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Here come the newcomer granules, better late than never Herbert Y. Gaisano Department of Medicine, University of Toronto, M5S 1A8, Toronto, Canada

Exocytosis in pancreatic b-cells employs Munc18/soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes that mediate the priming and docking onto the plasma membrane (PM) of insulin granules, called predocked granules, that sit on the PM until Ca2+ influx evokes fusion. This accounts for most of the initial peak secretory response. However, the subsequent sustained phase of glucose-stimulated insulin secretion arises from newcomer granules that have a minimal residence time at the PM before fusion. In this Opinion I discuss recent work that has begun to decipher the components of the exocytotic machinery of newcomer granules, including a Munc18/SNARE complex that is different from that mediating the fusion of predocked granules and which can potentially rescue defective insulin secretion in diabetes. These insights are applicable to other neuroendocrine cells that exhibit sustained secretion. Exocytosis in the pancreatic islet b-cell: is it like or not like the neuron? The pioneering contributors to the discovery of the machinery regulating vesicular traffic, particularly exocytosis (Figure 1), have just been awarded the Nobel Prize [1]. A considerable part of this work in exocytosis has been on neurons, elucidating the molecular machinery underlying docking, priming, and fusion of synaptic vesicles (SVs) [1,2]. SVs sit on the PM for an indefinite time (predocked vesicles, Figure 2A) within a readily releasable pool (RRP) (see Glossary) until Ca2+ influx across PM-bound Ca2+ channels (Cavs) triggers their fusion [2]. The pancreatic islet b-cell, with insulin packaged in dense core secretory granules (SGs), has long been studied as a secretory cell employing exocytotic machinery similar to that of the neuron (Munc18/SNARE complexes and accessory proteins; see below and Figure 1) to mediate fusion of predocked insulin SGs [3,4]. Fusion of predocked insulin SGs within the RRP has been postulated to account for most of the first phase (lasting10–15 min) of glucose-stimulated insulin secretion (GSIS) by the b-cell [5]. In type 2 diabetes (T2D), islet insulin secretory capacity is unable to meet the increasing insulin demand caused by insulin resistance and b-cells eventually decompensate with loss of firstCorresponding author: Gaisano, H.Y. ([email protected]). Keywords: SNARE proteins; newcomer granules; insulin secretion. 1043-2760/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2014.03.005

phase GSIS [6]. This loss of first-phase GSIS [6] is largely due to loss of the fusion competence of predocked SGs [7], in part from a severe reduction in b-cell levels of the putative Munc18/SNARE complex [8,9]. b-cells, unlike neurons, secrete for hours during and after a meal [6]. This lower-level but sustained second phase of GSIS, arising from SGs released from an elusive reserve pool [5], ensures continued assimilation of nutrients entering the bloodstream. This sustained secretory response was subsequently shown to be almost entirely attributable to continued recruitment of ‘newcomer’ SGs (Figure 2B), which do not sit on the PM for long or at all before fusion [7,10,11]. The sustained phase is only moderately diminished in T2D [6], consistent with the largely intact exocytosis of newcomer SGs observed in T2D rodents [7].

Glossary Closed conformation: when the Syn-1A N-terminal Habc trihelical domain folds back and binds the C-terminal helical SNARE motif (H3 domain) to block the H3 domain from binding the SNARE motifs of SNAP25 and VAMP to form the SNARE complex. Excitosomes: the complex formed by SNARE proteins including synaptotagmin and Ca2+ channels to tether SGs to the channels, positioning the SGs at sites of maximal Ca2+ entry to enable fusion. Exocyst: an octameric complex whose full assembly is initiated by RalAGTPase activation of Sec5. The exocyst tethers SGs loosely to the PM, which is postulated to prepare the SGs for docking onto the PM. Glucagon-like peptide-1 (GLP-1): a hormone secreted from intestinal L-cells after a meal that sensitizes b-cells to augment insulin release; acts through cAMP/PKA signaling pathways. Open conformation: when the Syn-1A H3 domain becomes unfolded from the Habc domain, thus exposing H3 to bind SNAP25 and VAMP to form the SNARE complex. Primary exocytosis: fusion of SVs with the PM to release the vesicles’ cargo to the cell exterior. Priming: the process of SGs entering the most advanced stage of fusion readiness. In the context of the SNARE Hypothesis, this occurs when v-SNAREs on SGs and t-SNAREs on the PM form a partially zippered trans-SNARE complex in the kinetically most advanced stage just before Ca2+ triggers fusion. Readily releasable pool (RRP): the small fraction of docked SGs that are primed and thus immediately available for release on stimulation. Reserve pool: a depot of SGs whose release is triggered only during intense or prolonged stimulation. This is believed to constitute most (80–90%) of the SVs in presynaptic terminals. Sec1/Munc18-like (SM) proteins: bind cognate Syntaxins to maintain the Syntaxins in an inactive, closed conformation. SM proteins were later found to bind both t- and v-SNAREs to organize the trans-SNARE complex zipper configuration required for fusion. Synaptosome-associated protein of 25 kDa (SNAP25): a t-SNARE protein that is on but not anchored to the presynaptic PM. Syntaxin (Syn): a t-SNARE protein anchored into the presynaptic PM. Total internal reflection fluorescence microscopy (TIRFM): enables visualization of fluorophore-tagged proteins within the evanescent wave reflected from the glass–water interface, which is typically 100 nm into the cell sample, at the PM. Vesicle-associated membrane protein (VAMP): a v-SNARE anchored to SVs and SGs.

