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Comparative Study
. 2011 Oct 5;31(40):14250-63.
doi: 10.1523/JNEUROSCI.1835-11.2011.

Piccolo Regulates the Dynamic Assembly of Presynaptic F-actin

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Free PMC article
Comparative Study

Piccolo Regulates the Dynamic Assembly of Presynaptic F-actin

Clarissa L Waites et al. J Neurosci. .
Free PMC article

Abstract

Filamentous (F)-actin is a known regulator of the synaptic vesicle (SV) cycle, with roles in SV mobilization, fusion, and endocytosis. However, the molecular pathways that regulate its dynamic assembly within presynaptic boutons remain unclear. In this study, we have used shRNA-mediated knockdown to demonstrate that Piccolo, a multidomain protein of the active zone cytomatrix, is a key regulator of presynaptic F-actin assembly. Boutons lacking Piccolo exhibit enhanced activity-dependent Synapsin1a dispersion and SV exocytosis, and reduced F-actin polymerization and CaMKII recruitment. These phenotypes are rescued by stabilizing F-actin filaments and mimicked by knocking down Profilin2, another regulator of presynaptic F-actin assembly. Importantly, we find that mice with a targeted deletion of exon 14 from the Pclo gene, reported to lack >95% of Piccolo, continue to express multiple Piccolo isoforms. Furthermore, neurons cultured from these mice exhibit no defects in presynaptic F-actin assembly due to the expression of these isoforms at presynaptic boutons. These data reveal that Piccolo regulates neurotransmitter release by facilitating activity-dependent F-actin assembly and the dynamic recruitment of key signaling molecules into presynaptic boutons, and highlight the need for new genetic models with which to study Piccolo loss of function.

Figures

Figure 1.
Figure 1.
Illustration of how percentage increase in EGFP-Synapsin1a dispersion was calculated. A, Graph depicting time course of EGFP-Syn dispersion for two sets of experiments from 2 different days, #1 (black) and #2 (red). Each curve represents the averaged values from >100 puncta. Note the pronounced week-to-week differences in absolute extent of EGFP-Syn dispersion observed (black curves vs red curves). Note also that wild-type curves (squares) always exhibit less dispersion relative to Pclo28 curves (triangles). The first step, indicated by gray “1),” for calculating percentage increase in EGFP-Syn dispersion (Pclo28 vs wild type) was to average the last five time points of each curve from a given day (values in gray boxes), producing the values shown. These values were then put into the equation [((avg Ft = 70–90 (Pclo28)/avg Ft = 70–90 (wt)) − 1)] × 100 (see Materials and Methods). B, Column graph depicting the final values for percentage increase in EGFP-Syn dispersion (Pclo28 vs wt) and their average (black line). Note that these ratioed values are reasonably similar when compared across different weeks (red circle vs black circle). C, Graph depicting time course of EGFP-Syn dispersion for one set of experiments from a single day, demonstrating how percentage increase in dispersion was calculated when there were multiple coverslips for each condition (wt 1, 2, 3 and Pclo28 1, 2, 3). The last five time points for all wild-type curves (black; in gray box) were averaged together to give a single value (−0.28). The last five time points for each Pclo28 curve (blue) were averaged to give a separate value for each coverslip. These values were each put into the above equation and plotted individually on the column graph (D).
Figure 2.
Figure 2.
