A Ca2+ channel differentially regulates Clathrin-mediated and activity-dependent bulk endocytosis

Clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE) are two predominant forms of synaptic vesicle (SV) endocytosis, elicited by moderate and strong stimuli, respectively. They are tightly coupled with exocytosis for sustained neurotransmission. However, the underlying mechanisms are ill defined. We previously reported that the Flower (Fwe) Ca2+ channel present in SVs is incorporated into the periactive zone upon SV fusion, where it triggers CME, thus coupling exocytosis to CME. Here, we show that Fwe also promotes ADBE. Intriguingly, the effects of Fwe on CME and ADBE depend on the strength of the stimulus. Upon mild stimulation, Fwe controls CME independently of Ca2+ channeling. However, upon strong stimulation, Fwe triggers a Ca2+ influx that initiates ADBE. Moreover, knockout of rodent fwe in cultured rat hippocampal neurons impairs but does not completely abolish CME, similar to the loss of Drosophila fwe at the neuromuscular junction, suggesting that Fwe plays a regulatory role in regulating CME across species. In addition, the function of Fwe in ADBE is conserved at mammalian central synapses. Hence, Fwe exerts different effects in response to different stimulus strengths to control two major modes of endocytosis.


Author summary
The arrival of an action potential at the nerve end induces synaptic vesicle (SV) exocytosis to allow the release of chemical neurotransmitters and the rapid transmission of signals. SV endocytosis is in turn elicited in order to rapidly replenish the vesicle pool in neurons. Therefore, tight coupling between exocytosis and endocytosis within these cells maintains constant synaptic transmission. Exocytosis and intracellular Ca 2+ elevation are known to be key prerequisites for the two main modes of SV endocytosis, Clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE), which are primarily triggered by moderate and strong nerve stimuli, respectively. However, how these two events cooperate to trigger endocytosis upon exocytosis remains unclear. In this study, we show that Flower (Fwe), an SV-associated Ca 2+  Introduction functions in response to two different stimuli that govern distinct modes of SV retrieval, thereby coupling exocytosis to endocytosis.

Results
Fwe promotes CME independently of Ca 2+ channeling We previously reported that the Fwe Ca 2+ channel promotes CME in the synaptic boutons of Drosophila neuromuscular junctions (NMJs) [24]. To investigate whether the Ca 2+ influx via Fwe plays a direct role in CME, we utilized the FweE79Q mutant whose channel activity is severely impaired [24] and assessed the ability to rescue CME defects associated with fwe mutants. We expressed UAS-flag-fwe-HA and UAS-flag-fweE79Q-HA with nSyb-GAL4, a panneuron GAL4 driver, in a strong loss of fwe background (fwe DB25 /fwe DB56 ) (S1A and S1B Fig) [24]. α-HA antibody staining was used to examine the distribution and expression of Fwe proteins in boutons. α-horse radish peroxidase (HRP) antibody staining labels the insect neuronal membranes, thereby outlining presynaptic compartments [25]. Both the wild-type Fwe and FweE79Q proteins are evenly distributed in boutons (S1A and S1B Fig). We then introduced a genomic HA-tagged fwe transgene to estimate the relative expression levels of UAS transgenes versus endogenous Fwe protein (S1C-S1F Fig). The proteins are expressed at~50% of the endogenous Fwe protein level in type Ib NMJ boutons (S1L-S1N Fig), whereas their expressions in type Is NMJ boutons correspond to~80% of the endogenous Fwe protein.
To estimate the efficacy of CME, we performed the FM1-43 dye uptake assay with moderate stimuli, i.e., 1-min 90 mM K + /0.5 mM Ca 2+ and 10-min 60 mM K + /1 mM Ca 2+ stimulations. The experimental paradigm is shown in S2A Fig. Both stimulation paradigms significantly elicit dye uptake in wild-type control larvae when compared to a resting paradigm (10-min incubation in 5 mM K + /0 mM Ca 2+ solution) (S2B- S2E Fig). We then performed a transmission electron microscopy (TEM) assay to assess the formation of bulk cisternae, a hallmark of ADBE [2,8]. No bulk cisternae were induced under these conditions (S2F- S2I Fig), showing that the strength of these stimuli is mild, which predominantly promotes CME. Upon 1-min 90 mM K + /0.5 mM Ca 2+ stimulation, loss of fwe impairs FM1-43 dye uptake (Fig 1A, 1B and  1H). It is possible that either a defect in SV endocytosis or exocytosis would reduce FM1-43 dye uptake in this case. To test the role of Fwe in SV exocytosis, we performed the FM1-43 dye loading/unloading assay. The experimental paradigm is indicated in S3A Fig indicating that Fwe plays a marginal or no role in SV exocytosis. Hence, the FM1-43 dye uptake deficit associated with fwe mutants mainly results from a defect in CME.
Reduced FM1-43 dye uptake in fwe mutants is completely rescued by the reintroduction of 50% Fwe protein (Fig 1C and 1H). However, a similar rescue was also observed when 50% FweE79Q is present (Fig 1D and 1H). Although the channel function of FweE79Q is mostly absent when analyzed in fly salivary glands [24], the remaining channel activity in this mutant might be sufficient to promote CME if enough proteins are present in the boutons. We therefore determined the minimal Fwe level required for CME to verify the channel function of Fwe. After surveying numerous GAL4 lines, elav-GAL4 and nSyb(w)-GAL4 were found to drive the expression of the transgenes at approximately 0% and 4% of the endogenous Fwe level, respectively (S1G-S1J, S1M and S1N Fig). As shown in Fig 1E, 1F and 1H, boutons expressing 10% or 4% Fwe can take up FM1-43 dye efficiently. We noted that the dye uptake with 4% Fwe expression is marginally reduced when compared to other Fwe-expressing larvae, indicating that this level of Fwe expression is near the minimal level for efficient CME.
Next, we assessed CME in 4% FweE79Q-expressing boutons (S1J, S1K, S1M and S1N Fig). Intriguingly, the efficiency of the dye uptake is not different between 4% Fwe and 4% Fwe E79Q-expressing boutons (Fig 1F-1H), suggesting that CME can occur despite the lack of a significant Ca 2+ influx via Fwe. We previously showed that loss of fwe results in a reduced SV number and enlarged SVs [24]. To examine the changes in SV ultrastructure, we performed TEM. The wild-type control bouton under the resting condition contains numerous SVs ( Fig  1I), whereas the number of SVs is decreased upon loss of fwe (Fig 1J and 1Q). This low SV number worsens following 10-min 60 mM K + /1 mM Ca 2+ stimulation (Fig 1M, 1N and 1Q). Either 4% Fwe or 4% FweE79Q expression rescues this SV loss (Fig 1K, 1L and 1O-1Q). In addition, enlarged SV sizes associated with fwe mutants are normalized under both expression conditions ( Fig 1R). Hence, these data indicate that Fwe triggers CME independent of Ca 2+ channeling.