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(A)

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Complexin TRENDS in Endocrinology & Metabolism

Figure 1. Stages of exocytosis. (A) Secretory granule (SG) tethering to the plasma membrane (PM). RalA binds Sec5 to promote assembly of the eight-subunit exocyst complex, which tethers the SG loosely to the PM. Munc18 binds Syntaxin in closed form, which is unable to form a complex with synaptosome-associated protein of 25 kDa (SNAP25) and vesicle-associated membrane protein (VAMP). (B) Priming. Munc18 is assisted by Munc13-1 in activating Syntaxin, which adopts an open conformation that can form a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex with SNAP25 and VAMP. This SNARE complex is bound to Ca2+ channels to form the excitosome, which positions SGs at sites of maximum Ca2+ influx. (C) Fully primed. The SG is held close to the PM by the SNARE complex, which is only partially zippered (note the space at the C terminus) due to being clamped by complexin. (D) Fusion. Ca2+ influx from open Ca2+ channels (relieved from binding the SNARE complex) acts on the Ca2+ sensor synaptotagmin, which removes complexin from the SNARE complex thus allowing full zippering of the SNARE complex. Synaptotagmin also promotes lipid mixing between SGs and the PM, leading to fusion pore formation. See also Box 1.

Contrary to previous thinking, newcomer SGs were later found to be also substantial contributors to first-phase GSIS [10,11], raising the possibility that they could be deployed to compensate for the defective predocked SGs in T2D [4,11]. It is only now that the molecular machinery of newcomer SGs is being unraveled. What is the current thinking on the exocytotic fusion machinery? The membrane fusion machinery requires two key components: SNARE and sec1/Munc18-like (SM) proteins [1,2] (Figure 1). The SNARE Hypothesis paradigm (Box 1) dictates that different fusion events are mediated by complexes comprising cognate vesicle (v-) SNAREs [vesicleassociated membrane proteins (VAMPs)] and target membrane (t-) SNAREs [syntaxins (Syns) and synaptosomeassociated protein of 25 kDa (SNAP25)]. Assembly of distinct SNARE complexes is regulated by cognate SM proteins to ensure not only their subcellular compartmental specificity, but also that fusogenic actions occur only in response to cellular needs and demand [2]. SM proteins initially act as fusion clamps, grabbing onto the fusogenicincompetent ‘closed’ conformation of Syn (Figure 1A). During SG priming, a SM protein acts as a clasp that holds onto both t-SNAREs (inducing Syn to adopt an activated ‘open’ conformation’) on the PM and v-SNAREs on SGs, thus forming a quaternary complex with a partial zipper 2

structure that brings the SGs into close proximity with the PM, into the predocked state. This priming process (Figure 1B) is assisted by priming factor Munc13-1 [12]. Why the partial zipper does not spontaneously zipper fully to effect fusion is attributable to regulators embedded in the SM/SNARE fusion machinery, including complexins acting as fusion clamps (Figure 1C) and synaptotagmins (syts) acting as Ca2+ sensors [2]. Fusogenic signal Ca2+ binds syt, which reverses the clamping action of complexin and allows complete zippering leading to fusion of the lipid bilayer (Figure 1D). Although still debated, this fusion was postulated to require only one SNARE, but three SNAREs are required to fully zipper and maintain the fusion pore in an open state for emptying cargo [13]. Super-high resolution imaging suggests that a cluster of Syn-1A molecules (probably 50–70) [14] (along with cognate SNAP25 [15]) is required for a SG to dock onto the PM, and when fusion occurs the t-SNARE cluster disperse. The t-SNAREs assume dynamic conformations in various stages of assembly and when complete enable rapid exocytosis; when less than fully assembled, their assembly can be driven to completion by Ca2+ influx but with slower kinetics of fusion [2,16,17]. This architecture enables exquisite precision at exocytic sites and is responsible for the differing rates or kinetics of fusion [16,17]. The above architecture of exocytosis in neurons has been used to investigate the secretory machineries of other

