RESEARCH ARTICLE

Calcium influx mediates the chemoattractant-inducedtranslocation of the arrestin-related protein AdcC in DictyosteliumLauriane Mas1, Adeline Cieren1,*, Christian Delphin2, Agnes Journet1 and Laurence Aubry1,‡

ABSTRACTArrestins are key adaptor proteins that control the fate of cell-surfacemembrane proteins and modulate downstream signaling cascades.The Dictyostelium discoideum genome encodes six arrestin-relatedproteins, harboring additional modules besides the arrestin domain.Here, we studied AdcB and AdcC, two homologs that contain C2 andSAM domains. We showed that AdcC – in contrast to AdcB –

responds to various stimuli (such as the chemoattractants cAMP andfolate) known to induce an increase in cytosolic calcium by transientlytranslocating to the plasma membrane, and that calcium is a directregulator of AdcC localization. This response requires the calcium-dependent membrane-targeting C2 domain and the double SAMdomain involved in AdcC oligomerization, revealing a mode ofmembrane targeting and regulation unique among members of thearrestin clan. AdcB shares several biochemical properties with AdcC,including in vitro binding to anionic lipids in a calcium-dependentmanner and auto-assembly as large homo-oligomers. AdcB caninteract with AdcC; however, its intracellular localization is insensitiveto calcium. Therefore, despite their high degree of homology andcommon characteristics, AdcB and AdcC are likely to fulfill distinctfunctions in amoebae.

KEY WORDS: Arrestin, Calcium signaling, Chemoattractant,C2 domain, SAM domain, Dictyostelium

INTRODUCTIONIn their environment, cells are constantly subjected to a variety ofstimuli that orient their behavior and/or fate in terms of motility,growth and differentiation. Sensing of these environmental cuesinvolves integral membrane proteins at the cell surface, amongwhich G-protein-coupled receptors (GPCRs) are clearly the moststudied example (Fredriksson et al., 2003; Lagerström and Schiöth,2008). In mammals, β-arrestins play a central role in the regulationof GPCRs and associated signaling by impeding their coupling toheterotrimeric G proteins, modulating the activation of downstreameffectors and controlling GPCR presence at the cell surface (Kendalland Luttrell, 2009; Gurevich and Gurevich, 2015; Tian et al., 2014;DeWire et al., 2007). These scaffolding proteins consist in a doublecrescent-shaped β-sandwich (arrestin N and C domains) thatrecognizes the receptor, and a short C-terminal tail (Han et al.,2001; Milano et al., 2002; Zhan et al., 2011; Vishnivetskiy et al.,

2004; Gurevich and Gurevich, 2004). This tail contains recruitmentsites for the endocytic machinery and becomes accessible uponβ-arrestin binding to the activated – and usually phosphorylated –receptor, and subsequent destabilization of a central polar core,allowing the internalization of the receptor (Kim and Benovic,2002; Laporte et al., 2002; Schmid et al., 2006). Besides theubiquitous β-arrestins and the retina-specific visual arrestins, allseemingly restricted to higher eukaryotes of the animal kingdom,the arrestin family also comprises structurally related proteins calledα-arrestins or arrestin-domain-containing proteins, discovered morerecently and which are present from protists to human (Alvarez,2008; Aubry et al., 2009). These novel members are all predictedto share the arrestin fold. However, the presence of a polar core isquestioned, the C-terminal tail as found in β-arrestins is absent andnovel extensions are present on either side of the arrestin domain,providing – or being likely to provide – different properties(Alvarez, 2008; Aubry and Klein, 2013; Becuwe et al., 2012a).Despite this structural diversity, the regulation of membrane cargotrafficking appears to be an evolutionarily conserved function of thearrestin clan, and the repertoire of known arrestin membrane targetsnow includes GPCRs, single-membrane span receptors, integrins,channels and transporters, as illustrated in mammals and yeast(Becuwe et al., 2012a; Kang et al., 2014; Kovacs et al., 2009;Lefkowitz et al., 2006; Lin et al., 2008).

In the soil amoeba Dictyostelium discoideum, six arrestin-relatedproteins (AdcA–AdcF) have been identified, but have so far beenpoorly studied (Aubry and Klein, 2013; Aubry et al., 2009).Recently, AdcC has been shown to localize to the plasma membranein response to cAMP and to bind the cAMP receptor, cAR1 (Caoet al., 2014). The GPCR cAR1 is key in the starvation-induceddevelopment of the amoeba, especially during the aggregationphase that allows this unicellular organism to reach multicellularityand enter the differentiation program (Aubry and Firtel, 1999;Parent and Devreotes, 1996). AdcC and its close homolog, AdcB,both exhibit C2 and sterile alpha motif (SAM) domains surroundingthe arrestin domain.Whereas SAM domains are mainly known to beprotein–protein interaction modules, C2 domains are commonphospholipid-binding domains, mostly present in proteins acting inmembrane trafficking/fusion events and signal transduction(Corbalan-Garcia and Gómez-Fernández, 2014). In many C2domain-containing proteins, lipid binding is conditioned bycalcium binding, resulting in a calcium-dependent associationwith membranes. Because cAMP activation of cAR1 induces a largearray of signaling events, among which a transient increase incalcium in the cytosol (Nebl and Fisher, 1997; Yumura et al., 1996),we investigated whether calcium might regulate AdcB and AdcCbehavior/function. Our work revealed that, in contrast to AdcB, anddespite shared biochemical properties, AdcC responds to variousstimuli (calcium, cAMP and folate), all inducing a calcium elevationin the cytosol, by transiently associating with the plasma membrane.We uncovered a mode of membrane targeting/regulation unusual forReceived 12 July 2017; Accepted 5 September 2018

1Universite Grenoble Alpes, CEA, INSERM, BGE U1038, F-38000 Grenoble, France.2Universite Grenoble Alpes, INSERM U1216, GIN, F-38000 Grenoble, France.*Present address: Faculty of Medicine, University of Geneva, CH-1211 Geneva 4,Switzerland.

‡Author for correspondence (laubry@cea.fr)

L.M., 0000-0003-0210-8966; L.A., 0000-0001-7640-1706

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members of the arrestin clan, requiring the C2 domain of AdcC andthe SAM extension involved in the oligomerization of the protein.The distinct behaviors of these two homologs in response tointernal calcium fluctuations are indicative of highly regulated andspecific functions.

RESULTSThe protein AdcC, but not AdcB, translocates to theplasma membrane in response to an increase inintracellular calciumAn in silico analysis of AdcB/AdcC predicted C2 moduleswas performed using the homology modeling server PHYRE2(http://www.sbg.bio.ic.ac.uk/phyre2/). The best hits were obtainedwith C2 domains from members of the PKC-C2 Pfam subfamily.This subfamily includes both calcium-dependent and -independentC2 domains with type I or type II topologies, depending on theorientation of the β-strands within the two four-stranded antiparallelβ-sheets forming the C2 domain (Nalefski and Falke, 1996).For AdcB and AdcC, modeling and sequence alignment indicated atype II topology, and conservation of the acidic residues implicatedin calcium binding in canonical calcium-dependent C2 domains(D20/D27/D76/D78 and D84 for AdcB; D20/D26/D72/E74 for AdcC),with the exception of one aspartate that is substituted by an argininein AdcC (R80) (Fig. 1A,B). AdcB and AdcC C2 domains alsoexhibit an overall conservation of the amino acids involved in PKCαin anionic phospholipid binding (Fig. 1B). The C2 module of bothAdcB and AdcC could therefore behave as a calcium-binding C2domain with a preference for anionic membrane environments.We investigated the in vivo dynamics of AdcB and AdcC in

response to calcium by fluorescence imaging of live cells. Bothproteins were tagged with GFP or RFP at the C-terminus andexpressed in the parental strain KAx-3 and in adcB or adcC nullcells (see Materials and Methods). To study the proteins at relevantstages of the D. discoideum life cycle, we first determined theirexpression profiles during development by western blot analysis

(Fig. S1A,B). Both AdcB and AdcC were detected in growingconditions (vegetative cells) and during the multicellular phasetriggered by starvation, with a decrease at later stages ofdevelopment. Microscopy studies were thus carried out onvegetative cells and cells engaged in the early phase of development.

