Plasma membrane rafts of rainbow trout are subject to thermal acclimation

Transcription

1 The Journl of Experimentl iology 26, The Compny of iologists Ltd doi:1.1242/je Plsm memrne rfts of rinow trout re suject to therml cclimtion John K. Zehmer* nd Jeffrey R. Hzel iology Deprtment, rizon Stte University, Tempe, Z 85287, US *uthor for correspondence (e-mil: jzehmer@imp4.su.edu) ccepted 28 Ferury 23 Rfts re cholesterol- nd sphingolipid-enriched microdomins of the plsm memrne () tht orgnize mny signl trnsduction pthwys. Interctions etween cholesterol nd sturted lipids led to ptches of liquid-ordered memrne (rfts) phse-seprting from the remining. Phse ehvior is temperture sensitive, nd cute chnges in temperture experienced y poikilotherms would e expected to pertur rft structure, necessitting n cclimtory response. Therefore, with therml cclimtion, we would expect compositionl chnges in the rft directed to offset this perturtion. Using differentil nd density grdient centrifugtion, we seprted from the livers of rinow trout cclimted to 5 C nd 2 C into rftenriched (rft) nd rft-depleted (). Compred with, the rft frctions were enriched in cholesterol, the β 2 -drenergic receptor nd denylyl cyclse, which re commonly used mrkers for this microdomin. Furthermore, cholesterol ws enriched in ll frctions from wrm- compred with cold-cclimted nimls, ut this increse ws 3.4 times greter in rft Summry thn in. We developed novel pproch for mesuring memrne moleculr interction strength (nd thus the tendency to stilize rft structure) sed on the susceptiility of memrnes to detergent. Specificlly, studies with model vesicles demonstrted tht the cpcity of memrne to ccommodte detergent prior to soluiliztion (sturtion point) ws good index of this property. The sturtion point of the isolted memrne preprtions ws temperture sensitive nd ws significntly different in 5 C- nd 2 C-cclimted when ssyed t 5 C nd 2 C, respectively. y contrst, this comprison in rfts ws not significntly different, suggesting compenstion of this property. These dt suggest tht compositionl chnges mde in the during therml cclimtion ct to offset therml perturtion of the rft ut not the structurl integrity. Key words: rft, plsm memrne, rft-depleted plsm memrne, temperture, cholesterol, detergent, therml cclimtion, rinow trout, Oncorhynchus mykiss. Introduction During therml cclimtion, poikilotherms must mke sustntil iochemicl chnges to their plsm memrnes (s) to preserve physiologicl function. These chnges include increses in cholesterol concentrtion nd cyl chin sturtion with cclimtion or dpttion to elevted tempertures (Hzel et l., 1991). The mintennce of memrne order/fluidity (homeoviscous dpttion) hs clssiclly een invoked to rtionlize these modifictions (Sinensky, 1974). However, these compositionl djustments would lso e expected to stilize the structure of rfts, microdomins of the implicted in orgnizing diverse signling pthwys (rown nd London, 1998). Therefore, we hve investigted the possiility tht the therml cclimtory response of the is directed to mintin rft structure. Fvorle interctions mong cholesterol nd the sturted hydrocron chins of lipids, especilly sphingomyelin, result in ptches of liquid-ordered (L o ) phse memrne (rft) seprting from the remining liquid-disordered (L d ) memrne (hmed et l., 1997). The L o phse is chrcterized y highly ordered hydrophoic region compred with the L d phse (rown nd London, 1998). Specific proteins re trgeted to, nd concentrted in, rfts sed on the greter soluility of their lipid nchors in L o over L d phse memrne (Wng et l., 2). This locliztion is clerly importnt since the dissolution of rfts, y stripping the memrne of cholesterol, results in the loss of function of the signling proteins normlly loclized there (Pike nd Miller, 1998). Rft structure is dependent on lipid phse ehvior (rown nd London, 1998), mking it temperture sensitive. Therefore, cute increses in temperture could result in rft dissolution while cute decreses could result in inpproprite ccretion of memrne components. The resulting disruption of signling ctivities would necessitte homeosttic response to stilize rft structure nd restore pproprite signling. The gol of this study ws to determine if rft structurl integrity is defended during therml cclimtion in the verterte poikilotherm rinow trout (Oncorhynchus mykiss). We mesured compositionl chnges in totl s well s in

2 1658 J. K. Zehmer nd J. R. Hzel the rft nd rft-depleted () su-domins of heptocytes from rinow trout cclimted to 5 C nd 2 C. Thermlly induced djustments in memrne lipid composition were greter in rft compred with. In ddition, we developed novel pproch for mesuring memrne moleculr interction strength (nd thus the tendency to stilize rft structure) sed on the susceptiility of memrnes to detergent. We present evidence tht moleculr interction strength is regulted in rft ut not in. These dt provide the first evidence for the homeosttic regultion of rft structure nd suggest the need to re-exmine memrne cclimtion from the perspective of sptilly heterogeneous. Mterils nd methods Mterils Cholesterol oxidse ws purchsed from Cliochem (Sn Diego, C, US). ll primry ntiodies were from Snt Cruz iotechnology (Snt Cruz, C, US) except nticveolin, which ws purchsed from D iosciences (Sn