Microscopic studies of fatty acid vesicles

Chem~try ~ d Phy~Czo[ Liptds 2~ (1977) 243-252 ©EIs~vier/Nort_h-HoUandScientific Publishers, Ltd.

MK OSCOPlC STUDIES OF FATTY ACID VESICLES M~ HlCKSand J.M. GEBICKI

~ lff~tO~B ~ # I

~Ct~ces, Mncquarie ~iveeffr),, North Ryde NSI¢ 2113, A tastralia

Recei~Ki Februa~ 14th, [977

accepted May 3rd, 1977

Microscopi¢~cs enelosedby membranes formed entirely of oteic or tinoleic acids (u famines) were studied by the f~eze-~tching and birefringence techniques. The results suggest the presence of one or more membtaMs a~ound the panicles, in which the fatty acid chains iie perpendicx,lar to the surface. Comparison with results obtained with phospholipid fipos~mes shows that both types of particle, are basically similar, although ufasomes have a tess regular structure.

I. Introduction Much of the information which led to the present view of the arrangement of biological membrane components was derived from electron microscopy of secho~,~ed biological spe¢im~..ns. It was usually recognised, however, that tile necessary" fixing alzd staining can produce distortion of these dehcate structures, with consequent loss of definition and formation of artifacts [ l ] . Such pi'oblems can sometimes be reduced by the use o f l e ~ harsh techniques, although the kind of information then obtained i~ likely to be somewhat different. One of the most successful methods applied to both natural and model membranes is the f~eeze.fracturc tech..Lique [ 2 - 7 ] . An even tess harsh method is the detection of birefdngence. Already in 1935 Schmidt [8] observed strong birefringence in membranes oi" the myelin aheath and suggested that it was caused by orientation of lipid mo|eculea with tbeit long axes perpendicular to the membrane surface. More recently, bitefrinE~ence was observed in suspensions of |iposomes, which are microscopic vesicles with phospholipid membranes [9, tot. We.have ¢ecently described the formation of a new type of vesicle, which was named ufasome, because it was enclosed by a membrane formed from unsaturated fatty acids [ t 1 ]. Ufasmtw~ proved to be osmotically active and capable of retaining trapped ~oltttes and it was concluded that the fatty acids were arranged with their long axes p e q 3 e a ~ t d a t l o the nq~mbfaae suff~tce [ 12]. As attempts to study ufasome structure by eleCUolt mifmseopy ~3f neptively stained specimens showed that they did not set~v~ ~he p ~ t o r y ~teps, we employ the gentler freeze.fracture and birefringence

methOdS This peper restarts our findings. 243

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M. Hicks and J.M. Gebicki, Micro#copic studies of[ally a¢id ~ericles

II. Materials and methods Oleic and linoleic acids were obtained from the Lipid Preparations Laboratory of the Hormel Institute, Minnesota. Their purity, stated to be over 99%, was Checked by gas chromatography after esterification with BF3-methanol reagent [13]. A PyeUnicam Series 104 instrument was used and a polyethylene glycol adipate'column. Both acids were at least 99.7% pure. Lecithin was obtained from Sigma Chemical Co., St. Louis, USA and purified on acid-treated florisil column [141. All other cherai~,'als were of analytical grade. Water was triply distilled in a stream of oxygen from acid dichromate and alkaline permanganate. The ufasomes were prepared from either oleic or linoleic acid as described pr,.'viously [ l 2]..Typically, 0.02 ml of a 10% solution of the acid in chloroform was dried in a test tube in a stream of high purity nitrogen. Buffer (0.2 ml ofO.l M Tris • HCI, pH 8) was then added and the tube shaken vigorously on a vortex mixer until all the fatty acid was taken up into a fine, opalescent suspension. Liposomes were prepared by dispersing a film of phosphotipid in water. All attempts to stain ufasomes with neutralized pc "tassium phosphotungstate for electron microscopy failed to produce specimens with any internal structure. For fracturing and etching, the ufasome suspensions were rapidly frozen on to copper helmets with freon and then stored in liquid nitrogen, Some suspensioffs were equilibrated with 17% glycerol for I0 rain before freezing [15, 16]. Fracturing was carried out in a Balzers (L~echtenstein) microtome at --110° C and at 2 . 1 0 ~ Torr pressure. For etching, the temperature was increased to -100°C for 1 rain. After cutting, a film of platinum and carbon was deposited on the fracture face to a thickness of 3 nm at an angle of 45°. The most successful of the several techniques used to clean the replicas was to float them off the metal helmet on to water, to which methanol was gradually added, until the solutk,n was 80% alcohol. It took 30 rain to retrieve all traces of fatty acid. The replicas were then examined in a Hitachi HS8 electron microscope. Observations. of birefringence were made on suspensions of ufason~s and liposoraes under phase contrast and between crossed points of an Olympus microscope. A first. order red compensator was used to enhance any birefringem:¢ pre~-nt [10], HI. Results and discussion