To test the effect of internal calcium variations on AdcB andAdcC, vegetative cells were stimulated by the addition of CaCl2 inthe extracellular medium, a condition known to trigger a cytosoliccalcium increase in most cell types, including Dictyostelium(Lombardi et al., 2008; Lusche et al., 2009). We recorded tagged-protein dynamics using spinning disk confocal microscopy over aperiod of 5–7 min. In untreated cells, both AdcBGFP and AdcCGFP

were exclusively found in the cytosol (Fig. 2A,E). Addition of2 mM CaCl2 led, within the next 1 min, to the enrichment ofAdcCGFP at the plasma membrane, with a concomitant decreaseof the fluorescent signal in the cytosol, in an oscillatory manner(Fig. 2B; Movies 1 and 2). The cytosol/total index (IC/T) (seeMaterials and Methods) was used as a measure of AdcC enrichmentat the plasma membrane in response to calcium (Fig. 2A,B). Similarbehavior was observed for AdcCRFP (Fig. 2C, lower panel) or anN-terminally GFP-tagged AdcC (data not shown). Cell response,in terms of AdcC translocation, was more homogeneous andprominent when cells were transferred in hyposmotic mediumprior to calcium addition, possibly due to an increase in calciumentry consecutive to membrane stretching (Lombardi et al., 2008)caused by cell swelling. This treatment was therefore favored,with the perspective to evaluate the behavior of truncated mutantsin conditions triggering efficient translocation. On average, inthese conditions, the first oscillations were observed within theinitial 30 s after stimulation and the number of oscillations variedfrom 0 to 13 with a mean of 7±3 (n=41) during the 7 min followingcalcium addition. These values are compatible with the work ofLombardi and colleagues on intracellular calcium fluctuationsin similar conditions (Lombardi et al., 2008). Under prolongedincubation (>10–15 min), AdcC progressively re-adopted a

Fig. 1. In silico analysis of AdcB andAdcCC2domains. (A) Homology-basedmodeling of AdcB (left) and AdcC (right) C2 domain structure by PHYRE2 serverunder intensive mode using the following PDB templates: c4v29B (Arabidopsis thaliana CAR4), c4npjA (Homo sapiens extended synaptotagmin 2), c3jzyA(H. sapiens intersectin 2), c4rj9A (Oriza sativa OsGAP1), c4ighB (H. sapiens dysferlin c2a variant 1) and d1gmia (Rattus rattus PKCε) for AdcB-C2; c4v29B,c4npjA, c5a52A (A. thaliana CAR2), c4rj9A, d1gmia and c2dmhA (H. sapiensmyoferlin C2-1) for AdcC-C2. For both proteins, ≥99% of the domain was modeledwith at least 99% confidence. Images of the structures were generated using PyMOL software. Aspartate and glutamate residues expected to bind calciumions are indicated in red; β1–β8, β-strands. (B) Sequence alignment of AdcB and AdcC C2 domains with PKCα (H. sapiens) calcium-binding C2 domainusing T.Coffee (http://tcoffee.crg.cat/). Gray, PKCα β-strands; blue, AdcC β-strands predicted from PHYRE2; red asterisks, PKCα acidic residues involved incalcium ion coordination and highly conserved in other calcium-binding C2 domains; orange outline boxes, amino acids mediating phosphatidylserine binding;green outline boxes, amino acids implicated in PI(4,5)P2 binding.

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stable cytosolic localization. To confirm that AdcC indeedresponded to cytosolic calcium variations, AdcCRFP-expressingcells were electroporated with Calcium Green dextran (a calciumindicator) and membrane translocation of AdcCRFP was imagedsimultaneously with the fluorescence variation of the calciumprobe. As shown in Fig. 2C, AdcCRFP recruitment to the plasmamembrane systematically paralleled intracellular calcium peaks.Cell treatment with the calcium-channel blockers Ruthenium Red(RR) and gadolinium (Gd3+) inhibited AdcC relocalization to themembrane, supporting a role for intracellular calcium in AdcCdynamics (Fig. 2D; Fig. S2A). A similar response to calcium wasobtained in amoebae subjected to starvation in phosphate buffer,except that AdcCGFP translocation from the cytosol to the plasmamembrane could be efficiently triggered with 10-fold lowerconcentrations of extracellular calcium (200–100 µM) comparedwith those of vegetative cells, owing to a higher sensitivity ofstarved cells to calcium (Movie 3). AdcC oscillations progressivelyattenuated with time as observed in vegetative cells.

Two chemoattractants, folic acid and cAMP, have been describedto trigger a transient elevation of cytosolic calcium in vegetative andaggregation-competent amoebae, respectively (Nebl and Fisher,1997; Yumura et al., 1996; Milne and Coukell, 1991). We thereforeexamined AdcC behavior upon folate and cAMP stimulation. As thechemoattractant-induced increase in calcium was shown to resultprimarily from a calcium influx across the plasma membrane, cellswere first pre-incubated in phosphate buffer containing 100 µMcalcium. In linewith our above data, such a concentration of calciuminduced some transient AdcC translocation to the plasma membrane(very weak in vegetative cells and marked in aggregation-competentcells; data not shown). Pre-incubation was thus prolonged untilAdcC had regained a stable cytosolic localization (Fig. 3A,B; Time0, top and middle rows). Subsequent stimulation of vegetative cellswith folate, but not with buffer alone, led to a rapid and transienttranslocation of AdcC to the plasma membrane in a timescaleconsistent with the reported kinetics of the folate-triggered calciumrise (Fig. 3A, top and middle rows). AdcC similarly responded to

Fig. 2. AdcC transiently associates with the plasma membrane in response to extracellular calcium. Cells were imaged by spinning disk confocalmicroscopy. (A,B) AdcCGFP localization in vegetative adcC null cells expressing AdcCGFP prior to (A) and after (B) 2 mM CaCl2 addition. Shown imageswere extracted from a timelapse acquisition at the indicated times poststimulation. AdcC response was quantified by measurement of the cytosol/total index(IC/T, see Materials and Methods) prior to (−) and after (+) calcium addition. Values are presented as mean±s.d. (n=21). (C) adcC null cells expressing AdcCRFP

and containing Calcium Green dextran were imaged after CaCl2 addition. Calcium Green dextran (cytosol) and RFP (cytosol and plasma membrane)fluorescence intensities were quantified using ImageJ software and plotted as a function of time. Quantification results from a representative cell responseare shown. (D) KAx-3 cells expressing AdcCGFP were pretreated with or without 20 µM Ruthenium Red (RR) prior to 2 mM CaCl2 addition. (E) Response ofvegetative adcB null cells expressing AdcBGFP to 2 mMCaCl2 stimulation.When no translocation was observed (D,E), illustrating images were chosen at arbitrarytimepoints. Scale bars: 10 µm.

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cAMP when added to aggregation-competent cells (Fig. 3B, topand middle rows), in agreement with the data from Cao et al.(2014). As described, AdcC translocation in response to cAMPwas lost in a mutant strain lacking the two cAMP receptorsexpressed during aggregation (car1/3 null cells), indicating thatcAMP operates through a cAMP receptor-dependent pathway(Fig. 3C). In both conditions of stimulation, the chemoattractant-induced translocation of AdcC was not observed in the absence ofcalcium in the external medium (Fig. S2B,C), or in the presence of

the calcium-channel inhibitors RR or gadolinium (Fig. 3A,B,bottom rows; Fig. S2D), supporting that the increase in calciumtriggered by folate and cAMP is essential for the recruitment ofAdcC to the plasma membrane in response to these chemoattractants.As car1/3 null cells are unable to induce a cytosolic calcium elevationin response to cAMP (Milne et al., 1997), we askedwhether causing acalcium elevation in aggregation-competent car1/3 null cells withexternal calcium could drive AdcC to the membrane. As shown inFig. 3D, AdcC responded equally well in the car1/3 null and parental

Fig. 3. Folate and cAMP trigger AdcC translocation to the membrane in a calcium-dependent manner. Cells were observed by spinning disk confocalmicroscopy. (A,B) KAx-3 cells expressing AdcCGFP, in vegetative conditions (A), or rendered aggregation-competent by a 4 h period of cAMP pulses (B),were pre-incubated in 12 mM NaK-phosphate buffer pH 6.2 (PB) containing 100 µM CaCl2. After restabilization of AdcC in the cytosol (time 0), vegetativecells were stimulated with 50 µM folate (A, middle row) and pulsed cells with 10 µM cAMP (B, middle row). Control experiments were performed with PB100 µM CaCl2 containing no chemoattractant (buffer alone) (A,B, top row). Addition of 20 µM RR prior to the addition of folate (A, bottom row) or cAMP(B, bottom row) prevented chemoattractant-induced AdcC translocation. (C,D) Ax2 (parent) and car1/3 null cells expressing AdcCGFP were pulsed in PBwith 100 nM cAMP for 4 h. Cells were either pre-incubated in PB containing 100 µM CaCl2 and then treated with 10 µM cAMP as described above (C) orstimulated directly with 200 µMCaCl2 (D). AdcC responsewas quantified by measurement of the IC/T. Values are presented as mean±s.d. (C, n=36; D, n=21).Statistical significance was assessed by unpaired two-tailed Student’s t-tests (**P<0.001; ns, nonsignificant). Shown images were extracted fromtimelapse acquisitions at the indicated times poststimulation. In the absence of translocation, images were chosen at arbitrary timepoints. Scale bars: 10 µm.

