Review

Adaptive regulation of membrane lipids and fluidityduring thermal acclimation in Tetrahymena

By Yoshinori NOZAWA*1,*2,†

(Communicated by Teruhiko BEPPU, M.J.A.)

Abstract: The free-living eukaryotic protozoan Tetrahymena is a potentially useful modelfor the thermoadaptive membrane regulation because of easy growth in the axenic culture,systematic isolation of subcellular organelles, and quick response to temperature stress. Exposure ofTetrahymena cells to the cold temperature induces marked alterations in the lipid composition andthe physical properties (fluidity) of various membranes. The increase in fatty acid unsaturation ofmembrane phospholipids is required to preserve the proper fluidity. In this homeoviscous adaptiveresponse, acyl-CoA desaturase plays a pivotal role and its activity is regulated by induction of theenzyme via transcriptional activation.

Keywords: Tetrahymena, homeoviscous adaptation, membrane fluidity, membrane lipidcomposition, fatty acid desaturase

Introduction

There is substantial evidence that poikilother-mic organisms possess the potential ability to modifythe fatty acyl composition of their membranephospholipids in response to changes in environ-mental temperature. The general trend is an increasein unsaturated fatty acids at lower growth temper-atures and an increase in saturated fatty acids athigher temperatures. Therefore, when exposed to adown-shift in the growth temperature, cells arerequired to increase the level of unsaturated fattyacids. Such compositional adaptation of membranelipids, which was called a homeoviscous adaptationprocess, serves to maintain the correct membranefluidity at the new growth temperature. Thishomeoviscous adaptation occurs in diverse micro-organisms and in plants.1)

The unicellular eukaryotic protozoan Tetra-hymena exerts marked changes in membrane lipid

composition when exposed to altered growth temper-atures. Thus this cell has proved to be a potentiallysuitable model system for studying the molecularmechanisms of the thermoadaptive control of mem-brane lipids and physical properties (fluidity).From data obtained by ultrastructural and physico-chemical techniques, it was indicated that, inTetrahymena cells, the temporal adjustment ofmembrane fluidity coincided with the proportionalchanges in the levels of unsaturated and saturatedfatty acids; i.e. increases in palmitoleic (C16:1"9),linoleic (C18:2"9,12) and .-linolenic (C18:3"6,9,12)acids and a decrease in palmitic acid (C16:0). Thisadaptive regulation of fatty acid composition canbe achieved by modifying the activity of "

9-desaturase, which catalyses the introduction of thefirst double bond into palmitic acid or stearic acid(C18:0). Upon exposure of Tetrahymena cells tolow-temperature, the activities of palmitoyl-CoAand stearoyl-CoA desaturases were increased. Theincreased activity of the desaturase enzyme wasprincipally due to enhanced expression of theenzymes, probably via induced transcription of itsmRNA. Membrane fluidity-mediated activation ofthe desaturase, possibly by a conformational changewas suggested to be involved in the early adaptiveresponse.

*1 Department of Health and Food Sciences, Tokai GakuinUniversity, Kakamigahara, Japan.

*2 Gifu International Institute of Biotechnology,Kakamigahara, Japan.

† Correspondence should be addressed: Y. Nozawa, GifuInternational Institute of Biotechnology, 1-1 Nakafudogaoka,Kakamigahara, Gifu 504-0838, Japan (e-mail: ynozawa@giib.or.jp).

Proc. Jpn. Acad., Ser. B 87 (2011) [Vol. 87,450

doi: 10.2183/pjab.87.450©2011 The Japan Academy

General description of Tetrahymena cell

The ciliate protozoan Tetrahymena, which isapproximately 60 µm in length and 20 µm in width,grows well axenically in lipid-free media and it can betherefore considered a closed system with respect tothe economy of its membrane components. All lipidsand proteins are synthesized de novo within the cell,primarily in one compartment, and are then dissemi-nated to various structurally and functionally differ-ent membranes for utilization. A wealth of structuraland biochemical details reveals Tetrahymena to bea typical cell.2) Tetrahymena cell contains the sub-cellular organelles typically found in eukaryotic cells,such as mitochondria, lysosomes, endoplasmic retic-ulum, peroxisomes, but no typical Golgi apparatus.The highly specialized surface structures are lesstypical (Fig. 1). Underlying the plasma membrane isan extensive anastomosing system of alveolar sacsof unknown function. The outer alveolar membraneis closely apposed to the plasma membrane andactually joined to it by cross bridges. This entiresystem of cortical membranes is referred to as thepellicle. Another membrane specialization of the cellsurface is the oral apparatus, which is a permanentlyfixed structure designed for the endocytosis. Being a

free-living unicellular organism, it furnishes the keyadvantages of bacteria with respect to rapid growthand easy manipulation. Thus, Tetrahymena hasbeen chosen as an appropriate model for the use instudying the structure and function of biologicalmembranes, particularly membrane biogenesis andmembrane adaptation (lipids and fluidy) to environ-mental stresses.3)–6)

