Fusion pore regulation by Epac2/cAMP controls cargo release during insulin exocytosis

Regulated exocytosis establishes a narrow fusion pore as the initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of larger peptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in type-2 diabetes and neurodegenerative disease. Here we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation leads to pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- mice. Conversely, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.


Introduction
Insulin is secreted from pancreatic β-cells and acts on target tissues such as muscle and liver to regulate blood glucose. Secretion of insulin occurs by regulated exocytosis, whereby secretory granules containing the hormone and other bioactive peptides and small molecules fuse with the plasma membrane. The first aqueous contact between granule lumen and the extracellular space is a narrow fusion pore (upper limit 3 nm 1 ) that is thought to consist of both lipids and proteins 2,3 . At this stage, the pore acts as a molecular sieve that allows release of small transmitter molecules such as nucleotides and catecholamines, but traps larger cargo [4][5][6][7] .
Electrophysiological experiments have shown that the fusion pore is short-lived and flickers between closed and open states, suggesting that mechanisms exist that stabilize this channel-like structure and restrict pore expansion 6,[8][9][10] . The pore can then expand irreversibly (termed full fusion), which leads to mixing of granule and plasma membrane and release of the bulkier hormone content 4,5,11 . Alternatively, the pore can close indefinitely to allow the granule to be retrieved, apparently intact, into the cell interior (termed kiss&run or cavicapture) 4,6,[12][13][14] . Estimates in β-cells suggest that 20-50% of all exocytosis in β-cells are transient kiss&run events that do not lead to insulin release 4,6 . However, kiss&run exocytosis contributes to local signaling within the islet because smaller granule constituents, such as nucleotides, glutamate or GABA, are released even when the fusion pore does not expand. Within the islet, ATP synchronizes -cells 15 , and has both inhibitory 16,17 and stimulatory 18 effects on insulin secretion. Within the islet, ATP suppresses glucagon release from αcells 19 , and activates macrophages 20 . Interstitial GABA leads to tonic GABA-A receptor activation and α-cell proliferation 21,22 , and glutamate stimulates glucagon secretion 23 .
Regulation of fusion pore behavior is not understood mechanistically, but several cellular signaling events affect both lifetime and flicker behavior. Elevated cytosolic Ca 2+ and activation of protein kinase C (PKC) accelerate pore expansion and full fusion 24,25 , while elevated extracellular Ca 2+ or cAMP, and PI(4,5)P2 favor longer pore lifetimes and kiss&run exocytosis 6,9,[26][27][28] . Both myosin and the small GTPase dynamin are involved in fusion pore restriction [29][30][31][32][33] , and assembly of filamentous actin promotes fusion pore expansion 34 , suggesting a link to endocytosis and the cytoskeleton. In β-cells of type-2 diabetics, upregulation of amysin leads to decreased insulin secretion because fusion pore expansion is impaired 35 , and the Parkinson's related protein α-synuclein promotes fusion pore dilation in chromaffin cells and neurons 36 , thus providing evidence for altered fusion pore behavior in human disease.
3 Inadequate insulin secretion in type-2 diabetes (T2D) is treated clinically by two main strategies. First, sulfonylureas (e.g., tolbutamide and glibenclamide) close the K ATP channel by binding to its regulatory subunit SUR1, which leads to increased electrical activity and Ca 2+ -influx that triggers insulin secretion 37 . Sulfonylureas are given orally and are first line treatment for type-2 diabetes in many countries. Second, activation of the receptor for the incretin hormone glucagon-like peptide 1 (GLP-1) raises cytosolic [cAMP] and thereby increases the propensity of insulin granules to undergo exocytosis. Both peptide agonists of the GLP-1 receptor (e.g., exendin-4) and inhibitors of the GLP-1 peptidase DPP-4 are used clinically for this purpose. The effect of cAMP on exocytosis is mediated by a protein-kinase A (PKA) dependent pathway, and by Epac2, a guanine nucleotide exchange factor for the Ras-like small GTPase Rap 38 that is recruited to insulin granule docking sites 39 . Epac2 is also thought to be activated by sulfonylureas 40 , which may underlie some of their effects on insulin secretion.
Here we have studied fusion pore regulation in pancreatic β-cells, using high resolution live-cell imaging. We report that activation of Epac2, either through GLP1-R/cAMP signaling or via sulfonylurea, restricts expansion of the insulin granule fusion pore by recruiting dynamin and amisyn to the exocytosis site. Activation of this pathway by two classes of antidiabetic drugs therefore hinders full fusion and insulin release, which is expected to reduce their effectiveness as insulin secretagogues.

