*Author ([email protected]) for correspondence Tiburon, CA94920-1205,USA. Environmental Studies, SanFrancisco State University, 3152Paradise Drive, Avenue, SanLuisObispo, CA93407-0401,USA. Center for Coastal MarineStudies, Environmental ProteomicsLaboratory, 1Grand 388 Received 11 August2014;Accepted8December 2014 1 Energy metabolism,Immuneresponse,Proteomics,Systemsbiology KEY WORDS:-bindingproteins,ATP-buffering, Clawmuscle, acute heatshockwithlimitedchangesinthemuscleproteome. temperature rangeoverwhichclawmusclewasabletorespond toan moderately fluctuatingtemperatures(10–20°C)broadened the and fluctuating10–30°Cshowedthegreatesteffects ontheproteome, Furthermore, althoughheatshockafteracclimationtoconstant 10°C fluctuations andsubsequentacuteheatshockinmuscletissue. restructuring andimmuneresponsesareallaffected bytemperature during heatshock.TheresultssuggestthatATP supply, musclefiber 10°C andfluctuating10–20°C,possiblyinhibitinganimmuneresponse prostaglandin reductaseincreasedduringheatshockfollowingconstant muscle fiberrestructuring.RAFkinaseinhibitorproteinand increased duringheatshockfollowing10–20°C,possiblypromoting actin severinganddepolymerizationproteins(gelsolincofilin) ), increasedduringheatshockfollowing10–30°C;however, Actin-binding ,whichstabilizeactinfilaments(filaminand whereas myosinlightandheavychaindecreasedwithheatshock. and myosinheavychaintype-Bincreasedinabundance,respectively, peptides. Followingconstant10°Candfluctuating10–20°C,paramyosin 10°C andfluctuating10–20°C,possiblyplayingaroleasantimicrobial hemocyanin fragmentsincreasedafterheatshockfollowingconstant increased abundancefollowingacclimationto10–30°C,but across allacclimationregimes.Full-lengthisoformsofhemocyanin isomerase andglyceraldehyde3-phosphatedehydrogenaseincreased andglycolyticenzymessuchasaldolase,triosephosphate increased inabundance.Followingheatshock,isoformsofarginine acclimation to10–30°C(measuredat10°C),enolaseandATP synthase to analyzetheproteomicchangesinclawmuscletissue.Following acclimation, animalswereexposedtoeither10°Cora30°Cheatshock 1 temperature fluctuationsbetween10and20°C(5 one ofthreetemperatureregimes:(1)constant10°C,(2)daily and subsequentheatshock,weacclimated biochemical changesaffected byincreasingtemperaturefluctuations which canincreasehabitattemperaturebyupto20°C.To identifythe during hightideandsaturatinghumidconditionsinairlowtide, mussel bedsinthemid-intertidalzonewhereitexperiencesimmersion The porcelaincrab Michael A.Garland following acclimationtodailytemperaturefluctuations eurythermal porcelaincrab The proteomicresponseofchelipedmyofibriltissueinthe RESEARCH ARTICLE © 2015.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2015)218,388-403doi:10.1242/jeb.112250 ABSTRACT California PolytechnicCalifornia Biological Sciences, State University, of Department

h down-rampto10°C)and(3)10–30°C(up-ramp30°C).After Petrolisthes cinctipes 1 , JonathonH.Stillman 2 Romberg Tiburon Centerfor lives underrocksandin P. cinctipes

h up-rampto20°C, 2 for 30 and LarsTomanek Petrolisthes cinctipes days to response toasubsequentacuteheatstress. fluctuations affect thephysiologyoforganisms anddetermine the acute heatstress,verylittleisknownabouthowdailytemperature physiological plasticityinresponsetoconstanttemperaturesand understanding ofthethermalrangeorganisms andtheir and Somero,1999).However, althoughwehaveagood in responsetotemperatureacclimation(Stillman,2003;Tomanek adjust cardiacthermalperformanceandheat-shockproteinsynthesis from thesubtidaltomid-intertidalzonediffer intheirabilityto that varygreatlyinthermalfluctuationsalongthephysicalgradient Somero, 2000)andcloselyrelatedspeciesoccupyingenvironments under eachcondition(BjeldeandTodgham, 2013;Tomanek and during emersionandimmersionasaresultofdiffering heatingrates organisms showdifferent physiologicalresponsestoheat stress recent thermalhistory, e.g.acclimatization.Forexample,intertidal evolutionary history, e.g.thethermalenvironmentitoccupiesand determined bytherateandamplitudeoftemperaturechangeits biochemical processestodifferent bodytemperaturesisinlarge part (Somero, 2012;Tomanek, 2010).Anorganism’s abilitytoadjust structures tocopewiththespecificthermalpropertiesoftheirhabitat organisms, whichhaveadaptedtheircellularprocessesand biochemical reactionsandcellularstructuresinectothermic Environmental temperaturehasaubiquitouseffect onratesof might occurunder fluctuatingtemperatureregimes, especiallynot knowledge therehasnotbeena studyoftheproteomicchangesthat temperature fluctuations(Podrabsky andSomero,2004).To our framework forthecellularprocesses thatmightbeaffected by of membraneintegritydiffer betweentheseconditions,providing a proliferation, molecularandchemical chaperonesandmaintenance daily temperaturesshowedthat genesinvolvedincellgrowthand ( transcriptomic changesinlivertissueofannualkillifish variable environments(Rocketal.,2009).A studyonthe diversity inmyosinisoformscongenersfromthermally more although astudyongammaridamphipodsshowedgreatermolecular differences intemperature fluctuationsarelargely unknown, trade-off betweenthetwo. Theevolutionaryadaptationsto physiological costsbutalsoincreasedstresstolerance,suggesting a Stillman, 2012).Thus,greaterfluctuationscomewithincreased increased temperaturetoleranceafterover15generations(Chen and fluctuations reducedmetabolicratesaftersixgenerations but (15°C, 15–25°Cand15–30°Cdaily)showedthatthegreatest of thewaterflea( to constanttemperatures(Widdows, 1976).A studyontheresponse filtration-feeding isexpandedincomparisontoanimalsacclimated rate istemperature-independentandthattemperaturerangeof fluctuating temperaturesbroadenstherangeoverwhichmetabolic INTRODUCTION Austrofundulus limnaeus)inresponsetoconstantandfluctuating Studies ontheintertidalmussel 1, * Daphnia pulex)tovarioustemperaturefluctuations to heatshock Mytilus show thatacclimationto

The Journal of Experimental Biology thermal tolerancesofthetemperate occupies themid-tohigh-intertidalzoneandhasoneofhighest al., 2010;Teranishi andStillman,2007). characterized inresponsetoacutetemperaturestress(Tagmount et of severaltissuesonespecies, humid (Teranishi andStillman,2007).Furthermore,thetrancriptome a tidalcycleunderconditionsthatrangefromsubmersiontofully where theyexperiencetemperaturefluctuationsofupto20°Cduring from theshallowsubtidaltomid-intertidalzoneofrockyshores known. fluctuations affect theproteomicresponsetoheatstressisnot 2012b; Tomanek andZuzow, 2010).However, howtemperature (Dillyetal.,2012;Fields2012a; presumably becauseoftheeffect ofreactiveoxygenspeciesonthe abundance ofoxidativestressproteinsandmolecularchaperones, temperature stressshowedthatincreasesthe non-model organisms inresponsetoacuteheatandchronicwarm number ofrecentproteomicanalysesgilltissueseveralmarine oxidative stress(Tomanek, 2011; Tomanek, 2014).Specifically, a responses ofmarineorganisms totemperature,osmotic,hypoxicand (EST) librariesandhaveaddedtoourunderstandingofthe genome sequencesandwell-annotatedexpressedsequencetag possible recentlybecauseoftheincreasingnumbercompleted fluctuating thermalconditions. for intertidalorganisms thataretypicallyexperiencinggreatly RESEARCH ARTICLE 14.4 kDa 21.5 kDa 31.0 kDa 45.0 kDa 66.2 kDa 97.4 kDa Species oftheporcelaincrabgenus Proteomic analysesofnon-modelorganisms havebecome o-o tandemtimeofflight ToF-ToF peptidemassfingerprint S S porcelaincrabarraydatabase principalcomponentanalysis PMF pI isoelectric tandemmassspectrometry PCAD PCA matrix-assistedlaserdesorption/ionization MS/MS point MALDI P immobilizedpHgradient LT IPG List of abbreviations List of 2 1 50 pI 4 198 53 164 slow phasicmusclefiber slow twitchmusclefiber median lethaltemperature 105 83 192 95 78 204 103 94 84 85 81 180 86 185 Petrolisthes cinctipes Petrolisthes 80 165 184 100 125 127 62 Petrolisthes 79 126 101 387 18 99 320 91 120 Petrolisthes cinctipes 123 179 119 119 congeners (Stillman 102 92 90 87 159 183 88 163 17 178 150 89 are distributed 52 194 161 162 200 203 , hasbeen 13 157 4 25 128 153 6 30 27 26 57 9 132 58 156 160 38 59 136 133 137 40 193 37 29 34 439 138 8 39 129 148 189 actin-binding proteins. separated intothinfilament(),thick()and cytoskeletal andsignalingproteins.Cytoskeletalproteinswere following categories:ATP buffering, energy metabolism,respiratory, with thefunctionofspecificproteins,wegroupedproteinsinto to behypothesesthatgenerallyrequirefurthervalidation. . With theselimitationsinmind,ourinterpretationsaremeant and thereforemightnotrepresentthecompleteabundanceofthis not representthefullcomplementofisoformsaparticularprotein interpretation isbasedonasubsetofproteinisoforms,whichmight modifications (PTMs),includingpartialproteolysis.Thus,our increase ordecreaseinsynthesis,degradationpost-translational Table available for73.6%(145)oftheseproteins(supplementarymaterial heat shock,ortheirinteraction;functionalannotationswere changed abundanceinresponsetotemperatureacclimation,acute 1),wedetected469proteinspots.Ofthesespots,42%(197) (Fig. acclimation andacuteheatshocktreatments.Inthisfusedgelimage We generatedafusedimagecomprisinggelsfromalltemperature thermal limits,e.g.LT and Somero,2000).Inaddition,itshowslimitedadjustmentsin on atwo-waypermutationANOVA ( significance foracclimation,heatshockorinteractioneffects, based variation inproteinabundancepatternsbasedonstatistical We conductedprincipalcomponentanalyses(PCAs)toassessthe tissue duringsubsequentheatstress. structures anddynamicstheimmuneresponseofclawmuscle fluctuations affect energy metabolism,oxygendelivery, actomyosin heat stress.Ourresultssuggestthatdifferent rangesofthermal three different rangesoftemperaturefluctuationsandasubsequent the proteomeofclawmuscletissuechangesduringacclimationto identification ofproteinswithmassspectrometry, weassessedhow with thetranscriptomicdatabaseasafoundationfor (Stillman, 2003).Thus,giventhethermalbiologyof in contrasttoitssubtidallow-intertidaltemperatecongeners RESULTS ANDDISCUSSION Principal componentanalysis 36 35 131 135 33 141 To discussthecontributionofcellularprocessesassociated 74 42 130 118 5 75