The lipid composition of the functionally differ-ent membranes has been studied extensively. In orderto examine the metabolic interrelationships amongthe different membrane types, it is highly desirable torecover in purified form as many organelles aspossible. Accordingly, we have developed the proce-dure,7) which separates cilia, pellicles, mitochondria,and microsomes as well as postmicrosomal super-natant. Table 1 shows the remarkable differencesin phospholipid composition among several organ-elles. The major phospholipids are phosphatidyl-choline (PC), phosphatidylethanolamine (PE), andthe rather unusual 2-aminoethylphosphonolipid(AEPL).8) Thirty percent of the phosphoglyceridescontain an ether-linked hydrocarbon side chain at theC-1 position in place of the more typical fatty acylgroup. The ether side chains are localized mainly inAEPL and PC. The resistance of the phosphonolipidwith ether linkage to attack by endogenous lipolyticenzymes has been proposed as the factor responsiblefor its accumulation in the metabolically isolatedsurface membranes. The assortment of lipids presentin a membrane is important in determining themembrane’s physical properties (fluidity). The prin-cipal neutral lipid is the pentacyclic triterpenoidtetrahymanol,9) which serves as a fluidity modulatorsimilar to cholesterol in mammalian cells. There is a9-fold higher molar ratio of tetrahymanol to phos-pholipids in ciliary membranes as compared withmicrosomal membranes. The stabilizing effect oftetrahymanol is reflected in the fluidity propertiesof these membranes.

Many analyses of Tetrahymena fatty acids havebeen reported.3),6),10) As in the case of phospholipidpolar head groups, each membrane maintains acharacteristic fatty acid pattern. Tetrahymena syn-thesizes large amounts of polyunsaturates, with .-linolenic acid (C18:3"6,9,12) being the predominantspecies present. As has been observed in many othertissues, each phospholipid class in Tetrahymena hasits own unique fatty acid distribution.

Of particular advantage is abundant infor-mation regarding the physical properities (fluidity)of Tetrahymena membranes. The most detailed

Fig. 1. General feature of ultrathin-sectioned T. pyriformis. PM,plasma membrane; OAM, outer alveolar membrane; IAM, inneralveolar membrane; CM, ciliary membrane; M, mitochondria;KS, kinetosome; MT, microtubule; AS, alveolar space; FV, foodvacuole. From Nozawa (1975).3)

Low-temperature acclimation of Tetrahymena membranesNo. 8] 451

comparative analysis of Tetrahymena intracellularmembranes has involved freeze-fracture electronmicroscopy. When cells were exposed to temperatureshift-down, the alveolar membrane displayed anobvious lateral movement of protein particles intodense aggregates, leaving behind large particle-free areas of lipids in the crystalline phase. Othermembranes, such as endoplasmic reticulum, plasmamembrane and nuclear envelope, also showed par-ticle-free domains, but pronounced lateral movementof particles was not distinct. On the other hand, theciliary membrane underwent no particle rearrange-ment. Kitajima and Thompson11) have developeda method for quantitative comparison of particlerearrangement in several membranes. The temper-ature at which each particular membrane firstshowed particle-free regions varied with the growthtemperature of the cells, but the relative ordersremained unchanged.

Fluidity-related parameters of isolated mem-brane fractions have been examined by severalphysico-chemical techniques, such as electron spinresonance (ESR), X-ray diffraction, fluorescencedepolarization and 31PNMR. The fact that generalagreement is found using these basically differentphysical-chemical tools inspires a high degree ofconfidence that each membrane type within aeukaryotic cell possesses very specific physical prop-erties determined largely by its lipid composition.ESR study with the use of the probe 5-nitrostearatedemonstrated that the membranes are more fluid inthe order, microsomes > pellicles > cilia10) (Fig. 2).The localization of certain lipids is striking inparticular Tetrahymena membrane fractions, which

perhaps reflects a diversity in the physical state ofthe membranes. Taking into consideration that thecontent of tetrahymanol increases in the order,cilia > pellicles > microsomes, one would expect thatthis specific sterol-like lipid might be involved instabilizing membrane fluidity, as observed in choles-terol of mammalian cells.

Another useful technique for probing the phys-ical state of Tetrahymena membranes is fluorescencelabeling. The fluidity or microviscosity was measuredfor isolated membranes by using the fluorescentprobe (DPH).12) There are pronounced differencesin the microviscosity of various membrane fractions,which are compatible with data from ESR.10) The

Table 1. Major phospholipids of various membrane fractions from T. pyriformis WH-14 cells

Membrane

fraction

Total phospholipids (mole%) Tetrahymanol

(moles/mole lipid

phosphorus)

Glyceryl ethers

(moles/100 moles

lipid phosphorus)Lyso-PC PC

Lyso-AEPL/

PEPE AEPL CL

Whole cells 2 33 0 37 23 5 0.057 29.7

Cilia 1 28 9 11 47 1 0.30 52.6

Ciliary supernatant 8 19 13 16 35 1 0.16 23.1

Pellicles 5 25 3 34 30 2 0.084 32.8

Mitochondria 2 35 0 35 18 10 0.048 24.7

Nuclear membranes 6 31 6 26 23 3 0.036 —

Microsomes 1 35 3 34 23 1 0.041 18.3

Postmicrosomal supernatant 5 34 4 30 22 2 0.016 27.4

Data from Nozawa and Thompson (1971)7) and Thompson et al. (1971).9)

PC, phosphatidylcholine; PE, phosphatidylethanolamine; AEPL, 2-aminoethylphosphonolipid; CL, cardiolipin.

CiliaPelliclesMicrosomes

Fig. 2. The order parameter of 5-nitroxide stearate label invarious membranes of T. pyriformis as a function of thereciprocal of the absolute temperature. Data from Nozawa et al.(1974).10)

Y. NOZAWA [Vol. 87,452

microviscosity of the ciliary membranes is markedlygreater than the microsomal and pellicular mem-branes. Such diversity in the microviscosity reflectsthe different lipid compositions of the three mem-brane fractions. The ciliary membranes contain aconsiderably higher level of tetrahymanol and 2-aminoethylphosphonolipid (AEPL), whereas themore fluid microsomes have the lowest content ofthese two lipids together with the greatest degreeof unsaturation in fatty acyl chains (Table 1).