cAMP-dependent fusion pore restriction is regulated by Epac but not PKA
To monitor single granule exocytosis, human pancreatic β-cells were infected with adenovirus encoding the granule marker NPY-Venus and imaged by TIRF microscopy. Exocytosis was evoked by local application of a solution containing 75 mM K + , which leads to rapid depolarization and Ca 2+ influx. Visually, exocytosis of individual granules occurred with two phenotypes. In the first, termed full fusion, fluorescence of a granule that was stably situated at the plasma membrane suddenly vanished during the stimulation (in most cases within <100 ms; Fig 1a-c, left panels). Since the EGFP label is relatively large (3.7 nm vs 3 nm for insulin monomers) this is interpreted as rapid pore widening that allowed general release of granule cargo. It is likely that most of these events involved the collapse of the granule membrane into the plasma membrane. In the second type, the rapid loss of the granule marker was preceded by an increase in its fluorescence that could 4 last for several seconds (flash events , Fig 1a-c, right panels). We and others have previously shown 41 that this reflects neutralization of the acidic granule lumen and dequenching of the EGFP-label, before the labeled cargo is released. Since this neutralization occurs as the result of proton flux through the fusion pore, the fluorescence timecourse of these events can be used to quantitatively study fusion pore behavior.
In the following, we will report two parameters that reflect fusion pore behavior, the fraction of exocytosis events with flash phenotype (indicating restricted pores, about 40% in control conditions ; Fig 1d), and fusion pore lifetimes estimated by fitting a discontinuous function to the fluorescence timecourse (see Fig 1c, lines and Fig 1e). The distribution of the lifetimes followed a mono-exponential function and was on average 0.87±0.12 s (186 granules in 26 cells) in control conditions (Fig 1e). We limited our analysis to granules that eventually released their peptide content, true kiss&run exocytosis events, corresponding to infinite pore dwell-times. Such events are increased by elevated cAMP 6,9 and likely other conditions that stabilize the fusion pore. Indeed, when forskolin (2 µM; fsk) was added to the bath solution we observed a 2-fold increase of exocytosis rate (Fig 1f), a 3-fold increase of fusion pore lifetimes (Fig 1e), and a nearly doubled fraction of events with restricted fusion pores (Fig 1d,f). The GLP-1 agonist exendin-4 (10 nM; Ex4) had comparable effects (Fig 1d-f). Effects similar to those observed for human -cells (Fig 1) were observed in the insulin secreting cell line INS-1 (Suppl Fig 1).
The effect of fsk on fusion pore behavior was mimicked by the specific Epac2 agonist S223 42 . Incubation with S223-acetomethoxyester (5µM) increased the fraction of flash events by 60% (Fig 1d), tripled average lifetimes (Fig 1e) and doubled the event frequency (Fig 1f); the effects of fsk and S223 were not additive. In contrast, the Epac-inhibitor ESI-09 decreased the exocytosis rate in the presence of fsk by 90% (Fig 1f), and the average fusion pore lifetime and the fraction of flash events were both reduced by 60% (Fig 1d-e). PKA inhibition with Rp8-Br-cAMPS 43 decreased exocytosis by 40% but not the fraction of flash events, although average fusion pore lifetimes were increased compared with fsk alone (Fig 1e). The results indicate that Epac rather than PKA is responsible for cAMP-dependent fusion pore regulation. Paradoxically, Epac activation increases the rate of exocytosis but slows the rate of peptide release from individual granules.