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Ax2 strains, suggesting that, in a wild-type context, the increase incytosolic calcium resulting from cAMP receptor activation might besufficient to recruit AdcC from the cytosol to the plasma membrane.Taken together, our data indicate that the arrestin-related protein

AdcC responds to a variety of stimuli known to elicit an elevationof cytosolic calcium, and that this calcium elevation is essential forAdcC membrane binding. In contrast to AdcC, AdcBGFP did notrespond to the addition of calcium in the external medium.The protein remained cytosolic in the range of extracellularcalcium concentrations tested (50 µM–40 mM) in vegetative- andaggregation-stage adcB null and KAx-3 cells (Fig. 2E; Movies 4–6).Tagging the protein at the N-terminus rather than C-terminally didnot modify AdcB response. In addition, no translocation wasobserved in response to cAMP or folate (data not shown). Thus,despite their similar architecture, the two proteins AdcB and AdcCharbor distinct sensitivity to calcium in vivo.

AdcC membrane targeting is dependent on its calcium-binding type C2 domainTo assess the contribution of the C2 domain to AdcC response tocalcium, GFP-tagged truncated forms of the protein were expressedin the adcC null strain. Deletion of the whole region upstream ofthe arrestin core (AdcCΔNtGFP), or of the first 101 amino acids,thus solely impairing the C2 domain (AdcCΔC2GFP), completelyabrogated AdcC response to calcium stimulation, indicating thatthe C2 domain is required for AdcC translocation to the plasmamembrane (Fig. 4A). Point mutations in the C2 domain of AdcCwere also generated, substituting D20 and D26 for asparagines,alone or together with D72 and E74 (AdcCD20N/D26N

GFP orAdcCN4

GFP) (Figs 1B and 4B). Although they interfer withcalcium binding, such substitutions are expected to mimic thecharge-neutralizing effect of calcium by removing the electrostaticrepulsion provided by the aspartate residues. As reported for

similar mutations in DOC2B C2A or copine 2, 6 and 7 C2Bdomains (Friedrich et al., 2008; Groffen et al., 2006; Perestenko et al.,2015), AdcCD20N/D26N

GFP and AdcCN4

GFP were found constitutivelylocated at the plasma membrane, further indicating that a functionalcalcium-sensing C2 domain is required for the detection ofintracellular calcium variations and appropriate trafficking of AdcC(Fig. 4B, left).

In contrast to several isolated calcium-binding C2 domains(Ananthanarayanan et al., 2002; Manna et al., 2008; Oancea andMeyer, 1998; Perisic et al., 1999), the C2 domain of AdcC is notsufficient to fully support membrane association of the protein on itsown, as neither AdcC-C2GFP nor AdcC-NtGFP, limited to the C2 andNt domains, respectively, were able to translocate to the plasmamembrane in response to calcium (Fig. 4A). Mutation in AdcC-Nt ofthe four acidic residues involved in calcium binding (AdcC-NtN4GFP)led to the constitutive membrane localization of the mutated protein,but with reduced efficiency comparedwith the equivalent mutation infull-length AdcC, AdcCN4

GFP (Fig. 4B, left). The value of the IC/Twas significantly higher for AdcC-NtN4GFP (0.82±0.05, n=25)compared with AdcCN4

GFP (0.48±0.14, n=25) and AdcCD20N/D26NGFP

(0.39±0.08, n=25) (Fig. 4B, right). Together, these results suggest that,besides the C2 domain, an additional region of the AdcC protein likelycontributes to its membrane recruitment.

AdcC and AdcB bind directly to phospholipids in vitro in acalcium-dependent mannerTo characterize the lipid-binding properties of AdcC, liposome-binding assays were performed in the presence of calcium or EGTA.Full-length AdcC and the N-terminal domain were expressed asMBPfusion proteins (MBP-AdcC and MBP-AdcC-Nt) and tested for theirability to bind phospholipid vesicles containing phosphatidylcholine(PC) alone, or a mix of PC and phosphatidylserine (PS). WhereasMBP exhibited no binding on any liposomes, MBP-AdcC was found

Fig. 4. AdcC translocation to the plasma membrane depends on a functional C2 domain. (A) adcC null cells expressing GFP-tagged AdcC, AdcCΔC2,AdcCΔNt, AdcC-C2 or AdcC-Nt were stimulated with 2 mM CaCl2 (arrowhead) and observed by confocal spinning disk microscopy. As constructs deletedof the C2-containing region or limited to the N-terminal domain failed to translocate to the plasma membrane, shown images were chosen arbitrarily at thesame time points as AdcC. (B) adcC null cells expressing GFP-tagged point-mutated proteins AdcCN4, AdcCD20N/D26N and AdcC-NtN4 were observed in theabsence of any calcium stimulation. Membrane binding efficiency of the different constructs was estimated by measurement of the IC/T. Values are presentedas mean±s.d. (n=25). Statistical significance was assessed by unpaired two-tailed Student’s t-tests (**P<0.001). Scale bars: 10 µm.

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to bind to PC/PS liposomes in a calcium-sensitive PS-dependentmanner, suggesting, as expected, a selectivity towards anionic lipids(Fig. 5A,B). Calcium titration experiments using nonlimiting PSconcentrations indicated an apparent dissociation constant (Kd) forAdcC of ∼16 µM. In contrast to MBP-AdcC, and in agreement withthe constitutive association of AdcCN4

GFP to the plasma membrane,MBP-AdcCN4 fully bound to PC/PS liposomes in the presence ofEGTA (Fig. 5C). In saturating conditions of calcium and PS, MBP-AdcC-Nt displayed no binding at all (Fig. 5A), further supporting thatthis domain is not sufficient for efficient membrane binding.However, when expressed as a GST fusion protein, AdcC-Nt wasable to bind PC/PS liposomes in a calcium-dependent manner. Incontrast to MBP, GST is known to form dimers (Maru et al., 1996;Tudyka and Skerra, 1997), and crosslinking experiments usingbis(sulfosuccinimidyl) suberate (BS3) indeed suggested a differentoligomeric state for GST- and MBP-tagged AdcC-Nt proteins(Fig. S3A,B). This GST-promoted oligomerization could increase the

avidity of the construct for anionic lipids, thereby favoring binding.These results, and the distinct behavior of the full-length versusN-terminal alone constructs in cellulo, further support the hypothesisthat an additional domain of AdcC participates directly or indirectlyin membrane association, together with the C2 domain.

Similar experiments on AdcB showed, unexpectedly, that full-length MBP-AdcB, as well as MBP-AdcB-Nt (but to a lowerextent), can associate with PS-containing liposomes upon calciumaddition (Fig. 5D,E). Similar to observations for MBP-AdcC,half-maximal binding of MBP-AdcB to 50% PC/50% PSliposomes was observed with a free calcium concentration of∼18.5 µM. As observed for AdcC-Nt, use of a GST tag alsoimproved calcium- and PS-dependent binding of the truncatedAdcB-Nt construct (Fig. 5D), again likely to be caused by theoligomerizing property of the GST (Fig. S3C). Therefore, the C2domain of AdcB can bind anionic lipids in a calcium-dependentmanner in vitro and qualifies as a bona fide calcium-sensitive C2

Fig. 5. Recombinant AdcB and AdcC bind liposomes in a calcium- and PS-dependent manner. (A) The full-length AdcC or its N-terminal domain wereexpressed as MBP or GST fusions and tested in liposome (100% PC or 50% PC/50% PS) co-sedimentation assays in the presence of 1 mM CaCl2 or EGTA.Protein distribution in the soluble (S) and pellet (P) fractions was analyzed by SDS-PAGE and Coomassie staining. MBP and GST were used as controls.(B) Calcium titration experiments were performed on MBP-AdcC using liposomes containing increasing concentrations of PS (10–60%). For each point, theamount of MBP-AdcC in the supernatant and in the pellet was quantified from the Coomassie-stained gel using ImageJ software. Data (mean±s.d., n=3)were fitted to the Hill equation. (C) Distribution of MBP-AdcCN4 in the presence of 1 mM EGTA and 100% PC or 50% PC/50% PS liposomes. (D,E) Similarexperiments to those in A and Bwere performed using AdcB constructs. (F) Chimeric proteins of AdcB and AdcCwere constructed by exchanging their N-terminaldomain (AdcB, light gray; AdcC, dark gray) and expressed in KAx-3 cells as GFP-tagged proteins. Their response to a 2 mM CaCl2 stimulation (arrowheads)was followed by confocal spinning disk microscopy over 5 min. As the chimera AdcB/AdcC does not translocate to the membrane, images used forillustration were taken arbitrarily at the same timepoints as for AdcC/AdcB. Scale bars: 10 µm.