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Box 1. The SNARE Hypothesis

(A)

Predocked SG Plasma membrane

Ca2+ Primary exocytosis

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Ca2+ Newcomer SG fusion (C)

Ca2+

Ca2+

SNARE proteins were originally found to be receptors for cytosolic N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (SNAP), which were purified and shown to be required for transport vesicle fusion in a cell-free system [2]. SNARE proteins were then found to be the specific substrates for proteolysis by botulinum (Syntaxin and SNAP25) [23] and tetanus (VAMP) [22] neurotoxins, which were long known to block SV exocytosis. The three SNARE proteins are: (i) VAMP, a v-SNARE anchored to SVs and SGs; (ii) Syntaxin, a t-SNARE anchored to the presynaptic PM; and (iii) SNAP25, a second t-SNARE protein located on but not anchored to the presynaptic PM. The SNARE Hypothesis is a concept that the three SNARE proteins on the vesicle and target membrane form a complex to bridge the two membranes for fusion [2]. This concept applies to all membrane fusion processes employing ubiquitous homologs of the three SNARE proteins [1,2]. For exocytotic fusion, Munc18 exhibits a clasping action to maintain Syntaxin in a folded, closed conformation whereby the trihelical Habc domains of the N-terminal half bind the SNARE motif in the C-terminal H3 domain, thus blocking the H3 domain from binding the cognate SNAREs [2]. On activation of Munc18 and Munc13, these ‘priming’ factors cooperatively act to unfold Syntaxin into an open conformation, whereby the SNARE motif of the Syn H3 domain binds the SNARE motifs of SNAP25 (one each from the N- and C-terminal halves) and VAMP, coming together to form a stable four-helix bundle, with three helices anchored on the target membrane and one helix on the transport vesicle to form a trans-SNARE complex. Assembly of this four-helix bundle proceeds progressively from the N termini away from the PM toward the C termini of the SNAREs in a zippering action that generates an inward force sufficient to pull the lipid bilayers together and force them into fusion. However, this zippering becomes stuck close to the C-terminal end by the binding of complexin at the v-/t-SNARE interface, thus serving as a fusion clamp [46]. This clamping action is relieved when Ca2+ acts on the Ca2+ sensor synaptotagmin, enabling it to compete with complexin for binding to the SNARE complex and allowing the zippering to be completed, leading to fusion. Synaptotagmin also promotes lipid mixing between SGs and the PM, facilitating fusion pore formation. After fusion, the fully zippered cis-SNARE complex is in the same membrane [2].

Compound exocytosis TRENDS in Endocrinology & Metabolism

Figure 2. Modes of exocytosis of secretory granules (SGs). (A) Predocked SG. A SG is docked on the PM, becomes primed, and sits on the PM for an indefinite time until Ca2+ influx mediates its fusion with the PM to release its cargo (primary exocytosis). The sec1/Munc18-like (SM)/soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that mediates the exocytosis of predocked SGs is Munc18a/[Syntaxin 1A/synaptosome-associated protein of 25 kDa (SNAP25)/vesicle-associated membrane protein (VAMP2)]. (B) Newcomer SGs. These SGs undergo fusion with minimal or no residence time on the PM. (C) Compound exocytosis. Several SGs first undergo fusion inside the cytoplasm, then one SG fuses with the PM to unload massive amounts of cargo from the compound granule. See also Box 2. Adapted, with permission, from [4].