Jose, C, US). Got nti-rit peroxidse conjugted secondry ntiody ws purchsed from iord (Hercules, C, US). Super Signl Enhnced chemiluminescent sustrte ws purchsed from Pierce (Rockford, IL, US). DNse ws type II from Sigm (St Louis, MO, US). Synthetic lipids were purchsed from vnti Polr Lipids (lster, L, US). Other iochemicls were from Sigm, nd ll other chemicls were of nlyticl grde. nimls Rinow trout (Oncorhynchus mykiss Wlum) were otined from the lchesy Ntionl Fish Htchery in Whiteriver, Z, US nd were mintined t the niml Resource Center of rizon Stte University. Fish were housed in recirculting freshwter quculture systems consisting of circulr fierglss tnks; wter tempertures were controlled using flow-through chillers. nimls were cclimted to 5 C or 2 C for t lest three weeks efore use in experiments. Fish were held under constnt 12 h:12 h L:D cycle nd were fed trout food (Rngen Inc., uhl, ID, US) to stition dily. Plsm nd rft memrne isoltions Plsm memrnes () were isolted from pproximtely 14 g of liver ccording to modifiction of the procedure of rmstrong nd Newmn (1985), s descried previously (Hzel et l., 1992). The ws resuspended in working uffer (W;.25 mol l 1 sucrose, 2 mmol l 1 tricine, ph 7.8, 1 mmol l 1 EDT) nd ws seprted into rft-depleted () nd rft-enriched (rft) using non-detergentsed method (Smrt et l., 1995). The memrne frctions were seprted sed on the lighter uoynt density of rft, compred with. riefly, the ws sonicted nd then djusted to 23% OptiPrep efore lyering 1 2% OptiPrep grdient on top (for totl of 11 ml in ech tue). fter centrifuging for 9 min t 72 8 g in eckmn SW 41-Ti rotor (OP1), the top 5.5 ml (rft) ws removed, mixed with 4 ml of 5% OptiPrep in fresh tue, cpped with 25 µl of 5% OptiPrep nd centrifuged for 9 min t 72 8 g (OP2). The rft memrne concentrted t the top of the OP2 tue ws collected with Psteur pipette, diluted three times with uffered sline nd centrifuged to pellet for 2 min t 2 8 g in refrigerted microcentrifuge (Eppendorf 5417 R). The ottom 5.5 ml from OP1 () ws diluted pproximtely four times with uffered sline nd ws centrifuged for 1 h t 23 7 g in eckmn J 3.5 rotor. Protein, phosphte nd cholesterol ssys Totl protein ws determined colorimetriclly y the method of rdford (1976). Phospholipid concentrtion ws mesured y colorimetric phosphte nlysis of totl lipid extrcts fter complete hydrolysis (mes, 1966). n verge moleculr mss of 75 ws ssumed for conversion to mss. coupled cholesterol oxidse fluorometric ssy ws used to mesure cholesterol (Crockett nd Hzel, 1995). Western lots Western lots were performed to determine the reltive undnce of specific proteins in the frctions. Smples (3 µg of totl homogente protein nd 15 µg of ll other smples) were mixed with n equl volume of 2 Lemmli uffer [62.5 mmol l 1 Tris, ph 6.8, 25% glycerol, 2% sodium dodecyl sulfte (SDS), 5% β-mercptoethnol], oiled for 3 min nd sujected to SDS polycrylmide gel electrophoresis (12% gel for cveolin, 7.5% gel for ll others). The resolved proteins were then trnsferred to.45-µm nitrocellulose memrnes nd locked for 1 h in 5% non-ft dry milk (NFDM) in phosphte-uffered sline (PS). The memrnes were immunoleled for 1 h in 5% NFDM/PS with n nticveolin polyclonl ntiody (p; 1:2), nti-β 2 drenergic receptor p (1:25), nti-denylyl cyclse p (1:4), ntiinsulin receptor β-suunit p (1:16) or nti-clthrin p (1:25). The lots were wshed three times in PS with.1% Tween-2 for 5 min nd once in PS for 5 min. The lots were then incuted for 1 h in got nti-rit horserdish peroxidse (HRP)-conjugted secondry ntiody (1:12 for cveolin, 1:1 for ll other westerns). The lots were then wshed four times in PS with.1% Tween-2 for 5 min, followed y detection using enhnced chemiluminesence nd X-ry film. t lest two lots were prepred for ech experiment, nd representtive films re shown. Stle plurilmellr vesicle preprtion Stle plurilmellr vesicles of synthetic lipids were prepred ccording to the method of Grüner et l. (1985) for use in detergent ssys. Detergent ssys ssy Detergent ssys were performed to mesure the moleculr interction strength of memrne smples. Vesicles or iologicl smples (in 2 µl W) were dispersed into 2.8 ml

3 Therml cclimtion of plsm memrne rfts 1659 ssy uffer (15 mmol l 1 NCl, 2 mmol l 1 Hepes, ph 7.4) nd mintined in thermlly controlled cuvettes. The quntities used for myristoylplmitoyl phosphtidylcholine (MPPC) vesicles, plmitoyloleoyl phosphtidylcholine (POPC)/ cholesterol vesicles nd iologicl smples were 47 nmol of lipid, 27 nmol of lipid nd 17 nmol of lipid, respectively. The turidity of the suspension ws mesured continuously s the verge opticl density etween 394 nm nd 44 nm in diode rry spectrophotometer (8452; Hewlett Pckrd, Plo lto, C, US). Triton X-1 ws dded s seril dditions of 12.5 µl nd llowed to stilize for 1 2 min (Fig. 1). The finl five dt points efore ech new detergent ddition were verged s the effect of the previous detergent ddition (e.g. Fig. 1, rrow). The resulting opticl density vlues were normlized s percentge of the initil opticl density nd were plotted ginst detergent/lipid (D/L) rtios, clculted s detergent concentrtion (% v/v) divided y µmol totl lipid (Fig. 1, inset). The resulting curves were fit with fourprmeter sigmoidl eqution using Sigm Plot 2 (most r 2 vlues were.996 or etter, with the