32 143 3 145 S1). Changesinproteinabundancemaybeattributedtoan 73 114 108 139 134 167 22 82 7 142 106 67 115 116 116 158 110 The JournalofExperimentalBiology(2015)doi:10.1242/jeb.112250 109 50 71 23 111 111 146 187 49 113 149 155 12 21 70 51 11 11 168 154 107 112 112 46 10 96 pI 7 50 , withincreasingacclimationtemperature, supplementary materialTable mass spectrometry(foridentification,see P treatments (two-waypermutationANOVA; response toacclimationandheatstress spots arethosethatchangedabundancein volumes foreachproteinspot.Numbered proteome maprepresentsaveragepixel porcelain crabPetrolisthescinctipes claw muscletissueoftheintertidal treatments depicting469proteinspotsfrom Fig. ≤ 0.02) andwereidentifiedusingtandem

.Proteomemapofgelsfromallsix 1. P ≤ 0.02). Principalcomponents

P. cinctipes S1). . The 389 and

The Journal of Experimental Biology 390 RESEARCH ARTICLE 30.5%), separatedboth10–30°Cacclimationtreatments(positive were significantforanacclimationeffect. Thefirstcomponent(PC1; based ontheirloadings. identifying proteinsthatcontributedthemosttoseparation, (PCs) thatseparatedthetreatmentswereanalyzedfurtherby C B A PC1 (26.3%) PC1 (23.0%) PC1 (30.5%) i.2A) The firstPCA (Fig. includes 83proteins(65identified)that PC2 (17.6%) PC2 (15.8%) PC2 (16.4%) values on fluctuations inducedacellularresponsethatpreparedtheanimalsfor shifting samplesalongPC2,suggestingthatthesetemperature shock followingthe10–20°Cacclimationonlyhadalimitedeffect on regimes onasubsequentheatshock(negativevalues (PC2; 16.4%)representedtheeffect of10°Cand10–30°Cacclimation 10–30°C Acclimation, 30°Cheatshock 10–20°C Acclimation, 30°Cheatshock 10°C Acclimation, 30°Cheatshock 10–30°C Acclimation, noheatshock 10–20°C Acclimation, noheatshock 10°C Acclimation, noheatshock x -axis) fromallothertreatments.Thesecondcomponent The JournalofExperimentalBiology(2015)doi:10.1242/jeb.112250 30°C heatshockplusa1 to 10°C(striated)ora5 (red) andeithersubsequentlyexposed (blue), 10–20°C(green)and10–30°C represents acrabacclimatedto10°C ANOVA; P effect (basedonatwo-waypermutation (B) heatshockand(C)interaction interaction effect. acclimation, heatshockor proteins thatshowedasignificant (PCA) oftreatmentsbasedon Fig. PCA. explanation ofPC1andPC2foreach explain areshown.Seetextfor (significant protein)datasetthey total variationoftheselected and PC2)thepercentageof Principal components1and2(PC1 collected afterrecoveryat10ºC). 10°C recovery(solidline)(samples

.Principalcomponentanalysis 2. ≤ 0.02). Eachsymbol (A) acclimation,

h up-ramptoa

y h down-ramp -axis). Heat

The Journal of Experimental Biology antimicrobial – seebelow)proteins(hemocyanin fragments) GAPDH), ATP-buffering, thinfilamentsandrespiratory(or PC1 (23%;positive)andPC2(15.8%; negative). shock treatmentsofallthreeacclimation regimesareseparatedalong heat shockincluded130(90identified) proteins during heatshock. shock). Additionally, aneurogenicnotchhomologplayedrole loadings (higherabundancesat10°Cheatshockand10–30°C heat proteins (threefilaminisoforms)contributedthemosttonegative ATP buffering (fivearginine kinaseisoforms)andactin-binding contributed themosttopositiveloadings all others.Thepay-off phase ofglycolysisandATP buffering 10–30°C acclimation(seebelow). length hemocyaninincreasedwhereasfragmentsdecreasedin the (spots 37and160)showednegativeloadings,suggestingthat full- (spots 58and59),othersthatarefragmentsofthefull-lengthprotein some full-lengthhemocyaninsshowedhighlypositiveloadings preparatory andthepay-off phaseofglycolysis.Similarly, although aldolase oraldolase)forPC1,suggestingdifferences betweenthe others showedhighlynegativeloadings(fructose1,6-bisphosphate highly positiveloadings(phosphoglyceratekinaseandenolase), fluctuations. Interestingly, whereassomeglycolyticproteinsshowed functions changedthemostwithextremedailytemperature abundances inthe10–30°Cacclimation),suggestingthatthese filament proteinsalsocontributedhighlynegativeloadings(lower 1).ATPthe 10–30°Cacclimationgroup;Table buffering andthin showed themostpositiveloadings(indicatinggreaterabundancesin (phosphoglycerate kinaseandenolase)thin(actin)filaments the clawmusclecouldrespondtoheatstress. moderate temperaturefluctuationsbroadenedtherangeoverwhich 10°C and10–30°Cacclimationregimesdidnotdothesame.Thus, a 30°Cheatshock,requiringlimitedchangesoftheproteome.The Compare tablewithresultsinFig.2A. Positive loadingsforacclimationeffect loading rank Component RESEARCH ARTICLE Table 1.Positiveandnegativeloadingsforprincipalcomponents12ofproteinssignificantanacclimationeffect Negative loadingsforacclimationeffect 8 7 6 5 3 4 8 1 2 10 9 4 7 6 5 3 2 1 10 9 Glycolysis (glyceraldehyde 3-phosphate dehydrogenaseor The PCA basedonproteinsthatsignificantlychangedwithacute PC2 separatedthe10°Cand10–30°Cheatshocktreatmentsfrom Proteins representingATP buffering (arginine kinase),glycolysis Hemocyanin subunit1(160) heavychaintypeB(71) Cardiac-like muscleactin(29) Protein (spotID) Arginine kinase(5) rcoe16bshsht loae(2)1.2073 Fructose 1,6-bisphosphatealdolase(320) α (18) Hemocyanin (37) Arginine kinase(100) rcoe16bshsht loae(2)–1.5319 Fructose 1,6-bisphosphatealdolase(320) Hemocyanin subunit1(160) Myosin heavychaintypeB(71) Cardiac-like muscleactin(29) α Principal component1 α Arginine kinase(5) Hemocyanin (37) Gelsolin (18) Arginine kinase(100) α / / / / β β β β -Actin (84) -Actin (84) -Actin (85) -Actin (85) Tbe1), (Table Fg 2 (Fig. similar toPC1. B). Theheat 1.1395 1.2030 1.1539 1.2112 –1.4944 –1.5179 odn au Protein(spotID) Loading value 1.2507 1.0963 –1.5089 1.3186 1.0984 –1.5332 –1.5935 –1.4520 –1.6349 –1.4927 1.0526 –1.4032 10–30°C acclimation regimeundercontrol,but not acuteheatshock. 3).Also,full-lengthhemocyanins increasedunderthe (Table (most negativeloadings)increased with10–30°Cplusheatshock buffering andthinthick(myosinheavychain)filament proteins loadings) increasedwithacclimation to10–30°C,whereasATP several thinfilamentandrespiratory proteins(mostpositive claw musclecanrespondtoan acuteheatstress.Morespecifically, that moderatetemperaturefluctuationsbroadentherangeoverwhich acclimation controlplusheatshockoverlapped,againsuggesting treatments. Incontrast,10–20°Cacclimationcontroland 10°C acclimationplusheatshock(mostnegative)fromall other 10–30°C acclimationregimethemost.PC2(17.6%)separated the (most positive)andtheheatshocknegative)treatmentsof the 2C).PC1(26.3%)separatedthecontrol the interactioneffect (Fig. and 138),filaminsarginine kinaseisoforms. and distinctheavychainisoforms),hemocyaninfragments(spots 12 heavy chain(butlowerabundancesofmyosinregulatorylight chain regimes. Thus,heatshockleadstohigherabundancesofmyosin during heatshockfollowingthe10°Cand10–30°Cacclimation Sonnenberg, 2001),alongPC2werebasedonhigherabundances binding proteinthatconnectsactinfilaments(vanderFlierand light chainisoforms.Negativeloadingsofthreefilamins,anactin- B) butlowerabundancesofdifferent myosinregulatoryheavyand to higherabundancesofmyosinheavychain(typeBandnon-type Combined withtheloadingsforPC1,itseemsthatheatshockleads chain isoformscoincidedwithlowerabundancesduringheatshock. be tosuppressinflammationduringheatshock(seebelow). which showedthesecondhighestpositiveloadingalongPC1,might abundances duringheatshock.Theroleofprostaglandinreductase, filaments (myosinnon-type-B)alongPC1coincidedwithlower loadings ofrespiratoryproteins(full-lengthhemocyanins)andthick B) isoformscontributedhighlypositiveloadings.Thenegative 2).Fourmyosinheavychain(threetype following heatshock(Table contributed highlypositiveloadingstoPC1,withhigherabundances Seventy-one proteins(55identified)contributedsignificantly to Along PC2,positiveloadingsbyfourmyosinregulatorylight The JournalofExperimentalBiology(2015)doi:10.1242/jeb.112250 Arginine kinase(23) ucemoi ev hi 10 1.2374 Hemocyanin (58) Muscle myosinheavychain(120) Enolase (51) -C (141) Enolase (149) Filamin-A (46) Filamin-A (142) Phosphoglycerate kinase(143) Hemocyanin (59) Arginine kinase(116) Principal component2 Enolase (50) Notch-type protein(139) Arginine kinase(102) Arginine kinase(107) Arginine kinase(109) α Arginine kinase(145) α Arginine kinase(114) / / β β -Actin (82) -Actin (39) 1.2576 1.0674 1.3506 1.2247 –1.9090 Loading value 1.2628 –1.7500 –1.7448 –1.8065 1.0536 –1.9344 –2.0273 –1.6090 1.4194 –2.1154 0.9337 –1.4448 1.0664 –1.6239 391