Effects of growth temperature on membranelipid composition and fluidity

Experimentation in many laboratories includingour own has established a firm base of informa-tion concerning the membrane biochemistry ofT. pyriformis. Some strains of this ciliate are capableof adapting to rather high growth temperatureswhile others are more temperature sensitive. Amoderately adaptable strain, WH-14, was used forthe earlier studies as above described, because of thecapability to alter its membrane lipid compositionand fluidity. However, we have discovered thatanother strain of T. pyriformis, which was isolatedfrom a natural hot spring in New Mexico by Phelps,A. (1961) and provisionally designated this strainNT-1,13) can adapt to higher temperatures and alterits membrane lipids in a much more pronouncedfashion than can strain WH-14. Because of itsrapid and extensive temperature response, we havechosen this thermotolerant strain for futher studiesof the molecular mechanisms underlying temperatureadaptation.

Tetrahymena NT-1 cells were grown isother-mally at 15, 24, and 39 °C, and the generation time atthese temperatures was 16, 4, and 3 h, respectively.The profound changes were observed in the fatty acidcomposition of phospholipids from several subcellularfractions.13) The principal effects of decreasingtemperature are an increase in linoleic and linolenicacid and a decrease in palmitic acid thus producinga generally higher degree of unsaturation (Table 2).For example, the ratios of total unsaturated tosaturated fatty acids of microsomal phospholipidsfrom cells grown at 15, 24, and 39 °C are 4.51, 3.97,and 2.66, respectively.

It was also rather surprising to observe thatthere were marked differences in the phospholipidshead group composition of cells grown at the threedifferent temperatures. There was a marked increasein phosphatidylethanolamines (PE) as the growthtemperature increased, and the relative concentra-tion of phosphatidylcholines (PC) remained constantin pellicles and microsomes. In contrast, the level ofAEPL was increased at the low temperature.14) Thepositional distribution of fatty acids shows a charac-teristic profile for each phospholipid. When comparedwith 39 °C-grown cells, the C-1 position of PC from15 °C-grown cells undergoes a large increase inpalmitoleic (C16:1"9) and .-linolenic (C18:3"6,9,12)acids with a corresponding decrease of myristic(C14:0) and palmitic (C16:0) acids. At C-2 positionof 15 °C-grown cells, linoleic (C18:2"9,12) and .-linolenic acids in PC, and linoleic acid in AEPLincrease with a large decrease of palmitoleic acid inboth phospholipids and of .-linolenic acid in AEPL.

Table 2. Lipid composition of various membranes from T. pyriformis NT-1 cells grown at different temperatures

Cilia Pellicles Microsomes

15 °C 24 °C 39 °C 15 °C 24 °C 39 °C 15 °C 24 °C 39 °C

Phospholipid composition

2-Aminoethylphosphonolipids (AEPL) 40.0 37.0 36.4 30.8 32.9 19.1 22.9 20.8 14.7

Phosphatidylethanolamines (PE) 20.8 21.4 20.0 33.2 36.2 49.2 34.1 35.5 43.9

Phosphatidylcholines (PC) 15.8 18.1 30.0 18.9 21.4 23.5 30.0 27.6 32.4

Fatty acid composition of phospholipids

C14:0 6.0 3.9 6.7 8.4 9.6 8.9 7.2 7.4 7.3

C16:0 16.8 19.4 16.8 12.7 15.8 16.4 8.9 10.4 13.2

C16:1"9 14.4 7.7 8.3 9.9 7.4 7.1 8.8 9.8 8.7

C18:0 2.5 3.8 6.1 0.7 2.0 3.0 0.5 1.4 2.2

C18:1"9 6.9 8.0 11.7 7.4 8.5 12.6 8.1 7.1 13.9

C18:2"9,12 9.0 9.7 8.1 16.2 13.8 10.5 19.5 17.7 13.0

C18:3"6,9,12 27.2 28.2 20.7 28.0 27.4 18.5 31.8 31.2 21.4

Data from Fukushima et al. (1976).13)

Low-temperature acclimation of Tetrahymena membranesNo. 8] 453

It can therefore be speculated that such pro-nounced alterations in the membrane lipid composi-tion play an important role in adapting the mem-brane fluidity to changed temperatures. Wunderlichet al.15) showed that the physical properties ofmembranes of T. pyriformis GL cells and the fattyacid composition of the cellular phospholipids weredependent upon the growth temperature of the cells,with apparent fluidity visualized by freeze-fractureelectron microscopy and fatty acid unsaturation bothbeing greater in cells grown at lower temperatures.We observed a similar but more distinct pattern ofchanging fatty acid composition using the thermoto-lerant T. pyriformis NT-1 cell and were able to inferby ESR analysis that the fluidity of membranes fromlow temperature-grown cells was greater than that ofhigh temperature cells measured at the same temper-ature.12) The fluidities of major membrane fractionsisolated from 15 °C- and 39 °C-grown cells weremeasured by ESR as a function of temperature.The membranes of cilia, pellicles,16) and microsomesfrom cells grown at 15 °C were more fluid than thosefrom cells grown at 39 °C.16) The effect of growthtemperature on the membrane fluidity was alsoexamined by wide-angle X-ray diffraction.17) Thesolid-to-fluid phase transition temperatures of phos-pholipids from mitochondria, microsomes and pel-licles were 21, 19 and 26 °C for cells grown at 39 °C,and !8, !3 and 6 °C for cells grown at 15 °C,respectively. All phospholipids were found in acompletely fluid state at these growth temperatures.