lowest r 2 =.987). nlysis Detergent memrne interctions proceed in two phses, oth of which re sensitive to moleculr interction strength. Initilly, detergent is incorported into memrne structure until sturtion, which is followed y soluiliztion s the memrne components re incorported into mixed micelles (Fig. 1; Lichtenerg et l., 1983). Vlues reflecting ech of these processes were derived from the dt. The D/L vlues corresponding to the peks of the 2nd derivtive of the fitted curve (Fig. 1C, inset, points nd ) define the onset nd completion of the high slope region of the curve (Fig. 1C, points nd ). The D/L vlue corresponding to point ws chosen s n index of the mximum cpcity of the memrne to ccommodte detergent (sturtion point; Fig. 1C). Further dditions of detergent result in memrne dissolution. The slope of liner regression of the fitted dt etween points nd provides n index of the mount of detergent required for soluiliztion (soluiliztion slope; Fig. 1C). Fourier trnsform infrred spectroscopy Infrred spectroscopy ws performed to estimte the gel fluid melting temperture of MPPC vesicles. The MPPC vesicle smple ws loded etween two CFl 2 crystls with 5.8 µm Teflon spcer nd plced in thermlly controlled smple lock. Using Fourier trnsform infrred spectrometer Rft OD % OD D/L (% µmol 1 ) Time (min) 1 Mixed micelles Mixed micelles % OD D/L (% µmol 1 ) Detergent lipid Rft lipid Cholesterol 2 C D/L (% µmol 1 ) Fig. 1. Detergent nlyses. () Digrmmtic representtion of detergent memrne interction. Loosely pcked rft-depleted plsm memrne () ccommodtes more detergent prior to sturtion (higher sturtion point; plte 1) thn tightly pcked rft (plte 2). fter sturtion occurs, mixed micelles form, dissolving the memrne. Lipids in re more redily incorported into micelles (steeper soluiliztion slope; plte 3) thn re rft lipids (plte 4). () Detergent ssy. Memrne dissolution is followed s chnges in opticl density (OD) over time. End points re recorded s opticl density just prior to detergent ddition (rrow). Inset: normlized end points s function of detergent/lipid (D/L) rtios with sigmoidl regression. (C) nlysis. Sigmoidl regression with D/L vlues of 2nd-derivtive peks mrked (points nd ) with the liner regression etween points nd superimposed. Inset: 2nd derivtive with peks mrked (points nd ). Sturtion point (i.e. where soluiliztion ecomes dominnt over detergent incorportion) is defined y point. Soluiliztion slope is defined s the slope of the liner regression etween points nd.

4 166 J. K. Zehmer nd J. R. Hzel (Spectrum 2; Perkin Elmer, Shellon, CT, US), the men of 75 scns etween the wvenumers of 37 nd 78 ws tken t 2 C intervls etween 2 C nd 5 C nd were ckground sutrcted. The instrument softwre pckge ws used to find the peks of the 2nd derivtive, identifying the sorption pek corresponding to the methylene symmetric stretching frequencies. This frequency ws plotted s function of temperture. The points were fitted using TleCurve 2D 5. (Systt Softwre, Inc., Richmond, C, US), nd the gel fluid melting temperture ws found s the pek of the 1st derivtive. Sttistics The protein, phospholipid nd cholesterol compositionl dt were expressed s rtios (e.g. cholesterol/protein). The dt were nlyzed y two-wy nlysis of vrince (NOV) with cclimtion group s fctor with two levels (cold nd wrm) nd memrne frction s fctor with three levels (, nd rft). Post hoc testing ws performed using Tukey s test. The soluiliztion slopes nd sturtion points from the detergent experiments were ech nlyzed using three-wy NOVs with cclimtion group s fctor with two levels (cold nd wrm), ssy temperture s fctor with two levels (5 C nd 2 C), nd frction s fctor with three levels (, nd rft). Where pproprite (significnt effect with no interction), the dt were collpsed cross one or two fctors for multiple comprisons using Tukey s test. The dt were lso nlyzed seprtely y frction (, nd rft) y one-wy NOV followed y Tukey s test. In ll cses, P vlue of.5 ws ccepted s indicting sttisticl significnce. Results Memrne frction composition Most cells contin cholesterol- nd sphingolipid-enriched microdomins in their plsm memrnes. Mny cell types, including dipocytes, myocytes nd epithelil cells, contin cveole, which re microdomins with distinct flsk-shped morphology nd cot mde from oligomers of the protein cveolin (Rzni nd Lisnti, 21). In rinow trout, cveolin is expressed in spleen nd is undnt in dipose tissue ut is sent from kidney nd liver tissue (Fig. 2). In cell types lcking cveolin (nd cveole), microdomins re generlly referred to s lipid rfts. Heptocyte plsm memrnes (s) isolted from rinow trout cclimted to 5 C nd 2 C were seprted into rftenriched (rft) nd rft-depleted () y soniction followed y density grdient centrifugtion. sed on the yield of protein, cholesterol nd phospholipid (estimted s phosphte), nd n ssumption tht the memrne consisted of only these components, we estimted tht the frctions tken s rft consisted of 39% nd 32% (y mss), respectively, of the totl memrne for the cold- nd wrm-cclimted nimls. Microdomins (rfts nd cveole) hve previously een descried in numerous systems to e enriched in proteins of C D E Cv Kid Hom Spl di β 2 R mny signl trnsduction cscdes, including the β 2 drenergic receptor (β 2 R; Xing et l., 22) nd denylyl cyclse (C; Ostrom et l., 21; Ryin