The Journal of Experimental Biology 392 RESEARCH ARTICLE Compare tablewith resultsinFig.2C. Protein(spotID) Positive loadingsforinteractioneffect loading rank Component Compare tablewithresultsinFig.2B. Protein(spotID) loading rank Component shock) effect Table 3.Positiveandnegativeloadingsforprincipalcomponents12ofproteinssignificantaninteraction(acclimation byheat Table 2.Positiveandnegativeloadingsforprincipalcomponents12ofproteinssignificantaheatshockeffect abundance with10°Cacclimationplusheatshock).Thissuggests GAPDH, showedhighlynegativeloadingsforPC2(higher acclimation plusheatshock),whereasanotherglycolyticprotein, positive loadingsalongPC2(decreasingabundancewith10°C 10°C acclimationplusheatshock.Two enolaseisoformsshowed suggesting thatsomeactinsandmyosindecreasedabundanceat acclimation. Thinandthickfilamentscontributepositiveloadings, increasing abundanceswithheatshockfollowingthe10°C Positive loadingsforheatshockeffect Negative loadingsforheatshockeffect Negative loadingsforinteractioneffect 1 3 rsalni euts 3)180 ysnrgltr ih hi 15 1.4420 Myosinregulatorylightchain(105) 1.8526 1.8708 Argininekinase(4) Prostaglandinreductase(30) 3 2 1 4 4 2 5 6 eoynn(2 .37Agnn iae()0.8186 Argininekinase(7) 0.8680 1.0045 1.7327 Myosinregulatorylightchain(198) Myosinregulatorylightchain(164) 1.7262 1.7344 1.8142 MyosinheavychaintypeB(26) Hemocyanin(12) Musclemyosinheavychain(126) MyosinheavychaintypeB(71) 8 7 6 5 8 7 eoynn(7 166 iai- 12 –2.1902 –2.2402 –2.1898 –2.0921 –2.0086 –2.0472 0.7329 Notch-typeprotein(139) Filamin-A (142) Filamin-C(141) Argininekinase(102) 0.7772 Argininekinase(109) Filamin-A (46) –1.6351 –1.6464 –1.6951 –1.6019 Argininekinase(110) –1.6073 MyosinheavychaintypeB(38) –1.6135 Myosinregulatorylightchain(96) Myosinheavychaintype1(200) Musclemyosinheavychain(119) 1.6357 1.7057 T (133) Hemocyanin(57) Hemocyanin(58) 7 6 MyosinheavychaintypeB(38) 5 Peroxiredoxin5isoformB(11) 4 3 2 1 10 9 9 eoynn(9 156 aaysn()–1.8089 –1.9705 –1.7964 Paramyosin(3) Hemocyanin(138) Argininekinase(116) –1.5660 –1.5669 –1.5277 Cardiac-likemuscleactin(148) Hemocyanin(59) Argininekinase(110) 10 9 8 1 10 2 3 4 5 6 7 9 8 10 ATP buffering alsocontributedhighnegativeloadingstoPC2by rnia opnn Principalcomponent2 α Principal component1 Hemocyanin (58) α α Hemocyanin (59) α Cardiac-like muscleactin(122) Hemocyanin (57) Hemocyanin subunit2precursor(62) Phosphoglycerate kinase(143) Arginine kinase(89) Cardiac-like muscleactin(42) α Myosin heavychaintypeB(27) GAP dehydrogenase(73) Filamin-C (141) α lwmsl ysnS ev hi 18 –1.0188 Arginine kinase(102) Slow musclemyosinS1heavychain (178) Arginine kinase(109) / / / / / / β β β β β β GAP dehydrogenase(8) rnia opnn Principalcomponent2 Principal component1 α α -Actin (128) -Actin (79) -Actin (136) -Actin (135) -Actin (39) -Actin (92) / / β β Atn(8)181 ysnrgltr ih hi 5)1.2316 Myosinregulatorylightchain(53) 1.8511 -Actin (185) Atn(3)–.96Agnn iae(1)–2.0012 Argininekinase(111) –1.5946 -Actin (132) 1.9067 odn au Protein(spotID) Loading value odn au Protein(spotID) Loading value 1.6991 1.7323 1.6032 1.4513 1.6647 1.4780 1.2545 1.2923 1.7158 1.4906 –1.4293 –1.7986 –1.3297 –1.2943 –1.3576 –1.2213 –0.9598 –0.7862 –1.1390 the transferofphosphorylgroups,e.g.arginine kinase,orinATP The majorityofmetabolicproteinsidentifiedareeitherinvolvedin 10–30°C acclimation. GAPDH alsoincreasesabundancewithheatshockfollowingthe in abundancewithheatshockforenolaseandGAPDH,respectively. shock followingacclimationto10°C:withadecreaseandincrease that theseglycolyticreactionsarerespondingdifferently toheat Energy metabolism Energy Sarcoplasmic Ca α α Cofilin (33) Arginine kinase(113) α Enolase (51) Arginine kinase(7) Muscle myosinheavychain(120) Enolase (50) α Sarcoplasmic Ca lwmsl ysnS ev hi 18 0.8298 Slow musclemyosinS1heavychain(178) GAP dehydrogenase(106) Arginine kinase(116) Notch-type protein(139) Arginine kinase(109) Arginine kinase(107) (168) Filamin-A (46) Arginine kinase(102) Paramyosin (118) α / / / / / The JournalofExperimentalBiology(2015)doi:10.1242/jeb.112250 β β β β β Atn(5 1.3084 -Actin (85) Atn(8 0.8182 -Actin (78) -Actin (78) -Actin (85) -Actin (121) 2+ 2+ bnigpoen(5 1.7940 -binding protein(95) -binding protein(95) Loading value Loading value 1.4294 0.7188 0.5270 0.8396 0.7623 0.9041 0.6708 0.9058 –2.1973 –2.1995 1.5237 –2.0801 –1.8695 –2.1907 –1.7318 –1.8568 –1.6388 –1.7689 –1.6839

The Journal of Experimental Biology cysteine residue reducesthepIby0.5pHunits (Ubukaetal.,1987). 2000; Wolosker etal.,1996).Glutathionylationof oneprotein oxidative stressandcanaffect creatinekinaseactivity(Reddy etal., nitrosylated orglutathionylated thiolgroupsthatarerelatedto its vertebratefunctionalhomolog, creatinekinase,whichinclude that arginine kinasehassimilarpost-translationalmodifications to modifications havebeenreported forarginine kinase,itis possible compartment (Udaetal.,2006).Althoughnopost-translational They maybeseparateisoformsforthecytosolicandmitochondrial transcripts inthePetrolisthes explained bytheexistenceoffourdifferent arginine kinase five (clusterI)tonine. (cluster III).Thenumberofarginine kinaseisoformsvariedfrom and II)orheatstress(clusterIV)aloneaninteractioneffect statistical results,indicatingsignificanceforacclimation(clusters I 3).Thesegroupingsaregenerallysupportedbythe (cluster IV; Fig. 10–30°C heatshock;clusterIII)orallthreeacclimationtreatments shock following10°Cacclimation(andsomeisoforms 10–30°C; clusterII)andthosewithhighabundanceduringheat following 10–20°Cheatshock(andlowabundance following the10–30°Cacclimation(clusterI),highabundance separated intofourgeneralgroups:thosewithhighabundance Thirty arginine kinaseisoformschangedabundanceandwere separately andthendescribepossiblelinks. production viaglycolysis,e.g.GAPDH.We willfirstdiscussthem protein abundance.Eachcolumnrepresentsanindividualmuscle,groupedbytreatment( greaterthanaverage Blue coloringrepresentsalowerthanaverageproteinabundance(standardizedvalues,normalizedvolumes),whereasyellow crabPetrolisthescinctipes temperature fluctuationsandsubsequentcontrol(10°C)heatshock(30°C)exposuresfromclawmuscletissueoftheporcelain Fig. RESEARCH ARTICLE ( isoelectric point(pI)islisted.Significancesforanacclimation(A),heatshock(HS)orinteraction(I)effect onatwo-waypermutationANOVA aretickedbased mass(MW)and abundances ofproteins,organizedbyclusterssimilarabundancechanges(C),thatareidentifiedtotherightandwhosemolecular Phosphotransfer proteins P ≤ The diversityofarginine kinase isoformscan,inpart,be 0.02).

.Hierarchicalclusteringofchanges inabundanceofphosphotransferproteins. 3. –2.26 4.47 Abundance change: 10°C 10°C Acclimation 30°C EST library(Tagmount etal.,2010). 10–20°C Acclimation 10°C 0.04 30°C 10–30°C Acclimation 10°C Fg 4). (Fig. abundance withheatshockafter acclimationto10°Cand10–20°C identification ofglycogen phosphorylase, whichincreased isoforms inregulatingglycogenolysis comesfromthe Griffiths, 1981).Supportforapossibleroleofarginine kinase buffer duringglycogenolysisandglycolysis(Ellington, 2001; breakdown ofglycogentoglucose-1-phosphate, and(2)aH serves as(1)asubstrateforglycogenphosphorylase,leading to the glycolytic ratesthroughthereleaseofinorganic phosphate,which 2001; Hochachka,2003).However, arginine kinasealsoaffects to maintainATP levelsandtherebyenergy homeostasis (Ellington, transfer ofphosphorylgroupsfromarginine phosphatetoMgADP arginine kinase’s mainfunctionunderthesecircumstancesisthe high andfluctuatingATP consumptionrates,weassumethat experiment probablyaffected reactionratesandthereforecaused isoforms. stress causeschangesintheabundanceofmultiplearginine kinase from hypoxicstress(Abeetal.,2007),suggestingthatenergetic isoforms increasingabundanceinmuscletissueduringrecovery analysis ontheprawn specific arginine kinaseisoforms.Another2-Dgelelectrophoresis propodus, carpusandmerus),butthereisnoevidenceformuscle- 3).Clawtissueincludesfourmusclesubtypes(dactyl, (Fig. isoforms ofclusterI,possiblyindicatingtwoglutathionylationsites are aboutonepHunitlowerthanthemajorityofarginine kinase abundance withheatshock(clustersIIIandIV)showlowerpIsthat Interestingly, severalarginine kinaseisoformsthatincrease Given thatthetemperaturefluctuationsappliedinour N 30°C =5-6 foreachtreatment).Therowsrepresentthestandardized Pearson’s correlation,inresponsetoacclimationdaily Furthermore, glycogen phosphorylaseisregulated by The JournalofExperimentalBiology(2015)doi:10.1242/jeb.112250 (184) (162) (165) (101) (183) (156) (107) (102) 6.64 35 (7) (108) Arginine kinase (110) Arginine kinase Protein ID (91) (89) (116) (111) (109) (90) (153) (113) (5) (154) (115) (158) (163) (159) (100) (4) (114) (145) (23) (134) A A A A A A A A A A A A A A A A A A A A A A A A A A A A A rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase denylate kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase rginine kinase Marsupenaeus japonicas W I A S I HS A C pI MW 3 5.17 23 5.60 22 5.21 20 5.39 22 5.69 29 6.17 29 5.42 29 5.71 31 6.81 40 6.86 38 6.84 35 6.80 35 5.48 19 5.45 29 6.65 28 5.57 21 5.65 28 5.23 17 5.93 31 6.79 33 6.47 31 6.58 28 5.93 35 6.86 32 6.63 31 6.63 33 6.57 46 6.66 46 6.63 37 5 6.66 35 III IV II I showed three