Adaptive membrane alterations inducedduring low-temperature acclimation

As observed above, fatty acids of Tetrahymenaphospholipids were considerably more unsaturatedwhen the cells were grown at low temperatures. Thelogical advantage to a cell of increasing fatty acidunsaturation with decreasing temperature is that itsmembrane fluidity can thereby be maintained at anoptimal level for metabolic function (homeoviscousadaptation).

In order to gain more insight mechanisms of thethermal membrane adaptation, we have done tem-perature shift-down (chilling) experiments. WhenTetrahymena NT-1 cells were exposed to a rapidtemperature drop, cell division was quickly arrested,and the cells remained motile but relatively inactivefor a period of several hours before eventuallyresuming growth at a slower rate characteristic oftheir new environmental temperature. Tetrahymenacells were grown initially at 39 °C and then shifted to

15 °C over a period of 30min. This cooling treatmentcaused a growth cessation for a period of 10 h. Duringthis lag period with little apparent metabolicactivity, the critically important alterations shouldoccur in membrane lipid dynamics, e.g. membranelipid composition and membrane fluidity.

Alterations in membrane lipid composition.Although, as described above, the large differencesin polar head groups were found to exist between 39and 15 °C-grown cells,13) rather surprisingly, thesechanges did not take place during the initial low-temperature acclimation period of 10 h followingchilling, when the most significant fatty acid changesoccur and the cells regain their capacity to divide.18)

Instead, there was a slow transition from thecharacteristic 39 °C pattern to the 15 °C patternduring the first few generations of cell growth at15 °C, with AEPL increasing drastically at theexpense of PE. It is thus assumed that the polarhead group changes constitute a secondary responseto the lowered temperature, thereby readjustingstructural associations resulting from the initial fattyacid changes.

On the other hand, a rapid response to thetemperature shift-down was observed in the fattyacid composition of membrane lipids. There wastypically a rapid rise in the percentage of palmitoleicacid (16:1"9) concurrent with a decrease in palmiticacid (16:0), followed by a slower decline of that fattyacid as other species gradually increase. The composi-tional changes accompanying low-temperature accli-mation do not take place concurrently in variousmembranes19) (Table 3). The immediate modifica-tions of fatty acids occur considerably more quicklyin microsomal membranes than in the peripheralmembranes, such as pellicles. The much slowermodification of the cell surface membrane fattyacid pattern was completed at roughly the sametime (10 h) that low-temperature acclimation wasachieved, as judged by the resumption of celldivision. The microsomal fatty acid compositionwas rapidly modified to the pattern observed inmicrosomes from 15 °C-grown cells, as compared tothe peripheral cellular membranes.

The fatty acid composition was also examinedfor each phospholipid class of whole cells at varioustimes after the temperature shift.20) The ratios ofunsaturated to saturated fatty acid content followingchilling (0, 2, 4, 10 h) were 1.1, 2.8, 2.4, 3.0 for PC;1.8, 3.9, 4.6, 5.7 for PE; 2.7, 4.7, 7.9, 14.0 for AEPL,respectively, indicating that all three phospholipidshave the maximum level of unsaturated fatty acid

Y. NOZAWA [Vol. 87,454

content at 10 h. It is generally accepted that thephysical state of a phospholipid or mixture ofphospholipids is sensitive not only to the nature oftheir fatty acid constituents, but also to the position-ing of fatty acids within the phospholipid molecules.In general, naturally occurring phospholipids containsaturated fatty acids (in some cases, ether sidechains) at C-1 position and unsaturated fatty acidsat C-2 position. Tetrahymena phospholipids showthis distributional pattern, particularly when grownat moderate to high temperatures. However, adramatic change in fatty acid positional specificityoccurs upon the temperature shift-down. This changeis reflected in the appearance of unusually high levelsof polyunsaturated fatty acids at C-1 position ofcertain phospholipids.21) Upon temperature shiftfrom 39 to 15 °C, palmitic acid at C-1 position beganto be replaced by .-linolenic acid in all 1,2-diacyl-types of PE, PC, and AEPL from microsomes andmitochondria, while pellicles did not exhibit suchrapid and pronounced incorporation of this mostunsaturated fatty acid into the C-1 position.22) Thisrather mild modification in pellicles might reflect theslow transfer from microsomes of phospholipids withmodified fatty acid compositions. On the other hand,as for alkyl-acyl types of AEPL and PC which areunique in containing a high level of .-linolenic acid,there were no marked differences between pellicles,mitochondria and microsomes in temperature-in-duced rearrangement of the fatty acid compositionat C-2 position. Of great interest was the tremendousincrease of .-linolenic acids in 1-O-hexadecyl-2-acyl-PC at C-2 position, which was compensated mainlyby the reduction of oleic and palmitoleic acids. Incontrast, there were only small or no significantchanges in 1-O-hexadecyl-2-.-linolenoyl AEPL.Thus, the renewal of two principal molecular species,

1-.-linolenoyl-2-linoleoyl-PE and 1-O-hexadecyl-2-.-linolenoyl-PC, plays a crucial role for adjustingrapidly the membrane fluidity to the newly exposedlow temperature.