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domain. However, swapping the N-terminal domain of AdcCin the AdcCGFP protein for the N-terminal domain of AdcB(AdcB/AdcCGFP chimera) inhibited AdcC translocation to theplasma membrane in vivo in response to 2 mM extracellular CaCl2(Fig. 5F, right). In contrast, AdcC/AdcBGFP chimera, in which theNt domain of AdcC replaced that of AdcB, responded with amassive and oscillatory membrane association similarly to AdcC(Fig. 5F, left), indicating distinct properties for AdcB and AdcCC2 domains in vivo.

TheSAMdomain-containing regionof AdcCcontributes to itsplasma membrane associationA preliminary analysis of AdcC and AdcB using SMART(http://smart.embl-heidelberg.de/) had revealed the presence of aputative SAM domain at the C-terminal end of the proteins (Aubryand Klein, 2013; Aubry et al., 2009). SAM domains are ∼70 aminoacid modules that exhibit limited sequence homologies but a well-conserved structure (Fig. 6A). They are most often found as singleunits, but sometimes as repeats, such as in liprins, CASKIN andAIDA proteins (Kurabi et al., 2009; Stafford et al., 2011; Wei et al.,2011). Based on the structure of liprin-α2 (PDB c3tadB), PHYRE2predicted the presence of a second SAM domain for AdcB andAdcC, just downstream of the arrestin domain. Fig. 6B and C showthe three-dimensional structure models obtained from PHYRE2under intensive mode using a set of SAM-containing templatesselected to maximize coverage and confidence. In contrast to

proteins containing SAMs in tandem, the linker separating the twoSAM domains is shorter in AdcB and AdcC, implying a possiblydifferent spatial organization of the domains relative to each other.To investigate a possible contribution of the SAM domain-containing region to AdcC response, C-terminally truncated formswere generated, removing the last (AdcCΔSAM2GFP) or both SAMs(AdcCΔSAM1/2GFP) (Fig. 6D). When expressed in adcC null orKAx-3 cells, these two cytosolic proteins responded to calcium bysome visible, but limited, membrane translocation (Fig. 6D in adcCnull cells). Recruitment to the plasma membrane was significantlyimpaired compared with full-length AdcC (Fig. 6E), indicating thatthis domain is required for efficient AdcC membrane translocation.

AdcB and AdcC homo-oligomerize in a SAM-dependentmannerBecause SAMdomains canmediate homo- or hetero-oligomerizationof SAM domain-containing proteins (Kim and Bowie, 2003; Qiaoand Bowie, 2005), we tested the possibility that AdcB and AdcCmight exist as stable oligomers. Analysis of the purified proteins innative conditions by blue-native (BN)-polyacrylamide gelelectrophoresis (PAGE) indicated that MBP-AdcB and MBP-AdcC(∼110 kDa) migrate as high-molecular-weight species (Fig. 7A).Their molecular weight, estimated at ∼1000–1200 kDa, suggestedthat the purified proteins self-interact in complexes of ≥10 subunits.To further characterize these complexes, MBP-AdcB and MBP-AdcC were examined by negative-staining electron microscopy.

Fig. 6. AdcC contains a double SAM domain necessary for its calcium-dependent translocation to the plasma membrane. (A–C) The C-terminalregions of AdcB (472-617) and AdcC (484-654) were modeled using PHYRE2 (intensive mode) based on the SAM domain-containing templates c3k1rB(H. sapiens Usher syndrome type-1G, shown in A to illustrate SAM domain structure), c1pk1A (Drosophila melanogaster ph-p CG18412), d1pk3a1(D. melanogaster Scm), c5f3xB (Mus musculus Anks4b Harp) and c3tadB (H. sapiens liprin-α2) for AdcB (B); and c2gleA (Rattus norvegicus neurabin 1),c1pk1A, d1pk3a1, d1kw4a (D. melanogaster Polyhomeotic), c5f3xB and c3tadB for AdcC (C). At least 97% of residues were modeled at >90% confidence.Images of the structures were generated using PyMOL software. (D) Scheme and localization of AdcCΔSAM1/2GFP and AdcCΔSAM2GFP after stimulation ofthe cells with 2 mM CaCl2 (arrowheads). (E) Estimation of membrane translocation efficiency by measurement of the IC/T. Histograms represent mean±s.d.with n=20, 20 and 21 cells for AdcC, AdcCΔSAM1/2 and AdcCΔSAM2, respectively. Statistical significancewas assessed by unpaired two-tailed Student’s t-tests(**P<0.001). Scale bars: 10 µm.

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This revealed a monodisperse preparation, with visible ring-shaped structures (∼20–30 nm diameter), which were absent fromthe MBP alone preparation, providing additional evidence thatMBP-AdcB and MBP-AdcC auto-assemble in organized structuresin vitro (Fig. 7B). Treatment of MBP-AdcB and MBP-AdcC withcalcium or EGTA did not affect the overall appearance of theoligomers in electron microscopy (Fig. S4), or the size of thecomplexes on nondenaturing gels (data not shown).AdcB-AdcB and AdcC-AdcC interactions were confirmed

in vivo in yeast two-hybrid assays (Fig. 7C), as well as inco-immunoprecipitation experiments using KAx-3 cells expressing

AdcCGFP or adcB null cells expressing AdcBRFP/AdcBGFP

(Fig. 7D,E). Endogenous AdcC or AdcBGFP were specificallypulled down with immunoprecipitated AdcCGFP and AdcBRFP,respectively. Calcium addition to the lysis buffer had no effect onAdcB or AdcC homotypic interactions (data not shown). Whereasdeletion of the N-terminal domain of AdcB or AdcC (ΔNtconstructs) did not noticeably affect pulldown of the full-lengthproteins, truncation of the C-terminal SAM (ΔSAM2 constructs) orof the SAM tandem (ΔSAM1/2 constructs) in AdcB or AdcCmarkedly interfered with binding (Fig. 7C–E). Accordingly,constructs limited to the Nt domains also failed to bind the

Fig. 7. See next page for legend.

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full-length proteins (Fig. 7D,E). These results therefore support arole for the SAM domain-containing region in the oligomerizationof the proteins.When expressed in Dictyostelium, the constructs limited to the

SAM-containing region of AdcB and AdcC (AdcB-SAM1/2 andAdcC-SAM1/2) could not be recovered in the soluble fraction aftercell lysis, precluding co-immunoprecipitation approaches to testthe implication of a SAM–SAM interaction in the oligomerization.As an alternative, a C-terminally S-tagged version of AdcC SAMtandem (AdcC-SAM1/2-Stag) was expressed in bacteria togetherwith an N-terminally His-tagged version (His-AdcC-SAM1/2).Purification of His-AdcC-SAM1/2 on Ni-NTA beads allowedthe co-purification of the S-tagged protein (Fig. 7F), suggestingthat AdcC homo-oligomerization might involve a SAM–SAM interaction.Analysis by BN-PAGE of a cytosolic fraction from vegetative

KAx-3 indicated that, in nondenaturing conditions, cytosolic AdcBand AdcC were exclusively found in high-molecular-weightcomplexes of ∼750 kDa (Fig. 7G). Their exact composition iscurrently unknown, and we cannot exclude a possible associationwith partners. However, in the hypothesis of homo-oligomericcomplexes, the molecular weight of ∼750 kDa estimated from thegel would again suggest the presence of ∼10 subunits, as obtainedfor the in vitro complex with theMBP-tagged counterparts. Similaranalyses were conducted on a plasma membrane-enriched fractionfrom KAx-3 cells after calcium stimulation to examine whethermembrane-associated AdcC is also part of a complex. Prior toelectrophoretic separation, the fraction was treated with 0.5%dodecylmaltoside, allowing the solubilization of ∼50% of theprotein. This pool of AdcC was mostly found associated with a

high-molecular-weight complex, similar in size to the one present inthe cytosol, and occasionally with much larger species, migrating assmears, that could correspond to precipitated protein (Fig. 7G). Totest whether the membrane complexes contain several AdcCentities, we analyzed the ability of AdcCRFP to bring to theplasma membrane the Nt-deleted construct, AdcCΔNtGFP, which isunable to reach the membrane on its own (Fig. 4A). Although GFPalone was insensitive to the presence of AdcCRFP at the membraneand remained cytosolic (Fig. 7H, right), expression of AdcCRFP

restored some AdcCΔNtGFP translocation to the plasma membraneupon calcium stimulation, which paralleled that of AdcCRFP

(Fig. 7H, left), indicating that AdcC associates with themembrane as an oligomeric complex.