393 . + –

The Journal of Experimental Biology 394 RESEARCH ARTICLE 10–30°C, increasing theabundanceoffive additional GAPDH affected theresponsetoacuteheatshockfollowingacclimation to they differ inpost-translationalmodifications.Thesechanges also isoforms ofenolasediffer mainlyinpIbutnotmass,suggesting that phosphoglycerate kinaseandF1-ATP synthase fluctuations of10–30°Cledtoan increaseinthreeenolaseisoforms, stress conditionsthroughglycolysis. Acclimationtoconstantdaily shock (clusterIII),possiblytosupportATP productionunderacute and GAPDH,arepotentiallymodifiedinresponsetoacute heat bisphosphate to1,3-bisphosphoglycerate,includingaldolase, TPI (cluster III).Thus,thethreereactionsfromfructose-1,6- acclimation to10–30°C(clusterI)and10°Caswell10–20°C acclimation to10–30°C(clusterII)andwithheatshockfollowing cluster thatischaracterizedbyanincreaseinabundance with cluster alsoincludesglycogenphosphorylase.Thus,wehave a during heatshockfollowingacclimationto10°Cand10–20°C. This by threeenzymes,GAPDH(threeisoforms),TPIandaldolase, 10–30°C acclimation.ClusterIIIshowedanincreaseinabundance three enolaseandonephosphoglyceratekinaseisoformsduring (PDH), respectively. ClusterIIshowedanincreaseinabundanceof phosphate isomerase(TPI),enolaseandpyruvatedehydrogenase the isoformsinthisclusterwereidentifiedasGAPDH,onetriose following the10–30°Cacclimationinnineproteinisoforms.Sixof showed increasedabundancesspecificallyduringheatshock shock, ledtothreemajorclustersofproteinabundances:clusterI Acclimation todifferent thermalconditions,followedbya30°Cheat 4). acute heatshockarepartoftheglycolyticpathway(Fig. Most ofthemetabolicproteinsthatchangedduringacclimationand adenylate kinaseinregulatingthesecellularpathways. fluctuating temperaturesandapossibleroleforarginine and shock afteracclimationtoconstant10°Candmoderately important roleforATP-buffering andglycogenolysisduringheat interaction effect ( conversion oftwoADP intoATP andAMP andshowedan are regulatedinpartbyadenylatekinase,whichcatalyzesthe historical accountofthistopic,seeFischer(Fischer, 2013)],which phosphorylation and,inaddition,AMP levels[foraninteresting cinctipes crab daily temperaturefluctuationsandsubsequentcontrol(10°C)heatshock(30°C)exposuresfromclawmuscletissueoftheporcelain Fig. Glycolysis

.Hierarchicalclusteringofchanges inabundanceofproteinsinvolvedenergymetabolism. 4. . Foradditionaldetails,seeFig. –1.75 4.56 Abundance change: 10°C 10°C Acclimation i.3). Fig. 30°C Together thesefindingssuggestan

3. 10–20°C Acclimation 10°C –0.29 β -subunit. Thethree 30°C 10–30°C Acclimation 10°C methylation (Zhouetal.,2010). addition, enolaseismodifiedbyphosphorylation,acetylationand and aldolase(Dalle-Donneetal.,2009;Fratelli2003).In which isknowntomodifyandaffect theactivityofGAPDH,TPI isoforms arepost-translationalmodifications,e.g.glutathionylation, isoforms andphosphoglyceratekinase. extreme fluctuationsrequireseveraladditionalenolaseandGAPDH (10°C) andmoderatetemperaturefluctuations(10–20°C),butmore response toacuteheatshockfollowingacclimationconstant the trianglebetweenaldolase,GAPDHandTPIcharacterize isoforms, TPIandenolase,aswellPDH.Thus,thereactionsof to itsroleasoxygen-carrier, hemocyaninshavebeen shown to protein ofarthropodsandmolluscs (Burmester, 2001).Inaddition We identifiedelevenisoformsofhemocyanin,anoxygen-carrying consumption (Hochachka,2003). maintain ATP concentrationsdespitegreatlyfluctuatingrates ofATP specifically themicrostructureofthosenetworksthatareable to dependent ontherestructuringofcellulararchitecture, further suggeststhattheenergetics oftemperatureacclimationare shock conditionsthattemporarilydemandhighATP turnover. This that itismodifiedduringtemperaturefluctuationsandacute heat identified suggeststhatsuchanetworkexistsinclawmuscle and tissue (DzejaandTerzic, 2003).Theproteomicfingerprintwe of thetransferphosphorylgroups,specificallyincardiacmuscle equilibrium metabolicreactions,therebyimprovingtheefficiency consuming withATP-producing processesbasedonnear- providing anefficient intracellular networkthatcouplesATP- (and phosphoglyceratekinase)arerecognizedasplayingarole in extension arginine kinaseaswelladenylatekinase)andGAPDH tissue duringtemperaturefluctuations.Creatinekinase(andby proteins andglycolysisinmaintainingenergy homeostasisinmuscle and heatshocksuggestanimportantroleforphosphotransfer e.g. GAPDH,intheproteomicresponsestotemperatureacclimation The prominenceofarginine kinaseandglycolyticproteinisoforms, Hemocyanin Energy metabolismconclusions A possibleexplanationforthenumber ofenolaseandGAPDH 30°C The JournalofExperimentalBiology(2015)doi:10.1242/jeb.112250 (106) GAP dehydrogenase Protein ID 15 roepopae smrs 3 6.85 55 6.68 31 54 6.79 Triose-phosphate(155) isomerase 6.61 54 6.30 43 6.56 40 6.87 34 (143) Phosphoglycerate kinase (50) Enolase 6.23 (51) Enolase 45 Pyruvate dehydrogenaseE1α-subunit (137) 23 6.56 (112) Triose-phosphate isomerase (130) GAP dehydrogenase (129) Enolase (75) GAP dehydrogenase 7)GPdhdoeae 15 6.79 33 5.46 6.13 68 (70) GAP dehydrogenase (87) Glycogen phosphorylase (193) F1-ATP synthase β-subunit 19 nls 5 6.72 53 28 6.24 6.55 20 5.44 15 (320) Fructose-1,6-bisphosphate aldolase (8) GAP dehydrogenase (167) GAP dehydrogenase (149) Enolase 17 6.52 16 6.45 15 6.44 (73) GAP dehydrogenase (74) GAP dehydrogenase (36) GAP dehydrogenase Pearson’s correlation,inresponsetoacclimation W I A S I HS A C pI MW 0 6.88 40 Petrolisthes III II I

The Journal of Experimental Biology myosin heavychain (MHC),myosinregulatory lightchain(MLC) We identified22thickfilamentproteins,including isoformsof binding proteins. categories: thickfilament(myosins), thinfilament(actins)andactin- fluctuations. We separatedtheseproteinsintothree functional cytoskeletal proteinschanginginresponsetotemperature that areinvolvedinmusclecontraction.We identifiedanumberof In myofibrillartissue,cytoskeletalelementscomposethesarcomeres cleaved off fromfull-length hemocyanins. oxygen. ClusterIImostlikelyrepresentsN-andC-terminalpeptides extreme dailytemperaturefluctuations,possiblytosupply more isoforms thatweremostlikelyincreasingabundanceinresponse to three isoformsincreasedwithheatshockafter10–30°C(spot 138). two oftheseisoforms(Havanapanetal.,2009).Onlyone these below forlinktoinflammation),explainingtherelativelylowpIof phosphorylated bytheERK1/2MAP kinasesignalingpathway(see represent peptidescleavedoff theC-terminusandmaybe isoforms ofintermediatemass(spots62,138and179)may sequence inwhiteshrimp(Havanapanetal.,2009).Hemocyanin in clusterIIarepeptidescleavedoff theN-terminalhemocyanin work showedthatisoformssimilartothosewithlowmolecularmass vannamei observed inhemocytesofthePacificwhiteshrimp cluster I.Analmostidenticalpatternofhemocyaninisoformswas masses between24and48 the full-lengthsequence.Incontrast,allsixisoformsofclusterIIhad in clusterIhadmassesequalorgreaterthan65 shock followingacclimationto10–30°C.Allhemocyaninisoforms 10°C and10–20°C,includingoneisoformthatincreasedwithheat cluster IIhassixisoformsthatincreasedwithheatshockfollowing showed higherabundanceswithacclimationto10–30°C,whereas major clusters(Fig. et al.,2001;Lee2003). terminal endiscleavedoff duringinfection(Destoumieux-Garzón Hemocyanins alsofunctionasantimicrobialpeptidesaftertheC- crustacean post-moltexoskeleton(DeckerandJaenicke,2004). in vivo undergo atransitiontofunctionascatecholoxidasesundercertain additional details,seeFig. fluctuations andsubsequentcontrol(10°C)heatshock(30°C)exposuresfromclawmuscletissueoftheporcelaincrab Fig. RESEARCH ARTICLE Thick filamentproteins(myosins) Cytoskeletal proteins Thus, themajorityofclusterIrepresentsfull-lengthhemocyanin Hierarchical clusteringofthehemocyaninisoformssuggeststwo

.Hierarchicalclusteringofchangesinabundancehemocyaninisoforms. 5. conditions, andthusplayaroleinthesclerotizationof –1.69 4.01 Abundance change: upon virusinfection(Chongsatjaetal.,2007).Subsequent 10°C 10°C Acclimation

5). ClusterIrepresentsfiveisoformsthat

3. 30°C

kDa, andhigherpIsthanisoformsfrom 10–20°C Acclimation 10°C –0.28

kDa, representing 30°C Penaeus 10–30°C Acclimation 10°C switching between fasttwitch(F),slow (S of thebeginninganincrease inproteinturnoverforfibertype heat-shock-specific, itispossible thatthesechangesareindicative from aseparateproteolytictarget. Becausethesechanges were also fromadifferent MHCtype,suggestingthattheyoriginated acclimation-induced MHCfragments (clusterIV),buttheywere (cluster IandII),thesewere evenlowerthanforthe10–30°C 2004), hadmassesbelowtheirpredictedrange.ForsomeMHCs decapod crustaceanstobeindicativeoffibertype(Medleret al., induced isoformsofMHCandparamyosin,whichareknown in shock inanacclimation-dependentmanner. Allheat-shock- abundances ofproteinsclustersI,IIandIIIincreasedwith heat be areasonablehypothesis. greater temperaturefluctuationsatcontrolconditions(10°C)would at 10°Cacclimation(relativetoheatshock),butanincrease with fragmentation pattern.Itseemsunlikelythatapoptosiswaselevated and notanacutetemperaturestresssignal,leadingto this reduced abundanceswithheatshock,suggeststhatitisachronic with acclimationtogreatertemperaturefluctuationsandshowed However, thefactthattheseisoformsshowedincreasedabundances proteolytic activityoftheproteasome(MyklesandHaire,1991). 2000) andcouldbetheresultofaheat-activatedincreasein during apoptosis,using2-Dgelelectrophoresis(Suarez-Hertaetal., gels. Myosinfragmentsofthissizehavebeenidentifiedtoform 205 and rangeinmassfrom75to108 increased abundancesduringacclimation,especiallyto10–30°C, et al.,1994).SeveralMHCisoformsfromclusterIV showed stabilization oftheMHCtailregion(Tohtong etal.,1995;VanBuren might negativelyaffect modulationofsarcomericpoweroutputand and fiveMHCisoforms,whichsuggeststhatacutethermalstress masses between20and24 cluster containsfiveisoformsoftheregulatoryMLCwithmolecular following acuteheatshock,regardlessofacclimationregime.This proteins inclusterIV showedsharplydecreasingabundances this isshownintheirhierarchicalclustering(Fig. 2),and contribute greatlytovariationintheheatshockeffect (Table invertebrates (Hooperetal.,2008;Squire,2009). et al.,2002).Paramyosinsarepartofthecorerodin bridges withactin,theC-terminusofMHCsmakesuprod(Clark MLCs makeuptheheadofthickfilamentthatformscross- and paramyosin(Fig. While clusterIV decreased inresponsetoheatshock, The PCA showedthatchanges inthickfilamentproteins kDa (Mykles,1997),amassabovetherangeofour2-D Pearson’s correlation,inresponsetoacclimationdailytemperature 30°C The JournalofExperimentalBiology(2015)doi:10.1242/jeb.112250 6) eoynnsbnt2peusr 6 5.72 56 Hemocyaninsubunit2precursor (62) Protein ID 1)Hmcai 38 6.94 6.28 45 (10) Hemocyanin (138) Hemocyanin 1)Hmcai 25 6.76 6.15 27 (12) Hemocyanin Hemocyanin subunit1 (160) 17 eoynn 4 6.75 24 24 6.30 79 6.10 78 6.06 5.47 79 65 6.01 (187) Hemocyanin (37) Hemocyanin Hemocyanin subunit4 (179) (59) Hemocyanin (58) Hemocyanin (57) Hemocyanin