Alterations in membrane fluidity. Loweringthe temperature of Tetrahymena cells induced therearrangement of membrane protein particles, asdetected by freeze-fracture electron microsco-py.5),18),23) When cells grown at 39 °C were cooled to15 °C over a 30-min period, the alveolar membranedisplayed a marked lateral movement of particles,thereby producing densely aggregated areas andparticle-free domains (Fig. 3). Other membranes,such as endoplasmic reticulum, plasma membrane,and nuclear envelope, also showed the perturbeddistribution of membrane particles, but the lateralmovement of particles was not distinct.11) Therefore,the alveolar membrane may serve as an effectivethermosensor to detect the environmental temper-ature changes and to trigger the rapid modification ofmembrane lipid composition. During the low-temper-ature acclimation, the altered distribution of alveolarmembrane particles was remodeled.18) The mem-brane particles induced the lateral movement withinthe plane of membrane, finally resulting in a random,homogeneous arrangement at 10 h after the temper-ature shift-down. Of particular note was that thecells regained their ability to grow and divide atabout this time, when the particle distribution wasrestored to the initial random distribution. Such arestoration of the distribution of membrane proteinparticles coincided with the modification of phospho-lipids fatty acid composition.

The temporal changes in membrane fluidityduring cold acclimation were also examined by ESRspectroscopy.19) The increase in fluidity in micro-somal and pellicular lipids during the temperature

Table 3. Temporal changes in fatty acid composition of various membrane fractions from T. pyriformis NT-1 cells during temperatureacclimation

Fatty acidsPellicles Mitochondria Microsomes

0 h 2 h 4 h 6 h 10 h 0h 2h 4 h 6h 10 h 0h 2 h 4h 6h 10h

C14:0 8.1 8.1 8.6 6.8 7.5 5.4 5.3 5.8 4.8 4.2 7.0 7.8 7.9 6.7 6.7

C16:0 17.6 15.4 13.4 10.4 10.9 12.4 10.9 10.1 7.7 6.6 15.5 12.7 10.9 8.1 7.6

C16:1"9 9.6 12.3 13.4 13.0 11.7 10.0 11.8 11.5 11.5 10.5 10.9 14.5 14.5 14.6 13.6

C18:1"9 11.9 8.5 7.4 7.4 7.8 9.4 7.4 6.8 7.0 6.0 12.4 6.5 6.6 6.1 6.0

C18:2"9,12 15.4 16.2 16.3 17.9 17.9 19.0 19.4 17.9 20.3 20.3 16.0 16.4 16.8 17.4 18.4

C18:3"6,9,12 19.7 20.9 20.8 25.3 25.1 27.2 28.9 29.5 32.7 36.2 20.5 21.8 23.4 26.8 27.9

U/S 1.60 1.76 1.82 2.43 2.24 2.62 2.92 3.04 3.79 4.38 1.94 2.00 2.22 2.71 2.97

U/S, A ratio of unsaturated to saturated fatty acids. Data from Yamauchi et al. (1981).19)

Low-temperature acclimation of Tetrahymena membranesNo. 8] 455

shift from 39 to 15 °C corresponded with the increasein the ratio of total unsaturated to saturated fattyacid content. However, unexpectedly, despite theincrease in the unsaturated to saturated fatty acidratio, the fluidity of mitochondrial lipids was found tobe rather constant within 10 h after the temperatureshift. A distinct difference in lipid compositionbetween mitochondria and the other two membranes(pellicles and microsomes) was the much highercontent of cardiolipin in mitochondria. Accordingly,it can be expected that this lipid may play a role inmaintaining a consistent fluidity. The correlationbetween the fluidity of three phospholipids (AEPL,PE, PC) and their fatty acid composition wasexamined at various intervals after the temperatureshift.20) The fluidity of AEPL increased within thefirst 10 h of the cold acclimation, with the content of.-linolenic acid in this phospholipid being highest. Incontrast, the fluidities of PE and PC showed gradualdecreases up to 24 h after the shift, although their .-linolenic acid contents were highest after 10 h of 15 °Cincubation. Thus, the fluidity changes of these twophospholipids could be due to the altered content ofother fatty acids as well as to .-linolenic acid.

Fluorescence polarization measurements showedthat upon the temperature shift from 39 to 15 °C over30min, rapid and pronounced alterations in the DPH

polarization occurred in the microsomal lipids, whiledistinct changes in polarization of pellicle lipids wereobserved after 1 h of a lag time.24) The decrease inpolarization of microsomes was much more rapidthan that of pellicular membranes, but it did notreach the level of the 15 °C-grown cells. The polar-ization of pellicles became almost identical withthe value of the 15 °C-grown cells at 4 h after thetemperature shift. The fluidity changes in the micro-somal lipids correlated well with the level of fattyacid unsaturation. On the other hand, fluidity ofmitochondrial lipids was kept constant up to 10 h,which was compatible with the result obtained byESR.19),25)

When examined by X-ray wide-angle diffraction,the phase transition temperature was lowered grad-ually in microsomal and pellicular phospholipids,whereas the transition temperature of mitochondrialphospholipids was unchanged for 10 h after chill-ing.26) Transition temperatures of microsomal, pel-licular, and mitochondrial phospholipids reached thegrowth temperature (15 °C) at 6, 10, and 24 h afterthe temperature shift, respectively. In the case of themembrane phospholipids extracted from cells grownat 39 °C, lowering the temperature increased the solidphase in all three membrane fractions. However, inthe case of the phospholipids from cells acclimated to

Fig. 3. Changes in distribution of intercalated-membrane particles of the outer alveolar membrane during low-temperature acclimation.The 39 °C-grown culture (thermotolerant T. pyrifomis NT-1) was shifted down to 15 °C over a 30-min period and continued to growat this low temperature. Samples were taken at indicated time intervals for freeze-fracture electron microscopy. a) 39 °C (before shiftto 15 °C); b) 0 h; c) 6 h; d) 11h after shift. Data from Nozawa and Kasai (1978).18)

Y. NOZAWA [Vol. 87,456

15 °C for 10 h, the increase in the solid phase wassignificantly smaller in mitochondrial phospholipidsthan in the phospholipids of two other membranefractions.

