2022.12.24.521856 doi: bioRxiv preprint 11 capture, matches prior control responses with GFP imaging (Shakiryanova et al., 2005(Shakiryanova et al., , 2006Wong et al., 2015;Cavolo et al., 2016). Thus, the phosphoincompetent mutant fully rescues Ca 2+ dependent synaptic DCV exocytosis.
Then cAMP-evoked release was measured in the absence of extracellular Ca 2+ . Figure 6B shows that the ANF-GFP release response to forskolin is abolished in Cpx S126A animals: with the mutant the minor change in fluorescence that occurred with forskolin matched the photobleaching control. Finally, FAP imaging was used to measure the impact of Cpx S126A on DCV full fusions ( (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. ; https://doi.org/10. 1101/2022 to preserve presynaptic DCVs, which can only be replaced very slowly by axonal transport, but posed the question of whether large DCV cargos can permeate through fusion pores. FAP imaging with a series of PEGylated fluorogens provided a means to determine the conduction properties of synaptic DCV fusion pores without cargo specific confounds that affect release (e.g., binding to luminal DCV constituents). The experimental data presented here establish that the cutoff for protein passage through presynaptic DCV fusion pores falls between 32 and 53 kDa, but this limit is bypassed by cAMP-induced Ca 2+ -independent full fusions. The resultant efficient release of large cargos that are important for synaptic plasticity and growth (such as the protease TPA) may contribute to developmental growth and refinement of NMJ terminals, which depend on cAMP (Zhong et al., 1992;Koon et al., 2011;Vonhoff and Keshishian, 2017).
cAMP-evoked DCV emptying (Fig. 3) requires PKA-R2 and its anchor Rugose (Fig. 5). cAMP also stimulates spontaneous release from SSVs at the Drosophila NMJ (Yoshihara et al., 1999;Zhang et al., 1999;Cho et al., 2015), but the role of Rugose has not been examined for SSVs. However, Rugose affects larval NMJ morphology and a variety of adult behaviors including associative learning (Volders et al., 2012;Wise et al., 2015). Because Rugose and its mammalian ortholog Neurobeachin, a candidate autism gene (Castermans et al., 2003), are localized near the trans Golgi network (Wang et al., 2000;Volders et al., 2012), effects on synapses and behavior have been proposed to originate from perturbing the somatodendritic compartment (e.g., by affecting Golgi function, postsynaptic receptors and dendritic spines) (Niesmann et al., 2011;Volders et al., 2012;Gromova et al., 2018;Repetto et al., 2018). However, the effect of acute disruption of the PKA-R2 anchoring on cAMP-induced full fusions at . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. ; https://doi.org/10.1101/2022.12.24.521856 doi: bioRxiv preprint evoked fusion pores are released. Thus, diverse triggers (Ca 2+ elevation induced by activity and complexin phosphorylation induced by rugose-anchored PKA) evoke release of distinct DCV cargos by opening differentially dilated fusion pores.

Imaging.
Drosophila melanogaster third instar larvae were filleted and imaged in Ca 2+free HL3 in which 1.5 mM Ca 2+ was substituted with 0.5 mM EGTA, a Ca 2+ chelator (70 mM NaCl, 5 mM KCl, 0.5 mM Na3EGTA, 20 mM MgCl2, 10 mM NaHCO3, 5 mM trehalose, 115 mM sucrose, 5 mM hemi-sodium HEPES, pH 7.25). For electrical stimulation experiments they were transferred to HL3 saline that contained (in mM) 70 mM NaCl, 5 KCl, 1.5 CaCl2, 20 MgCl2, 10 NaHCO3, 5 trehalose, 115 sucrose, and 5 hemi-sodium HEPES, pH 7.25 supplemented with 10 mM L-glutamate to prevent muscle contractions. Nerve terminals were stimulated at 70 Hz for 1 minute via segmental nerves with a suction electrode (Shakiryanova et al, 2005). Dilp2-FAP data were acquired in muscle 6/7 type I boutons of with an upright Olympus microscope equipped with a 60×1.1 NA water immersion objective, a Yokogawa CSU-X1 spinning disk confocal head, a Coherent Obis 640 nm laser for fluorogen illumination, a Ludl filter wheel with an ET 700/75 emission filter and a Teledyne Photometrics Prime 95B sCMOS camera. ANF-GFP type Ib bouton data were acquired as described previously (Levitan et al, 2007;Wong et al., 2015). PACα photoactivation in synaptic boutons was achieved by illuminating synaptic boutons with 488 nm light for 300 ms at 0.33 Hz for the whole duration of FAP imaging (4 minutes). Quantification of fluorescence intensity was performed with ImageJ software (https://imagej.nih.gov/ij/) as previously described . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 Anantharam A, Bittner MA, Aikman RL, Stuenkel EL, Schmid SL, Axelrod D, Holz RW.
A new role for the dynamin GTPase in the regulation of fusion pore expansion. Mol Biol Cell. 22, 1907Cell. 22, -18 (2011. Baranes D, et al  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 Neuropharmacology 38, 645-57 (1999).
Zhong Y, Budnik V, Wu CF. Synaptic plasticity in Drosophila memory and hyperexcitable mutants: role of cAMP cascade. J Neurosci.12, 644-51 (1992).  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 dynamics. Numbers on images indicate times in seconds. Scale bar, 1 µm. Normalized time courses of representative individual (B) kiss and run and (C) full fusion sites.  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 expressing Epac null without FSK (-FSK, n=11) and with FSK (+FSK, n=17), ***p < 0.001.
(D-F) Frequency of kiss and run fusions with conditions corresponding to A-C. Note that there were no significant differences in kiss and run fusions for the experimental conditions presented here.  . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 29, 2022. ; https://doi.org/10.1101/2022.12.24.521856 doi: bioRxiv preprint (A) Release indicated by loss of ANF-GFP fluorescence is induced by a 1 minute 70 Hz stimulation (indicated by the horizontal bar) in elav-GAL4 UAS-ANF-GFP;;UAS-Cpx S126A , Cpx SH1 animals (n=4). (B) Forskolin evoked ANF-GFP release in 7 minutes is abolished in elav-GAL4 UAS-ANF-GFP;;UAS-Cpx S126A , Cpx SH1 animals. WT, wild type Cpx; PB Con, photobleaching control. n=4-6, ****p < 0.0001, Tukey's test following ANOVA. (C) Frequency of kiss and run (filled columns) and full fusions (open columns) in boutons from elav-GAL4;UAS-Dilp2-FAP;UAS-Cpx S126A , Cpx SH1 /Cpx Delta2 flies in the absence and presence of forskolin (-FSK (n=7) and +FSK (n=14), respectively). Cpx SH1 and Cpx Delta2 are null mutants. Note that forskolin does not have a statistically significant effect (ns, not significant by t-test) on full fusion frequency (open bars). Likewise, no effect was produced on kiss and run frequency.
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Frequency (1/min)
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