AdcB is a partner of AdcCGiven the presence of SAM domains in both AdcB and AdcC, wetested a possible interaction between the two proteins. In KAx-3 cells,the endogenous proteins were found to co-immunoprecipitate,independently of the antibody used for immunoprecipitation(anti-AdcB or anti-AdcC) (Fig. 8A). Binding of AdcB to AdcC islikely to be direct, as AdcB and AdcC also strongly interacted witheach other in yeast two-hybrid assays (Fig. 8B). The interactionwas maintained upon truncation of the AdcB-Nt domain, but wascompletely abolished by deletions removing the SAM domains ofeither protein (Fig. 8B,C), indicating that the SAM-containing regionsare essential for the formation of the hetero-oligomer. Despite thisinteraction, co-expression of AdcCRFP together with AdcBGFP

was not sufficient to efficiently recruit AdcB to the plasmamembrane upon calcium stimulation (Fig. S5). Barely visiblemembrane staining, if any, was observed with AdcBGFP in cellsharboring membrane-associated AdcCRFP, indicating that AdcBis not part of the AdcC membrane complex, or is in a verylimited amount compared with AdcC. BN-PAGE analyses ofDictyostelium cytosolic fractions showed that deletion of adcBor adcC did not visibly alter the size or the abundance of theAdcC- and AdcB-positive complexes, respectively (Fig. 7G),suggesting that the AdcB- and AdcC-positive complexes detectedaround 750 kDa are unlikely heteromeric. It cannot be excluded,however, that in the single-null background, the remaining proteinsubstitutes for the disrupted one, allowing the formation ofcomplexes of a similar size to those in the wild-type context.

DISCUSSIONA direct role for calcium in AdcC targeting to theplasma membraneArrestin proteins respond to a variety of external signals, amongwhich a large panel of GPCR ligands. Although cytosolic in restingconditions, these adaptor proteins translocate to their GPCR targetsat the plasma membrane upon receptor ligand activation, andmodulate their fate and associated signaling. For most GPCRs,arrestin binding involves recognition, by different regions of thearrestin core, of the activated conformation of the receptor andits phosphorylated state, even though requirement for receptorphosphorylation is not absolute (Zhou et al., 2017; Gurevich andGurevich, 2013; Tobin, 2008). In this work, we established that thearrestin-related protein AdcC ofDictyostelium is transiently targetedto the plasma membrane in response to various stimuli (externalcalcium, cAMP and folate), all inducing a cytosolic calciumelevation, and we provide evidence that calcium is a direct regulatorof AdcC localization. In contrast to mammalian canonical arrestins,AdcC displays a C2 domain located at the N-terminal extremity ofthe protein and a C-terminal SAM domain-containing extension.

Fig. 7. AdcB and AdcC homo-oligomerize in a SAM-dependent manner.(A) Purified MBP, MBP-AdcB and MBP-AdcC were separated by BN-PAGE on3–12% Bis-Tris acrylamide gels followed by western blot analysis using anti-MBP antibodies. (B) Purified MBP, MBP-AdcB and MBP-AdcC were observedby electron microscopy after uranyl acetate staining. Higher magnification viewsof MBP-AdcB andMBP-AdcC are shown in the smaller images to the right of themain images. (C) AdcB and AdcC were tested for their interaction with AdcB,AdcBΔSAM1/2 and AdcBΔSAM2, or AdcC, AdcCΔSAM1/2 and AdcCΔSAM2,respectively, by yeast two-hybrid assays. Interaction was assessed by the bluecoloration of the colonies (insets) and/or measurement of β-galactosidaseactivity expressed as a percentage of AdcB-AdcB or AdcC-AdcC, respectively.Histograms represent mean±s.d. (n=4, except n=3 for negative controls).(D) Immunoprecipitation experiments were performed on adcB null cellsco-expressing AdcBGFP and RFP-tagged forms of AdcB using an RFP-TrapMA kit (left) or anti-RFP antibodies plus protein G agarose beads (right).Co-immunoprecipitation of AdcBGFP was analyzed by western blotting usinganti-GFP antibodies. HC, heavy-chain antibody. (E) Immunoprecipitationexperiments were performed on KAx-3 cells expressing GFP-tagged AdcC ortruncated forms using a GFP-Trap MA kit. Co-immunoprecipitation ofendogenous AdcC was analyzed by western blotting using anti-AdcCantibodies. (F) His6-tagged and S-tagged AdcC-SAM1/2 domains wereco-expressed in bacteria. His-AdcC-SAM1/2 was purified on Ni-NTA beads.Co-elution of the AdcC-SAM1/2-Stag construct was followed by western blotting.FT, flow-through on the Ni-NTA resin; S, soluble fraction prior to purification;W, wash; 1–4, successive elution fractions. Bacteria solely expressing AdcC-SAM1/2-Stag were used as controls. (G) KAx-3, adcB, adcC and adcB/C nullcells were stimulated with or without calcium and lysed through a 3-µm pore filterin the presence of calcium or EGTA, respectively. The cytosolic fraction (S) ofEGTA-treated lysates and the plasma membrane-enriched pellet fraction (P) ofcalcium-treated cells/lysates were analyzed by BN-PAGE and western blotting.The arrowhead indicates high-molecular-weight structures containingendogenous AdcB or AdcC; the asterisk indicates nonspecific band obtainedwith the anti-AdcB antibody. (H) adcC null cells co-expressing AdcCRFP andAdcCΔNtGFP were observed after stimulation with 2 mM CaCl2 (left). Imageswere extracted from timelapse acquisitions. Cells co-expressing AdcCRFP andGFP were used as controls (right). Scale bars: 10 µm.

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We showed here that the C2 domain confers the protein calcium-sensing and lipid-binding properties, and that a functional C2domain is essential for the translocation of AdcC to the plasmamembrane: (1) deletion of the C2 domain prevents AdcCmembranetranslocation in response to external calcium; (2) recombinantAdcC, as well as its N-terminal domain alone, can bind anioniclipids in a calcium-dependent manner in liposome sedimentationassays, provided that the N-terminal domain is expressed with thedimerizing GST tag that partially substitutes for the oligomerization-mediating SAM domain in the full-length protein; and (3) interferingwith calcium binding through point mutations in the C2 domain(AdcCN4) modifies AdcC properties both in vivo and in vitro.So far, the only known membrane target of AdcC is the high-

affinity cAMP receptor cAR1 (Cao et al., 2014). This GPCR-typereceptor is essential during the early stages of Dictyosteliumdevelopment. At this stage, pulses of cAMP in the nM range,released by starving cells, orchestrate their chemotactic aggregation.As cells reach mound stage, they are subjected to higherconcentrations of cAMP, leading to the activation of cell-typedifferentiation-specific pathways. The repertoire of cAMP receptorspresent at the plasma membrane is modified, with a partialreplacement of cAR1 by lower-affinity members of the cAMPreceptor family (Kim et al., 1998; Ginsburg et al., 1995; Sergé et al.,2011). Stimulation of cAR1 by cAMP (as well as other cARs) hasbeen shown to trigger a transient calcium influx, the extent of which

depends on ligand concentration (Nebl and Fisher, 1997; Yumuraet al., 1996; Milne and Devreotes, 1993). In this work, we establishedthat calcium entry and subsequent cytosolic elevation play a key rolein the cAMP-induced recruitment of AdcC to the plasma membrane,as no translocation is observed in the absence of external calcium or inthe presence of calcium channel inhibitors. Whether calcium-dependent membrane binding (via the C2 domain) occurssimultaneously to, or precedes, receptor recognition (likely throughthe arrestin domain) is not yet clarified. Our results showing thecalcium-dependent lipid-binding properties of recombinant AdcC invitro, and the fact that an increase in external calcium, and thus incytosolic calcium, is sufficient to trigger transient AdcC membranebinding in the absence of cAMP, or in a car1/3 null mutant, argue infavor of a model in which the calcium increase caused by thechemoattractant is the primary signal driving AdcC recruitment fromthe cytosol to the plasma membrane. This hypothesis is alsosupported by data from Cao et al. (2014), showing that althoughAdcC preferentially interacts with the cAMP-activatedphosphorylated form of cAR1 in co-immunoprecipitationexperiments, its translocation to the plasma membrane in responseto cAMP is not visibly perturbed in a car1/3 null strain expressing thecm1234 version of cAR1, which is nonphosphorylatable but is stillcapable of causing calcium influx (Milne et al., 1995). Nonetheless, inmammals, receptor phosphorylation is not a systematic prerequisite toarrestin recruitment (Tobin, 2008). In addition, besides GPCRs,