5
We studied next the effect of Epac2 overexpression on fusion pore regulation. INS-1 cells were cotransfected with EGFP-Epac2 and NPY-tdmOrange2 and fluorescence was recorded simultaneously in both color channels. Epac2 overexpression had no effect on the overall exocytosis rate in either absence or presence of fsk (Fig 2a), but increased the rate of flash events (Fig 2b-c), supporting our finding, based on manipulation of the endogenous Epac2 activity, that Epac2 is involved in fusion pore regulation (Fig 1). Fusion pore lifetimes in cells overexpressing Epac2 increased 3-fold in the absence of fsk, and were similar to controls in presence of fsk (Fig 2d). This indicates that a high Epac concentration can achieve sufficient activity to affect insulin secretion even at basal cAMP level, likely because cAMP acts in part by increasing the Epac concentration at the plasma membrane 39 .

ATP release is accelerated upon Epac inhibition
To test if cAMP-dependent fusion pore restriction affects release of small transmitter molecules, we quantified nucleotide release kinetics from individual granules using patch clamp electrophysiology. The purinergic receptor cation channel P2X 2 , tagged with RFP (P2X 2 -RFP), was expressed in INS-1 cells as an autaptic nucleotide sensor (Fig 3a). The cells were voltage-clamped in whole-cell mode and exocytosis was elicited by including a solution with elevated free Ca 2+ (calculated 600 nM) in the patch electrode. In this configuration, every exocytosis event that co-releases nucleotides causes an inward current spike, similar to those observed by carbon fiber amperometry (Fig 3a- In the presence of cAMP, but not cAMP+ESI-09, they were markedly widened as indicated by on average 20% longer half-widths (Fig 3d), 30% longer decay constants (τ ,   Fig 3e), and 40% slower rising phases (25-75% slope, Fig 3f), compared with control. This indicates that nucleotide release is slowed by cAMP, likely because of changed fusion pore kinetics. Since the effect is blocked by ESI-09, we conclude that the cAMP effect probably is mediated by Epac.

cAMP-dependent fusion pore regulation is absent in Epac2 -/β-cells
Since ESI-09 blocks all Epac isoforms 44 , we characterized fusion pore behavior in isolated β-cells from Epac2 -/mice, in which all subforms of Epac2 are deleted 45 . Cells from WT or Epac2 -/mice were infected with 6 adenovirus encoding the granule marker NPY-tdmOrange2 and challenged with 75 mM K + (Fig 4a-b). In the absence of forskolin, exocytosis was significantly slower in Epac2 -/cells than WT cells, and the fraction of flashassociated exocytosis events was five-fold lower (Fig 4c-e). This was paralleled by strikingly shorter fusion pore life-times in Epac2 -/cells compared with WT (Fig 4f). As expected, forskolin increased both exocytosis (Fig 4e) and the fraction of flash events (Fig 4c) of WT cells. In contrast, exocytosis was not accelerated by forskolin in Epac2 -/cells, and the fusion pore lifetimes and fraction of flash events were similar with or without forskolin . We conclude therefore that the effects of cAMP on fusion pore behavior are mediated specifically by Epac2.

Sulfonylureas delay fusion pore expansion through the same pathway as cAMP
Sulfonylureas have been reported to activate Epac 40 , in addition to their classical role that involves the sulfonylurea receptor (SUR). We tested therefore if sulfonylureas (SUs) could affect fusion pore behavior. INS-1 cells expressing NPY-EGFP were tested with three types of SUs, with different relative membrane permeability (tolbutamide<glibenclamide<gliclazide). In addition, diazoxide (200 µM) was present to prevent electrical activity. Exocytosis was not observed under these conditions, but could be triggered by local application of elevated K + (75 mM). In the absence of fsk, all three sulfonylureas accelerated K + -stimulated exocytosis 2-3fold over that observed in control (Fig 5b, left), which is consistent with earlier findings that sulfonylureas augment insulin secretion via intracellular targets 46,47 . This effect was entirely due to an increase in flashassociated exocytosis events (Fig 5b-c) and the average fusion pore lifetime increased accordingly in the presence of sulfonylurea (Fig 5d). In the presence of fsk, which strongly stimulated both flash-associated and full fusion exocytosis in absence of sulfonylurea (Fig 5b-c, middle), the addition of sulfonylureas decreased strongly full-fusion exocytosis, but had no additional effect on the frequency of flash-associated events (Fig 5b, middle). Accordingly, fusion pore lifetimes were elevated compared with control (no fsk), and only marginally longer than with fsk alone (Fig 5d, right). Similar results were obtained in human β-cells, where glibenclamide increased exocytosis in the absence of fsk (P=0.01, n=13 cells) but not in its presence (P=0.80, n=7 cells; data not shown). The data indicate that sulfonylureas restrict fusion pore expansion through the same intracellular pathway as cAMP, which may counteract their stimulating effect on exocytosis by preventing or delaying peptide release.