A. Electron microscopy Unetched, freeze-fractured preparations~showed particles which were sphcrival or ellipsoidal in shape, with diameters between 0.1 and 2/am. Such size heterogeneity has been noted in liposomes prepared by mechanical agitation [17]. Fig, I s h ~ onot-ehed ufasomes protruding slightly above the gener,'~L~uffac¢, The small irregular flragcmcnts are due to contamination. The much target parti~le~in flg~2 re~ah someoftts |/Rernal structure. This appears to'consist largely of .roughly ¢oncentric,'irregular layer, shnilar

kL Hicks and £M. Gebieki, Microscopic studies o f fatty acid vesicles

245

t.ig. !. Unetcited tYccze-t~aclmedprepztratkmo! olci¢ ,ttad utasomes suspended in tt~s buffer, pit 8. The bat in this and in all other electron micrographscorresponds to I urn.

to the phospholipid planes seen in liposomes J5,.18]. There was no difference m the appearance of ufasomes made from oteic or from linolelc acids. Since ufasome preparations conta/ned a large proportion of water, much of the fre.czefftactured face consisted of ice, which often had a very irregular surface. This led to uneven deposition of the carbon-metal film and formation of fragile replicas, which readily fragmented during cleaning. Early attempts to remove the residual fatty acids by a ehlotoform-~-methanol solution resulted in roiling up of the replicas. Satisfactory conditions for complete cleaning were established by comparing the appearance of the water.n~thanot washed replicas with fragments cleaned up by the more harsh chioroform-methanol or bleadh-wate,-acid treatment. Etching of the surface, especially if the ufasomes were pre-cquilibrated in glycerol, produced a marked difference in appearance between ~ce and the particle surface. This is c!¢arly shown in fig. 3, where cutting and etching broke off a thick layer from the surt~:e of the sphere. The exposed outer and inner fatty acM surfaces are smooth, while the surrounding ice is typically granular. The space between the membranes is also roush, mggesting that it was fiiled with water. Ftg. 4 shows the interior tqueous spaces d¢ady. |I¢~ the two larger particles have only a thin outer membrane region

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M. Hicks and J ~ . Gebicki, Microscopic studies o f fatty acid vesicles

Fig. 2. A large unetched finoleic acid ufasom¢ showing the e~anceatvI: layered membrane a r r a ~ t .

which encloses a large water-filled space containing sn~ller tl'apped ufasomes. Equilibration in glycerol ofte~ led to formation of very anifonn particles, with eve~dy spaced membranes (fig. 5). It is not possible, evon in such cases, to estimate accurately the intermembrane spacing, becau~ of the geometry of the particle surface. However, if we assume that in fig. 5 the 45 ° shadowing just n,issed the 2 or 3 outermost sm face layers on the lower left hand edge of the ufasome, th,~ calculated vertical distance between the layers is 20-25 nm~ or about tO times greater than the length of a fully extended 18 carbon fatty acid chain [19]. Thus, in this particle, the intermembrane st-ace may be made up of 80% aqueous and 20% fatty ~¢id phases. The parallel ridges in fig. 5 were caused by the microtome knife and the fl agment obscuring part of the ufasome was broken off the frozen sample. Fine surface features, which can help to understand how the ufasome fraclures~ were seen in numerous freeze-etched preparations. Sim:e the etchingprocess did not change the appearance of the surface~it was inferred that ufa~)mes have no etchable material at the fracture face. Similar observations made eadie, for Iiposmms were ~sed as evidence that in phospholipid membranes the fractut~ passes through the middle of the lipid bitayer rather than through the water-lipid interface [7] .We have postulated in an earlier paper [12] that ufasome membranes a~ stabilized by head group

M, iticks and J.M. Gebicki, Mtcro,reopic studies o f fatty acid ~,estclcs

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Fig. 3. An etched uftu|omewith a thick outer layer partly removed. Thisexample contrasts the relativelysmooth particle membranes(O and the roughice surface (iL

hydrogen bonding with water, complex formation between ionized and neutral acid molecules and by hydration of the dissociated carbo×yl groups. In addmon, the hydrocarbon regions of the fatty acids are held together by precisely the same dispersion and hydrophobic interactions which stabilize niicelles and the interior regions of membranes [20], It seems likely therefore that the arguments used to locate t.he ~i'~cture plane in the middle of the phospholipid membranes apply equally to those formed from long chain fatty acids alone. ,Figs. 6 and 7 show how an ufasome might fracture according to this reasoning. The appearance ofth¢ pmtiek surface along the line AB is interpreted in the schematic diagram, t-kre the fracture runs through ice and through the middle of the fatty acid bilayer (7(a)). On etehiag(7(b))some of the outer membrane surface on the edge of the particle i~extl~gd by the towering of the ice level. The thin ridge thus produced a~ound the pat'tide is unshadowed on the side exposed to the metal-carbon vapour but becomes coated on the oppo~te side if the.surface curvature is not too great. The bottom ~¢tkm (7(c)) r e p ~ t t t s the distribution of shadow over the face of such a particle. Thin ridges at junctiom of expend bilayers and monolayers were previously obset~d in plant cell vacuo!~ 13] and in l i p o ~ e preparations [41.