Fwe initiates ADBE upon intense stimulation
Upon mild stimulation, CME retrieves the membrane that corresponds in size to an SV. In contrast, in response to intense stimulation, ADBE takes up large quantities of fused SVs from the plasma membrane to form bulk cisternae. It has been shown that both SV exocytosis and intracellular Ca 2+ elevation are essential for ADBE to proceed around the periactive zones [7,18,26]. This raises the possibility that Fwe may play a role in ADBE. High K + and Ca 2+ -containing solutions have been widely used to elicit ADBE at several different synapses, including fly NMJ boutons [8,[27][28][29][30]. To assess the role of Fwe in ADBE, we applied 10-min 90 mM K + / 2 mM Ca 2+ stimulation to induce ADBE and examine the formation of bulk cisternae using a TEM assay. The TEM image of control boutons reveals numerous cisternae (>80 nm in diameter, red arrows) elicited by this stimulation paradigm (Fig 2A, 2B and 2G). These processes, however, are dramatically suppressed by loss of fwe (Fig 2C, 2D and 2G). In unstimulated conditions, bulk cisternae are also less abundant in fwe mutants than in controls ( Fig 2G). This ADBE defect indeed results from the fwe mutation, as 50% Fwe expression rescues this ADBE phenotype (Fig 2E-2G). Interestingly, the average size of the few bulk cisternae observed in fwe mutants is comparable to that observed in control boutons (Fig 2H), suggesting that Fwe acts at the initiation step of ADBE rather than during a late membrane invagination process. Furthermore, following high K + stimulation, the accumulation of early endocytic intermediates was observed around the periactive zone in fwe mutant boutons (Fig 2D-2D1 and 2I, yellow arrows) when compared to wild-type controls and 50% Fwe-rescued larvae (Fig 2B-2B1, 2F-2F1 and 2I). Since optimal SV exocytosis is shown as a prerequisite for triggering ADBE [7,26], we therefore estimated the total SV area per bouton area under the resting condition. No difference between controls and fwe mutants was found (Fig 2J), showing that the ADBE defect associated with fwe mutants is not due to insufficient supply of exocytic SV membrane upon stimulation. Moreover, following 90 mM K + /2 mM Ca 2+ stimulation, the strength of SV exocytosis determined by the FM1-43 dye loading/unloading assay is comparable between controls and fwe mutants (S3H-S3N Fig). Collectively, these results reveal that Fwe is responsible for initiating ADBE during intense activity stimulation.
Acute inactivation of the components involved in CME, such as Clathrin, AP180, and Dynamin, elicits bulk membrane invaginations [31][32][33][34], suggesting that CME suppresses ADBE or that ADBE is the result of membrane expansions. To assess the role of Fwe in this process, we treated larvae with 200 μM chlorpromazine to inhibit Clathrin coat assembly [31],

Fig 2. Loss of fwe impairs Activity-Dependent Bulk Endocytosis (ADBE) in high K + stimulation. (A-F)
Transmission electron microscopy (TEM) images of neuromuscular junction (NMJ) boutons were obtained from FRT80B control larvae (A, B, and B1), fwe mutant larvae (fwe DB25 /fwe DB56 ; C, D, and D1), and 50% Fwerescued larvae (E, F, and F1). Samples were processed under the resting condition (10-min incubation in 5 mM K + /0 mM Ca 2+ solution; A, C, and E) or after 10-min 90 mM K + /2 mM Ca 2+ stimulation (B-B1, D-D1, and F-F1). Red arrows indicate bulk cisternae larger than 80 nm in diameter. White arrowheads indicate the border of the active zone. Loss of fwe impairs the formation of high K + -induced bulk cisternae. This deficit is rescued by 50% Fwe expression. Enlarged images of B, D, and F (white dashed boxes) are shown in B1, D1, and F1. Yellow arrows indicate endocytic intermediates formed around the periactive zone. (G) Data quantifications of the number of bulk cisternae per bouton area. The data in resting controls are derived from S2I Fig. Type Ib boutons (at rest: control, n = 17; fwe mutant, n = 31; and 50% Fwe, n = 11. Ten-minute 90 mM K + /2 mM Ca 2+ stimulation: control, n = 29; fwe mutant, n = 22; and 50% Fwe, n = 27) derived from !3 larvae followed by 10-min 90 mM K + / 2 mM Ca 2+ stimulation in the presence of FM1-43 dye. As shown in Fig 2K-2N, large membranous invaginations enriched with FM1-43 dye were detected in the controls. In contrast, these structures decrease upon loss of fwe. In summary, these results reinforce the functional importance of Fwe in ADBE.