Fig. 8. AdcB and AdcC are partners. (A) AdcB and AdcC were immunoprecipitated from KAx-3 cells using anti-AdcB and anti-AdcC antibodies, respectively.The co-immunoprecipitation of endogenous AdcC and AdcB was assessed by western blotting. The adcB and adcC null strains were used as controls. (B) AdcBand AdcC were used as bait in yeast two-hybrid assays to test the interaction with AdcC, AdcCΔSAM1/2 and AdcCΔSAM2, or AdcB, AdcBΔSAM1/2 andAdcBΔSAM2, respectively. Interaction was assessed by the blue coloration of the colonies (insets) and/or measurement of the β-galactosidase activity.Histograms represent mean±s.d. (n=4, except n=3 for negative controls). (C) Immunoprecipitation experiments were performed on adcC null cells co-expressingAdcCGFP and RFP-tagged forms of AdcB using an anti-RFP antibody. Co-immunoprecipitation of AdcCGFP was assessed by western blotting using anti-GFPantibodies. HC, heavy-chain antibody. IP, immunoprecipitation.

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members of the arrestin clan are known to regulate a variety ofother receptor and nonreceptor membrane proteins (Lefkowitz et al.,2006; Becuwe et al., 2012a). Stimulation of car1/3 null cells(and parental strains as well) with external calcium could not onlyincrease internal calcium but also ‘activate’ some other membranetargets of AdcC, which could contribute to the recruitment of thearrestin to the plasma membrane, together with the C2 domain/lipidinteraction. Identification of the complete repertoire of AdcC targetsand further analysis of the chronology of the events following cAMPstimulation are needed to fully understand the modalities ofrecruitment and the functioning of this unconventional arrestin inthis cAMP context.The involvement, in Dictyostelium, of a calcium-dependent

component in the recruitment of the arrestin AdcC at the plasmamembrane raises the question of the gain of such a calcium-dependent regulation, compared with the situation in mammals. Inthe context of cAMP signaling, membrane docking of AdcC inresponse to the increase in calcium triggered by cAR1 stimulationcould contribute to amplify the effect of cAMP and facilitatereceptor recognition of AdcC by concentrating the arrestin in thereceptor-close environment. As this calcium influx is dependent onthe cAMP concentration, we propose that intracellular calcium, byreflecting the level of external cAMP, allows a spatiotemporalcontrol of AdcC activities by setting the conditions (such as timeand amount) of recruitment of the arrestin to the plasma membraneas a function of the stimulus intensity, thereby adjusting the effect ofthe arrestin on its membrane target fate and/or associated signalingdifferently depending on the developmental stage. Cao et al. (2014)have established that disruption of adcC and of its homolog adcB(see below) affects the development of the double-null strain, withaltered Erk2 phosphorylation kinetics during aggregation andreduced internalization of cAR1 postaggregation. To pursue thefunctional characterization of AdcC, it would have been interestingto test the ability of truncated and point mutants of AdcC tosubstitute for the wild-type protein and complement the adcC/B nulldefects. In our hands, the KAx-3 cells devoid of adcC or adcB/C(this work), and the double null strain (Ax2 background) from Caoand colleagues obtained from the DictyStock Center, displayedno obvious defect regarding development, Erk2 phosphorylationkinetics and internalization of cAR1 (L.A. and L.M., unpublished),precluding complementation assays. The reasons for the discrepancybetween our observations and their published data are currentlyunclear, but they could – in part – reside in differences in cultureconditions or experimental procedures.Given that AdcC also responds to folate, it is possible that the

recently identified GPCR-type folate receptor fAR1 (Pan et al.,2016) is also a target of AdcC. This receptor is required for both cellchemotaxis towards folate-secreting bacteria and their phagocytosis(Pan et al., 2016). As in the case of cAMP, cell stimulation withfolate has been shown to induce an increase in intracellularcalcium that depends on folate concentration in the extracellularmedium (Nebl and Fisher, 1997; Yumura et al., 1996). Dependingon fAR1 activation level, and on the intracellular calcium signalgenerated, AdcC could intervene in the fate or functions of thereceptor, and adjust the cell response to the chemotactic and/orphagocytic contexts.In addition to the direct role of calcium on AdcC localization, we

identified three calcium-binding proteins among the putativeinteractors of AdcC in a yeast two-hybrid screen (L.M. and L.A.,unpublished), suggesting that calcium might additionally impactAdcC-dependent signaling by modulating its interaction withpartners and/or the resulting outcomes.

AdcB and AdcC: alike but differentDespite high structural similarities, the present study unveiled amajor difference between AdcC and its homolog, AdcB. In contrastto AdcC, AdcB was never observed at the plasma membrane inresponse to a calcium-induced elevation of cytosolic calcium.This result is coherent with AdcB insensitivity to folate andcAMP (our own observations; Cao et al., 2014). Our data usingchimeric proteins indicate that the inability of AdcB to bindmembranes in response to calcium likely resides in the N-terminaldomain. However, in vitro characterization of the AdcB C2domain in liposome-based sedimentation assays revealed agenuine calcium-dependent C2 module. For many C2 domain-containing proteins with a preference for anionic lipids,phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] contributes totheir membrane association, together with PS, by binding to adistinct surface, the basic β-groove region (Cho and Stahelin,2006; Guerrero-Valero et al., 2009; Guillén et al., 2013). Somepreliminary experiments indicated that the addition of 5%PI(4,5)P2 to 20% PS-containing liposomes significantlyincreases binding of both AdcC and AdcB in a similar manner,without modifying their calcium affinity, suggesting that thedistinct behavior of the two proteins is not a consequence of adifferent response to that particular phosphoinositide. Theopposite response of the two proteins in vivo could ratherresult from distinct activatory/inhibitory post-translationalmodifications or partners targeting the C2 domain, therebyimpacting calcium and/or membrane binding as shown for severalmembers of the synaptotagmin and PKC protein families (Lee et al.,2004; Pepio and Sossin, 2001; Roggero et al., 2005). Independentlyof the mechanism(s) responsible for this behavioral difference, ourdata argue against a full functional redundancy between AdcB andAdcC, at least for the functions fulfilled by AdcC at the plasmamembrane. In this context, identification of AdcB as a partner ofAdcC raises the question of the functional role of such interaction.Additional experiments will be required to establish whether theassociation of AdcB with AdcC allows a specific set of functions orplays some regulatory role, for example in controlling a pool ofcytosolic AdcC, available for membrane targeting.

AdcC and AdcB: large polymeric platformsWe have shown here that AdcB and AdcC exist mainly as partof high-molecular-weight complexes in vivo. Their composition,which could include several partners, is currently unknown, but ourresults support the presence of several monomers per complex:(1) purified proteins form large size homo-oligomers in vitro;(2) two-hybrid and co-immunoprecipitation experiments establishedthat AdcB and AdcC are able to self-interact in cellulo; and, (3) inthe case of AdcC, the calcium-triggered membrane-bound formcontains several copies of AdcC. Our data indicate that the SAMmodule is involved in oligomerization, and a direct SAM-SAMinteraction, as shown for AdcC on truncated domains, could verywell account for the organized structures observed by electronmicroscopy with recombinant full-length proteins. Many isolatedSAM domains can produce helical polymeric assemblies (fibers)through head-to-tail interactions (Harada et al., 2008; Kimet al., 2002; Knight et al., 2011; Thanos et al., 1999). Such fiberswere not present in our preparations of MBP-tagged full-lengthproteins, but were sometimes observed with the isolated MBP-tagged SAM region of AdcC (C.D. and L.A., unpublished). In thefull-length proteins, polymerization might be restrained by aspecific arrangement of the SAM tandem and/or the presence ofthe long N-terminal extension. Besides the SAM region, oligomer

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formation could also involve the arrestin core, as described formammalian β-arrestins and visual arrestin-1 (Chen et al., 2014).β-arrestins, which are devoid of SAM domains, are able to self-associate as dimers or tetramers through their N and C domains in anIP6-dependent manner, and their oligomeric status has been shownto impact their subcellular localization and functions (Chen et al.,2014; Hanson et al., 2008; Milano et al., 2006). AdcB and AdcCarrestin domains alone were found to be poorly expressed inDictyostelium and bacteria, but their capacity to multimerize andtheir regulation by IP6 would certainly be worth investigating, allthe more because this inositol polyphosphate, which is highlyabundant in the amoeba (Martin et al., 1987), might modulate lipidbinding by the C2 domain, as shown for the mammaliansynaptotagmin 1 C2B domain (Joung et al., 2012; Lu et al., 2002).Oligomerization could serve different purposes. In the case of