Molecular mechanisms for adaptive regulationof membrane phospholipid fatty

acyl unsaturation

As described above, a critical cellular response tothe low-temperature acclimation is increased propor-tion of unsaturated fatty acids in membrane phos-pholipids and this is thought to compensate forthe direct ordering effects of the cold exposure. Thefluidity of membrane phospholipids is largely aproperty of their unsaturation level and it is generallyaccepted that the fluidity of membrane lipids arerelated to the number of unsaturation bonds. How-ever, there is also strong evidence to suggest that theintroduction of the first unsaturation bond has thegreatest effect on membrane physical state andsuccessive bonds have progressively smaller effects.This initial reaction can be achieved by modifyingthe activity of the enzyme, "

9-desaturase, whichincorporates the first double bond into palmitic orstearic acid at the (C9–C10) position, resulting inproduction of plamitoleic or oleic acid. The character-ization of this regulatory mechanism is thereforeof great importance for better understanding thelow-temperature acclimation in Tetrahymena cells.

Fatty acyl-CoA desaturase system in Tetra-hymena cell. Numerous studies of various micro-organisms have provided substantial evidence thatboth molecular oxygen and NADH or NADPH areessential for the microsomal desaturase system,which catalyzes unsaturation of long-chain fattyacids. In addition, an obligatory requirementhas been established for flavoproteins (NADH-cytochrome b5 reductase and NADPH-cytochrome creductase) and cytochrome b5 in the oxidativedesaturation of fatty acids in microsomal mem-branes. The activity of the terminal component ofthe desaturase system changes in parallel with thetotal fatty acid desaturase, while the flavoproteinsand cytochrome b5 of this system are not affectedin mammalian cells,27) suggesting that the terminalcomponent is a rate-limiting enzyme involved inregulation of the overall fatty acid desaturationsystem. The electrons provided by NADH (orNADPH) are mediated by NADH-cytochrome b5reductase (or NADPH-cytochrome c reductase) andcytochrome b5 as sequential electron carriers andthey are finally transported to the terminal compo-

nent. Regarding the topology of the microsomalenzyme system, it was shown that three integralmembrane components of the microsomal fatty aciddesaturase system, including NADH-cytochrome b5reductase, cytochrome b5, and the terminal compo-nent, are localized on the cytoplasmic surface of theendoplasmic reticulum.

On the other hand, it has been shown inTetrahymena that, in parallel with large changes inthe fatty acyl (palmitoyl, stearoyl)-CoA desaturaseactivity of microsomes in the acute stage (2 h) of low-temperature acclimation, a concurrent, pronouncedalteration in the activity of the terminal componentof the desaturase system occurred.28),29) However,smaller change was observed in the activity ofmicrosomal NADH-cytochrome c reductase. We havepurified a b-type hemoprotein, cytochrome b560ms,which was functionally similar to but not identicalto mammalian cytochrome b5, from Tetrahymenamicrosomes.30),31) Cytochrome b560ms was reduced byNADH in the presence of an NADH-cytochrome b5reductase purified from rat liver microsomes, suggest-ing that the pathway of electrons to the cytochromeb560ms-linked fatty acyl-CoA desaturase in Tetra-hymena microsomes is mediated via microsomalNADH-cytochrome b560ms reductase.

Regulatory mechanisms of acyl-CoA desa-turase activity. The three independent regulatorymechanisms, which were thought most likely tooperate for adaptively increased desatuse activity,have been proposed and evaluated experimentally; 1)the control of fatty acid desaturation by level ofdissolved O2 as a co-substrate, 2) induced synthesisof fatty acid desaturase enzyme, and 3) membranefluidity-mediated activation of pre-existing desatur-ase enzyme.

It was proposed that the much greater solubilityof O2 in aqueous environments at low temperaturewould automatically raise the concentration of the O2

co-substrate for the desaturation reaction. In fact,the good correlation was observed in some plantsbetween O2 level and desaturation activity. However,when the O2 levels in cultures of Tetrahymena wereexperimentally changed over the range of values thatthe cells might encounter in nature at high and lowtemperature, there was no significant effect on thedegree of phospholipid fatty acid desaturation.32)

Also, at the constant O2 tension, the temperatureshifts did induce the expected changes in fatty acidcomposition. Thus, the O2 tension may not beimplicated in the increased desaturase activity bycold exposure.