secretory cells [1,2], including b-cells [3,18] (Figure 1). Specifically, Munc18a/(Syn-1A/SNAP25/VAMP-2) is the putative SM/SNARE complex mediating exocytosis of predocked insulin SGs [3,19]. Munc18a [20] and Syn-1A [21] gene deletion in mice and botulinum toxin-induced proteolysis of SNAP25 [22] and VAMP2 [23] in b-cells uniformly abrogated exocytosis of insulin SGs from the RRP (Figure 1A,B). Employing knockout (KO) mice, Munc131 was shown to be the major priming factor [24,25] (Figure 1B) and syt-7 the putative Ca2+ sensor [26] (Figure 1D) for insulin SGs in the RRP. For more information on how this SM/SNARE complex and associated proteins mediate predocked insulin SGs within the RRP (Figure 2A and Box 2), see [2,3,19]. An interesting aspect

requiring further study is that when predocked SGs sit for too long on the PM, some lose exocytosis competence, which has been attributed to an undefined ‘aging’ process [27]. A recent report using time-resolved genetic tagging of insulin SGs showed that as SGs come closer to the PM, they lose mobility and fusion competence; by contrast, younger SGs containing newly synthesized insulin situated further from the PM exhibit high mobility and are preferentially released [28]. It would be interesting to determine the molecular signatures of these two functionally distinct pools of SGs. What is the molecular machinery of newcomer SGs? b-Cells express all three Munc18 isoforms – Munc18a [20], Munc18b [29], and Munc18c [30] – along with their cognate SNARE proteins, including Syn-1A to -4, SNAP25, and VAMPs-2 and -8. Intuitively, these distinct SM/SNARE complexes and their respective regulators should confer distinct exocytoses (Figure 2 and Box 2). Here I discuss recent work that has begun to identify the components of the exocytotic machinery of newcomer insulin SGs (Figure 2B). The SM/SNARE complex of newcomer SGs VAMP8. VAMP8, first identified as an endosomal SNARE (endobrevin) [31], was implicated in mediating exocytosis 3

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Opinion Box 2. Types of exocytosis Predocked SGs Because of the work done on the docking and priming of SVs in neurons, much attention on other secretory cells, including b-cells, has focused on predocked SGs, which, like neuronal SVs, are morphologically docked (within 50 nm) on the PM. This is due to SNARE complexes pulling SGs close to the PM where they are primed for fusion but in a predocked state such that they do not proceed to fusion but sit on the PM indefinitely, constituting the RRP. Predocked SGs undergo fusion only when triggered by an influx of Ca2+ through Ca2+ channels. It is thus believed that predocked SGs sit on Ca2+ channels bound to SNARE complexes to form ‘excitosomes’, an architecture that optimizes Ca2+ influx to sites of exocytosis [54]. Newcomer SGs Newcomer SGs move rapidly toward the PM and undergo fusion with minimal or no residence time on the PM before fusion, consistent with the emerging view that SG docking is not a prerequisite for but a temporal constraint on fusion [73]. Newcomer SGs were initially thought to come from a reserve pool that replenishes SGs for the low-level sustained phase of GSIS [21,64] after the RRP supply of primed, docked SGs that effect most of the first phase of GSIS has been depleted [5]. Newcomer SGs were later shown to also contribute to a substantial portion of first-phase GSIS [10,11]. Compound exocytosis Compound exocytosis originally referred to SGs undergoing fusion with each other in the cytosol, then fusing with the PM rapidly to empty all of their cargo. This occurs in certain types of secretory cell, such as mast cells [39], eosinophils, and basophils, that are capable of large numbers of SG–SG fusions to effect a massive release of SG contents over a short time. Compound exocytosis also occurs in slower secretory cells such as pancreatic acinar cells [71,74] secreting digestive enzymes and gastric parietal cells secreting acid, both of which secrete over several hours for food digestion. Compound exocytosis also occurs in many neuroendocrine cells that, like b-cells, secrete hormones over several hours. In these secretory cells exhibiting sustained secretion, the kinetics of compound exocytosis are slow (compared with mast cells); SGs first fuse with the PM, then become targets for further SGs to fuse with the exocytosing SGs. This has been referred to as sequential SG fusion [29,36,71]. Fusion pores between these compound SGs sometimes remain open for a long time for slow emptying of cargo [74].

in several cell types, including glucose transporter (GLUT)-4 vesicles [32] and pancreatic acinar cells [33]. Total internal reflection fluorescence microscopy (TIRFM) (which very finely tracks fusion of insulin SGs with the PM [21]) of the exocytosis of glucagon peptide-1 (GLP-1) [which activates cAMP/protein kinase A (PKA) signaling-potentiated glucose stimulation of b-cells from VAMP8 KO mice] demonstrated that VAMP8 is the v-SNARE that mediates the recruitment and exocytosis of newcomer SGs (Figure 2B), contributing to both first- and second-phase GSIS but having no effect on predocked SGs [34]. Syn-3. TIRFM also showed severe reductions in the recruitment and exocytosis of newcomer SGs in GLP-1/glucose-stimulated b-cells with no change in predocked SGs when t-SNARE Syn-3, which is abundant in insulin SGs, was depleted [35], underlying the inhibition of biphasic GSIS. Munc18b. In assessing the SM/SNARE complex formed by these cognate v-/t-SNARE proteins, Munc18b overexpression was found to facilitate the formation of Munc18b/ (Syn-3/VAMP8/SNAP25) SNARE complexes when b-cells 4