et l., 2). Therefore, we exmined the distriution of β 2 R nd C in the crude homogente,, nd rft frctions. oth proteins were depleted in the frction, compred with the, while they were sustntilly enriched in the rft frction (Fig. 2,C). The insulin receptor hs een reported s sent from microdomins in the mjority of the literture (Mstick et l., 1995; Ohir et l., 2), ut some reserchers hve found it to e microdomin ssocited (Vinio et l., 22). In our system, the insulin receptor β suunit ws depleted in the rft, compred with the or, frctions (Fig. 2D). Clthrin, protein involved in receptor-medited endocytosis, is not ssocited with rfts or cveole (Rzni nd Lisnti, 21). Surprisingly, we found clthrin to e evenly distriuted mong the, nd rft frctions (Fig. 2E). cclimtion temperture effects on composition To further chrcterize the frctions, we mesured the concentrtions of cholesterol, phospholipid phosphte nd protein in the frctions from cold- nd wrm-cclimted trout to ssess compositionl chnges ssocited with therml cclimtion. The dt were expressed s mss rtios (Fig. 3 C) nd s mss percentges (Tle 1; the tle ws constructed with the ssumption tht the memrne consisted Liv Rft C IRβ Clth Fig. 2. Western lots. () Distriution of cveolin in trout kidney (Kid), spleen (Spl), dipose (di) nd liver (Liv). ll lnes, 15 µg totl protein. ( E) Smples loded: 3 µg crude homogente (Hom) nd 15 µg plsm memrne (), rft-depleted () nd rft-enriched (rft). Proed with () ntiodies ginst β 2 drenergic receptor (β 2R), (C) denylyl cyclse (C), (D) insulin receptor β suunit (IRβ) nd (E) clthrin (Clth).

5 Therml cclimtion of plsm memrne rfts 1661 Tle 1. Percentge composition of memrne frctions from cold- nd wrm-cclimted nimls % Composition niml Memrne frction Cholesterol Phospholipid Protein Wrm-cclimted 9.5± ± ±.8 1.±.4 23.± ±1.3 Rft 16.1± ± ±1.2 Cold-cclimted 8.5± ± ±.7 7.3± ± ±1.1 Rft 11.5± ± ±2.2 Vlues re mens ± S.E.M. (N=6) nd re sed on the ssumption tht cholesterol, phospholipid nd protein content dd up to 1% of the memrne., plsm memrne;, rft-depleted plsm memrne. of only protein, phospholipid nd cholesterol). Wheres the cholesterol/protein (Ch/Pr) rtios were similr in nd in oth cclimtion groups, this rtio ws significntly higher in rft frctions, eing elevted 66% nd 14% over in cold- nd wrm-cclimted fish, respectively (Fig. 3). Furthermore, the Ch/Pr of rfts from wrm-cclimted fish ws significntly greter (P<.5, N=6) thn tht from coldcclimted fish y 33%. Ch/Pr (mg mg 1 ) PL/Pr (mg mg 1 ) Ch/PL (mg mg 1 ) C,,c Cold Rft Fig. 3. Compositionl nlyses of memrne frctions. () Compositionl comprisons of memrne frctions from cold-cclimted (open rs) nd wrm-cclimted (filled rs) fish expressed s rtios. () Cholesterol/protein (Ch/Pr). () Phospholipid/protein (PL/Pr). (C) Cholesterol/phospholipid (Chl/PL). Vlues re mens ± S.E.M., N=6. rs with common lower-cse letter do not differ significntly., plsm memrne;, rft-depleted plsm memrne.,c,d,d,, c Wrm c d Rft Rfts from cold-cclimted fish hd significntly higher (P<.5, N=6) phospholipid/protein (PL/Pr) rtios thn ll other frctions, which did not differ from one nother (Fig. 3). This represented 65% increse over the from cold-cclimted fish. There ws non-significnt 42% increse of PL/Pr in rft, compred with, from wrmcclimted fish. The Ch/PL rtio ws 32% greter in from wrmcompred with cold-cclimted nimls, while this increse ws 63% in rfts (Fig. 3C). Furthermore, the increse in cholesterol concentrtion ssocited with cclimtion to 2 C ws 3.4 times greter in rft thn in (Tle 1). Detergent ssys vesicles We developed novel pproch for mesuring memrne moleculr interction strength sed on the susceptiility of memrnes to detergent. We followed oth the cpcity of memrne to incorporte detergent into the ilyer [sturtion point (SP)] nd the mount of detergent required to soluilize the memrne (soluiliztion slope). More tightly pcked memrnes were expected to sturte with less detergent (low SP) ut to require more detergent to soluilize them (shllow soluiliztion slope) compred with loosely pcked memrnes (Fig. 1; see Discussion). We first nlyzed severl model vesicle preprtions to evlute the efficcy of this pproch. Moleculr interction strength ws mnipulted y vrying ssy temperture of myristoylplmitoyl phosphtidylcholine (MPPC) vesicles nd cholesterol content of plmitoyloleoyl phosphtidylcholine (POPC) vesicles. Fourier trnsform infrred spectroscopy indicted tht MPPC vesicles melted from the tightly pcked gel phse to the loosely pcked fluid phse t 33 C (T m ; Fig. 4, circles). SP vlues for MPPC decresed etween 25 C nd 3 C s T m ws pproched, efore incresing ove T m (Fig. 4, rs). Sustntilly more detergent ws required to rech sturtion ove the T m, reflecting the looser pcking of the fluid-phse lipids. Vesicles of POPC lone or with 3 mol% cholesterol were nlyzed t 25 C in the fluid phse. Cholesterol hs condensing effect on fluid phse memrnes, cusing