7
To test how sulfonylureas affect exocytosis through direct stimulation of SUR1 and K ATP channels, we applied sulfonylureas also acutely, to avoid their accumulation in the cytosol (Fig 5e). Reduced diazoxide (50 µM) prevented glucose-dependent exocytosis but still allowed acute stimulation of exocytosis by sulfonylureas.
Under these conditions, for all three sulfonylureas the fraction of flash-associated exocytosis events (Fig 5f-g) and the fusion pore lifetimes (Fig 5h) was similar to control (stimulation with elevated K + ). Taken together, the data suggest that sulfonylureas must enter the cytosol to affect fusion pore behavior, and that this effect is not mediated by the plasma membrane SUR. We excluded the possibility that sulfonylureas affect the fluorescence signal indirectly, by altering granule pH (Suppl Fig 2). Moreover, an EGFP-tagged SUR1 (EGFP-SUR1) expressed in INS-1 cells did not localize to exocytosis sites or affect fusion pore behavior (Suppl Fig 3). We therefore conclude that sulfonylureas affect fusion pore behavior through Epac2.

Dynamin and amisyn-controlled restriction of the fusion pore is cAMP-dependent
The proteins dynamin and amisyn 30,35 have previously been implicated in fusion pore regulation in β-cells.
To understand how these proteins behave around the release site EGFP-tagged dynamin1 (Fig 6a) or mCherrytagged amisyn (Fig 6b) were expressed together with a granule marker in INS-1 cells, and exocytosis stimulated with elevated K + . In the presence of fsk both of the two fluorescent proteins were recruited to the granule site during membrane fusion (Fig 6c,f), and their overexpression markedly prolonged the fusion pore lifetimes (Fig   6d;g) and increased the fraction of flash-associated exocytosis events (Fig 6e,h). In the absence of fsk, overexpression of the two proteins had no effect on fusion pore behavior and amisyn (but not dynamin1) was recruited to the exocytosis site (Fig 6i-n). The data suggest that dynamin1 and amisyn are acutely recruited to the exocytosis site, where they contribute to fusion pore restriction in a cAMP-dependent manner.