Low-temperature acclimation of Tetrahymena membranesNo. 8] 457

The classical studies of desaturase regulationduring cold exposure in some bacteria have providedevidence for increased expression of the desaturaseenzyme, as an acclimatory response. Induction ofthe microsomal desaturase enzyme in Tetrahymenahas been suggested to be caused by environmentaltemperature shift-down. When cells were pulse-labeled with 14C-palmitic acid (C16:0) at intervalsafter the temperature shift-down (39 to 15 °C), the14C-pamitoleic acid (C16:1"9) level immediately roseafter the shift, reaching its maximal level 2 h there-after.18) The maximum of the conversion rate of 14C-palmitic to 14C-palmitoleic acid was comparable tothe palmitoyl-CoA desaturase activiy. After thetemperature shift to 15 °C, there were rapid increasesin the activities of palmitoyl- and stearoyl-CoAdesaturases in microsomes (Table 4).28) Unlike thesituation in mammalian cells, the temperature shift-down also produced small but significant increasesin NADH-cytochrome c reductase activity andcytochrome b560ms content. The pre-treatment withcycloheximide prevented the increases in the desa-turase and reductase activities after the temperatureshift, suggesting that the increase in overall desatur-ase activities during the cold acclimation may bedue to the induction of of NADH-cytochrome b560ms

reductase and cytochrome b560ms protein, as well asthe terminal component of the desaturase system.Similar findings have also been obtained in thestarved Tetrahymena cells which were deprived of

the pre-existing desaturase enzyme.33) Although thestarved cells have little or no desaturase activity,they showed a lowered temperature-induced rise inthe desaturase activity to the level of control-chilledcells. This provides evidence to support the conceptthat the increased level of total desaturase activityresults from induction of the desaturase enzymeprotein.

In order to gain further insight into induction ofdesaturase enzyme, we have isolated a gene that en-codes "

9-fatty acid desaturase from T. thermophilaand examined its expression during cold acclima-tion.34) The nucleotide sequence indicates that the1.4 kbp gene encodes a polypeptide of 292 amino acidresidues which shows marked sequence similarity to"

9 acyl-CoA desaturase from other sources, e.g. rat,mouse, and Saccharomyces. This desaturase proteinhas three histidine-cluster motifs (one HXXXXH andtwo HXXHH), and two hydrophobic regions whichare conserved among "

9 acyl-CoA desatuases. Thelevel of the desaturase mRNA was sensitive todecreasing the temperature of the culture media,and was close to maximum immediately after thetemperature was shifted down from 35 to 15 °C(0.8 °C/min) (Fig. 4). Thereafter, the amount ofmRNA gradually decreased with time, but remainedabove the control level for at least 5 h. Furthermore,during the cooling process to 15 °C, the increasedexpression of the desaturase mRNA became evidentat 27 °C. Nuclear run-on analysis and actinomycinD chase experiments revealed that the elevation ofthe mRNA level was due to increases in both tran-scription and mRNA stability. These results suggest

Table 4. Palmitoyl-CoA and stearoyl-CoA desaturase activitiesin T. pyriformis NT-1 microsomes from control and cyclo-heximide-treated cells after temperature shift-down

Growth

condition

nmol/min/mg protein

Palmitoyl-CoA

desaturase

Stearoyl-CoA

desaturase

Control Cycloheximide Control Cycloheximide

39 °C-cells 0.90 0.65 6.31 4.80

After shift

(39 °C! 15 °C)

0 h 2.29 0.74 12.25 4.65

1 h 3.40 0.39 15.50 6.20

2 h 3.90 0.24 17.84 4.10

4 h 2.07 0.15 4.90 2.65

6 h 0.54 0.08 2.70 1.30

10 h 0.32 0.05 1.50 0.70

24 h 0.77 — 5.85 —

15 °C-cells 0.59 5.00

Data from Umeki et al. (1982).28)

A) B)

Fig. 4. Changes in the level of dasaturase mRNA after (A) orduring (B) temperature shift-down (39 °C to 15 °C). T. thermo-phila grown at 35 °C were cooled to 15 °C over 25min. After (A)or during (B) the shift-down to 15 °C, total RNA was extractedat the indicated time intervals. Data from Nakashima et al.(1996).34)

Y. NOZAWA [Vol. 87,458

that the enhanced desaturase activity during the coldacclimation is controlled, at least in part, at thetranscriptional level. However, the molecular mech-anisms for signal transduction mediated by the coldstress leading to increased desaturase gene expressionremain to be disclosed.

On the other hand, much attention has beenpaid to the direct influence of the membrane lipidfluidity on desaturase activity in endoplasmic retic-ulum. Decreased membrane fluidity has been corre-lated, along with enhanced desaturase activity, withthe increased incorporation of unsaturated fattyacids into membrane phospholipids and the conse-quent adaptive increase in fluidity. Thompson andcoworkers35),36) suggested that a chilling-inducedcompositional modification of fatty acids in Tetra-hymena was due to the increased activity of pre-existing desaturase enzymes by the decreased fluidityof the microsomal membrane. There is experimentalevidence to indicate that the fluidity of the Tetrahy-mena microsomal membrane lipids is an importantfactor in regulating the activity of fatty aciddesaturases. Supplementation of polyunsaturatedfatty acids (C18:2, C18:3) leading to increasedfluidity offset the increased desaturase activity inthe control chilled cells.23),37) Furthermore, exposureto chimyl alcohol (1-O-hexadecylglycerol), the pre-cursor to alkyl-ether-containing phospholipids, in-duced an enhancement of unsaturated fatty acids anda large increase in fluidity in membrane phospho-lipids.38) Subsequent cooling of the cells induced littleor no change in desaturase activity. These resultsindicate that when the chimyl alcohol-treated cells,with more-fluid membranes, were chilled to a lowtemperature, enhancement of desaturase activity tothe extent observed in untreated control cells was notrequired. Accordingly it is reasonable to consider thatdecreasing fluidity would have a stimulatory effect onfatty acid desaturases activity, possibly by inducingconformational change of the desaturase enzyme.Moreover, when membrane fluidity was modifiedisothermally by several procedures, the microsomalfatty acid desaturase activity was altered in afluidity-dependent fashion. The fluidizing com-pounds, such as methoxy fatty acids39) and anes-thetics,40),41) reduced fatty acid desaturation in a waywhich should restore membrane fluidity back into theoptimal level. Decreased fluidity was almost invar-iably accompanied by increased desaturase activityand vice versa. It was thus suggested that fluidity-mediated regulation would be involved in the earlyadaptive response.