6). WhereastheN-terminusofMHCsand

kDa (withoneexception),aspredicted,

kDa, belowthepredictedmassof Petrolisthes cinctipes W I A S I HS A C pI MW

6). Forexample, II I . For 1 ) andslow

395

The Journal of Experimental Biology 396 RESEARCH ARTICLE additional details, see Fig. fluctuations andsubsequent control(10°C)andheatshock (30°C)exposuresfromclawmuscle tissueoftheporcelaincrab Fig. phasic (S additional details,seeFig. fluctuations andsubsequentcontrol(10°C)heatshock(30°C)exposuresfromclawmuscletissueoftheporcelaincrab Fig. tropomyosin (seebelow),suggestingashifttoS the 10–30°Cacclimationplusheatshockincreasedslow-twitch entire actomyosincomplex. moderate temperaturefluctuationscausesarestructuringofthe below), possiblyindicatingthata30°Cheatshockfollowing 1997), increasedat10–20°Cacclimationplusheatshock(see filaments andrepresentanapoptoticsignal(Kothakotaetal., Interestingly, gelsolinfragments,whicharestillabletoseveractin

.Hierarchical clusteringofchangesinabundancethinfilamentproteins. Pearson’s correlation,inresponsetoacclimationdailytemperature 7. Hierarchicalclusteringofchanges inabundanceofthickfilamentproteins. 6. 2 –1.86 4.96 Abundance change: –1.83 4.80 Changes inabundance: ) inresponsetoheatshock.Insupportofthishypothesis, 10°C 10°C 10°C Acclimation 10°C Acclimation

3. 3. 30°C 30°C 10–20°C Acclimation 10–20°C Acclimation 10°C 10°C –0.27 –0.24 30°C 30°C 1 fiber type. 10–30°C Acclimation 10–30°C Acclimation 10°C 10°C are homologoustoboth isoforms, withsixcardiac-likemuscleisoformsand24that abundant proteinsinmuscletissue.We identified30different Thin filamentsaremadeof Thin filamentproteins(actins) specific totheheart,skeletalmuscleandcytoplasm(Varadaraj etal., cellular-compartment-specific localization,includingvariants crustacean actin,isoformdiversitystemsinpartfromtissue-and of Scylla paramamosain.Inhistochemicalstudiesthatexamine Pearson’s correlation,inresponsetoacclimationdailytemperature 30°C 30°C The JournalofExperimentalBiology(2015)doi:10.1242/jeb.112250 Protein ID (25) Paramyosin 3 aaysn(ogfr) 69 6.40 (3) Paramyosin (long form) 13 ysnhaycantp 7 5.50 75 4.19 24 (123) Myosin heavychaintype1 6.40 41 6.19 67 Myosinregulatorylightchain (53) 5.36 6.08 20 13 (131) Fast myosinheavychain form) (long (189) Paramyosin (126) Muscle myosinheavychain MyosinheavychaintypeB (27) 49 ysnhaycantp 18 6.10 108 5.63 95 5.53 95 6.86 46 5.49 95 (439) Myosin heavychaintypeB 6.68 4.12 44 20 (200) Myosin heavychaintype1 4.14 21 (119)Muscle myosinheavychain 4.20 Myosinregulatorylightchain 21 (96) 5.55 81 (120) Muscle myosinheavychain (198) Myosin regulatorylightchain (164) Myosin regulatorylightchain (105) Myosin regulatorylightchain (194) Myosin heavychaintype1 6.68 13 (118) Paramyosin 5.51 6.10 66 16 6.09 14 (178) Slow musclemyosinS1heavychain MyosinheavychaintypeB (71) MyosinheavychaintypeB (38) MyosinheavychaintypeB (26) (81) α/β-Actin Protein ID 7)αβAtn 51 5.28 6.21 53 54 6.64 5.10 20 (79) α/β-Actin Cardiac-like muscleactin (148) 17 6.29 57 4.62 5.77 25 23 5.78 (82) α/β-Actin (185) α/β-Actin 6.03 31 14 6.26 5.75 (35) α/β-Actin 41 (78) α/β-Actin (161) α/β-Actin (13) α/β-Actin Cardiac-like muscleactin (6) Cardiac-like muscleactin (150) (39) α/β-Actin 8)αβAtn 37 4.84 39 4.85 (85) α/β-Actin (84) α/β-Actin 37 /-ci 5 5.28 58 6.11 53 6.19 50 6.27 50 6.09 50 5.90 50 4.91 53 53 5.12 (387) α/β-Actin 17 6.27 (132) α/β-Actin (136) α/β-Actin (135) α/β-Actin 6.22 21 (128) α/β-Actin 32 (203) α/β-Actin 5.56 (180) α/β-Actin (80) α/β-Actin 6.27 12 Cardiac-likemuscleactin (40) 36 (34) α/β-Actin 5.76 28 5.43 Cardiac-likemuscleactin (29) 46 5.22 (88) α/β-Actin 6.31 13 (52) α/β-Actin (92) α/β-Actin Cardiac-likemuscleactin (42) (99) α/β-Actin α -actin of α - andβ Homarus americanus Petrolisthes cinctipes Petrolisthes cinctipes -actins andarethemost W I A S I HS A C pI MW W I A S I HS A C pI MW 16 5.94 16 45 4.97 III IV III IV II II I I . For .For and

β

-actin

The Journal of Experimental Biology additional details, see Fig. fluctuations andsubsequent control(10°C)andheatshock (30°C)exposuresfromclawmuscle tissueoftheporcelaincrab Fig. capping (gelsolins),G-actinbindingproteinsthatregulateF-actin (tropomyosin, Tm;filaminandsarcomeric categorized intofourgroups:actinfilamentstabilizing monomers andfilamentsalsochangedinabundance.Theycanbe Actin-binding proteins(ABP)thatregulatethedynamicsofactin a molecularmassof45and46 correlation withpatternsofmolecularmass. abundance canbegroupedintofourclustersthatshowedaregular tissue (Götzetal.,1999;Cayenne2011). Changesin separate studyhasverifiedthistobethecasein involved incolocalizationofenergy metabolismenzymesanda the predicted42 found 18isoformswithamassofatleast37 Varadaraj etal.,1996;Kim2009).Inthepresentstudy, we Gecarcinus lateralis Homerus americanus 1996; Kimetal.,2009). RESEARCH ARTICLE Actin-binding proteins 58 actin isoforms(oneacardiac-likemuscleactin)rangingfrom50to full-length isoform.ClusterIV showedincreasedabundancesof11 between 17and32 actins withmassesfrom17to57 cardiac muscleactinsthatrangedfrom12to41 increased abundancesfollowing10–20°Cplusheatshock,withfour abundances after10–30°Cplusheatshock.ClusterIIIshowed showed threeisoformswithmassesbelow28 following acclimationto10°Cand10–30°C(Fig. isoforms inseasquirtsofthegenusCiona a similarheat-shock-inducedpatternofpartialproteolysisforactin under which dependsonseveralactin-bindingproteinsandATP levels, protocol butisratherduetoalowthermalstabilityintrinsicactin, occurrence offragmentsisnotanartifactoursamplepreparation shock treatmentsdidnotshowpartialproteolysissuggeststhatthe vivo showed increasedabundanceofactinfragments,indicatepartial and interactionclusters(IIIV).ClustersIIIII,which Cluster Ishowedincreasedabundancesoftwoactinisoformswith We candividetheclustersintoacclimation(I),heatshock(III) kDa afteracclimationto10–30°C.

.Hierarchical clusteringofchangesinabundanceactin-bindingproteins. Pearson’s correlation,inresponsetoacclimationdailytemperature 8. proteolysis duringheatshock.Thefactthatgelsfromnon-heat- in vivo –1.77 4.82 Abundance change: 10°C 10°C Acclimation conditions (Levitskyetal.,2008).Also,weobserved

kDa mass.Thecytoskeletonisknowntobe

kDa, amassrangethatisunlikelytorepresent (Macias andSastre,1990;Ortegaetal.,1992;

3. has atleast12,and8havebeenfoundin Artemia 30°C

kDa, closetothepredicted42 have atleast10isoformsofactin,

kDa, includingfiveisoforms 10–20°C Acclimation 10°C (Serafini etal.,2011). α

-), severingand kDa, whichiscloseto –0.32

kDa withincreased

kDa, andtenα/β- P. cinctipes 30°C

7). ClusterII

kDa, claw 10–30°C Acclimation in 10°C of actinin(spot127only),severalfilamins,andtropomyosin and 10–30°Cbutnotto10–20°C,basedontheabundancepatterns involved instabilizingactinfilamentsfollowingacclimationto10°C 10°C acclimation. tropomyosin alsoincreasedinresponsetoheatshockfollowingthe during acuteheatshockonly. Actininandfilaminsbutnot acclimation andthusrepresentsastrategytostabilizeactinfilaments abundances inresponsetoanacuteheatshockbutnotthe10–30°C Flier andSonnenberg, 2001).Interestingly, thisclusterincreased whereas filaminsmayanchororbundleactinfilaments(vander filaments totheZ-lineinstriatedmuscles(Clarketal.,2002), Tropomyosins stabilizefilaments(Wegner, 1982),actininsanchor abundance following10–30°CandheatshockinclusterIV. monomeric actintocontrolfilamentgrowth. the needforanchoringorbundlingactinfilamentsandbindingof filamin andprofilin,at10°Cacclimationplusheatshock,suggesting III showedanincreaseintwoofthethreeproteinscluster, regulation ofthetropomyosin–actininteraction,respectively. Cluster involve anchoringorbundlingofactinfilaments,severingand abundances offilamin,gelsolinandtroponin(Fig. of actinfragments(Fig. Interestingly, thisclustercoincides withanincreaseinthenumber strengthen anchoringoffilamentstotheZ-linethrough Dominguez, 2010).Moderatetemperaturefluctuationsmayalso and therebyactivateactinfilamentrestructuring(Lee fluctuations plusheatshockseveranddepolymerizeactinfilaments Thus, wehypothesizethatchronicmoderatetemperature (Clark etal.,2002),following10–20°Cacclimationplusheatshock. troponin T, whichorganizes theactinfilamentregulatorycomplex severing proteingelsolin,theanchoring abundances ofcofilin(oractindepolymerizationfactor),theactin- across severalclusters,clusterIwasalsocharacterizedbyincreased most ABP (withcofilinandtropomyosinbeingtheexceptions) grouped intofourclusters(Fig. The proteinisoformsweidentifiedasbelongingtothesecategories (troponin T, TnT)(Clarketal.,2002;LeeandDominguez,2010). filament length(profilinandcofilin)regulatoryproteins Overall, heatshocktriggeredincreasedabundancesforABP All threeactin-stabilizingproteinsshowedanincreasein Cluster IIisthe10–30°Cacclimationclusterwithincreased Although 10°Cacclimationplusheatshockledtoanincreasein 30°C The JournalofExperimentalBiology(2015)doi:10.1242/jeb.112250 (33) Cofilin Protein ID 4)FlmnA 68 6.87 6.76 9 5.82 28 18 5.67 (46) Filamin-A (22) (157) Troponin T (17) Gelsolin 16 iai 8 6.82 83 63 6.68 (146) Filamin (49) Filamin-C (86) Sarcomeric α-actinin 12 iai- 6 6.65 61 6.53 66 6.80 52 5.21 63 5.03 4.51 6.15 90 47 53 4.48 47 Tropomyosin(204) (slowmuscle isoform) Tropomyosin(192) (slowmuscleisoform) (142) Filamin-A (141) Filamin-C 18 5.38 (127) Sarcomeric α-actinin (168) Troponin T (133) Troponin T (125) Gelsolin (18) Gelsolin

7; clusterIII). 35 4.84

8). Petrolisthes cinctipes W I A S I HS A C pI MW 19 6.45 8). Thefunctions III IV II I α -actinin and . For

α -actinin.