The Ca 2+ influx via Fwe sustains presynaptic Ca 2+ levels during strong stimulation
To assess whether Fwe mediates an intracellular Ca 2+ increase to initiate ADBE upon intense activity stimulation, we expressed the lexAop2 transgene of the fast-decay version of the genetically encoded Ca 2+ indicator GCaMP6, GCaMP6f [35], in the presynaptic terminals with vglut-lexA, a glutamatergic neuron driver. We have shown previously that fwe mutant boutons display low resting Ca 2+ levels [24]. Similarly, decreased GCaMP6f fluorescence was observed upon loss of fwe ( Fig 3A, 3B and 3E, white arrows). This indicates a reduction in the resting Ca 2+ levels, as the expression level of GCaMP6f in boutons is higher in fwe mutants than in controls (S4A, S4B and S4E Fig). Next, we stimulated boutons with 90 mM K + /2 mM Ca 2+ solution for 10 min, which elicits ADBE (Fig 2A and 2B), and measured GCaMP6f fluorescence in the 6th and 10th min. In controls, the intracellular Ca 2+ concentrations in response to stimuli are substantially increased (Fig 3A-3A3 and 3F), whereas loss of fwe significantly impedes these Ca 2+ elevations (Fig 3B-3B3 and 3F). Hence, Fwe sustains presynaptic Ca 2+ levels upon strong stimulation.
To assess the role of Fwe-derived Ca 2+ influx in regulating presynaptic Ca 2+ level, we traced GCaMP6f fluorescence in 4% Fwe-and 4% FweE79Q-expressing boutons under the resting and high K + stimulation conditions. In both conditions, the levels of GCaMP6f are expressed similarly to that in controls (S4C- S4E Fig). We found that 4% Fwe but not 4% FweE79Q expression restores normal resting Ca 2+ levels in fwe mutants (Fig 3C-3E). Furthermore, a defect in high K + -induced Ca 2+ elevation associated with fwe mutants is partially reversed by 4% Fwe expression (Fig 3C-3C3 and 3F). In contrast, 4% FweE79Q expression fails to rescue this Ca 2+ defect (Fig 3D-3D3 and 3F). However, when we examined the presynaptic Ca 2+ changes following 1-min 90 mM K + /0.5 mM Ca 2+ stimulation, which prevalently elicits CME (S2 Fig), the Ca 2+ increases among all genotypes are quite similar (Fig 3G). Hence, these results indicate that Fwe triggers a Ca 2+ influx specifically in response to strong stimuli.
for each genotype were analyzed. (H) Data quantifications of the size of bulk cisternae. Bulk cisternae (control, n = 275; fwe mutant, n = 38; and 50% Fwe, n = 153) obtained from !20 type Ib boutons were analyzed. Boutons were derived from !3 larvae for each genotype. (I) Data quantifications of the number of endocytic intermediates per periactive zone length. Following high K + stimulation, more endocytic intermediates were observed in fwe mutant boutons when compared to control and 50% Fwe-rescued boutons. Type Ib boutons (at rest: control, n = 13; fwe mutant, n = 25; and 50% Fwe, n = 20. Ten-minute 90 mM K + /2 mM Ca 2+ stimulation: control, n = 26; fwe mutant, n = 18; and 50% Fwe, n = 25) derived from !3 larvae for each genotype were analyzed. (J) Data quantifications of the ratio of total synaptic vesicle (SV) area to bouton area. Type Ib boutons (control, n = 14; and fwe mutant, n = 16) derived from 3 larvae for each genotype were analyzed. (K-L) Confocal Z-projection images of NMJ boutons labeled with fixable FM1-43 dye were obtained from FRT80B controls (K) and fwe mutants (L). Larval fillets were treated with 200 μM chlorpromazine for 30 min, followed by 10-min 90 mM K + /2 mM Ca 2+ /200 μM chlorpromazine stimulation in the presence of FM1-43 dye. Bulk membranous invaginations loaded with high levels of FM1-43 dye are indicated by red arrows. (M-N) Data quantifications of the number of bulk cisternae per bouton area and the ratio of total area of bulk membrane invaginations to bouton area. After chlorpromazine treatment, bulk membrane invaginations are impeded upon loss of fwe. Type Ib boutons derived from A2/A3 muscles 4 or 6/7 were counted, and NMJs (control, n = 25; fwe mutant, n = 23) derived from 5 larvae for each genotype were analyzed. A Student's t test was used for analyses in G, I, J, M, and N. A one-way ANOVA test was used in H. p-Value: ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.01; ****, p < 0.001. Error bars indicate the standard error of the mean. Scale bar: 500 nm in A-F; 125 nm in B1, D1, and F1; 5 μm in K-L. The underlying data can be found in S1 Data.  , the Ca 2+ increase at 10 Hz is slightly higher than that in controls, and the Ca 2+ increase at 20 Hz is similar to that in controls. In contrast, at 40 Hz, loss of fwe significantly impairs the evoked Ca 2+ increase (Fig 3H and 3I). Moreover, 50% Fwe expression normalizes this deficit (S4H, S4K and S5C-S5C4 Figs and Fig 3H and 3I, black circle), whereas a partial restoration by 50% FweE79Q expression was observed (S4I, S4K and S5D-S5D4 Figs and Fig 3H and 3I, orange circle). Furthermore, we observed similar effects on rescuing low resting Ca 2+ levels associated with fwe mutants (S5F Fig). These results support the finding that Fwe does not mediate a Ca 2+ influx under a moderate stimulation condition that predominantly induces CME. Instead, it conducts a Ca 2+ influx when neurons undergo intense stimulation.
To rule out that the defect in evoked Ca 2+ increase may be attributed to slow CME associated with fwe mutants, we applied the same stimulation protocol to dap160 mutants, which exhibit a similar CME defect [36][37][38]. As shown in S5E- S5E4 Fig and Fig 3H and 3I (green circle), in dap160 mutant boutons, the Ca 2+ concentrations at 40 Hz are elevated to wild-type levels, although the Ca 2+ increase at 10 and 20 Hz is higher than that observed in controls and fwe mutants. In addition, the fluorescence level but not the expression level of GCaMP6f under the resting condition is reduced upon loss of dap160 (S4J, S4K, S5E and S5F Figs), suggesting that dap160 mutants display low resting Ca 2+ levels. Therefore, our results argue that a defective D). The vglut-lexA/lexAop2-GCaMP6f larvae expressed detectable amounts of GCaMP6f in boutons when the weak nSyb (w)-GAL4 driver was used to drive expression of UAS-fwe transgenes. Therefore, the use of this binary system allowed us to compare the Ca 2+ imaging results between controls, fwe mutants, 4% Fwe-rescued larvae, and 4% FweE79Q-rescued larvae. Boutons were subjected to 10-min 90 mM K + /2 mM Ca 2+ stimulation. Representative GCaMP6f images were captured at rest, in the 6th min, in the 10th min, and after stimulation. White arrows indicate type Ib boutons. (E) Data quantifications of the absolute unit (A.U.) of the resting GCaMP6f fluorescence intensity. Loss of fwe impairs the resting Ca 2+ levels, which is normalized by expressing 4% Fwe but not 4% FweE79Q. Type Ib boutons of A3 muscles 6/7 were counted, and NMJs (control, n = 13; fwe mutant, n = 16; 4% Fwe, n = 17; and 4% FweE79Q, n = 14) derived from !6 larvae for each genotype were analyzed. (F) Data quantifications of the increases in GCaMP6f fluorescence in the 6th or 10th min. The increase in the intracellular Ca 2+ level in response to high K + stimulation is perturbed by loss of fwe. The expression of 4% Fwe but not 4%FweE79Q partially rescues these defects. Type Ib boutons of A3 muscles 6/7 were counted, and NMJs (6th min: control, n = 5; fwe mutant, n = 6; 4% Fwe, n = 8; 4% FweE79Q, n = 6. 10th min: control, n = 7; fwe mutant, n = 6; 4% Fwe, n = 10; and 4% FweE79Q, n = 6) derived from !5 larvae for each genotype were analyzed. (G) Larvae of the indicated genotypes were subjected to 1-min 90 mM K + /0.5 mM Ca 2+ stimulation. The GCaMP6f images were taken between 30-60 s. The increase in GCaMP6f fluorescence is shown. The Ca 2+ increase is similar among all genotypes. Type Ib boutons of A3 muscles 6/7 were counted, and NMJs (control, n = 6; fwe mutant, n = 6; 4% Fwe, n = 6; and 4% FweE79Q, n = 6) derived from 6 larvae for each genotype were analyzed. Loss of Fwe or its channel activity does not affect the Ca 2+ increases evoked at 10 Hz and 20 Hz but impairs the Ca 2+ increases evoked at 40 Hz. Type Ib boutons of A3 muscles 6/7 were counted, and NMJs (control, n = 20; fwe mutant, n = 16; 50% Fwe, n = 14; 50% FweE79Q, n = 12; and dap160 mutant, n = 16) derived from !6 larvae for each genotype were analyzed. A one-way ANOVA test was used for statistical analysis. p-Value: ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.01; ****, p < 0.001. Error bars indicate the standard error of the mean. All images were captured in the same scale. The underlying data can be found in S1 Data.
https://doi.org/10.1371/journal.pbio.2000931.g003 CME does not account for presynaptic Ca 2+ dysregulation in fwe mutants. In addition, we found no evidence for the changes in the distribution and expression of Cacophony, the major VGCC located at the active zone (S6 Fig).