6 1662 J. K. Zehmer nd J. R. Hzel Sturtion point (% µmo l 1 ) Temperture ( C) POPC POPC + 3 mol% cholesterol Fig. 4. Effects of temperture nd cholesterol content on sturtion point. () Symmetric methylene virtionl rtes of myristoylplmitoyl phosphtidylcholine (MPPC) vesicles s function of temperture (circles). Sturtion points of MPPC vesicles s function of temperture (rs). Vlues re mens ± rnge, N=2. () Sturtion points of plmitoyloleoyl phosphtidylcholine (POPC) nd POPC + 3 mol% cholesterol vesicles t 25 C. Vlues re mens ± rnge, N=2. memrne lipids to pck more tightly (Phillips, 1972). The ddition of 3 mol% cholesterol to the POPC vesicles cused sustntil reduction in the SP, reflecting this tighter pcking (Fig. 4). In nlyzing the soluiliztion slopes produced from these sme vesicle preprtions, we expected memrnes with tightly pcked lipids to produce more shllow slopes (more detergent required to complete soluiliztion). Contrry to our prediction, soluiliztion slopes for MPPC vesicles were sustntilly steeper t tempertures elow the T m compred with those ove the T m (Fig. 5). However, s expected, POPC vesicles with 3 mol% cholesterol hd more shllow soluiliztion slope thn did POPC vesicles lcking cholesterol (Fig. 5). These results suggest tht SP is more relile indictor of moleculr interction strength thn is soluiliztion slope. Detergent ssys iologicl memrnes Sturtion points Representtive detergent soluility curves of, nd rft re presented in Fig. 6. Note tht the curve hs more pronounced sigmoidl shpe, reflecting higher SP, Wvenumer (cm 1 ) Slope (%OD µmol % 1 ) Temperture ( C) POPC POPC + 3 mol% cholesterol Fig. 5. Effects of temperture nd cholesterol content on soluiliztion slope. Vlues re mens ± rnge, N=2. solute vlues of slopes of ner-liner regions etween 2nd-derivtive peks of fitted sigmoidl curves for () myristoylplmitoyl phosphtidylcholine (MPPC) vesicles s function of temperture nd () plmitoyloleoyl phosphtidylcholine (POPC) nd POPC + 3 mol% cholesterol vesicles t 25 C. % OD 1 Rft D/L (% µmol 1 ) Fig. 6. Detergent soluility curves. Decline in normlized opticl density (OD) s function of detergent/lipid (D/L) rtios. Vlues re mens ± S.E.M., N=4. Cold-cclimted smples ssyed t 5 C (other dt not shown)., plsm memrne;, rft-depleted plsm memrne. compred with the more flttened curve produced y rft. The SPs for ll smples were nlyzed y three-wy NOV (cclimtion group ssy temperture frction). There ws significnt effect of frction nd ssy temperture, with no

7 Therml cclimtion of plsm memrne rfts 1663 Sturtion point (% µmo l 1 ) Cold 5 2 effect of cclimtion group, nd no significnt interctions. However, the frction cclimtion group interction term ws ner significnce (P=.75, N=4). Therefore, the dt were collpsed cross ssy temperture for comprisons of frctions from the two cclimtion groups (Fig. 7). Within cclimtion groups, the SPs of the smples were significntly greter (P<.5, N=4) thn oth the nd rft smples (Fig. 7). The SP of nd rft from wrmcclimted fish did not differ significntly. In the smples from cold-cclimted fish the SP of the ws significntly greter thn tht for the rft. It should e noted tht the SPs for the cold-cclimted rft smples were, on verge, negtive. The second-derivtive pek ws chosen s convenient index of sturtion; the negtive vlues re product of this pproch nd simply reflect very low SP. The SPs of ll of the frctions were sensitive to ssy temperture. Incresing the ssy temperture from 5 C to 2 C cused significnt (P=.11, N=24) men reduction in SP of 38% (Fig. 7). The uncollpsed SP dt, grouped nd nlyzed y frction, re presented in Fig. 8. The nd displyed similr ptterns. When ssyed t common temperture, smples hd similr SPs, regrdless of their cclimtion group. s result, c Rft,c Wrm ssy temperture ( C) Fig. 7. Sturtion points. rs with common lower-cse letter do not differ significntly. () Effects of cclimtion group nd frction (dt collpsed cross ssy temperture). Cold-cclimted, open rs; wrm-cclimted, filled rs. Vlues re mens ± S.E.M., N=8. () Effects of ssy temperture (dt collpsed cross frction nd cclimtion group). Vlues re mens ± S.E.M., N=24., plsm memrne;, rft-depleted plsm memrne.,c Rft Sturtion point (% µmol 1 ) Rft,,c c ssy temperture ( C) Fig. 8. Sturtion points, ll comprisons. Vlues re mens ± S.E.M., N=4. Cold-cclimted, open rs; wrm-cclimted, filled rs. Dots denote mesurements mde t physiologicl tempertures., plsm memrne;, rft-depleted plsm memrne. wrm-cclimted smples hd lower SPs thn cold-cclimted smples when ssyed t their respective physiologicl tempertures (Fig. 8, dots). y contrst, the SPs of the rft smples from wrm-cclimted fish were up-shifted reltive to those from cold-cclimted fish, the source of the nersignificnt cclimtion group frction interction term in the three-wy NOV. s result, the SPs of the rfts from the two cclimtion groups do not differ t their respective physiologicl tempertures. Three of the cold-cclimted rft smples showed reduced SP when ssyed t 2 C compred with t 5 C, while one smple displyed drmticlly higher SP. sed on the dt collected on the MPPC vesicles (Fig. 4), this suggested tht 2 C ws ner the L o L d phse trnsition temperture for these smples. Therefore, we repeted the ssy t 28 C to determine if melting ws detectle (Fig. 9). Neither cold- nor wrmcclimted showed significnt increse in SP when ssyed t 28 C. y contrst, the SPs of rfts from oth cclimtion groups incresed significntly t 28 C compred with 2 C. Soluiliztion slopes The region of the detergent soluility curves etween points nd in Fig. 1C ws nerly liner nd represented the shift from ilyer morphology to mixed micelles (soluiliztion). The slopes from liner regressions fitted to these regions were compred with three-wy NOV. The nlysis found n effect of cclimtion group nd frction ut no effect of ssy temperture. There ws significnt interction etween cclimtion group nd frction ut no other significnt interctions. Therefore, the dt were collpsed cross ssy temperture (Fig. 1). In oth cclimtion groups, the rft soluiliztion slopes were intermedite in vlue etween those of the nd. The slopes were significntly greter thn those of the y 87% nd 46% for the cold- nd wrmcclimtion groups, respectively. In the cold-cclimtion group, the slope for the ws significntly greter