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were stimulated [29,34]. On two-photon confocal microscopy [36,37], Munc18b-depleted rat islet b-cells exhibited a 40% reduction in primary exocytosis (SG–PM), which was likely to involve mostly newcomer SGs, although this finding needs to be confirmed by TIRFM; this accounted for a similar reduction in biphasic GSIS [29]. Consistently, Munc18b overexpression amplified Ca2+-evoked insulin exocytosis [38]. Because this SM/SNARE complex is the putative mechanism for exocytosis in many secretory cells throughout the body (i.e., airway epithelial cells [39], mast cells [39], pancreatic acini [40]), the current work and future investigations in this area will have broad applications. Although Munc18b and VAMP8 also bind Syntaxin-2 to form a SM/SNARE complex [29], the exocytotic function of Syn-2 per se has not been elucidated and requires further study. Potential regulators of the newcomer SG SM/SNARE complex Munc13-1. Munc13-1 is the dominant substrate for the cellular phorbol ester diacylglycerol signal that acts as a major priming factor, assisting the activation of Syn-1A in priming predocked SVs [12] and insulin SGs [24,25,41] for fusion (Figure 1B). A later study by TIRFM of Munc13-1+/ mouse b-cells showed that Munc13-1 deficiency caused a reduction in the exocytosis of both predocked and newcomer SGs [42]. Application of phorbol ester partially rescued biphasic GSIS in Munc13-1-deficient islets by preferentially enhancing the recruitment of newcomer SGs. This suggests that newcomer SGs are more sensitive to the priming action of Munc13-1 than predocked SGs [42]. cAMP substrates RIM2a and GEF2. GLP-1 and similar compounds act predominantly on cAMP and PKA pathways in b-cells and are currently used clinically to improve GSIS in T2D patients [43]. The major exocytotic substrates for GLP-1 in b-cells are the cAMP sensor guanyl nucleotide exchange factor, isoform 2 (GEF2) (also called Epac2A), which acts downstream of RAP1 [10,44] in a PKA-independent manner, and PKA-dependent activation of Rab-3 interacting molecule, isoform 2 (RIM2a) acting on Rab3 [45]. Epac2a and RIM2a, which have been extensively studied by Seino and coworkers with KO mice, were shown to mediate the recruitment of newcomer SGs [10,45]. However, Epac2a/Rap1 signaling was postulated preferentially to influence newcomer SG recruitment to the RRP, contributing to first-phase GSIS, whereas RIM2a preferentially influenced newcomer SG recruitment from the reserve pool to effect second-phase GSIS [11]. Because GLP-1 stimulation can bypass insulin-secretory defects in T2D [43], this has prompted further work into assessing the mechanisms of exocytosis mediated by GLP-1 in rescuing GSIS in T2D. Seino’s group reported that RIM2a activates Munc13-1, enabling the Munc13-1–Syn-1A interaction that induces Syn-1A to adopt an open conformation capable of forming the SNARE complex [45] (Figure 1B). GLP-1 was found to rescue exocytosis in Munc13-1-deficient mouse b-cells (Munc13-1+/ mice with islet Munc13-1 levels reduced by 50%), which was accounted for by a large increase in recruitment of newcomer SGs that exceeded and therefore