Fwe triggers a Ca 2+ influx to initiate ADBE during intense activity stimulation
The above-mentioned results prompted investigations into the role of Fwe-driven Ca 2+ influx in ADBE. We showed previously that 4% Fwe is sufficient for CME. We therefore addressed if this level of Fwe is sufficient to promote ADBE. When boutons are expressed with 10% or 4% Fwe, ADBE elicited by 10-min 90 mM K + /2 mM Ca 2+ stimulation efficiently produces bulk cisternae (Fig 4A, 4B, 4D, 4E and 4K). Thus, a partial Ca 2+ influx by 4% Fwe (Fig 3F) is sufficient for initiating ADBE. Consistently, 50% FweE79Q, which induces a fractional Ca 2+ influx ( Fig 3H and 3I), robustly triggers ADBE after high K + stimulation (S7 Fig). However, high K +induced bulk cisternae are significantly reduced in 4% FweE79Q-rescued larvae (Fig 4G, 4H and 4K). Notably, the number of high K + -induced bulk cisternae between 4% FweE79Q-rescued and fwe mutant larvae is comparable (fwe mutant larvae, 1.06 ± 0.4, n = 22, versus 4% FweE79Q-rescued larvae, 1.66 ± 0.42, n = 26, [Student's t test, p = 0.31]). Similar to loss of fwe, there is an increase in the level of endocytic intermediates formed around the periactive zone in 4% FweE79Q-rescued boutons after high K + stimulation when compared to 4% Fwe-rescued boutons (Fig 4L), thus supporting an important role of Ca 2+ influx via Fwe in ADBE. At rest, the total SV membrane area per bouton area is also comparable between 4% Fwe-and 4% FweE79Q-rescued boutons ( Fig 4M). Therefore, both expression conditions yield equal SV membranes available for SV exocytosis. These data suggest that Fwe triggers ADBE mainly through fluxing Ca 2+ . Furthermore, after chlorpromazine treatment, bulk membrane invaginations are less abundant in 4% FweE79Q-rescued boutons when compared to 4% Fwe-rescued boutons, also documenting a role of Fwe-derived Ca 2+ influx in chlorpromazine-induced bulk membrane invagination (Fig 4N-4P). If a suboptimal Ca 2+ level in the presynaptic terminals results in impaired ADBE phenotype in 4% FweE79Q-rescued larvae, then we expected that increasing overall intracellular Ca 2+ concentrations either via the remaining channel activity of FweE79Q or the other Ca 2+ channels during stimulation might compensate for this low intracellular Ca 2+ and rescue the defective ADBE. We therefore raised the Ca 2+ concentration from 2 mM to 5 mM in our 90 mM K + stimulation solution. When we applied a 10-min 90 mM K + /5 mM Ca 2+ stimulation to 4% FweE79Q-rescued boutons, the bulk cisternae number is significantly rescued (Fig 4I and 4K). Similarly, 5mM Ca 2+ also rescues the ADBE deficit associated with fwe mutants (Fig 4J and  4K). In contrast, this treatment does not increase the number of bulk cisternae further in 4% or 10% Fwe-rescued larvae when compared to the 10-min 90 mM K + /2 mM Ca 2+ stimulation condition ( Fig 4C, 4F and 4K). Hence, this rescue effect might be due to increased Ca 2+ levels rather than enhanced ADBE in the presynaptic compartments. These data further support the role of Ca 2+ influx via Fwe in triggering ADBE during intense activity stimulation.
Rodent Fwe isoform 2 is associated with SVs and can substitute the endocytic functions of Drosophila Fwe Fwe homologs are found in most eukaryotes [24], but their role in SV endocytosis in vertebrates has not been established. The mouse Fwe (mFwe) gene can generate at least six alternative mRNA splicing isoforms [41], producing five different mFwe isoforms (S8 Fig). The mFwe isoform 2 (mFwe2) is the most similar to Drosophila Fwe. Moreover, the mFwe2 and rat Fwe isoform 2 (ratFwe2) share~99% amino acid identity (170/172). In adult rat brain, ratFwe2 mRNA is widely expressed (Fig 6A). In the lysates of mouse neuroblastoma Neuro 2a (n2a) cells, our antisera against the C-termini of both mFwe2 and ratFwe2 (α-m/ratFwe2) recognize a~18 kDa protein band, corresponding to the predicted molecular weight of mFwe2 ( Fig 6B). This signal is significantly decreased when mFwe2 is knocked down by mFwe-microRNAi (miRNAi) (Fig 6B), showing antibody specificity. mFwe2 is expressed in postnatal as well as adult mouse brains (Fig 6B). Similarly, ratFwe2 was detected in rat brain and cultured rat hippocampal neurons (Fig 6C).
To determine if ratFwe2 is enriched in SVs, we purified SVs from adult rat brain using a series of centrifugations [42]. As shown in Fig 6D, ratFwe2 was specifically detected in the SV (Lysate pellet 2 [LP2]) fraction, marked by the presence of Synaptophysin (Syp), an abundant SV protein. ratFwe2 is also present in the SV fractions of adult rat brain separated with sucrose gradients (Fig 6E). To assess the subcellular localization of ratFwe2, we performed immunostaining in cultured neurons. Although staining with our antisera can visualize the expression of ratFwe2 in the cell bodies (S9B Fig), we failed to obtain specific staining in the presynaptic terminals. We therefore expressed HA-tagged mFwe2 in ratFwe knockout neurons (see below for details) and determined the SV localization of mFwe2-HA using α-HA staining. As shown in Fig 6F and 6G, mFwe2-HA protein is enriched in the presynaptic terminals and largely colocalized with Syp. The biochemical data combined with the in vivo localization data provide compelling evidence that ratFwe2 is associated with SV proteins, similar to Drosophila Fwe.