O0

M. Iticks and J.M. GebickJ, Microscopic studies of yat~ acid vestcles

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iqg. 6. The replica o! the ulasome on ti~e Ictt ~a~ plt, bably formed on the hydrophob~c interior ul the fatty acid membrane split by l'tacturing. The rim around t.he edge was produced by the lall of the level of surrounding ice during etching which exposed pal t of the outer membrane. All particles appearing convex have such a rim, whid~ is absent from couture impressions such as the one on the

right. 13. 8irefringence Fig. 8 shows some tiposomes under phase contrast (81 a)) and the same parttcles between crossed potars (8(b)). A relatively high propurtion of tlle ve~lcles are birefnngent, as reported earlier by severat authors [9, I0]. Although ufasomes also show birefringenee, this appears to be confined to the larger par~lcles (fig. 8(c) and (d)). The difference in the frequency o f birefdngent particles c~n be explained by the wide variability o f intermembrane distances commo,~dy observed in tt~e ufasomes (figs. 2 and 4). Bireftingel~ee of the type observed in multilameUar particles is made up of an "intttnsie"- component, usually positive in sign, which ~_~.se~from the perpendicular orientation of lipid molecules to the membrane surface, and a n,~gztive " f o r m " comFg~ 4. ~.l'wocxamptcs of ~tched ufasom©s with a thin oute~ membrane ~¢gionenclosing large aqueott~ ~pa~s and s ~ ¢ ~tmller trapped ufa.~mcs. t'ig. 5, Part of the interior of a ufs~m¢ equilibrated with 17~ ~dycero|before freeze-fracturing and etching. ~la~¢fatty acid n~embranelayers are evenly spaced with a ~paratlon of about 20 nm ( ~ text).

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M. Hicks and J.M. Gebieki, Microscopic studies o[1atty acid vesicles

fill "L _--_--._" ---

(b) of s h o d o w

,•le

rnl

{cj A

B

Fig. 7. Scheme for fracture of the ufasome shown in fig, 6 along the line AB. In (~), tile knife (k)

cutting through the ice (i) breaks the fatty acid bilayer producing a fracture face (ff) and exposing the membrane interior (mi). After etching fb), the ice levelfalls, exposing a rim of membrane exterior (me). A section of the particle surface in the direction of shadowing would appear as in (c). portent which Is due to the parallel arrangement of adjacent membranes [9]. As distance between neighbounng membranes increases, the intensity of birefringence decreases, owing to an increase in the negative component balandng out some of the intrinsic hire fringence [ I0]. Bangham et al. [9] found that uncimrged lecitin hposomes showed strong birefringence. Introduction of a net positive or negative charge caused separation of membranes with a corresponding decrease of observed birefringence. Our inspection of several dozen freeze-etched ufasome preparations showed clearly that the irregular multimembrane particles or large water-filled spheres are much more common than the symmetrical particles (fig. 5) which would be expected to give rise to stlong birefringence. The observations of the structure of ufasomes presented in this paper, together w~th the studies of their properties [ 12], leave little doubt that in many respects the fatty acid vesicles closely resemble the phospholipid liposomes. While we l,ave no dtrect proof of the manner in which the components of the ufasome membranes are arranged. any orientation of the fatty acids other than perpendicular to the interface seems unlikely, Further work with these particles will show whether their chemical sm~pti¢ity offers advantages not possessed by the much studied hposomes,

Acknowledgements We are greatful to Dr. DJ, Goodchild and his coiteagues at the C.S,I.R.O. Division of Plant Industry for the preparation of freeze-fractured samples and to Mrs. J.M, Gregory for help with the electron microscopy. This work wa,~ supported by a Macquarie University Research Grant.

M. fhcks and J.M Gehtckt, 3hcrose~¥~ic studtes ol iatty acid re~h'le;

251

|:ig, 8. A stt~i~n,~ioa ~,f hpo~ne$~a} and a uJa~ome #b)under phase etmtta,~ and the ~ame particles between ~,rosscd polars ~c) and td). The bat cu~responds to t0 ~tm.

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M. Hicks and J.M. Gebgckt,M~'roseopi:~studies of yatty acid w~s!cJes

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