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Opinion compensated for the reduced effects of phorbol ester acting on the residual Munc13-1 [42]. Because newcomer SGs are reported to be mediated by Syn-3 [35], this raises the possibility that GEF2–RIM2–Munc13-1 interactions [25,45] on newcomer SGs might also activate Syn-3 (rather than only Syn-1A) to form newcomer SG Munc18b/Syn-3 SNARE complexes; this requires further study. Complexins and syts. Complexins and syts are the putative proteins involved in unclamping the partial SNARE complex zipper to induce fusion [46,47] (Figure 1C,D). Of the many syts examined, syt-7 is the major Ca2+ sensor so far shown to regulate GSIS in b-cells and is suggested to act on predocked SGs [26]. However, deletion of syt-7 reduced not only first-phase but also second-phase GSIS [26], the latter implicating a reduction in newcomer SGs as well, which has not yet been demonstrated. Complexin I (which is reported to regulate GSIS in insulinoma cell lines [48]) and complexin II bind Syn-3 SNARE complexes in basophils [49]. This raises the possibility of similar actions of complexins on newcomer SGs in b-cells. Further work is required to assess whether syt-7 (or other syts) and complexins can bind and modulate newcomer SG SNARE complexes to influence the zipper mechanism leading to fusion of newcomer SGs (Figure 1C,D). Snapin, which is reported to bind SNAP25, facilitated GLP-1-potentiated GSIS [50]. Because SNAP25 is the t-SNARE partner for all exocytotic Syns [19,29], snapin might modulate Munc18b/ SNARE complex assembly to influence newcomer SG fusion. Other regulators of newcomer SGs Exocyst and Sec5. The fact that newcomer SGs are not docked but are nonetheless situated close to the PM (Figure 2A) – either very loosely tethered to would-be exocytosis sites or about to enter releasable pools – suggests that the exocyst might play a predocking role [51,52] (Figure 1A). Indeed, the octameric exocyst complex has been implicated in the delivery of SGs to be loosely tethered to the PM, a step that occurs before SG priming by the SM/SNARE complex [51,52]. Sec5 is a major component of the octameric exocyst complex that promotes RalA (a GTPase)–exocyst interactions as an initiating step in full exocyst assembly [52] (Figure 1A). Sec5 (which is localized to insulin SGs in human and rodent islet b-cells) when depleted caused a reduction in GSIS attributable to a reduction in the recruitment and exocytosis of newcomer SGs, with surprisingly little effect on predocked SGs [53]. RalA and excitosomes. Insulin SGs have been postulated to be tethered to Cavs, specifically L- (Cav1.2, Cav1.3) and perhaps R- (Cav2.3) type a-subunits via SNARE complexes, in excitosomes [54,55] (Figure 1B), where maximal Ca2+ influx occurs to effect first- or second-phase GSIS, respectively [56,57]. RalA depletion of INS-1 cells and rat b-cells caused a reduction in Ca2+ currents arising from L(Cav1.2, Cav1.3) and R- (Cav2.3) type channels that was restored by re-expression of RalA [58]. Surprisingly, instead of binding the pore-conducting a-subunits of these Cavs [59], RalA coprecipitated with the common a2d-1 auxiliary subunit that binds these Cav a-subunits. RalA

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depletion inhibited trafficking of a2d-1 to insulin SGs, suggesting that RalA plays a role in tethering predocked and newcomer SGs to L- and R-type Cavs, with exocytosis at these sites culminating in first- or second-phase GSIS, respectively [58]. Actin cytoskeleton. Glucose stimulation induces remodeling of the actin cytoskeleton, which enables insulin SGs to mobilize to t-SNARE proteins, affecting predominantly second-phase secretion [60]; the latter suggests these to be newcomer SGs. However, the SM/SNARE complex examined in these studies was postulated to be Munc18c/ Syn-4 [60] (see below); more studies are required to assess whether the actin cytoskeleton interacts with the newcomer SM/SNARE complex. Munc18c/Syn-4 complex. Munc18c and Syn-4 have been well studied in their role in GLUT-4 vesicle exocytosis in adipocytes and muscle and to a lesser extent in insulin exocytosis (reviewed in [19]). Heterozygous Munc18c+/ mice showed a reduction in second-phase GSIS [30], whereas Syn-4+/ mice showed impaired first-phase GSIS [61], which seems inconsistent because these are partner proteins expected to influence the same exocytotic machinery and fusion events. Unequivocal elucidation of the endogenous function of the Munc18c/Syn-4 complex will require total ablation of these exocytotic proteins, preferably in a bcell-specific manner, because perturbed glucose homeostasis caused by Munc18c and Syn-4 ablation in insulinsensitive tissues [62,63] is likely to have had secondary effects on b-cell insulin secretion. The Munc18c/Syn-4 complex was suggested to mediate fusion of predocked insulin SGs [19], redundant to Munc18a/Syn-1A [20]. The precise exocytotic events mediated by the Munc18c/ Syn-4 SM/SNARE complex nevertheless remains to be definitively demonstrated by exocytosis imaging. The fact that Munc18c depletion affected second-phase GSIS [30] and Syn-4 overexpression increased both first- and secondphase GSIS [61] suggests that the Munc18c/Syn-4 SNARE complex might also affect newcomer SGs. Why is priming of newcomer SGs faster than that of predocked SGs? This is an interesting question. It is likely that newcomer and predocked SGs are not morphologically distinct, but somehow become functionally segregated, in part by the assembly of distinct SM/SNARE complexes that select entry into either the RRP or the reserve pool, which seem to be temporally assigned to first- and second-phase GSIS, respectively. Because docking of SVs onto the PM is followed by priming to render the SVs extremely receptive to triggered release [2] (Figure 1), it seems counterintuitive that, as insulin SGs approach and dock onto the PM, they paradoxically lose (not gain) fusion competence [28,64] and that this worsens the longer they sit on the PM [27]. Insulin SGs more distant from the PM, probably mostly newcomer SGs, exhibit the following features of increased fusion competence: (i) higher sensitivity to the priming factor Munc13-1 [42], the cAMP effectors GEF2 and Rim2a [11], and the SG tethering factor Sec5 [53]; (ii) higher Ca2+ sensitivity [64]; (iii) younger, with newly synthesized 5