8 1664 J. K. Zehmer nd J. R. Hzel Sturtion point (% µmol 1 ) Cold Cold rft * Wrm Wrm rft ssy temperture ( C) Fig. 9. Sturtion points, elevted ssy temperture. Vlues re mens ± S.E.M., N=4. sterisks denote significnt difference from vlues t 2 C (P<.5)., rft-depleted plsm memrne. (P<.5, N=8) thn tht for the rft y 59%, while these frctions did not differ in the wrm-cclimtion group. Discussion Plsm memrnes (s) were isolted from trout using comintion of differentil nd density grdient centrifugtion. The frction otined y this method hs een previously chrcterized. It comprised % of the totl cell protein, regrdless of cclimtion group. The picl domin mrker 5 nucleotidse ws enriched y fctor of 11.14±2.3 (2 C cclimted trout) nd 1.96±1.51 (5 C trout) while the solterl domin mrker N + /K + -TPse ws enriched y fctor of 38.64±9.26 (2 C trout) nd 52.53±6.72 (5 C trout) (Hzel et l., 1992). Thus, oth mjor domins of the re present nd the preprtion is resonly representtive of the cell memrne. We used soniction nd density grdient centrifugtion to seprte the into low nd high uoynt density frctions. Liver from rinow trout does not express the protein cveolin (Fig. 2). Nevertheless, the low density frction ws enriched in molecules chrcteristic of lipid microdomins, including cholesterol (Fig. 3; Tle 1) nd oth the β 2 drenergic receptor nd denylyl cyclse (Fig. 2,C). These three molecules hve een reported to e lipid microdomin enriched in other systems (Ostrom et l., 21; Ryin et l., 2; Xing et l., 22). Furthermore, the low density frction ws sustntilly depleted in the β suunit of the insulin receptor, which is lso consistent with previous oservtions (Mstick et l., 1995; Ohir et l., 2). The even distriution of the protein clthrin mong, nd rft in our system is surprising since it is not ssocited with rft or * Slope (%OD µmol % 1 ) , c Cold, Rft cveole microdomins (Rzni nd Lisnti, 21). However, unlike ll other proteins exmined in this study, clthrin is not n integrl memrne protein nd cn e seprted from memrnes y gentle dissocition (Keen et l., 1979). Therefore, it is possile tht the soniction step used in seprting the frctions cted to redistriute the protein mong ll memrne prticles present in the preprtion. Regrdless, the weight of evidence suggests tht this method seprtes trout liver into iochemiclly distinct frctions, including low uoynt density frction consistent with lipid microdomins. s this memrne frction lcks cveole, we will refer to it opertionlly s rft-enriched (rft) nd the high uoynt frction s rft-depleted (). The discovery tht the of most cells is not homogenous, ut contins microdomins with distinct compositions, presents n opportunity for the re-exmintion of memrne therml cclimtion. In response to chnging environmentl tempertures, poikilotherms sustntilly lter the lipid composition of the (Hzel, 1997). Mny studies hve sought to rtionlize these compositionl chnges s mechnisms of homeostsis for specific physicl properties, notly hydrophoic region fluidity/order (homeoviscous dpttion), with the impliction tht criticl functions re sensitive to these properties (Hzel, 1997). However, these studies hve exmined the s whole. It is possile tht the compositions of different regions of the re ltered in different wys during therml cclimtion. Furthermore, microdomins form s result of seprtion of liquid-ordered (L o ; rft) nd liquid-disordered (L d ; ) phses within the memrne (hmed et l., 1997). Since lipid phse ehvior is temperture sensitive, it is resonle to hypothesize tht cute chnges in temperture could disrupt rft structure nd the function of rft-trgeted proteins. Consequently, cclimtory compositionl chnges of the my e imed t stilizing rft structure ginst therml perturtion. Therefore, we Wrm Fig. 1. Slopes. solute vlues of slopes of ner-liner regions etween 2nd-derivtive peks of fitted sigmoidl curves (dt collpsed cross ssy temperture). Cold-cclimted, open rs; wrm-cclimted, filled rs. Vlues re mens ± S.E.M., N=8. rs with common lower-cse letter do not differ significntly., plsm memrne;, rft-depleted plsm memrne., Rft