Pclo28 phenotypes in dissociated hippocampal neurons. A, EGFP-Synapsin1a (EGFP-Syn) dispersion induced by 10 Hz, 90 s stimulation (at t = 0, 30, 60, and 90 s) in lentivirus-infected neurons expressing EGFP-Syn in the absence or presence of Pclo28 shRNA. Note the more pronounced dispersion of EGFP-Syn puncta in the Pclo28 background. Scale bar, 10 μm. B, Time course of EGFP-Syn dispersion in wild-type (black) and Pclo28-expressing (gray) boutons during 10 Hz, 90 s stimulation. Each time point represents an average of three curves obtained on the same day, each from a single coverslip containing ∼200 EGFP-Syn puncta. Rate of fluorescence loss (expressed as FoFt/Fo for each time point t) is fit by a single exponential. Error bars show SEM (n = 3). C, Average extent of EGFP-Syn dispersion, expressed as percentage increase in EGFP-Syn dispersion for Pclo28 versus wt (n = 24 coverslips; >4 batches of neurons). The extent of EGFP-Syn dispersion in Pclo28 boutons is 55% greater than in wt boutons, as denoted by black line (***p < 0.0001, t test). D, FM4-64 destaining at EGFP-Syn puncta in wild-type or Pclo28 background. The arrows denote colocalized EGFP-Syn and FM4-64 puncta. FM destaining is more complete in Pclo28-expressing boutons. Scale bar, 10 μm. E, Time course of FM4-64 destaining at wild-type (black) and Pclo28 (gray) boutons during 10 Hz, 90 s stimulation. Each time point represents an average of two coverslips, each containing ∼200 EGFP-Syn puncta, all imaged the same day. Rate of fluorescence loss is fit by a single exponential. SEM bars are shown. F, Average extent of FM4-64 destaining, again expressed as percentage increase for Pclo28 versus wild type (n = 15 coverslips; >4 batches of neurons). The average extent of FM destaining is 15% greater in Pclo28 boutons than in wt boutons (**p < 0.005, t test).
Figure 3.
Figure 3.
Aberrant Synapsin1a phosphorylation does not cause the Pclo28 phenotypes. A, Schematic of Synapsin1a showing its seven phospho-sites and the kinases responsible for phosphorylation. B, Time course of dispersion for wild-type and S23A mutant Synapsin1a in the Piccolo background, in the presence or absence of KN62. KN62 attenuates the dispersion of both wild-type and S23A Synapsin1a. SEM values are shown (n = 2 experiments/condition). C, Average extent of EGFP-Syn dispersion in the absence or presence of KN62, expressed as percentage decrease in EGFP-Syn dispersion for S23A versus wild-type EGFP-Syn in the Pclo28 background (n = 2 coverslips for EGFP-Syn plus KN62, 3 for S23A, 3 for S23A plus KN62; 2 batches of neurons). The dashed line denotes “complete rescue” of Pclo28 phenotype, defined as the percentage decrease in EGFP-Syn dispersion observed in wild-type neurons versus Pclo28 neurons (=33%). D, Time course of dispersion for wild-type, S1A, S23A, and S12346A EGFP-Syn constructs in the Pclo28 background. S12346A partially rescues the EGFP-Syn dispersion phenotype, with 8.4% less dispersion than wt EGFP-Syn in Pclo28 boutons (*p < 0.05, t test). E, Average extent of EGFP-Syn dispersion, expressed as percentage decrease in EGFP-Syn dispersion for phosphomutants versus wild-type EGFP-Syn in the Pclo28 background. The dashed line denotes complete rescue of the Pclo28 phenotype as in C. F, Time course of FM destaining at boutons expressing wild-type, S1A, S23A, and S12346A EGFP-Syn constructs in the Pclo28 background. SEM values are shown (n > 2 experiments/condition). G, Average extent of FM destaining, expressed as percentage decrease in FM destaining for phosphomutants versus wild-type EGFP-Syn in the Pclo28 background (n = 5 coverslips for S1A, 7 for S23A and S12346A; 3 batches of neurons). The dashed line denotes complete rescue of Pclo28 phenotype, defined as the percentage decrease in FM destaining observed in wild-type neurons versus Pclo28 neurons (=11.6%). S1A and S23A both rescue the enhanced FM destaining (by 17.6 and 17.9%, respectively), but S12346A does not (*p < 0.05; **p < 0.005, t test). H, Time course of EGFP-Syn dispersion in the Pclo28 background, in the presence of PKA (KT5720) and MAP kinase (PD98) blockers. SEM values are shown (n > 3 experiments/condition). I, Average extent of synapsin dispersion in the presence of PKA and MAPK inhibitors, expressed as in C and E. Neither has a significant effect on EGFP-Syn dispersion.
Figure 4.
Figure 4.