AdcC, we proposed that oligomerization increases the avidity of thecomplex for membrane lipids by gathering multiple C2 domains,thereby indirectly participating in AdcC membrane binding. Giventhe multimodular organization of the protein, it could also directlycontribute to the assembly of signaling platforms or specificarchitectures in the vicinity of activated membrane targets, andthereby modulate/facilitate receptor downstream signaling orinternalization in the endocytic pathway.To conclude, our study characterized the AdcC protein of the

Dictyostelium amoeba as a novel calcium sensor, showing thatcalcium plays a key role in AdcC recruitment to the plasmamembranein response to various stimuli, such as the chemoattractants cAMPand folate. It revealed an unusual mode of regulation for a protein ofthe arrestin clan that involves a C2 domain with calcium-dependentmembrane-targeting properties, and a double SAM domain involvedin AdcC oligomerization, which is necessary for its association withthe membrane and interaction with the calcium nonresponsivehomolog AdcB. Despite high homology and common properties,our data suggest distinct roles and specific regulatory mechanisms forthese two arrestin-related scaffolding proteins.

MATERIALS AND METHODSStrains and cell cultureMost experiments were conducted on the D. discoideum parental strainKAx-3 from the R. Firtel laboratory (University of California San Diego,La Jolla, CA, USA) and derived knockout or overexpressing strains. Thecar1/3 null cells and their parent Ax2 cells were obtained from P. Devreotes(Johns Hopkins University School of Medicine, Baltimore, MD, USA).All cells were grown in Petri dishes or in shaking culture at 21°C inmaltose-containing HL5 medium. Overexpressors were selected byaddition of G418 (20 µg/ml) or hygromycin (40 µg/ml), depending onthe expression vector. The adcB and adcC null mutants were selected inthe presence of blasticidin (7.5 µg/ml). When required, cells were washedand resuspended in 12 mM NaK-phosphate buffer pH 6.2 (PB), thenstarved for 2–4 h (starved cells) or pulsed with 100 nM cAMP every 6 minfor 4 h (aggregation-competent cells).

Plasmid constructsThe single-null adcB−, adcC− and double-null adcB−/adcC− mutants weregenerated by homologous recombination using the Cre-Lox pLPBLP vector(blasticidin resistance BsR cassette) (Faix et al., 2004). Genomic DNAfragments corresponding to bp 223–545 (ClaI-HindIII) and 814–1160(BamHI-SpeI) for AdcB and bp 58–426 (ClaI-HindIII)/451–951 (BamH1-EcoRI/SpeI) for AdcC were amplified by PCR with oligonucleotidescontaining the mentioned restriction sites and subcloned in pLPBLP on eachside of the BsR cassette. The resulting plasmids were linearized with ClaIandClaI/EcoRI prior to electroporation in KAx-3 cells. After selection, cellswere cloned by plating on SM agar plates in association with Klebsiellaaerogenes. To obtain the adcB−/adcC− mutant, the adcC− null mutant

was transformed with the pDEX-NLS-Cre vector for BsR cassette extraction(Faix et al., 2013), and used to introduce the second disruption construct asdescribed above. Gene knockouts were confirmed by PCR, Southernblot and/or western blot analyses (Fig. S1A) of individual clones. Foroverexpression purposes, the following AdcB- and AdcC-derived constructswere generated by subcloning PCR fragments in the vectors pExp4+(G418R), pDM1045 and/or pDM1043 (HygromycinR): AdcB [amino acids(a.a.) 1–617], AdcB-Nt (a.a. 1–174), AdcBΔC2 (a.a. 106–617), AdcBΔNt(a.a. 166–617), AdcBΔSAM2 (a.a. 1–547), AdcBΔSAM1/2 (a.a. 1–472),AdcC (a.a. 1–654), AdcCD20N/D26N, AdcCD20N/D26N/D70N/E74N (AdcCN4),AdcC-C2 (a.a. 1–120), AdcC-Nt (a.a. 1–169), AdcC-NtN4, AdcCΔC2(a.a. 101–654), AdcCΔNt (a.a. 160–657), AdcCΔSAM1/2 (a.a. 1–485)and AdcCΔSAM2 (a.a. 1–567), and the chimeric proteins AdcB(a.a. 1–168)/AdcC (a.a. 162–654) and AdcC (a.a. 1–161)/AdcB(a.a. 169–617). For biochemical analyses, AdcB, AdcB-Nt, AdcC,AdcC-Nt and AdcCN4 were expressed as GST and/or MBP fusion proteinsfrom pGEX-KG or pMAL-C2 plasmids. AdcC-SAM1/2 (a.a. 485–654) andAdcC-Nt were expressed as His6-tagged proteins using the plasmids pET-duet1 and pET22, respectively. AdcC-SAM1/2 corresponding cDNA wasalso subcloned in the second site of pET-duet1 to co-express His-tagged andS-tagged versions of the domain. Constructs requiring PCR amplificationwere verified by sequencing (Beckman Coulter).

Recombinant protein expression and purificationRecombinant proteins were expressed in Bl21-DE3 Escherichia colias described (Becuwe et al., 2012b), but with a 2% ethanol treatment.For GST- and MBP-tagged proteins, bacterial pellets were resuspendedin PBS (pH 7.4), 5 mM EDTA-containing protease inhibitors and1 mg/ml lyzozyme. Bacteria were sonicated after addition of 1% TritonX-100 and three volumes of lysis buffer A (PBS, 10 mM EDTA, 1 mMEGTA, 3 mM DTT, protease inhibitors). After clearing of the lysate(30 min, 16,000 g), recombinant proteins were purified on glutathionesepharose (GE Healthcare) or amylose resin (New England Biolabs)according to the manufacturer’s instructions. After several washes inbuffer A containing 1% Triton X-100 and a wash in PBS, GST- and MBP-tagged proteins were eluted in 10 mM glutathione, 50 mM Tris pH 8.0 or10 mM maltose, 20 mM Hepes pH 7.4, 100 mM NaCl, 1 mM EGTA,respectively. Proteins were dialyzed against 20 mM Hepes pH 7.4, 100 mMNaCl and quantified before use. The His-tagged proteins were purified on Ni-NTA beads in the presence of 20 mM imidazole as described in Guetta et al.(2010) and eluted in 150 mM imidazole.

Production of antibodies and western blot analysisAntibodies against AdcB and AdcC were raised in Dunkin Hartley guineapigs and New Zealand White rabbits, respectively (Covalab, Villeurbanne,France). Purified GST-AdcB-Nt and His6-AdcC-Nt proteins were used asantigens, and antibodies were, respectively, purified on immobilized GST-AdcB-Nt (after removal of anti-GST antibodies) and GST-AdcC-Nt asdescribed (Harlow and Lane, 1988). Antibody specificity was assessed bywestern blotting using protein extracts from adcB and adcC null mutantsas controls. Anti-GFP (7.1/13.1, 1:1000), anti-RFP (5F8 or 3F5, 1:1000),anti-His (27471001, 1:1500), anti-Stag (71549-3, 1:5000) and anti-MBP(E8032, 1:10,000) were from Roche, ChromoTek, GE-Healthcare, Milliporeand New England Biolabs, respectively. Western blots were performed onpolyvinylidene difluoride (PVDF) membranes blocked in 1% bovine serumalbumin, or as suggested by the manufacturers.

Co-immunoprecipitationApproximately 1–2×107Dictyostelium cells were resuspended in lysis buffer(10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP40 andprotease inhibitors). After 10 min on ice, the lysates were centrifuged at16,000 g for 10 min. The appropriate antibodies (anti-GFP 7.1/13.1, 1:500;anti-RFP 5F8, 1:500; anti-AdcC, 1:200; or anti-AdcB, 1:200) and protein Aor G agarose (from Roche) were added to the supernatants. After 1 h ofincubation andwashes in lysis buffer, the proteins were recovered by additionof denaturing buffer. Alternatively, immunoprecipitation was performedusing the GFP- or RFP-Trap MA kit (ChromoTek) on whole-cell NP40extracts as described by the manufacturer.