397

The Journal of Experimental Biology 2008). resistant topartialproteolysisthanglobularactin(Levitskyetal., that filamentousactinismuchmorethermallystableandtherefore proteolysis duringheatshock.Thisisconsistentwiththeobservation severing mayincreasethesusceptibilityofactintopartial gelsolin, itseemslikelythatgreaterfilamentdepolymerizationand tropomyosin (Fig. cofilin (actindepolymerizationfactor),gelsolin,actininand acclimation to10–20°Cisaccompaniedbyincreasedabundancesof greater fragmentationofactinduringheatshockfollowing and therebypossiblyreducefragmentationduringacuteheatshock. stabilize actinfilamentsandenhancetheiranchoringtotheZ-line daily temperaturefluctuationsleadtoanincreaseinproteinsthat and therebyreducedfragmentation.Thus,extremebutnotmoderate acclimation to10–30°Conlyandpossiblystabilizedactinfilaments Thus, allthreeoftheseABPsincreasedduringheatshockfollowing 398 RESEARCH ARTICLE additional details, see Fig. fluctuations andsubsequent control(10°C)andheatshock (30°C)exposuresfromclawmuscle tissueoftheporcelaincrab Fig. protein (vanderFlierandSonnenberg, 2001)(Fig. Wegner, 1982)andfilamin,anactin-anchoringoractin-bundling tropomyosin, whichstabilizesactinfilaments(Clarketal.,2002; anchors actintotheZ-line(Clarketal.,2002;Wegner, 1982), This patterncorrespondstoincreasedabundancesofactinin,which proteolysis duringheatshockfollowingacclimationto10–30°C. cluster IIIversusII),indicatingagreaterresistancetopartial at 10–20°Cplusheatshockthan10–30°C(Fig. Thin filaments(actins)showedamuchgreaternumberoffragments regulatory complexoftheactomyosininteractions. filaments, greaterratesofseveringandmodificationsthe temperature fluctuationsrequireincreasedbundlingofactin filamin C,gelsolinandtroponinT, suggestingthatchronic one 10–30°Cacclimationcluster(II)withhighabundancesof complex ofactinfilaments(Clarketal.,2002).Finally, thereisonly 2008). Thereby, troponinT isthoughttoorganize theregulatory the ‘swivel’ effect inmuscles(Myersetal.,1996;Murakami complex, andinconjunctionwithactin,isresponsibleforproducing respectively. Troponin T anchorstropomyosintothetroponintrimer acclimation to10°C,10–20°Cand10–30°Cplusheatshock, length. Threedifferent troponinT isoformswereabundantfollowing cofilin, respectively, andtherefore limitedactinfilamentgrowthand severing anddepolymerization,basedonchangesingelsolin acclimation to10–20°Cplusheatshockcausedactinfilament (10–30°C plusheatshockonly).Ourinterpretationsuggeststhat Thin filamentsandactin-bindingproteins In apatternthatcomplementstheofactinstabilization,

.Hierarchical clusteringofchangesinabundancesignalingproteins. Pearson’s correlation,inresponsetoacclimationdailytemperature 9. –2.13 5.31 Abundance change: 10°C 10°C Acclimation 8; clusterI).Atleastinthecaseofcofilinand

3. 30°C 10–20°C Acclimation 10°C –0.19

8; clusterIV). 30°C 10–30°C Acclimation

7, 10°C it duringthepost-moltsclerotizationprocess(Ahearnetal.,2004). the freshwatercrayfish separate positions.ThreeSCP isoformsarepossiblesplicevariantsin peptides forbothSCP spots,anddidnotresolvewhytheseSCPshave 10°C acclimation.OurMS/MSanalysisidentifiedthesamethree inhibiting developmentalprocessesduringheatshockfollowingthe methyltransferase andanotchhomolog,possiblysuggestingrolein 10–20°C (Fig. the otherone(spot94)adecreaseinabundanceafteracclimationto a lowerlevelwithheatshockafteracclimationto10°Cand10–30°C, 22 1995). We identifiedtwoisoformsofSCPswithestimatedmasses invertebrates (Whiteetal.,2011; Gaoetal.,2006;HermannandCox, EF-hand calcium-bindingproteinsandarefoundonlyamong Changes inCa from theCaCO Ca contraction inmyofibrillartissues(Clapham,2007).Incrustaceans, from transcriptionalactivityinthenucleustochangesmuscle membrane fluidity(HochachkaandSomero,2002). complex similartothoseknownforenzymeactivitiesand temperature acclimationtomaintainpropertiesoftheactomyosin may beundergoing adjustments,e.g.compensations,during under controlconditions.We proposethatactinfilamentdynamics 10–30°C plusheatshockindicatesamorerigidactomyosincomplex conditions, andwhethergreaterfilamentstabilizationfollowing reflects amoredynamicactomyosincomplexundercontrol severing underthe10–20°Cacclimationplusheatshockconditions whether theputativegreatersensitivityforincreasedfilament proteases andthereforeincreasespartialproteolysis.Itisunclear unfolds morereadilyduringheatshock,increasesaccessto which, becauseG-actinisthermallymorelabileandsupposedly during heatshock(30°C)andthereforegreaterG-actinformation, increase inactinfilamentdepolymerizationandseveringproteins acclimation tomildtemperaturefluctuations(10–20°C)inducesan filamentous versusglobularactin.We furtherhypothesizethat cofilin andgelsolin),inpartthroughthegreaterthermalstabilityof depolymerization andseveringduringheatshock(noincreasein fluctuations (10–30°C)inducesresistancetoactinfilament Calcium ionregulation Signaling proteins Sarcoplasmic Ca We hypothesizethatacclimationtoextremetemperature 2+ kDa (spot94)and2195).Oneisoform95)showed regulation playsakeypartinthemoltcycle,depletingcalcium 30°C The JournalofExperimentalBiology(2015)doi:10.1242/jeb.112250 2+ 3 (95) Sarcoplasmic Ca Protein ID 1) eoieoi 2 6.78 25 6.00 (94) Sarcoplasmic 28 Ca 12 PEBP/RAFkinaseinhibitorprotein (9) 19 5.84 (30) Prostaglandin reductase/LTB4DH Peroxiredoxin5B (11) (21) FK506-binding protein (103) 14-3-3 ζ 11 6.39 (67) Proteasome β-subunit (32) Phosphohistidine phosphatase 19 oc-yepoen 7 6.62 67 4.56 43 (139) Notch-type protein FarnesoicacidO-methyltransferase (83) 9). Spot94clusterswithfarnesoicacidO- cuticle duringproecdysis(pre-molt)andrestoring concentration affect cellularprocesses,ranging 2+ -binding proteins(SCPs)belongtothefamilyof Procambrus clarkii

2+ 2+ bnigpoen 2 4.68 22 -binding protein 4.70 21 -binding protein Petrolisthes cinctipes (White etal.,2011). W I A S I HS A C pI MW 36 4.66 24 6.71 III II I . For

The Journal of Experimental Biology troponin CduringcontractionthroughslowCa Thus ,andpresumablySCP, limitscompetitionwith myosin. RegulationofcytosolicMg increased againfollowing10–30°C(Fig. acclimation to10°C,decreasedinresponsefollowing10–20°C,and abundance ofFaMeT increasedwithheatshockfollowing tissues, includingmuscle(Nagaraju,2011; Ruddelletal.,2003).The Transcripts forFaMeT havebeendetectedinmultiplecrustacean reproduction andmolting(Kuballaetal.,2011; Nagaraju,2011). sesquiterpenoid thatisinvolvedincrustaceanmorphogenesis, conversion fromfarnesoicacidtomethylfarnesoate,a Farnesoic acid RESEARCH ARTICLE environmental stress,becauseachangeinMg important mechanismsfordealingwithtemperature-related it slowlyreleasesMg binding tomyosinuntiltroponinCtakesupCa associated withcoldshockin IP stabilizes theclosedstateoftwocalciumchannels,ryanodineand characterized bythebindingofimmunesuppressantFK506;it Ca it ispossiblethatanincreaseinSCP leadstoasequesteringofmore et al.,2011; Berchtoldetal.,2000;Wang andMetzger, 2008).Thus, increases efficiency oftheactomyosincontractileapparatus(White resting state,parvalbuminbindsmostlyMg contraction, similartothefunctionofparvalbumin.During tonic muscletissueandserveasintracellularCa et al.,2011), theyaremorehighlyexpressedinfasttwitchthanslow following acclimationto10°Cand10–20°C. suggests thatmusclecontractionisinhibited,withheatshock Sesquiterpenoid metabolism 3 FK506-binding protein(FKBP)isaprolylisomerase Although theexactfunctionofSCPsiscurrentlyunknown(White 2+ G-protein (Dumaz andMarais,2003) receptor (MacMillan,2013).Itsabundanceincreased,which and thereforeachangeintheinteractionbetweenactin Inactive RAF RAF 14-3-3 Phosphohistidine phosphatase O P -methyltransferase (FaMeT)catalyzesthe 2+ PKA to takeupCa P Prostaglandin G

Phospholipase A Prostaglandin E Arachidonic acid

P. clarkii RAF MEK G-protein ERK RAS kinase GPCR COX

(Yeung etal.,1999) GPCR

2+ 2+ . Tropomyosin blocksactin (White etal.,2011). 2 2 PGE synthase LOX and Ca 2+ 15-PGDH

9). Gilltissueshowed , butduringcontraction

2+ HPETE

2+ RAF kinaseinhibitor 2+ (Clapham, 2007). 2+ 2+

concentration is protein (RKIP)

uptake, which buffers during could alsobe RKIP- 15-keto PGE 13-prostaglandin phosphorylation (Corbitetal.,2003) Phosphokinase C-dependent (Lorenz etal.,2003) Inflammation reductase LTB P LTA 4 DH/ 4