Discussion
cAMP-dependent signaling restricts fusion pore expansion and promotes kiss&run exocytosis in β-cells 9 and neuroendocrine cells 27,48 (but see 49 ). We show here that the cAMP-mediator Epac2 orchestrates these effects by engaging dynamin and perhaps other endocytosis-related proteins at the release site (Fig 7). Since the fusion pore acts as a molecular sieve, the consequence is that insulin and other peptides remain trapped 8 within the granule, while smaller transmitter molecules with para-or autocrine function are released 4,6,7,13,[50][51][52] . Incretin signaling and Epac activation therefore delays, or altogether prevents insulin secretion from individual granules, while promoting paracrine intra-islet communication that is based mostly on release of small transmitter molecules.
Paradoxically, two clinically important classes of antidiabetic drugs, GLP-1 analogs and sulfonylureas, activate Epac in β-cells and caused restriction of the fusion pore. Sulfonylureas have long been known to stimulate insulin secretion by binding to SUR1, which results in closure of K ATP channels and depolarization 37 .
The drugs also accelerate PKA-independent granule priming in β-cells, which may involve activation of intracellularly localized SUR1 53 .
Our data indicate that sulfonylureas exert a third mode of action that leads to the restriction of the fusion pore and therefore limits insulin release. Two pieces of evidence suggest that SUR1 is not involved in the latter. First, acute exposure to sulfonylureas had no effect on fusion pore behavior, although it blocks K ATP channels (indicating SUR1 activation). Only long-term exposure to sulfonylurea resulted in restricted fusion pores, likely because it allowed the drugs to enter the cytoplasm. Second, we could not detect enrichment of SUR1 at the granule release site, which precludes any direct role of the protein in fusion pore regulation. Sulfonylurea compounds have been shown to allosterically stabilize the cAMP-dependent activation of Epac 54,55 . Our finding that sulfonylurea caused fusion pore restriction in the absence of forskolin indicates that basal cAMP concentrations are sufficient for this effect. It can further be speculated that the competing stimulatory (via exocytosis) and inhibitor effects (via the fusion pore) of sulfonylureas on insulin secretion, contribute to the reduction in sulfonylurea effectiveness with time of treatment. Moreover, long term treatment with GLP-1 analogs disturbs glucose homeostasis 56 , and combination therapy of sulfonylurea and DPP4 inhibitors (that elevate cAMP) has been shown to lead to severe hypoglycemia 57 , an effect that likely depends on Epac 58 .
Epac mediates the PKA-independent stimulation of exocytosis by cAMP 59 . This effect is rapid 53 , suggesting that Epac may be preassembled at the site of the secretory machinery. Indeed, Epac concentrates at sites of docked insulin granules 39 , and forms functionally relevant complexes with the tethering proteins Rim2 and Piccolo 60,61 . However, the amount of Epac2 present at individual release sites did not correlate with fusion pore behavior, which may indicate that the protein acts indirectly by activating or recruiting other proteins.
Indeed, we show here that recruitment of two other proteins, dynamin and amisyn, depends on cAMP.
Another known target of Epac is the small GTPase Rap1, for which Epac is a guanine nucleotide exchange 9 factor (GEF). Rap1 is expressed on insulin granules and affects insulin secretion both directly 62 , and by promoting intracellular Ca 2+ -release following phospholipase-C activation 63 . By altering local PIP 2 levels, the latter could affect both exocytosis through C2 domain proteins such as Munc13 64 , and PIP 2 -dependent recruitment of dynamin to the plasma membrane 65 .
An unresolved question is whether pore behavior is controlled by mechanisms that promote pore dilation, or that instead prevent it. Dynamin causes vesicle fission during clathrin-dependent endocytosis 66 , and since dynamin is present at the exocytosis site and required for the kiss&run mode 29,30,67 , it may have a similar role during transient exocytosis. An active scission mechanism is also suggested by the finding that granules loose some of their membrane proteins during transient exocytosis 68 . Capacitance measurements have shown that fusion pores initially flicker with conductances similar to those of large ion channels, before expanding irreversibly 69 . This could result from pores that are initially stabilized through unknown protein interactions and that eventually give way to uncontrolled expansion. However, scission mechanisms involving dynamin can act even when the pore has dilated considerably beyond limit of reversible flicker behavior 4,14,51 , and even relatively large granules retain their size during fusion-fission cycles 6,8 . Separate mechanisms may therefore operate, one that prevents pore dilation by actively causing scission, similar to the role of dynamins in endocytosis, and another by shifting the equilibrium between the open and closed states of the initial fusion pore. Curvature-sensitive proteins are particularly attractive for such roles since they could accumulate at the neck of the fused granule; such ring-like assemblies that have indeed been observed for the Ca 2+ -sensor synaptotagmin 70 . Active pore dilation has also been proposed to be driven by crowding of SNARE proteins 71 and α-synuclein 36 .
Insulin granules contain millimolar concentrations of a variety of small transmitter molecules (serotonin, ATP, GABA) and ions (zinc, calcium) that, once released, result in biologically meaningful signals within the islet 72 . GABA release activates both autocrine and paracrine signaling in islets. Activation of GABA-A Cl --channels in α-cells inhibits glucagon secretion 73,74 , whereas depolarization in β-cells leads to enhanced insulin secretion 72,75 . Tonic GABA signaling is important for the maintenance of β-cell mass, by promoting α-to-β-like cell conversion 22,75 and by suppressing T-cell dependent autoimmune responses 75,76 . Changes in the local release of GABA could therefore play an important role in the development of type-1 diabetes. Interestingly, not all of a β-cell's granules contain GABA 77 , either due to differential expression of GABA transporters or, more likely, kiss&run exocytosis that results in granules without GABA content. The latter effect is well-documented in chromaffin cells, where catecholamine content varies considerably amongst granules 78 . The release of ATP and other nucleotides from β-cells acts as autocrine positive feedback signal, leading to depolarization and intracellular Ca 2+ -release that result in enhanced insulin secretion [79][80][81] , but also negative effects have been reported 16,17 . Paracrine ATP signaling coordinates Ca 2+ signaling among -cells 15 . In dogs, purinergic signaling stimulates secretion of somatostatin from δ-cells 82 . Other likely targets of ATP signaling are the islet vasculature, and islet-resident macrophages as part of the immune system 20 . The latter may play a role for the autoimmune attack in type-1 diabetes, the pro-inflammatory milieu associated with type-2 diabetes 83 , and βcell development during embryogenesis 84 . Finally, human β-cells also secrete serotonin (5hydroxytryptamine), which inhibits glucagon secretion from α-cells 85 . The effects of Epac activation by cAMP and sulfonylureas on fusion pore behavior are therefore likely to have profound consequences for intra-islet signaling.