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Liposome binding assayFor co-sedimentation assays, sucrose-loaded liposomes were generated frommixtures of L-α-phosphatidylcholine (PC) and/or L-α-phosphatidylserine(PS) (P3556 and P7769 from Sigma-Aldrich) as described (Mosior andEpand, 1993), except that lipid rehydration was performed in 20 mMHepespH 7.4, 180 mM sucrose, and liposomes were centrifuged for 30 min at20,000 g (22°C), and then washed and resuspended in 20 mM Hepes pH7.4, 100 mM NaCl (Hepes/NaCl buffer). Proteins (∼0.5 µM) wereincubated with liposomes (0.9 mg/ml) and EGTA (1 mM) or CaCl2 at thedesired concentration, for 30 min at room temperature. Liposomes werepelleted as above, generating a supernatant and a pellet that was washed andresuspended in Hepes/NaCl buffer with EGTA or CaCl2 prior to transfer to anew tube. Equivalent amounts of supernatant (free protein) and pellet(bound protein) were analyzed by sodium dodecyl sulfate (SDS)-PAGE.Coomassie-stained proteins were quantified using ImageJ software (https://imagej.nih.gov/ij/). Data were fitted to the Hill equation y=a[Ca2+]n/(Kdn+[Ca2+]n), where ‘n’ is the Hill coefficient, ‘a’ an arbitrarynormalization constant and ‘Kd’ the apparent dissociation constant. Ca2+/EGTA buffers with free calcium in the range of 0.1–1000 µM wereprepared according to MaxChelator (http://maxchelator.stanford.edu).

Live imagingCells were allowed to adhere to eight-well Labtek chambered coverglassesin HL5 culture medium (vegetative cells) or in PB (starved or pulsed cells).Prior to imaging, the medium was exchanged for PB or distilled water(vegetative cells) or PB (starved or pulsed cells). In the case of vegetativecells, water or PB was used, as the cell response to calcium was stronger inhypo-osmotic conditions. After several minutes, cells were treated withCaCl2 in the same medium and imaged within the next 6–8 s on a confocalspinning disk inverted microscope (Nikon TI-E Eclipse) equipped with aYokogawa motorized confocal head CSUX1-A1 and an Evolve EMCCDcamera (1 frame/2.5 s). For folate and cAMP stimulation, vegetative andpulsed cells, respectively, were first placed in PB containing 100 µM CaCl2,as external calcium was shown to be required for the chemoattractant-induced increase in calcium. Because calcium induces a transient phase ofoscillations of AdcC at the plasma membrane (see Results), cells were left inbuffer until AdcC regained a stabilized cytosolic localization. Cells werethen stimulated with 50 µM folate or 10 µM cAMP and imaged. ForRuthenium Red (RR) treatment, cells were exposed to 20 µMRR for severalminutes prior to the addition of the appropriate stimulus. Image acquisitionwas performed at a median z-plan every 2.5 s for 5–7 min using Metamorphsoftware. Fluorescence quantification was performed using ImageJsoftware. Mean total (T) and cytosolic (C) fluorescence density valueswere measured on a median z-plan at time points of maximal membranelabeling on regions of interest (ROIs) corresponding to the whole cell or tothe cytoplasm, respectively. Nuclei and large-size cytoplasmic vesiclesvisible on the z-plan were purposely removed from the selections to restrainthe measurement to the cytosol contribution only. The mean backgroundvalue obtained from ROIs taken outside of the cells was subtracted from theT and C values, leading to corrected C and corrected T values. The IC/Tcorresponding to the ratio (corrected C/corrected T) was used as anestimation of membrane translocation (or membrane binding in the case ofconstitutive binding) efficiency of the constructs. AdcC-binding sites at theplasma membrane were visibly not saturated in our conditions of proteinexpression.

For Calcium Green-1 dextran (CG-1) imaging, 6×106 cells wereresuspended in 50 µl PB containing 50 mM sucrose and 8 mg/ml CG-1and electroporated (1 pulse, 500 V, 3 µF, 0.2 cm gap cuvette) before transferto HL5medium. After cell adhesion, the mediumwas replaced by fresh HL5and by PB 1 h later. Imaging was performed within the next 1–2 h. Tocompare CG-1 variations of fluorescence intensity and AdcCRFP behavior,corrected mean fluorescence density values were measured as a function oftime on ROIs corresponding to the cytoplasm only for CG-1, or to thecytoplasm and to the plasma membrane for AdcCRFP.

Yeast two-hybrid interaction and β-galatosidase assaysTwo-hybrid interaction assays were performed using the LexA two-hybridsystem (Gyuris et al., 1993) in the EGY48 S. cerevisiae strain containing the

LacZ reporter construct pSH18-34. The vectors pEG202 and pJG4-5 (GAL1promoter) were used to express the bait and prey constructs, respectively.Interactions were examined on plates under high stringency selectionconditions [synthetic dropout (SD) medium containing 2% galactose (Gal)instead of glucose and lacking Ura, His, Trp and Leu] and confirmed byre-streaking the colonies on Gal-SD lacking Ura, His and Trp, supplementedwith 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal). Theβ-galactosidase activity was quantitatively assayed in liquid mediumusing O-nitrophenyl β-D-galactopyranose as a substrate. The backgroundactivity was obtained from the strain expressing the bait constructs andcontaining empty pJG4-5.

Transmission electron microscopyFor negative staining, 2 µl containing 30 ng purified MBP-AdcB or MBP-AdcC was loaded in a carbon-mica interface. The carbon layer was floatedon 2% uranyl acetate solution, recovered with a 400-mesh copper grid (AgarScientific), air dried and observed with a JEOL 1200EX transmissionelectron microscope at 80 kV. Images were taken with a digital camera(Veleta, Olympus) at 100,000× magnification.

BN-PAGEApproximately 5×107 cells were resuspended in 500 µl 50 mMTris, pH 7.5,50 mM NaCl plus protease inhibitors and stimulated with or without 2 mMCaCl2 for 5 min at 21°C in shaking conditions. Cells were directly brokenthrough a 3-µm pore polycarbonate filter in the presence of CaCl2 or 2 mMEGTA for nonstimulated cells. Lysates were centrifuged for 10 min at1000 g to recover the AdcC- and plasma membrane-enriched fraction(pellet, P) in the CaCl2 condition, or for 30 min at 100,000 g to obtain theAdcC-enriched soluble fraction (supernatant, S) in the EGTA condition.The pellet was resuspended in 500 µl of the same buffer containing 0.5%dodecylmaltoside, left for 1 h on ice with regular vortexing, and centrifugedat 16,000 g for 20 min to remove nonsolubilized material. All samples werestored at −20°C in 15% glycerol. Soluble and membrane samplescorresponding to 2×105 and 4×105 cells, respectively, were analyzed byBN-PAGE on 3–12% Bis-Tris native gels (Novex, Thermo FisherScientific) according to the manufacturer’s instructions. BN-PAGE gelswere soaked in transfer buffer (25 mM Tris, 192 mM glycine, 0.1% SDS,20% EtOH pH 8.3) for 20 min prior to protein transfer to PVDFmembranes.For analysis of purified MBP-tagged proteins, 1–5 ng of material wasloaded on the native gel and separated as above.

AcknowledgementsWe thank F. Letourneur and A. Bouron for critical reading of the manuscript; thegroup of C. Picart for advice on liposome binding assays; T. Rabilloud, E. Faudry,R. Dumas and F. Parcy for discussions; T. Soldati for pDM1045 and pDM1043plasmids; R. Firtel for the KAx-3 cells; and P. Devreotes for the car1/3− and parentAx2 strains. We also acknowledge the µLife cell imaging platform of the Biosciencesand Biotechnology Institute of Grenoble and the Electron Microscopy Facility ofGrenoble (MEC).

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: L.A.; Methodology: L.M., C.D., A.J., L.A.; Validation: L.M., A.C.,C.D., A.J., L.A.; Formal analysis: L.M., A.J., L.A.; Investigation: L.M., A.C., C.D., A.J.,L.A.; Resources: C.D.; Writing - original draft: C.D., A.J., L.A.; Writing - review &editing: L.M., A.C., C.D., A.J., L.A.; Visualization: L.M., C.D., A.J., L.A.; Supervision:L.A.; Project administration: L.A.; Funding acquisition: L.A.

FundingThis work was supported by the Commissariat a l’Énergie Atomique et aux ÉnergiesAlternatives, Institut National de la Sante et de la Recherche Medicale, UniversiteGrenoble Alpes, Centre National de la Recherche Scientifique and AgenceNationale de la Recherche [DYNOTEP ANR-12-BSV6-0016-01]. L.M. was therecipient of a fellowship from the Ministere de l’Education Nationale, del’Enseignement Superieur et de la Recherche/Universite Grenoble-Alpes.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.207951.supplemental

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