To determine whether rodent Fwe2 functions equivalently to Drosophila Fwe, we expressed UAS-flag-mFwe2-HA transgene in fwe mutants using nSyb(w)-GAL4. Overexpressed mFwe2 is localized to SVs in the boutons (Fig 6H-6H2) and rescues the FM1-43 dye uptake defect (Fig 6I-6K), as well as the reduced number of SVs (Fig 6L) in fwe mutants, showing that mFwe2 can promote CME in flies. Furthermore, the expression of mFwe2 corrects the ADBE deficit caused by loss of fwe (Fig 6M and 6N). Hence, mFwe2 promotes ADBE as well. We also observed that the early lethality of fwe mutant larvae is rescued by mFwe2 expression. Our results therefore suggest a conserved role of Fwe in SV endocytosis in mammals. To verify the role of ratFwe2 in SV endocytosis, we knocked out ratFwe in cultured rat hippocampal neurons using CRISPR/Cas9 technology [43]. We designed a specific guide RNA (gRNA; m/ratFwe-gRNA) that targets the first intron/second exon junction of both mFwe and ratFwe genes. To estimate the knockout efficiency, we transfected the gRNA construct into mouse neuroblastoma n2a cells and established a mFwe knockout n2a cell line. To assess the efficacy of CME, we elicited exocytosis of Synaptophysin-pHluorin (SypHy) [5] by delivering 200 action potentials at 20 Hz and monitored its retrieval via SV endocytosis. It has been documented that this mild stimulation paradigm prevalently induces CME [12,15,44]. In control neurons (Fig 7A, black line) bathed at room temperature, repeated exocytosis in response to 20-Hz stimuli increases SypHy fluorescence, followed by a gradual fluorescence decay caused by the reacidification of SVs formed via CME. However, in ratFwe knockout neurons (Fig 7A, red line), the decay rate of SypHy fluorescence is much slower (Fig  7A and 7C). To verify whether this defect is specific to loss of ratFwe2, we expressed mFwe2-HA in ratfwe knockout neurons. mFwe2-HA properly localizes in the Golgi (S9E Fig) as well as in SVs (Fig 6F and 6G). This protein further normalizes the slow SypHy fluorescence decay (Fig 7A and 7C, blue line). Recent studies have revealed distinct properties of SV endocytosis under physiological conditions [15,45,46]. We found similar results when these ratFwe2, mFwe2 was detected in the lysates of mouse neuroblastoma Neuro 2a (n2a) cells. This blotting signal is further reduced by expressing either one of two different mFwe-microRNAi (miRNAi). This isoform is also present in postnatal day 16 and adult mouse brains. Actin was used as the loading control. (C) In the immunoblots with α-m/ratFwe2, ratFwe2 was detected in the embryonic rat brain as well as in different days in vitro (DIV) cultured rat hippocampal neurons. Actin was used as the loading control. (D) The subcellular fractions were obtained from adult rat brain extracts using a series of centrifugations. ratFwe2 was found in the synaptosomal plasma membrane (lysate pellet 1 Flower Ca 2+ channel in CME and ADBE recordings were performed at physiological temperatures (Fig 7B and 7D). A slow decay of SypHy fluorescence is possibly due to either impaired CME or inefficient SV reacidification or both. To distinguish these hypotheses, we performed an acidic quenching assay [47,48]. As shown in S10 Fig, upon perfusion of an acidic buffer, the newly recycled SVs in both control and ratFwe knockout neurons are efficiently acidified. Hence, our data suggest that ratFwe2 promotes CME at mammalian central synapses.
To assess the role of ratFwe2 in ADBE, we performed a dextran dye uptake assay in the control and ratFwe knockout neurons. We triggered ADBE with a stimulation of 1,600 action potentials delivered at 80 Hz in the presence of 40 kDa tetramethylrhodamine (TMR)-dextran [15,49]. As shown in Fig 7E-7H, in the control axons marked with green fluorescent protein (GFP) expression, the presynaptic terminals filled with dextran dye (red puncta) were observed frequently. In contrast, removal of ratFwe significantly diminishes dextran dye uptake. This phenotype is specific to loss of ratFwe2, as the reintroduction of mFwe2-HA corrects this dye uptake defect. Hence, ratFwe2 is indispensable for ADBE. In summary, Fwe promotes CME and ADBE in mammalian neurons, thereby coupling exocytosis to two major modes of endocytosis.