9 Therml cclimtion of plsm memrne rfts 1665 sought to determine wht generl compositionl chnges occur in rft nd portions of the during therml cclimtion. We mesured the cholesterol, phospholipid nd totl protein content of rft, nd totl of livers from cold- nd wrm-cclimted trout. Rfts from oth cclimtion groups re sustntilly depleted in totl protein compred with or (Tle 1). The protein is primrily replced y n enrichment in phospholipids in the cold-cclimted rft (lthough cholesterol is lso enriched). y contrst, cholesterol ccounts for most of the lipid enrichment in wrm-cclimted rfts (Fig. 3,; Tle 1). s result, the cholesterol/phospholipid (Ch/PL) rtio is highly elevted in wrm-cclimted rft while the Ch/PL is not chnged in cold-cclimted rft compred with its source (Fig. 3). The Ch/PL rtio is proly the most importnt mesure for rft stiliztion since it is n interction etween cholesterol nd specific lipids, nd not cholesterol concentrtion per se, tht fvors rft formtion. This suggests tht there re importnt differences in how rfts re stilized in cold- nd wrm-cclimted nimls. The dt lso demonstrte tht the increse in cholesterol concentrtion is lrger in rft thn in with cclimtion to 2 C (Tle 1). Collectively, these dt suggest tht the compositionl chnges ssocited with therml cclimtion re t lest prtilly relted to properties specific to rft memrne. If therml cclimtory compositionl djustments cn e explined in terms of rft stiliztion, we would expect tht moleculr interction strength (nd thus the tendency to form the L o phse) would e regulted in rfts to offset therml perturtion. Memrne soluiliztion y detergent hs previously een used to mesure this property (hmed et l., 1997), ut most nlyses hve filed to pprecite or ccount for the different stges of detergent memrne interction. Detergent molecules dded to memrne suspension re initilly incorported into the ilyer structure, suppressing the free detergent monomer concentrtion elow the criticl micelle concentrtion (CMC; Fig. 1, pltes 1, 2). s the memrne sturtes, detergent monomer concentrtion in solution exceeds the CMC, mixed micelles form nd the memrne egins to e soluilized (Fig. 1, pltes 3, 4) (Lichtenerg et l., 1983). The cpcity of memrne to incorporte detergent monomers is inversely proportionl to the lipid lipid interction strength in the memrne (Lichtenerg et l., 1979). For exmple, Lngmuir trough study demonstrted tht smll increses in lterl pressure, with concomitnt increses in lipid cohesion forces, resulted in lrge decreses in the incorportion of Triton X-1 (Nyholm nd Slotte, 21). We found this interprettion to e well supported in model vesicle studies. The memrne condensing effect of n ddition of 3 mol% cholesterol to plmitoyloleoyl phosphtidylcholine (POPC) vesicles ws esily detected s drmtic reduction in the SP (Fig. 4). Furthermore, myristoylplmitoyl phosphtidylcholine (MPPC) vesicles sturted with less detergent in the tightly pcked gel phse thn in the fluid phse (Fig. 4). Similr results hve previously een reported for sphingomyelin vesicles (Ptr et l., 1999). reduction in SP ws oserved with n increse in temperture up to, ut remining elow, the cyl chin melting temperture in MPPC vesicles (Fig. 4), sphingomyelin vesicles (Ptr et l., 1999) nd iologicl memrnes (Fig. 7). Prior to the drmtic decreses in pcking density ssocited with phse trnsition, increses in temperture within phse cuse incresed memrne lterl pressure (de Kruijff, 1997). This my explin the lowered SP oserved in these cses. Memrne moleculr interction strength lso influences the detergent/lipid vlues necessry to disrupt ilyer morphology nd incorporte memrne molecules into mixed micelles (soluiliztion slopes). For exmple, reltively high concentrtion of detergent ws required to fully dissolve the vesicles mde of POPC nd 3 mol% cholesterol, producing shllow soluiliztion slope (Fig. 5). s predicted, the soluiliztion slope produced during dissolution of the more loosely pcked POPC vesicles ws comprtively steep. However, this mesure tends to e less informtive thn SPs since this process is much more sensitive to vriles other thn moleculr interction strength. For exmple, the CMC of Triton X-1 is temperture sensitive (Elworthy et l., 1968) nd, contrry to prediction, the soluiliztion slopes for MPPC vesicles re much steeper in the gel phse compred with the fluid phse (Fig. 5). Furthermore, the ility of detergent micelles to incorporte lipids, property known s mximum dditive concentrtion, is strongly dependent on the moleculr species to e soluilized (Elworthy et l., 1968). This complictes the interprettion of these dt collected on iochemiclly distinct frctions. Nevertheless, differences in soluiliztion slope etween rft nd re proly responsile for the detergent resistnce of rfts, which is widely oserved nd commonly used in their isoltion. The most commonly used detergentsed rft isoltion method exposes isolted to Triton X- 1 t 4 C nd flots the remining undissolved rft-enriched memrne on sucrose density grdient (rown nd Rose, 1992). In the current study, rft memrnes produced lower normlized opticl densities thn either or t most detergent concentrtions (Fig. 6). This reflects the lrge differences in the SPs of these memrnes ut seems contrdictory to the use of detergent s mens of rft isoltion since it suggests tht rft would dissolve more redily thn. However, this is esily resolved when considering the soluiliztion process of s mixture of rft nd memrnes. The smple s whole would ecome sturted with detergent efore the process of soluiliztion ecme dominnt. This would effectively remove the differences in SPs etween rft nd memrnes, nd the soluiliztion process would e primrily defined y the process mesured y the soluiliztion slopes of the nd rft. The frctions hd steeper soluiliztion slopes thn the rft or in oth cclimtion groups (Fig. 1). Therefore, memrne would e more rpidly soluilized, leving rft-enriched frction ehind.