Jasplakinolide rescues the Pclo28 phenotypes. A, EGFP-Synapsin1a dispersion and FM4-64 destaining for EGFP-Syn/Pclo28 or EGFP-Syn/Pclo28 in the absence or presence of 5 μm jasplakinolide. The arrows denote colocalized EGFP-Syn and FM4-64 puncta. Scale bars, 15 μm. B, Time course of EGFP-Syn dispersion at boutons lacking Piccolo, in the absence (black) or presence (gray) of jasplakinolide. SEM bars are shown (n = 2 experiments/condition). C, Average extent of EGFP-Syn dispersion at Pclo28-expressing boutons treated with jasplakinolide (n = 10 coverslips). Complete rescue (dashed line) is defined as in Figure 2C. Jasplakinolide decreases the extent of dispersion by 24.1% (***p < 0.0001, t test). D, Time course of FM4-64 destaining at boutons lacking Piccolo, in the absence (black) or presence (gray) of jasplakinolide. SEM values are shown (n = 2 experiments/condition). E, Average extent of FM destaining at Pclo28 boutons treated with jas (n = 6). Complete rescue (dashed line) defined as in Figure 3G. Jasplakinolide decreases the extent of FM destaining by 23.3% (**p < 0.005, t test). F, Time course of EGFP-Syn dispersion in wild-type neurons in the absence (black) or presence (blue) of jasplakinolide. SEM bars are shown (n = 3 experiments/condition). G, Time course of FM destaining in the absence (black) or presence (blue) of jasplakinolide. SEM bars are shown (n = 3 experiments wt, 5 experiments plus jasplakinolide). Wild-type boutons treated with jasplakinolide exhibit no change in either EGFP-Syn dispersion or FM destaining.
Figure 5.
Figure 5.
Colocalization of EGFP-actin puncta with presynaptic markers. A, Colocalization of axonal EGFP-actin puncta, induced by either high-K+ stimulation or jasplakinolide, with synaptophysin immunostaining (indicated by arrows). Scale bar, 10 μm. B, Quantification of EGFP-actin puncta colocalization with Piccolo or synaptophysin immunostaining, or with FM4-64. Approximately 60% of EGFP-actin puncta (induced by high-K+ stimulation for these experiments) are presynaptic based on colocalization with all three markers (n = 6 fields of view for Piccolo, 5 for synaptophysin, 9 for FM4-64). C, Quantification of EGFP-actin puncta colocalization with synaptophysin or Bassoon immunostaining in wild-type (black) or Pclo28-expressing (blue) neurons. Here, clustering was induced with jasplakinolide. Note that 70% of wild-type EGFP-actin puncta colocalize with synaptophysin and 73% with Bassoon, versus 27 and 32% of puncta, respectively, in Pclo28 neurons (n = 4 fields of view for each condition; ***p < 0.0005). Error bars show SEM. D, High-resolution images of high-K+-induced EGFP-actin puncta, taken using STED microscopy. Colocalization of EGFP-actin with synaptophysin and Piccolo immunostaining (acquired in confocal mode) are depicted in merged images. Note that EGFP-actin does not directly colocalize with Piccolo and synaptophysin puncta, but appears to surround them, indicating that a majority of presynaptic F-actin filaments form a meshwork encircling the active zone and SV pool. Scale bar, 5 μm.
Figure 6.
Figure 6.
Depolarization-induced F-actin assembly is impaired in boutons lacking Piccolo. A, EGFP-actin in control and latrunculin A-treated wild-type axons before (pre) and after (post) stimulation with 90 mm KCl Tyrode's buffer plus FM4-64. The arrows indicate presynaptic sites based on FM4-64 labeling. EGFP-actin becomes more punctuate after stimulation; latrunculin A largely blocks this effect. B, EGFP-actin in wild-type or Pclo28-expressing axons before and after high-K+ stimulation. The arrows indicate presynaptic sites. Note the lack of activity-induced clustering in Pclo28 axons. C, EGFP-actin in control and jasplakinolide-treated Pclo28-expressing neurons. High-K+ stimulation does not induce EGFP-actin clustering in the absence of Piccolo, but jasplakinolide does. Scale bars: A–C, 15 μm. D, Percentage increase in EGFP-actin fluorescence intensity at presynaptic boutons following various treatments (wild type, n = 17 experiments; wild type plus latrunculin A, n = 8; Pclo28, n = 12; Pclo28 plus jasplakinolide, n = 7). Presynaptic EGFP-actin fluorescence increases 47.8% in wild-type neurons, but only 18.2% in the Piccolo knockdown and 10.5% in the presence of latrunculin (***p < 0.0001, t test). Jasplakinolide significantly increases EGFP-actin fluorescence intensity in boutons lacking Piccolo (42.3%; **p < 0.01, t test). E, Number of EGFP-actin puncta/unit axon length before (pretreat) and after (posttreat) high-K+ stimulation or jasplakinolide treatment (same n values as D). Axons lacking Piccolo have fewer EGFP-actin puncta before stimulation than wild-type axons (0.16 vs 0.43 puncta/pixel, respectively). High K+ induces new EGFP-actin puncta in wild-type neurons (from 0.43 to 0.68 clusters/pixel; ***p < 0.0001, paired t test), but not those treated with latrunculin A or lacking Piccolo. Jasplakinolide also induces significant EGFP-actin clustering in neurons expressing Pclo28 (from 0.16 to 0.44 clusters/pixel; ***p < 0.0001, paired t test).