Discussion
A tight coupling of exocytosis and endocytosis is critical for supporting continuous exocytosis of neurotransmitters. CME and ADBE are well-characterized forms of SV endocytosis triggered by moderate and strong nerve stimuli, respectively. However, how they are coupled with exocytosis under distinct stimulation paradigms remains less explored. Based on the present data, we propose a model as shown in Fig 7I. When presynaptic terminals are mildly stimulated, SV release leads to neurotransmitter release and the transfer of Fwe channel from SVs to the periactive zone where CME and ADBE occur actively [7,26,38]. Our data suggest that this channel does not supply Ca 2+ for CME to proceed. However, intense activity promotes Fwe to elevate presynaptic Ca 2+ levels near endocytic zones where ADBE is subsequently triggered. Thus, Fwe exerts different activities and properties in response to different stimuli to couple exocytosis to different modes of endocytosis.
ADBE is triggered by intracellular Ca 2+ elevation, which has been assumed to be driven by VGCCs that are located at the active zones [18,26]. However, our data strongly support a role for Fwe as an important Ca 2+ channel for ADBE. First, following exocytosis, Fwe is enriched at the periactive zone where ADBE predominates [7,24,26]. Second, Fwe selectively supplies Ca 2+ to the presynaptic compartment during intense activity stimulation (Fig 3), which is highly correlated with the rapid formation of ADBE upon stimulation [8,50]. Third, 4% FweE79Q expression, which induces very subtle or no Ca 2+ upon strong stimulation, fails to rescue the ADBE defect associated with loss of fwe (Figs 3 and 4). Fourth, treatment with a low concentration of La 3+ solution that specifically blocks the Ca 2+ conductance of Fwe significantly abolishes ADBE (Fig 5). Lastly, the role of Fwe-derived Ca 2+ influx in the initiation of ADBE mimics the effect of Ca 2+ on ADBE at the rat Calyx of Held [7]. As loss of fwe does not completely eliminate ADBE, our results do not exclude the possibility that VGCC may function in parallel with Fwe to promote ADBE following intense stimulation.
Interestingly, Ca 2+ influx via Fwe does not control SV exocytosis during mild and intense stimulations (S3 Fig). How do VGCC and Fwe selectively regulate SV exocytosis and ADBE, respectively? One potential mechanism is that VGCC triggers a high, transient Ca 2+ influx around the active zone that elicits SV exocytosis. In contrast, Fwe is activated at the periactive zone to create a spatially and temporally distinct Ca 2+ microdomain. A selective failure to increase the presynaptic Ca 2+ level during strong stimulation is evident upon loss of fwe. This pinpoints to an activity-dependent gating of the Fwe channel. Consistent with this finding, an increase in the level of Fwe in the plasma membrane does not lead to presynaptic Ca 2+ elevation at the Calyx of Held when the presynaptic terminals are at rest or subject to mild stimulation [23]. However, we previously showed that, in shi ts terminals, blocking CME results in the accumulation of the Fwe channel in the plasma membrane, elevating Ca 2+ levels [24]. It is possible that Dynamin is also involved in regulating the channel activity of Fwe or that the effects other than Fwe accumulation associated with shi ts mutants may affect intracellular Ca 2+ handling [51,52]. Further investigation of how neuronal activity gates the channel function of Fwe should advance our knowledge on the activity-dependent exo-endo coupling.
Although a proteomic analysis did not identify ratFwe2 in SVs purified from rat brain [53], our biochemical analyses show that ratFwe2 is indeed associated with the membrane of SVs. Our data show that 4% of the total endogenous Fwe channels efficiently promotes CME and ADBE at the Drosophila NMJ. If a single SV needs at least one functional Fwe channel complex during exo-endo coupling, and one functional Fwe complex comprises at least four monomers, similar to VGCCs, transient receptor potential cation channel subfamily V members (TRPV) 5 and 6, and calcium release-activated channel (CRAC)/Orai1 [24,40,54,55], then we anticipate that each SV contains~100 Fwe proteins (4 monomers × 25). This suggests that Fwe is highly abundant on the SVs. It is unlikely that many SVs do not have the Fwe, as a 25-fold reduction of the protein is enough to ensure functional integrity during repetitive neurotransmission. Finally, our results for the SypHy and dextran uptake assays at mammalian central synapses indicate the functional conservation of the Fwe channel in promoting different modes of SV retrieval. In summary, the Fwe-mediated exo-endo coupling seems to be of broad importance for sustained synaptic transmission across species.