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Box 3. Outstanding questions 2+

 How does a SM/SNARE complex of predocked SGs lose its Ca sensitivity and fusion competence in b-cells but gain or retain these properties in neurons? Could this be due to differences in the architecture of the fusion apparatus between these two cell types, such as fusion clamps, Ca2+ sensors, or priming factors acting on their predocked SG/SV SNARE complexes?  Why is the priming of newcomer SG SM/SNARE complexes so rapid in effecting fusion, such that newcomer SGs do not have to dock on the PM before fusion? What are the additional regulators and cell signals acting on the common Munc18b/Syn-3 SNARE complex that predestine SGs to become fast-moving, fast-fusing newcomer SGs versus the slower compound fusion? Could the slow compound fusion observed as sequential SG fusion in bcells compared with the massive and rapid compound exocytosis of mast cells be due to reduced abundance of this SM/SNARE complex or priming factors, differences in Ca2+ sensors (i.e., fast versus slow) or local Ca2+ release, or, more intriguingly, involvement of undefined fusion clamps, inhibitory SNAREs, or components of the actin cytoskeletal mesh where the reserve SG pool purportedly resides?  Can we apply these insights into the exocytotic machinery to tailor novel treatment strategies for diseases pertaining to secretory cells that exhibit an excess (i.e., mast cells in anaphylactic reactions) or deficiency (b-cells in diabetes) of secretion?

insulin [27,28]; and (iv) greater motility [28]. I speculate that some of these features may preferentially promote the formation of newcomer SG SM/SNARE complexes and that their loss during the approach of SGs to dock on the PM might divert the SGs toward formation of predocked SG SM/SNARE complexes. The less Ca2+-sensitive predocked SGs (suggesting that the SM/SNARE complex is less Ca2+ sensitive) have to land on Cavs (L-type), forming excitosomes [54], to obtain enough Ca2+ to effect fusion, whereas more Ca2+-sensitive newcomer SGs (suggesting that the SM/SNARE complex is more Ca2+ sensitive) could respond to bulk Ca2+ influx arising from R-type Cavs [57,64]. This might in part explain why, in T2D, predocked insulin SGs are far more likely to losing fusion competence altogether than newcomer SGs [7]. The question raised here is how does the SM/SNARE complex of predocked SGs in b-cells lose Ca2+ sensitivity and fusion competence but gain or retain these properties in predocked SVs in neurons (Box 3)? Conceptually (Figure 1C,D) it would seem that the partial fusion zipper of predocked insulin SGs may be in a different architecture that easily becomes ‘stuck’ compared with the predocked SV fusion zipper, which zippers reliably and rapidly. Could these differences in the architecture of the fusion apparatus between the two cell types be due to differences in fusion clamps, Ca2+ sensors, or priming factors (Munc13, Munc18) acting on the predocked SG/SV SNARE complex? Compound exocytosis in b-cells: too little to care about? Although compound exocytosis (Box 2) in b-cells (Figure 2C) occurs rarely during GSIS, is limited to the fusion of two or three SGs, and accounts for only 2–3% of total secretion [29,36,37], GLP-1 potentiation via cAMP signaling [29,65] or cholinergic-evoked massive elevation of cytosolic Ca2+ [66] could upregulate compound exocytosis, and the latter has been shown to increase GSIS by >40% [66]. This included an increased frequency of and 6