Acknowledgement
We thank J. Saras, P.-E. Lund, and A. Thonig (Uppsala University) for expert technical assistance, and D.  S.B. conceived the study. S.B. and A.G. wrote, and all authors critically reviewed the manuscript.

Declaration of Interests
The authors declare no competing interests.            , Kolmogorov-Smirnov test). 213 (CTR) and 320
(i) As in c, but without forskolin present; n=37 flash events, n=39 full fusion events.
(j-k) As in (d-e), but for 38 (CTR) and 76 (dynamin1) events in the absence of forskolin (l) As in f, but without forskolin present; n=65 flash events, n=73 full fusion events. Collagenase P (1 mg/ml) and cut into small pieces before mechanical dissociation (7 min at 37 °C). BSA was added immediately and islets were washed 3X with ice cold Ca5 with BSA. Islets were dispersed into single cells using Ca 2+ -free cell dissociation buffer (supplemented with 10% (v/v) trypsin) and gentle agitation.
The cells were plated onto 22-mm polylysine-coated coverslips, and were transduced the next day using adenovirus (human & mouse cells) or transfected the same day with plasmids (INS1 cells, using Lipofectamine2000, Invitrogen) encoding the granule markers NPY-Venus, NPY-EGFP or NPY-tdOrange.

Constructs
The open reading frame of human amisyn (NM_001351940.1) was obtained as a synthetic DNA fragment (Eurofins, Germany) and was cloned into pCherry2 C1 (Addgene, plasmid nr 54563) by seamless PCR cloning.

Image analysis
Exocytosis events were identified manually based on the characteristic rapid loss of the granule marker fluorescence (1-2 frames). Events were classified as flash events if they exhibited apparent increase in the fluorescence signal before the rapid loss of the granule fluorescence. The fusion pore lifetimes were obtained for both types of events by non-linear fitting with a discontinuous function in Origin as described previously 41 .
Protein binding to the release site (F/S) was measured as described previously 88 .

Electrophysiology
ATP release was measured in INS1 cells expressing RFP-tagged P2X 2 receptor 4 . Cells were voltage-clamped in whole-cell mode using an EPC-9 amplifier and PatchMaster software (Heka Elektronik, Lambrecht, Germany) with patch-clamp electrodes pulled from borosilicate glass capillaries that were coated with Sylgard close to the tips, and fire-polished (resistance 2-4 MΩ). The free [Ca 2+ ] was calculated to be 600 nM (WEBMAXC standard) and elicited exocytosis that was detected as P2X 2 -dependent inward current spikes. Currents were filtered at 1 kHz and sampled at 5 kHz. Spike analysis was performed using automated program for amperometric recordings in IGOR Pro 89 , with the threshold set at eight times the RMS noise during event-free section of recording.

Statistics
Data are presented as mean ± SEM unless otherwise stated. Statistical significance was tested (unless otherwise stated) using Students t-test and is indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001).
Ratios of flash events were tested using Mann-Whitney u-test and fusion pore lifetimes were tested with Kolmogorov-Smirnov test.