Drosophila strains and genetics
Most of the experiments used y w; FRT80B isogenized fly, which was used for the generation of the fwe DB25 and fwe DB56 mutations [56] as the controls. Larvae were reared in standard fly food or on grape juice agar covered with yeast paste at 22˚C. The genotypes of flies used in the experiments are described below.

Molecular cloning
The pCasper4-genomic HA-fwe construct was constructed by inserting a HA sequence to the site after the translational start codon of fwe-RB in the context of the pCasper4-genomic fwe construct [24]. To obtain the pUAST-flag-mFwe2-HA construct, the fwe-RB fragment of the pUAST-flag-fwe-RB-HA construct [24] was replaced with the mFwe2 coding region, which was amplified from total mRNA of the adult mouse brain. P-element-mediated transgenesis was achieved by the standard procedure. The introduction of these genomic fwe transgenes to the fwe mutant background rescues the early lethality associated with fwe mutants, demonstrating that fused tags do not affect normal functions of Fwe. To generate the mFwe-miRNAi constructs, the sequences of miRNAs were designed according to Invitrogen's RNAi Designer.

Reverse-transcription PCR (RT-PCR)
Total RNA of different regions of the adult rat brain were extracted with TRIZOL reagent according to the manufacturer's instructions. Five μg of total RNA was mixed with oligo-dT primer in 20 μl of reverse transcription reaction solution. One μl of this mixture was used to amplify the cDNA of ratFwe2 mRNA with specific primers (forward primer: GAAGATCTAT GAGCGGCTCGGTCGCC; reverse primer: CGGAATTCTCACAGTTCCCCCTCGAATG). Twenty-five PCR cycles were used to allow exponential PCR amplification. The PCR products were sequenced to validate the identity of ratFwe2 mRNA.

Antibody generation
GST-fused polypeptides comprising seven tandem repeats of the C-terminus of Drosophila Fwe-PB isoform [24] were injected in guinea pigs to obtain GP100Y antisera. To generate αm/ratFwe2 antisera (GP67), the DNA fragment encoding seven tandem repeats of ratFwe2 Cterminus (a.a. 140-172) was subcloned to pET28a plasmid. His-fused polypeptides were purified and then injected into guinea pigs. Antibody generation was assisted by LTK BioLaboratories (Taiwan). Specific antibodies were further purified by antigen-conjugated affinity columns.
The staining intensities of all type Ib boutons from the same muscles 6 and 7 in one image were averaged to obtain each data value. Image processing was achieved using LSM Zen and Image J.

Generation of mFwe knockout neuroblastoma n2a cells
Mouse neuroblastoma n2a cells were transfected with pSpCas9(BB)-m/ratFwe-gRNA-2A-GFP plasmid. GFP-positive cells were sorted out using flow cytometry, and the cells were plated in a 96-well plate in which each well included approximately one cell. After 3-wk culture, single cell-driven colonies were subjected to immunostaining and immunoblotting for mFwe2 to verify the knockout of mFwe2. One of the confirmed mFwe knockout neuroblastoma n2a cell lines was used in S9A Fig.

FM1-43 dye uptake
To induce CME, the third instar larvae were dissected in 0 mM Ca 2+ hemolymph-like (HL)-3 solution at room temperature (70 mM NaCl, 5 mM KCl, 10 mM MgCl 2 , 10 mM NaHCO 3  imaged to indicate "loading." Subsequently, the dye loaded in SVs was unloaded by stimulation using 90 mM K + /0.5 (or 2) mM Ca 2+ solution for 1 min. Released dye was removed by several washes with a 0 mM Ca 2+ HL-3 solution. The remaining dye in boutons was imaged to indicate "unloading." The final FM1-43 dye intensity in the boutons was calculated by subtracting the dye fluorescence intensity in the surrounding muscles from the dye fluorescence intensity within the boutons. The dye fluorescence intensities of at least ten type Ib boutons from the same muscles 6 and 7 were averaged to obtain each data value. The dye unloading efficiency was indicated as (F load -F unload )/F load . Images processing was achieved using Image J and LSM Zen.

Ca 2+ imaging
GCaMP6f imaging. The third instar larvae were dissected in 0 mM Ca 2+ HL-3 at room temperature and incubated in 2 mM Ca 2+ /5 mM K + /7 mM glutamate solution (70 mM NaCl, 5 mM KCl, 10 mM MgCl 2 , 10 mM NaHCO 3 , 5 mM trehalose, 5 mM HEPES [pH 7.2], 115 mM sucrose, 2 mM CaCl 2 , and 7 mM monosodium glutamate). Glutamate treatment would desensitize postsynaptic glutamate receptors, thus reducing muscle contraction upon stimulation. GCaMP6f fluorescence was then measured to indicate the resting Ca 2+ levels. To image GCaMP6f in high K + stimulations, larval fillets were subsequently stimulated with 90 mM K + /2 mM Ca 2+ /7 mM glutamate solution. High K + and Ca 2+ lead to bulk Ca 2+ influxes into the muscles and cause dramatic contractions. The boutons were manually focused and simultaneously imaged in the 6th and 10th min every 1 s. After 10-min stimulation, larval fillets were rinsed with 2 mM Ca 2+ /5 mM K + /7 mM glutamate solution and imaged again. Similarly, boutons subjected to 1-min 90 mM K + /0.5 mM Ca 2+ /7 mM glutamate stimulation were manually focused and imaged between 30-60 s every 1 s. All images were captured from muscles 6 and 7 of abdominal segment 3. Each larva was only used for one recording. The images of clearly focused boutons were further used for data quantifications. The GCaMP6f fluorescence intensities in type Ib boutons and the surrounding muscles (served as the fluorescence background) were measured. Final GCaMP6f fluorescence intensity was calculated by subtracting the background fluorescence intensity in the surrounding muscles from the GCaMP6f fluorescence intensity in the boutons. The GCaMP6f fluorescence intensities of at least ten type Ib boutons from the same muscles 6 and 7 at a given time period were averaged to obtain each data value. For electric stimulation, the larval axonal bundle was aspirated and delivered with 10-40 Hz stimulations via a glass capillary electrode. The stimulus was fixed at 5 mV and 0.5 ms duration by pClamp 10.6 software (Axon Instruments). The images were captured every 2 s using MetaMorph software and an ANDOR iXon3 897 camera. All images were captured from muscles 6 and 7 of abdominal segment 3. Each larva was only used for one recording. The GCaMP6f fluorescence intensities in type Ib boutons and the surrounding muscles (which served as the fluorescence background) were measured. The final GCaMP6f fluorescence intensity was calculated by subtracting the background fluorescence intensity in the surrounding muscles from the GCaMP6f fluorescence intensity in the boutons. The GCaMP6f fluorescence intensities of at least five type Ib boutons from the same muscles 6 and 7 at a given time period were averaged to obtain each data value. Images processing was achieved using Image J and LSM Zen.
Ca 2+ imaging in salivary glands. Briefly, the gland cells of the third instar larvae were dissected in 0 mM Ca 2+ HL-3 solution. The cells were subjected to loading of 4 μM Fluo-4 AM (Invitrogen) in 100 μM Ca 2+ solution. For La 3+ treatment, the cells were incubated in 100 μM LaCl 3 /100 μM Ca 2+ solution. The images were captured every 10 min during dye loading using MetaMorph software and an ANDOR iXon 897 camera. The fluorescence intensities in the salivary gland cells and surrounding cover slips were measured. The final fluorescence value was calculated by subtracting the fluorescence intensity in the gland cells from the dye fluorescence intensity in the coverslips. The fluorescence intensity of one salivary gland was used for each data value. Image processing was achieved using Image J and LSM Zen.