G-protein LTB prostaglandin E inflammatory prostanoidsfromarachidonicacid,including leukotriene B protein orPEBP)(GranovskyandRosner, 2008),NADP-dependent protein or(RKIP;alsoknownasphosphatidylethanolamine-binding suppressing inflammation(Figs We identifiedfiveproteinsofdiversefunctionsthatmaybe environmental stress. an importantroleforFaMeT intheresponseofcrustaceanstoacute maenas testicular developmentinaMF-dependentfashion stress (ourunpublisheddata).Both,temperatureandsalinityaffected changes intwoFaMeT isoformsinresponsetopHandemersion recruitment (Marksetal.,2009;Tai, 2011). Inadditiontobeing metabolites, therebysuppressinganimmunereactionandhemocyte leukotrienes, suchasleukotrieneB also functionsasLTB (LOX). Furtherdownstream,prostaglandinreductase(PGR),which inactivation oftheinflammatorymediatorleukotrieneB prostaglandin reductase,aproteindirectlyinvolvedinthe phosphohistidine phosphatase(PHP)-likejanusprotein,14-3-3 Inflammation phospholipase A Yeung etal.,1999)andtherebythephosphorylationof inhibits theMEK-ERK1/2MAP kinasemodule(Yeung etal.,2000; of signalingpathwaysthatregulateinflammation(Fig. 10–20°C butnot10–30°C. shock suppressesinflammationafteracclimationto10°Cand PHP) and10°C(III:RKIP and14-3-3).We hypothesizethatheat with heatshockfollowingacclimationto10–20°C(II:PGRand inflammation fallintotwoclustersthatshowincreasingabundances FKBP (seeabove).Thefourproteinspossiblyinhibiting 13,14-dihydro-15-keto-PGE (nucleus, active) RKIP possiblyrepresentsacentralnodeinhypotheticalnetwork Proteasome degradation 4 +NF I κ kinase GPCR GPCR B- κ U6 (Nagaraju andBorst,2008).Together, theseresultssuggest pro-inflammatory B P

Inactivation of

mediators I 2-keto-LTB κ The JournalofExperimentalBiology(2015)doi:10.1242/jeb.112250

B ( (IκK) Yeung etal.,2001) 4 hydroxydehydrogenase (LTB 2

2 , directlyinhibitingthesynthesisofpro- (cytosol, inactive) 4 (PGE), viacyclooxygenase(COX)and I κ 4 B–NF DH, catabolizesPGEandLTB 2 κ B

9 and10):RAFkinaseinhibitor rapidly acceleratedfibrosarcoma. E; PKA,proteinkinaseA;RAF, dehydrogenase; PGE,prostaglandin kappa B;PGDH,prostaglandin kinase; NF mitogen-activated proteinkinase LTA kappa B;LOX,lipooxygenase; hydroperoxide; I HPETE, arachidonicacid5- G-protein coupledreceptor; signal-regulated kinase;GPCR, cyclooxygenase; ERK,extracellular For details,seetext.COX, represent immuneresponse. signaling proteins;redcrosses yellow circles.Redtextindicates phosphorylation isindicatedwith response. Putativeprotein inhibit aninnateimmune signaling proteinsthatmight changes inabundanceofseveral cellular processesaffectedby hypothetical pathwaysand Fig. B leukotriene B 4 4 hydroxydehydrogenase; MEK, 4

0 A schematicshowing 10. , leukotrieneA (LTB κ 4 4 B, nuclearfactor ) vialipooxygenase DH) alsoknownas 4 ; LTB κ B, inhibitorof 4 4 ; LTB DH, leukotriene 4 into inactive

10). RKIP 4 4 Carcinus , (LTB ζ and 399 4 ),

The Journal of Experimental Biology 400 RESEARCH ARTICLE actin filaments (filamin, actininandtropomyosin). and actin)proteinsaswell actin-binding proteinsthatstabilize PDH), onerespiratory(hemocyanin) andseveralfilament(MHC ATP-buffering, different metabolic(TPI, GAPDH, enolaseand (MHC andactin)proteins.A subsequent30°Cheatshockincreased kinase andATP synthase),respiratory(hemocyanin)and filament increased ATP-buffering, metabolic(enolase,phosphoglycerate (Fig. acclimation to10–30°Cwassimilarlystressful,basedonourPCAs proteins thatmightsuppressanimmuneresponse.Heatshock after paramyosin andMHCisoforms),anumberofABP andsignaling glycolysis (oneGAPDH),actomyosinrestructuring (two shock causedastrongincreaseinATP-buffering (Fig. stressful ofourtreatments,asindicatedbyPCA (Fig. 2014). are lessabundantthantheproteinswedetected(Carberryet al., treatments, withtheexceptionofFKBP, mostlikelybecausethey that wedidnotdetectanymolecularchaperonesinof the processes onlytoalimitedextent.Inthiscontext,itisworthnoting structure andpossibledynamicswhilechangingothercellular instead primedtodealwithheatshockbychangingactinfilament shock followingacclimationto10–20°Csuggestthatorganisms are the actin-bindingproteinscofilin,gelsolinandactininwithheat acclimation regimeperse.Increasinglevelsofactinfragmentsand However, therearefewchangesaccompanyingthe10–20°C acclimation increasedresistancetoasubsequent30°Cheatshock. limited, suggeststhattheproteomicchangesoccurredduring the responsetoheatshockfollowingacclimation10–20°Cwas acclimate toadailyoccurrenceof30°Cheatshock.Thefactthat acclimation regimewassevereenoughthatporcelaincrabsdidnot 10–30°C incomparisonto10–20°C(Fig. response toheatshockisgreaterfollowingacclimation10°Cand PCAs basedonthesignificantlychangingproteinsshowedthat following acclimationto10°C. of thechangesinabundanceglycolyticenzymeswithheatshock Landor etal.,2011), andthusmightbeinvolvedinregulatingsome glycolysis (CiofaniandZúñiga-Pflücker, 2005;Grazianietal.,2008; to modulatecellularmetabolism,specificallyglucoseuptakeand notch andNFκ 2009). Also,thereisevidenceforasynergistic interactionbetween expression bytheNF cleavage oftheintracellulardomainnotch,whichactivatesgene ligands fromothercellsduringdevelopment,triggeringtheproteolytic phosphatase (PHP)(Lorenzetal.,2003). subunit oftheG-proteinisasubstratephosphohistidine activates theERK1/2module.Furthermore,itispossiblethata et al.,2004;Lorenz2003).Interestingly, itisaGPCRthat protein–GPCR complexandprolongedG-proteinsignaling(Keller kinase (GPCRorGRK),leadingtothedissociationofaG- Finally, phosphorylatedRKIP bindstoG-protein-coupledreceptor of NFκ I B (NFκ activation ofthepro-inflammatorytranscriptionfactornuclearkappa 3-3 (DumazandMarais,2003).RKIP alsointerfereswiththe A (PKA)andsubsequentlysequesteredfromthemembraneby14- module, canbeinhibitedifitgetsphosphorylatedbyproteinkinase inhibited byRKIP, RAFkinase,aspartoftheERK1/2MAP kinase Conclusion κ B throughIκ Heat shockfollowingacclimationto10°Cispresumablythemost Notch isatransmembranereceptorproteinthatactivatedby 2A,B). Acclimationtodaily10–30°C(measuredat10°C) B) byinhibitingthephosphorylationofinhibitorprotein B–Iκ B toanactiveformofNF B (Barbaruloetal.,2011). Furthermore,notchseems B kinase,which,inturn,inhibitsthedisassociation κ B-like transcriptionfactorCSL (Marksetal., κ B (Yeung etal.,2001).

2). Thus,the10–30°C

3, clusterIII),

2C). Heat β - using a1:4ratio of ice-cold10%trichloroaceticacid inacetonewithan during acclimationandfollowingtheacuteheatshocktreatment. N 2012; Tomanek etal.,2011) aredescribedbelow. We analyzedtissuesfrom al., 2012b;Serafiniet2011; Tomanek andZuzow, 2010;Tomanek etal., statistical analysesfollowingpublishedprotocolsandprocedures(Fields et Luis Obispo,CA,USA)ondryiceforproteomicanalysis. and transportedtotheEnvironmentalProteomicsLaboratory(CalPoly, San Four clawsegments(dactyl,propodus,carpus,andmerus)weredissected at 10°Cbeforebeingflashfrozeninliquidnitrogenandstored 30°C heatshockover5 randomly placedineitheraconstant10°Cimmersionorgivenramp-up to 1 5 that experiencedheatramping,temperaturewasgraduallyincreasedovera acclimations hadtemperatureprecisionofatleast±0.5°C.Inthetwogroups ramps from10–20°C(‘moderate’)or10–30°C(‘extreme’).Allthermal groups experiencedacclimationateither10°C(‘constant’)ordailyheat crabs wererandomlyplacedintothreetreatmentgroups.Over30days,these constant immersionat10°Cfor30 USA), wheretheyweremaintainedinacommongardenacclimationunder for EnvironmentalStudies(SanFranciscoStateUniversity, Tiburon, CA, October 2008.ThecrabsweretransportedtotheRomberg Tiburon Center intertidal zoneofFortRoss,CA,USA (38°30 16,100 Piscataway, NJ,USA)],solubilizedat20°Cfor1 and 0.5%immobilizedpH4–7 gradient (IPG)buffer (GE Healthcare, homogenization buffer [7 Claw tissuewaslysedinground glasshomogenizersina1:4ratioof Adult (amidosulfobetaine)-14, 40 temperature ranges. broad modificationsoftheproteomeassociatedwithsuchshiftsin fluctuations willbephysiologicallycostlybecauseofthedailyand animals forasubsequentgreaterheatshock,moreextreme experiencing greattemperaturefluctuations,whicharepreparingthe Our resultssuggestthatwhereasintertidalcrustaceansarealready above thethresholdatwhich shock. Thedailyoccurrenceofanacute30°Cheatshockisthus proteomic changesinresponsetoasubsequentacute30°Cheat modifications andacclimationto10–30°Crequiredintermediate changes, acclimationto10–20°Crequiredfewadditionalproteomic of thesechanges.Acclimationto10°Ccausedthegreatestproteomic and inflammation.Acuteheatshockisalwaysthedominantdriver transport, thestructureanddynamicsofactomyosincomplex by modifyingproteinsinvolvedinATP production,oxygen making crustaceansmoresusceptibletopathogens. properties. Finally, acuteheat shock inhibitsinflammation,possibly changes causeactomyosinfiberstomodifytheirfunctional the proteinsthatstabilizeactinfilaments.Itispossibleboth turnover rates,whereasextremetemperaturefluctuationsincrease filaments, possiblytorestructureactomyosinfibersmodifyATP- characterized byproteinsthatdepolymerizeandseveractin filaments. Mostimportantly, moderate temperaturefluctuationsare temperature fluctuationsrequiremodificationsinthickandthin ATP synthase,aswell an increaseinhemocyanins.Chronic greater fluctuationsrequiremodificationsofglycolyticproteinsand Homogenization Animal collection,maintenanceandexperimental design MATERIALS ANDMETHODS

=5–6 animalspertreatment(Figs h periodtotherespectivepeaktemperature,andthencooled10°Cover h. Afterthesetemperatureacclimations,crabsfromeachtreatmentwere The preparationofthemuscletissuesamplesandexploratory The proteomeofclawmuscleadjustedtotemperaturefluctuations Thus, temperaturefluctuationsalwaysrequireATP buffering and Petrolisthes cinctipes g for 30 The JournalofExperimentalBiology(2015)doi:10.1242/jeb.112250 min at20°C.Proteinswereprecipitated fromthesupernatant

h. Allindividualsweregivena1

mmol mol (Randall, 1839)werecollectedfromthe l P. cinctipes −

1 l

− 3–9). Therewasnomortalityrecorded

1 days. Followingthecommongarden, urea, 2 Tris-base, 40 mol is abletofullyacclimate. ′ 51″ N, 123°14′ h, andthencentrifugedat l − 1 mmol thiourea, 1%ASB

h recoveryperiod

l − 1 dithiothreitol 34″ W) in − 80°C.