number of SGs in each compound fusion [29,65,66], which is reminiscent of the compound exocytosis occurring in mast cells [67]. In mast cells, compound exocytosis is extremely rapid with very extensive multi-SG fusions, accounting for the high secretory efficiency that effects immediate and massive release of SG contents. Compound fusion in b-cells occurs at a much slower rate than in mast cells even when upregulated, often appearing in sequential fashion whereby an insulin SG first fuses to the PM, then a second SG comes from behind to fuse with the first, and so on [29,36,37]. This enables a more metered, reduced, but sustained release of insulin over several hours. This relatively infrequent and inefficient exocytosis in b-cells makes it an excellent model for determining the molecular machinery that could upregulate compound exocytosis [20]; the insights gained from such studies are not inconsequential and have broad implications. Increasing the efficiency of compound exocytosis in b-cells to treat diabetes and applying those insights to reduce the efficiency of compound exocytosis in mast cells for the treatment of anaphylaxis and asthma have obvious therapeutic implications. Although little is known about the exocytotic machinery of compound exocytosis, new findings are emerging. The first step is to determine the SM/SNARE complex for compound SG fusion. Increased frequency and extent of compound fusion in b-cells could be effected by overexpression of Munc18b (fusion of up to six to eight SGs) [29] or Syn-3 (up to five SGs) [35]. These are the same Munc18b and Syn-3 that are reported to increase recruitment of newcomer SGs [29,35]. SNAP25, unlike syntaxin and VAMP, is not PM anchored and has been shown to translocate from the PM to sequentially fusing insulin SGs [36]. This suggests that SNAP25 may translocate from binding Syn-1A on the PM to binding Syn-3 on sequentially fusing SGs. With these limited insights into compound exocytosis, what further questions can be asked? First, what is the vSNARE? If it is VAMP8, major overlap potentially exists between the molecular signatures of compound SG and newcomer SG fusions [29,35]. The transition of SGs engaged in compound fusion to the much more rapid kinetics of exocytosis of newcomer SGs raises many questions. What are the additional regulators and cell signals acting on the common Munc18b/Syn-3 SNARE complex that predestine SGs to become newcomer SGs rather than undergo compound fusion (Box 3)? Could the slower sequential SG fusion in b-cells (compared with mast cells) be due to reduced abundance of the SM/SNARE complex or priming factors, differences in Ca2+ sensors (i.e., fast versus slow [68]) or local Ca2+ release, or, more intriguingly, involvement of undefined fusion clamps (i.e., tomosyns [69]), inhibitory SNAREs (which prevent rather than promote fusion) [70], or components of the actin cytoskeletal mesh where the reserve SG pool resides [60]? Concluding remarks and future perspectives This Opinion discusses recent work that has begun to decipher the components of the exocytotic machinery of newcomer SGs, which account for a substantial portion of first-phase and all of second-phase GSIS. I suggest that a complex different from that mediating the fusion of

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Opinion predocked granules is a critical component of this exocytotic machinery, which I propose to comprise Munc18b activation of the VAMP8/Syn-3/SNAP25 SNARE complex. More work is required to determine why the priming of this newcomer SG SM/SNARE complex is so rapid in effecting fusion without having to dock on the PM. This is of relevance to many neuroendocrine cells in the body that exhibit similar sustained secretion, probably employing the same newcomer SG SM/SNARE complex. Comparative assessment of priming (or loss of priming) of predocked SGs between bcells and neurons, and the distinct efficiencies of compound fusion involving b-cells and secretory cells that exhibit more- (mast cells) [39,67] or less- (pancreatic acinar cells) [40,71] rapid fusion kinetics, yet employ similar SM/SNARE complexes, will reveal important insights into the modulation of these fusion events. Such insights could help tailor novel treatment strategies for diseases pertaining to each of these cell types (Box 3). In T2D, islet levels of the exocytotic proteins of predocked insulin SGs are reduced by 70–90% in rodents [8,72] and humans [9], which underlies the nearcomplete loss of exocytosis of predocked SGs (Figure 2A). Because GLP-1, acting on newcomer SGs (Figure 2B) and compound fusion (Figure 2C) [65], can compensate for and rescue insulin secretory deficiency in T2D [43], future studies could be focused on how GLP-1 signaling facilitate the assembly of these SM/SNARE complexes (and associated proteins) to effect their zipper mechanism and achieve fusion (Figure 1). Acknowledgments This work was supported by grants to H.Y.G. from the Canadian Institute of Health Research (MOP 86544 and MOP 69083).

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