Figure 7.
Figure 7.
CaMKIIα recruitment to presynaptic boutons is impaired in Piccolo knockdown neurons. A, YFP-CaMKIIα in control, latrunculin A-treated, and Pclo28-expressing axons, before (pre) and after (post) stimulation with 90 mm KCl Tyrode's buffer plus FM4-64. The arrows indicate presynaptic sites based on FM4-64 labeling. In wild-type axons, YFP-CaMKIIα becomes more punctuate after stimulation; latrunculin A and Pclo28 shRNA block this effect. Scale bar, 15 μm. B, Percentage increase in YFP-CaMKIIα fluorescence intensity at presynaptic boutons following high-K+ stimulation. Fluorescence increases 45% in wild-type neurons, but only 17% in the Piccolo knockdown and 7% in the presence of latrunculin (n = 8 for wt, 6 for Pclo28, 3 for latA; **p < 0.005, t test). C, Number of YFP-CaMKIIα puncta/unit axon length before (pretreat) and after (posttreat) high-K+ stimulation or latrunculin treatment (same n values as B). Pclo28-expressing axons have fewer YFP-CaMKIIα puncta before stimulation than wild-type axons (0.12 vs 0.20 puncta/pixel, respectively). High K+ induces new puncta in wild-type neurons (from 0.20 to 0.36 puncta/pixel; **p < 0.005, paired t test), but not those treated with latrunculin A or lacking Piccolo. Error bars show SEM.
Figure 8.
Figure 8.
Characterization of Pfn380 single knockdown and Pclo28/Pfn380 double knockdown. A, Left, Western blot of lysates from hippocampal neurons infected with soluble EGFP alone (FUGW) or EGFP plus Pfn380, probed with Profilin2 and tubulin antibodies. Pfn380 shRNA eliminates the majority of Profilin2 from these neurons. Right, Western blot of lysates from hippocampal neurons infected with soluble EGFP plus scrambled Pclo28 and Bsn16 shRNAs (FUGW/SC) or EGFP plus Pclo28 and Pfn380 shRNAs (Pclo28/Pfn380), probed with Piccolo, Profilin2, and tubulin antibodies. Pclo28 and Pfn380 eliminate the majority of Piccolo and Profilin2, respectively, from neurons. B, Quantification of the number of primary dendrites (labeled with MAP2 immunostaining) for neurons infected with EGFP-Syn alone (wt, black), plus Pfn380 (red), or plus Pclo/Pfn (purple). No significant difference was observed between wt and Pfn380 single or Pclo/Pfn double knockdowns, indicating that dendritic morphology is not affected by these manipulations. SEM bars are shown (n = 10 cells for wt, 7 for Pfn380, 6 for Pclo/Pfn). C, Quantification of dendritic spine density, measured as number of spines per pixel along MAP2-positive processes, for neurons infected with EGFP-Syn alone (wt, black), plus Pfn380 (red), or plus Pclo/Pfn (purple). No significant differences were observed between wild-type and Pfn380 single or Pclo/Pfn double knockdown, indicating that spine formation is not affected by these manipulations. SEM bars are shown (n = 4 fields of view for wt, 5 for Pfn380, 6 for Pclo/Pfn). D, Images depicting colocalization between EGFP-Syn and FM4-64 for wt and Pclo/Pfn-expressing neurons (indicated by arrows). Scale bar, 10 μm. E, Images depicting colocalization between EGFP-Syn and Homer for wt and Pclo/Pfn-expressing neurons. A subset of colocalized puncta is indicated by arrows. Merged image also contains MAP2 immunostaining to label dendrites (blue). Scale bar, 10 μm. F, Quantification of EGFP-Syn colocalization with FM4-64 for neurons expressing EGFP-Syn alone (wt, black), plus Pfn380 (red), or plus Pclo/Pfn (purple). Pfn380 and Pclo/Pfn boutons have a similar degree of colocalization with FM as wt boutons, indicating that SV exo/endocytosis is not significantly inhibited by Profilin2 or Pclo/Pfn knockdown. SEM bars are shown (n = 10 fields of view for each condition). G, Quantification of EGFP-Syn colocalization with Homer1 for neurons expressing EGFP-Syn alone (wt, black), plus Pfn380 (red), or plus Pclo/Pfn (purple). No significant differences were observed between the three conditions, indicating that the presynaptic localization of EGFP-Syn is not affected by Profilin2 or Pclo/Pfn knockdown. SEM bars are shown (n = 7 fields of view/condition for wt and Pfn380, 6 for Pclo/Pfn).