Electrophysiology
The third instar larvae were dissected in 0 mM Ca 2+ HL-3 at room temperature and then bathed in 1 mM Ca 2+ HL-3 solution for 5-10 min before the recording. The mean value of the resistance of the recording electrode was~40 MΩ when the electrode was filled with a 3M KCl solution. All recordings were obtained from muscle 6 of abdominal segment 3. Each larva was only used for one recording. Recordings from the muscles that hold resting membrane potentials at less than −60 mV were used for further data quantifications. EJPs were evoked by stimulating the axonal bundle via a glass capillary electrode with an internal diameter of~10-15 μm (Harvard Apparatus Glass Capillaries GC120F-15) at 0.2 Hz. Stimulus pulses were fixed at 0.5 ms duration (pClamp 10.6 software, Axon Instruments). To obtain maximal EJP amplitude, 3-5 mV electric stimuli were applied. EJPs were amplified with an Axoclamp 900A amplifier (Axon Instruments, Foster City, California) under bridge mode and filtered at 10 kHz. EJPs were analyzed by pClamp 10.6 software (Axon Instruments). For the EJP amplitude at 0.2 Hz, the mean of the EJP amplitude was averaged from the amplitudes of 80 EJPs in one consecutive recording.

TEM
Larval fillets were dissected in 0 mM Ca 2+ HL-3 medium at room temperature. To trigger CME, samples were stimulated with 90 mM K + /0.5 mM Ca 2+ HL-3 solution for 1 min or 60 mM K + /1 mM Ca 2+ HL-3 solution for 10 min. To induce ADBE, larval fillets were subjected to stimulation of a 90 mM K + /2 mM or 5 mM Ca 2+ HL-3 solution in the presence or lack of 10 μM La 3+ for 10 min. Subsequently, the samples were fixed for 12 h in 4% paraformaldehyde/1% glutaraldehyde/0.1 M cacodylic acid (pH 7.2) solution and then rinsed with 0.1 M cacodylic acid (pH 7.2) solution. They were subsequently fixed in 1% OsO4/0.1 M cacodylic acid solution at room temperature for 3 h. The samples were subjected to a series of dehydration from 30% to 100% ethanol. After 100% ethanol dehydration, the samples were incubated in propylene, a mixture of propylene and resin, and pure resin. Lastly, they were embedded in 100% resin. The images of type Ib boutons were captured using Tecnai G2 Spirit TWIN (FEI Company) and a Gatan CCD Camera (794.10.BP2 MultiScan) at !4,400 × magnifications. The size of the SVs and the bulk cisternae and the area of type Ib boutons were measured using Image J. We identified type Ib boutons by multiple layers of subsynaptic reticulum. The radius of the bulk cisternae was calculated from A(area) = πr 2 . Isolated membranous structures larger than 80 nm in diameter were defined as bulk cisternae.

SypHy imaging and dextran uptake assays
For SypHy imaging, DIV7 cultured rat hippocampal neurons were transfected with pSpCas9 (BB) and pCMV-SyphyA4 (addgene#24478) plasmids [5].  [47,48]. Next, the imaging buffer was perfused to allow surface SypHy to be fluorescent. Experimental temperatures were maintained at physiological temperatures. The final SypHy fluorescence intensities in the presynaptic terminals were calculated by subtracting the background fluorescence intensity on the surrounding coverslip from the SypHy fluorescence intensity within presynaptic terminals. Each data value was obtained from a single terminal. For 40 kDa TMR-dextran uptake assays, DIV13-15 neurons transfected with pSpCas9 (BB) plasmids were stimulated by a train of 1,600 action potentials delivered with an 80 Hz electric field stimulation (50 mA, 1-ms pulse width) in the imaging solution (144 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl 2 , 2.5 mM MgCl 2 , 10 mM HEPES [pH 7.5], 10 μM CNQX, and 50 μM AP-5) [49] in the presence of 50 μM 40 kDa TMR-dextran (Invitrogen). Subsequently, neurons were perfused with the same buffer for 5 min to remove excess dextran dye. Experiments were performed at room temperature. Imaging was achieved through MetaMorph software and an ANDOR iXon 897 camera. Image processing was achieved using Image J and LSM Zen.

Statistics
Paired and multiple data sets were compared by Student's t test and one-way ANOVA statistical analyses, respectively. All data analyses were achieved using GraphPad Prism 7.0. The numerical data used in all figures are included in S1 Data.