The Journal of Experimental Biology manufacturer’s instructions. was performedusingthe2-DQuantKit(GEHealthcare)accordingto To determinetheproteinconcentrationineachsample,aassay CA, USA). then scannedwithanEpson1280transparencyscanner(Epson,LongBeach, destained throughrepeatedwashingwithmilliQwaterover48 stained usingcolloidalCoomassieBlue(G-250)overnightandthen 7) buffer and100 phenoxylpolyethoxylethanol)-40, 0.002%BromophenolBlue,0.5%IPG(4- (cholamidopropyldimethylammonio-propanesulfonic acid),2%NP (nonyl Protein solutionwasabsorbedintoeachIPGgelstripover5 in onewellofanisoelectricfocusingcell(Bio-Rad,Hercules,CA,USA). rehydration buffer and200 with 65 2% SDS,0.002%BromophenolBlue)at20°Cfortwo15 Billerica, MA,USA).To obtain (MALDI-ToF-ToF) massspectrometer(UltraFlexII;Bruker Daltonics,Inc., using amatrix-assistedlaserdesorption/ionizationtandemtime-of-flight and Zuzow, 2010).Briefly, peptidemassfingerprints(PMFs)wereobtained according topreviouslypublishedmethods(Fieldsetal.,2012;Tomanek Gel plugswerepreparedforanalysisusingmassspectrometry (MS) the gelimage. quantified bynormalizationagainstthetotalspotvolumeofallproteinsin background subtraction,therelativespotvolumeofeachproteinwas individual gels,establishingproteinspotparametersforeachgel.Following spot boundariesdetectedonthefusedimageweretransferredbackto gels fromalltreatmentsweremultiplexedintoasinglefusedimage.The Decodon, Greifswald,Germany)(Berthetal.,2007).To detectproteinspots, Digitized 2DgelimageswereanalyzedusingDelta(version3.6; Healthcare). Proteinsamplesweredilutedtoaconcentrationof2 their isoelectricpoints(pI),usingIPGgelstrips(pH4–7,11 cm;GE For firstdimensionelectrophoresis,proteinswereseparatedaccordingto overnight incubationat RESEARCH ARTICLE Dodeca; Bio-Rad)for55 strips werethenplacedover11.8% polyacrylamidegelsandrun(Criterion focusing ofproteinswasperformedbyrunninggelstripsat500 rehydration followedby12 search (MOWSE) scorewas42orhigherforPCAD and 34orhigherforthe Search resultswere deemedsignificant( precursor-ion masstolerancewassetat 0.6 cleavage wasallowedduringsearches. FortheMS/MSanalysis, the borealspidercrab could notbeidentifiedwithPCAD weresearchedagainstanEST libraryfor contains 19,000uniquesequences (Tagmount etal.,2010).Spectrathat Porcelain CrabArrayDatabase(PCAD), anEST libraryfor PMFs andtandemmassspectrawerecombinedsearchedagainst the identified usingMascot(version2.2;MatrixScience,Inc.,Boston, MA). Internal masscalibrationwasperformedusingporcinetrypsin.Proteins were peptide peaks,usingasignal-to-noiseratioof6forMSand1.5MS/MS. 2010). FlexAnalysis(version3.0;BrukerDaltonic,Inc.)wasusedto detect previously publishedmethods(Fieldsetal.,2012;Tomanek and Zuzow, were selectedfortandemMS.Spectralanalysisofpeptidesfollowed rehydration buffer [7 After brieflydryinginair, theproteinpelletswerere-suspendedin according tothepreviousparametersandsupernatantwasdiscarded. pellet washedwithice-coldacetone.Sampleswereagaincentrifuged for 15 Mass spectrometry analysis Gel image Two-dimensional gelelectrophoresis 1000 equilibration buffer (6 prepared forseconddimensionSDS-PAGE by incubatingthemin strips werefrozenat voltage changesoccurredinrapidmode).Afterisoelectricfocusing,gel V for1 min at4°C,afterwhichthesupernatantwasdiscardedandprotein mol l − h andthen8000 1 dithiothreitol andthenwith135 mmol − moll 80°C. Afterfreezingforatleast1

Hyas araneus l mol −

− min at200 20°C. Thesampleswerecentrifugedat18,000 1 μl (400g)wasloadedontoeachIPGgelstrip dithioerythritol] andsolubilizedat20°Cfor1

h ofactiverehydrationat50 −1 l − 1 urea, 375 mmol

b V for2.5 - andy urea, 2

V. Followingelectrophoresis,gelswere -ion parameters,atleastsixpeptides (Harms etal.,2013).Onemissed P mol h (maximumcurrent50 ≤ 0.05) iftheirmolecular weight

l l Da (defaultMascotvalue). −

−1 1 mol Tris base,30%glycerol, thiourea, 2%CHAPS l − 1 iodoacetamide. Gel

min intervals,first

V. Separationand h, gelstripswere P. cinctipes

h. Gelswere h ofpassive μg V for1 μA; all μl − that 1

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g umse,T. Burmester, A.E. Todgham, and B. E. Bjelde, aen,A . aet .adSila,J.H. Stillman, and B. Gabert, A.P., Cayenne, K. Ohlendieck, and D. Swandulla, M., Zweyer, S., Carberry, lpa,D.E. Clapham, M.A.G. andL.T. analyzedthedataandwrotemanuscript. M.A.G., J.H.S.andL.T. designedtheexperimentandanalysisofsamples. The authorsdeclarenocompetingorfinancialinterests. during theexperimentandDrChristinaVasquez forhelpfuleditorialcomments. We thankClaudiaTomas-Miranda andPaulaRobinsonforinvaluableassistance performed atwo-wayANOVA (P linking ofindividualgels(Delta2D)usingaPearsoncorrelationmetric.We We comparedproteinabundances betweentreatmentgroupsviaaverage identifications thatincludedtwomatchedpeptidesequenceswereaccepted. H. araneus http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.112250/-/DC1 Supplementary materialavailableonlineat (MCB-1041225 toJ.H.S.andEF-1041227L.T.). and Technology (COAST)toM.A.G.andNationalScienceFoundationgrants The studywassupportedbyagrantfromtheCouncilofOceanAffairs, Science ectl,M . rnmir .adMnee,M. Müntener, and H. Brinkmeier, M.W., Berchtold, M., Pelullo, G., DiMario, D., Bellavia, A.F., Campese, P., Grazioli, A. A., Barbarulo, Mandal, and P. K. Mandal, G.A., Ahearn, S. Okada, and S. Hirai, H., Abe, lr,K . clin,A . ekre .C n rgro C. Gregorio, and M.C. Beckerle, A.S., McElhinny, K.A., Clark, Krittanai, and V. Thongboonkerd, C.F., Lo, A., Bourchookarn, P. O., Chongsatja, J.H. Stillman, and X. Chen, ifn,M n úiaPlce,J.C. Zúñiga-Pflücker, and M. Ciofani, contribution tosampleseparationalongagivencomponent. component loadingvalueswhicharequantificationsofaprotein’s with MS.OftheproteinsthatwereidentifiedMS,weassigned profiles significantlychanged,regardlessofwhethertheywereidentified treatments, weusedPCA (Delta2D)basedonproteinswhoseexpression proteins incharacterizingtheproteomicresponsetodifferent temperature into hierarchicalclusters.To betterunderstandtheimportanceofparticular versus acuteheatshockeffects andgroupedsignificantlychangingproteins Author contributions Competing interests Acknowledgements statistical analysis Exploratory References Supplementary material Funding al-on,I,Rsi . oob,G,Gutrn,D n izn,A. Milzani, and D. Giustarini, G., Colombo, R., Rossi, I., Dalle-Donne, Mol. Biol.Evol. Lottia digitalis 1215-1265. dystrophic skeletalmuscle. proteomic analysisofthecontractile-protein-depletedfractionfromnormalversus muscle: itscrucialroleformusclefunction,plasticity, anddisease. Petrolisthes cinctipes interacting withlactatedehydrogenaseinclawmuscleoftheporcelain crab 881-888. at thebeta-selectioncheckpointbyregulating cellularmetabolism. 137A crustaceans duringthemoltcycle:areviewandupdate. Immunol. etal. L. Frati, A., Vacca, NF-kappaB signalingpathwayscooperativelyregulateFoxp3transcription. S., Colantoni, A., Ciuffetta, Comp. Biochem.Physiol. expression underhypoxicstressofthekurumaprawn Biochem. Sci. Protein S-glutathionylation: aregulatorydevicefrombacteria tohumans. Biol. muscle cytoarchitecture:anintricateweb offormandfunction. C. salinity tolerancein salinity variabilityaffects onmetabolicrate,generationtimeandactuethermal 3601. vannamei (2007). Proteomicanalysisofdifferentially expressedproteinsin 18, 637-706. , 247-257. 186 hemocytes uponTaura syndromevirusinfection. EST. ForresultswithasignificantMOWSEscore,onlypositive The JournalofExperimentalBiology(2015)doi:10.1242/jeb.112250 (2001). Molecularevolutionofthearthropodhemocyaninsuperfamily. , 6199-6206. (2007). Calciumsignaling. under emersionandimmersion. 34, 85-96. 18, 184-195. Daphnia pulex . Comp.Biochem.Physiol. 146A Anal. Biochem. (2012). Multigenerationalanalysisoftemperatureand , 40-46. (2007). Metabolicresponsesandargininekinase . J.Therm.Biol. (2013). Thermalphysiologyofthefingeredlimpet ≤ (2005). Notchpromotessurvivalofpre-T cells 0.02) comparingthermalacclimation Cell 446 131 J. Exp.Biol. , 108-115. 6D, 393-398. , 1047-1058. 37, 185-194. (2011). Identificationofproteins (2004). Calciumregulationin (2000). Calciumioninskeletal (2011). Notch3andcanonical Marsupenaeus japonicus Comp. Biochem.Physiol. 216 , 2858-2869. Proteomics Annu. Rev. CellDev. (2014). Comparative Physiol. Rev. Nat. Immunol. (2002). Striated Penaeus 7 , 3592- (2009). Trends 401 80, 6 J. , .

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