Figure 9.
Figure 9.
Profilin2 knockdown phenocopies Piccolo knockdown. A, EGFP-actin in axons of wild-type or Pfn380 neurons before (prestim) and after (poststim) 90 mm KCl plus FM4-64. The arrows indicate presynaptic sites based on FM4-64 labeling. Note the lack of activity-induced clustering in axons expressing Pfn380. B, Percentage increase in presynaptic EGFP-actin fluorescence intensity following high-K+ stimulation for wild-type, Pclo28, and Pfn380 boutons. Piccolo and Profilin2 knockdowns exhibit significantly attenuated presynaptic EGFP-actin fluorescence increases compared with wild-type neurons (47.8% for wt, n = 17, data from Fig. 6D; 18.2% for Pclo28, n = 12, data from Fig. 6D; 20.6% for Pfn380, n = 8; ***p < 0.0001, t test). C, Number of EGFP-actin puncta per unit axon length before (prestim) and after (poststim) high-K+ stimulation for wild-type, Pclo28, and Pfn380 boutons (same n values as B). Stimulation induces new EGFP-actin puncta in wild-type neurons (data from Fig. 6E), but not those lacking Piccolo (data from Fig. 6E) or Profilin2 *p < 0.05. D, EGFP-Syn dispersion in control, Pclo28, or Pfn380 neurons. Scale bars, 10 μm. E, Time course of EGFP-Syn dispersion in wild-type neurons (n = 2) and those lacking Piccolo (n = 2) or Profilin2 (n = 2). SEM bars are shown. F, Average extent of EGFP-Syn dispersion at boutons lacking Piccolo (n = 21) or Profilin2 (n = 21), expressed as percentage increase in dispersion versus wild type. Here, Pclo28-expressing boutons have a 52.3% increase in EGFP-Syn dispersion and those expressing Pfn380 have a 28.4% increase (***p < 0.0001, t test). G, Time course of FM destaining in wild-type neurons and those lacking Piccolo or Profilin2 (same n values as E). SEM bars are shown. H, Average extent of FM destaining at boutons expressing Pclo28 (n = 13) or Pfn380 (n = 15). Both exhibit similarly enhanced levels of FM destaining compared with wild type (24.5% for Pclo28, 22.7% for Pfn380; ***p < 0.0001, t test).
Figure 10.
Figure 10.