10 1666 J. K. Zehmer nd J. R. Hzel The difference in pcking strength etween rft nd indicted y the soluiliztion slopes is lso evident in the SP dt (Fig. 7). The more shllow soluiliztion slope nd lower SP of rft, compred wiht, proly reflects the L o phse of this microdomin. Proteins cn e trgeted to the L o phse rfts y modifiction with sturted lipids tht re more solule in the tightly pcked L o thn in the L d phse (Melkonin et l., 1999). Since the SP (nd pcking strength) of memrne in either phse is cutely temperture sensitive (Fig. 7), it is possile tht perturtions of this property could influence the trgeting of proteins to rfts. Furthermore, differences in pcking strength my hve functionl consequences for the proteins emedded in the memrne. Integrl memrne proteins my require specific rnge of lterl pressure to llow pproprite flexing without llowing the protein to relx into non-functionl conformtions (de Kruijff, 1997). Finlly, sufficiently lrge increse in temperture could drive phse trnsition, effectively melting L o phse rft nd resulting in mixing with the memrne. Conversely, lrge decrese in temperture could result in ccretion of lipids nd proteins normlly excluded from rfts. Therefore, we exmined the frctions from oth cclimtion groups t oth ssy tempertures to determine if memrne pcking strength ws conserved during therml cclimtion (Fig. 8). In oth the nd, smples ssyed t common temperture gve similr SPs. s result, the wrmcclimted smples ssyed t 2 C were different from the cold-cclimted smples ssyed t 5 C (physiologicl comprisons; Fig. 8, dots). However, this perturtion ws not seen in the rft memrnes. The SPs of the wrm-cclimted rfts were sustntilly up-shifted with respect to the coldcclimted rfts such tht there ws little difference in this property when exmined t physiologicl tempertures. To our knowledge, this is the first evidence of n pprent regultion of physicl property of sudomin of the. Such regultion would e expected to mintin rft structurl integrity nd pcking properties despite differing therml environments experienced y the orgnism. n increse of ssy temperture ove the gel fluid phse trnsition ws detected s drmtic increse in SP for the MPPC vesicles (Fig. 4). Our oservtion tht n increse in ssy temperture from 5 C to 2 C resulted in n increse in SP in one rft smple from cold-cclimted fish suggested tht rfts from these nimls were close to their L o L d melting temperture t 2 C. To test this hypothesis, ll smples were ssyed t 28 C (Fig. 9). Since is in the L d phse, n increse in temperture should not cuse n increse in SP. Indeed, there were only smll nd non-significnt SP increses in from oth cclimtion groups. y contrst, the SP of the rft smples ssyed t 28 C ws significntly greter thn those mesured t 2 C in oth cclimtion groups. The drmtic increse in SP t 28 C suggests tht the rft memrnes from oth cclimtion groups were ove their L o L d melting temperture t this temperture. In the plsm memrne, clustered rft proteins nd lipids would e expected to mix rndomly with molecules under these conditions. In conclusion, there is pprent sptil heterogeneity in the response of the to therml chnge. Compositionl chnges ssocited with therml cclimtion re different in the rft nd portions of the. Specificlly, the rtio of cholesterol to phospholipid is elevted in rfts from wrm-cclimted, ut not from cold-cclimted, nimls compred with their sources. Furthermore, there is conservtion of memrne pcking strength in the rft, ut not the rft-depleted, regions of the. This is consistent with regultion of this physicl property, which my hve functionl consequences for phsedependent protein trgeting. Furthermore, the detergent dt suggest tht elevted temperture cn olish the L o /L d phse seprtion responsile for rft structure. The rft-ssocited compositionl chnges oserved my lso function to stilize these microdomins. The coopertion of the personnel of the lchesy Ntionl Fish Htchery s well s the diligent efforts of Jmes dmn in mintining the fish in the lortory re gretly pprecited. Support from the Ntionl Science Foundtion is grtefully cknowledged (grnts IN nd IN 26614). References hmed, S. N., rown, D.. nd London, E. (1997). On the origin of sphingolipid/cholesterol-rich detergent-insolule cell memrnes: physiologicl concentrtions of cholesterol nd sphingolipid induce formtion of detergent-insolule, liquid-ordered lipid phse in model memrnes. iochemistry 36, mes,. N. (1966). ssy of inorgnic phosphte, totl phosphte nd phosphtses. Methods Enzymol. 8, rmstrong, J. M. nd Newmn, J. D. (1985). simple, rpid method for the preprtion of plsm memrnes from liver. rch. iochem. iophys. 238, rdford, M. M. (1976). 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