Deacylation and reacylation. As describedabove, the temperature shift-down exerted a pro-found modification of the membrane phospholipidmolecular species. For this adaptive process, thedeacylation–reacylation plays an important role,which can be assayed by changes in radioactivitywhose fatty acyl chain and glycerol-backbone arelabeled with different radioisotopes. When 39 °C-grown Tetrahymena cells prelabeled with both[32P] Pi and [14C] palmitic acid were shifted to 15 °Cand then linolenic and .-linolenic acids, which areknown to increase during cold acclimation, wereadded to the culture medium, the (14C/32P) ratios ofmajor phospholipids were progressively decreaseduntil 5 h after the temperature shift. This suggeststhat preexisting fatty acids of membrane phospho-lipids are deacylated and then, after the adequatemodification by desaturation and/or elongation, arereacylated into lysophospholipids.42) This concept issupported by the finding that the active replacementof the phospholipid fatty acyl chains occurs at the C-1 position; the principal saturated fatty acid, palmiticacid is replaced by polyunsaturated fatty acids,mainly .-linolenic acid.22) In this context, it is to benoted that phospholipase A1(PLA1) rather thanPLA2 was rich in T. pyriformis NT-1 cells.43) ThisPLA1 activity is Ca2D-dependent and cleaves fattyacyl chains from the C-1 position of phospholipids,giving rise to free fatty acids and 2-acylysophospho-lipids. Usually, the reacylation of lysophospholipidsoccurs at the 2-position and plays a crucial role indetermining molecular species of phospholipids inmammalian cells. However, in Tetrahymena cells,a marked alteration of fatty acid composition isobserved at the C-1 position of phospholipids duringtemperature acclimation. The reacylation process ismediated by acyltransferases. We have shown thatthere are reacylation activities not only for acyl-GPC (glycerol-3-phosphorylcholine) and 1-acyl-GPE(glycerol-3-phosphorylethanolamine), but also for 2-acyl-GPC and 2-acyl-GPE, using various acyl-CoAsas donors.44) The specificities for acyl-CoAs were verysimilar between 2-acyl-GPC acyltranserase and 2-acyl-GPE acyltransferase, which could transfer notonly saturated fatty acyl-CoAs but also unsaturatedones, despite enrichment of saturated fatty acids atthe C-1 position of PC and PE. Since it was observedthat the specificity of acyltransferases for variousacyl-CoAs was unchanged during temperature shift-down, the contribution of acyltranserase to thethermal adaptive modification of membrane phos-pholipid fatty acid composition, may be small.

Low-temperature acclimation of Tetrahymena membranesNo. 8] 459

In the light of these observations, we mayconclude that the fatty acyl-CoA desaturase plays apivotal role in the cold acclimation of membranephospholipid acyl chain composition; its enhancedactivity supplies sufficient amounts of unsaturatedfatty acids and subsequently they are utilized toproduce the newly adapted molecular species ofmembrane phospholipids.

Acknowledgments

I would like to express sincere thanks to allcolleagues, especially Prof. Guy A. Thompson (Uni-versity of Texas at Austin) who were involved in thisTetrahymena membrane adaptation project, and alsoto Prof. Tamio Yamakawa, M.J.A., for encouragingme to contribute this review article.

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positional distribution of phospholipid acyl chainsin plasma membranes, mitochondria and micro-somes. Biochim. Biophys. Acta 711, 229–244.

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(Received Apr. 20, 2011; accepted July 21, 2011)

Low-temperature acclimation of Tetrahymena membranesNo. 8] 461

Profile

Yoshinori Nozawa received MD (1962) and PhD (1966, Biochemistry) from GifuPrefectural Medical University. He then joined the Department of Biochemistry(Professor Yuki Ito) as a research associate and started his research on the biochemistryof the surface structures of the pathogenic fungal cells. He was appointed to Lecturer(1968) and an Associate Professor (1969). He spent two years (1969–l971) at theUniversity of Texas at Austin as a research associate where he worked with ProfessorGuy A. Thompson on the membrane biogenesis and the membrane adaptationmechanisms during thermal acclimation in the ciliate protozoan Tetrahymena cell.After return, he has extended these studies to investigate the membrane dynamicstructure (fluidity) by using electron spin resonance, wide-angle X-ray diffraction etc.,mainly in this suitable model Tetrahymena cell and also some mammalian cells. In 1978he was promoted to Professor and served as Dean of Gifu University School of Medicine (1996–1999). Afterretirement (1999) he became a Professor Emeritus of Gifu University, and moved to Gifu International Institute ofBiotechnology as Vice-President and then appointed to the President in 2002 (to present), where besides themembrane lipid signaling, he started the new research project studying the molecular mechanisms ofphytochemicals including polyphenols as the cell signaling modulator. In 2007, he was appointed to a Vice-Director of the Research & Development Foundation of Gifu prefecture. In 2009, he was appointed to a Professor,Department of Food and Health Sciences, Tokai Gakuin University. He has received the Hutner Award (AmericanSociety of Protozoology, 1982), the Doctor Honoris Causa from Semmelweis University (Hungary, 1990), theJapanese Society of Medical Mycology Award (1990) and Tokai Yomiuri Award in Medicine (1999).

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