Piccolo and Profilin2 could lie in the same molecular pathway for F-actin assembly. A, EGFP-Syn dispersion in control neurons and those expressing Pclo28, Pfn380, or Pclo28/Pfn380 (Pclo/Pfn). Scale bars, 10 μm. B, Average extent of EGFP-Syn dispersion at boutons lacking Piccolo (n = 21), Profilin2 (n = 21), or both proteins (n = 7), expressed as percentage increase in dispersion versus wild type. Pclo28-expressing boutons have a 52.3% increase in EGFP-Syn dispersion, those expressing Pfn380 have a 28.4% increase, and those expressing Pclo/Pfn have a 40.9% increase (***p < 0.0001, t test). C, Average extent of FM destaining at boutons expressing Pclo28 (n = 13), Pfn380 (n = 15), or Pclo/Pfn (n = 9). All exhibit similarly enhanced levels of FM destaining compared with wild type (24.5% for Pclo28, 22.7% for Pfn380, 15% for Pclo/Pfn; ***p < 0.0001, *p < 0.05, t test). D, EGFP-Profilin2 in axons of wild-type and Pclo28 neurons before (prestim) and after (poststim) high-K+ stimulation. In wild-type neurons, EGFP-Profilin2 becomes more punctate after stimulation; this effect is absent in neurons lacking Piccolo. Scale bar, 15 μm. E, Percentage increase in presynaptic EGFP-Profilin2 fluorescence intensity following high-K+ stimulation for wild-type (n = 12) or Pclo28-expressing neurons (n = 13). Presynaptic EGFP-Profilin2 fluorescence shows little increase in neurons lacking Piccolo compared with wild-type neurons (15.0 vs 26.9%; *p < 0.05, t test). F, Number of EGFP-Profilin2 puncta per unit axon length before (prestim) and after (poststim) high-K+ stimulation for wild-type or Pclo28-expressing neurons (same n values as E). Note that axons lacking Piccolo exhibit fewer EGFP-Profilin2 puncta before stimulation than wild-type axons (0.14 vs 0.24 puncta/pixel). Stimulation induces new EGFP-Profilin2 puncta in wild-type neurons (from 0.24 to 0.42 puncta/pixel; **p < 0.005, paired t test), but not those lacking Piccolo. Error bars show SEM.
Figure 11.
Figure 11.
PcloΔEx14 mice exhibit normal presynaptic F-actin assembly. A, Schematic diagram of Piccolo depicting its multiple domains [Q, two Zinc finger (Zn), three coiled-coil (CC), PDZ, C2A, and C2B] and regions targeted by Pclo28 shRNA, exon 14 deletion, and the 44aII antibody. B, Western blot of brain homogenates from wild-type (+/+), heterozygous (+/−), and PcloΔEx14 (−/−) mice, probed with the 44aII antibody. The black arrowheads indicate major immunoreactive bands (∼560, 500, and 400 kDa) that are absent in −/− mice. C, Western blot of lysates from rat hippocampal neurons infected with EGFP-Synapsin1a in the absence or presence of Pclo28, probed with 44aII antibodies. Note the disappearance of all immunoreactive bands in lysates expressing Pclo28. D, EGFP-actin in axons of 10 DIV +/+ and −/− hippocampal neurons, in the absence or presence of Pclo28 shRNA, treated with high K+ (1 min) and immunostained with 44aII antibodies. For both genotypes, extensive axonal EGFP-actin clustering is induced by high K+, and clusters colocalize with presynaptic Piccolo immunoreactivity (arrows). Pclo28 eliminates EGFP-actin clustering and Piccolo immunoreactivity for both genotypes (arrowheads). Image gain is set approximately five times higher for EGFP-actin (but not Piccolo) in Pclo28-infected axons to enable its visualization, as it is largely diffuse. Scale bar, 10 μm. E, Intensity of high-K+-induced EGFP-actin puncta at presynaptic boutons, expressed as percentage increase in bouton/axon fluorescence (+/+, n = 5 images from 2 animals; −/−, n = 14 images, 2 animals; +/+/Pclo28, n = 8, 2 animals; −/−/Pclo28, n = 14, 2 animals). For both genotypes, presynaptic EGFP-actin fluorescence is >40% increased versus axon fluorescence, but only ∼20% increased in the presence of Pclo28 (***p < 0.0001, t test). F, Number of EGFP-actin puncta/unit axon length (n values same as E). For both genotypes, high-K+ stimulation induces EGFP-actin puncta densities of ∼0.6/pixel axon length, and Pclo28 reduces this value threefold (***p < 0.0001, t test). G, Fraction colocalization of EGFP-actin puncta with Piccolo immunostaining (n values same as E). For both genotypes, EGFP-actin puncta exhibit a similar degree of colocalization with Piccolo (∼0.65). Although Piccolo immunoreactivity is substantially weaker in −/− neurons, it is still readily detectable with 44aII antibodies. Pclo28 significantly reduces Piccolo immunoreactivity, and hence colocalization with axonal EGFP-actin clusters, in both genotypes (to <0.11; ***p < 0.0001, t test). Error bars show SEM.

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