The Lateral Diffusion of Probes in the Surface Membrane of Schistosoma mansoni

Michael Foley,* Andrew N. MacGregor,* John R. Kusel,* Peter B. Garland,* Thomas Downie,§ and Iain Moore§ * Department of Biochemistry, University of Dundee, Dundee DD1 4HN, Scotland, U.K.; ~Department of Biochemistry, University of Glasgow, Glasgow, G12 8QQ, Scotland, U.K.; and §Department of Pathology, Western Infirmary, University of Glasgow, G12 8QQ, Scotland, U.K. Dr. Garland's present address is Unilever Research, Colworth Laboratory, Colworth House, Sharnbrook, Bedford, MK44 1LQ, England.

Abstract. The technique of fluorescence recovery after membrane blebs allowed us to measure the lateral photobleaching was used to measure the lateral diffusion of in the membrane without the diffusion of fluorescent lipid analogues in the surface influence of underlying cytoskeletal structures. The re- membrane of Schistosoma mansoni. Our data reveal stricted diffusion found on the normal surface mem- that although some lipids could diffuse freely others brane of mature parasites was found to be released in exhibited restricted lateral diffusion. Quenching of membrane blebs. Quenching of fluorescent lipids on lipid fluorescence by a non-permeant quencher, trypan blebs indicated that all probes were present almost en- blue, showed that there was an asymmetric distribution tirely in the external monolayer. Juvenile worms ex- of lipids across the double bilayer of mature parasites. hibited lower lateral diffusion coefficients than mature Those lipids that diffused freely were found to reside parasites: in addition, the lipids partitioned into the mainly in the external monolayer of the outer mem- external monolayer. The results are discussed in terms brane whereas lipids with restricted lateral diffusion of membrane organization, cytoskeletal contacts, and were located mainly in one or more of the monolayers biological significance. beneath the external monolayer. Formation of surface

CmSrOSOMA MANSONI is one of the parasites responsi- A double outer membrane is an invariable and unique fea- ble for the widespread chronic tropical disease bilhar- ture of intravascular trematodes (27). The double outer mem- zia. The life cycle of this species of Schistosoma in- brane is also present in schistosomula, having developed by volves excretion of eggs liberated by adult worms in the about 3 h after skin penetration by cercariae (19). Since primary (human) , infection of aquatic snails (the sec- young schistosomula are susceptible to immune attack (39), ondary host), emergence from the snails of infective water- the double outer membrane is not of itself sufficient to confer borne cercariae, which penetrate human skin and then de- protection, although it may well be necessary. Other possible velop into schistosomula, and finally adult worms. Infection mechanisms for evasion of the immune response include the is chronic, and the adults can reside for many years in the masking of surface antigens (39) and continuous sloughing hepato-portal system of the host. The host-parasite combina- of the outer membrane into the blood stream (32, 33). A fur- tion exhibits the phenomenon of concomitant immunity, the ther possibility, indicated by our earlier work (22) and ex- essence of which is that chronically infecting adult worms plored more fully in this present study, is that the compo- elicit but resist an immune response that is nevertheless nents of the outer membrane of adult schistosomes have capable of partially protecting against subsequent infection restricted lateral diffusion in the plane of the membrane. (41, 42). Such restriction of lateral diffusion could inhibit immune at- The molecular basis for concomitant immunity is not tack in several ways (see Discussion). known, but it is generally thought that the evasion of immune The study reported here describes the measurement of the damage is related to some property or properties of the lateral diffusion coefficients of a variety of fluorescent lipid schistosome surface. The outermost layer of schisto- probes inserted into the outer membrane of Schistosoma somes is a 2-4-ttm thick known as the tegument. mansoni, using the fluorescence photobleaching recovery The innermost membrane of the tegument is a conventional (FPR) 1 method (2, 23). We also explored the location of bilayer (the basal membrane), whereas the outer membrane various probes in the bilayers of the outer membrane using on the exterior of the syncytium consists of two tightly ap- a non-permeant fluorescence quencher, trypan blue. We have posed bilayers that make a tetralayer (17, 18, 20). We shall refer to the two bilayers of the outer membrane as the inner 1. Abbreviations used in this paper: DE, lateral diffusion coefficient; FPR, and outer bilayers, each of which is composed of inner and fluorescence photobleaching recovery. Abbreviations for lipid probes are in outer monolayers. Materials and Methods.

© The Rockefeller University Press, 0021-9525/86/09/807/12 $1.00 The Journal of Cell Biology, Volume 103, September 1986 807-818 807 studied the outer membrane not only in intact worms but also probe, it was added to 1.0 trd of Eagle's medium as 10 I.tg of FI-PE in 5 pl after it has been lifted clear of underlying structures by for- of CHC13. The solution was then ultrasonicated for 5-10 s before adding the parasites, and the procedure then followed that used with the other mation of blebs (24). There is strong evidence, reviewed by fluorescent lipid probes. For microscopic examination, the labeled parasites Webb (48), that cytoskeletal interactions with membrane can were placed on a microscope slide in 80 ~tl of Eagle's medium containing impose severe constraints on the lateral diffusion of mem- 50 ~tg of the paralyzing agent carbamycholine chloride (Sigma Chemical brane . It has been demonstrated that the formation Co., St. Louis, MO) to prevent worm movement. The solution on the slide of blebs or vesicles in certain eucaryotic systems such as was confined by a ring or square of silicone grease, and finally sealed by addition of a coverslip. and lymphocytes releases these constraints thus enabling the lateral diffusion coefficient (DL) and the Vesiculation Procedure mobile fraction to approximate to the high values observed in simple model systems such as liposomes (44). We have ob- Membrane vesicles or blebs were prepared by applying the hypertonie sa- served a similar release of restricted lateral diffusion of lipid line procedure of Kusel et al. (24) to schistosomula and adults. Briefly, the procedure involves incubation of the parasite for 1 h at 4°C and then an addi- probes in surface blebs of S. mansoni. A possible mecha- tional 1 h at 25°C in 1.19 M NaCI in 25 mM Tris-HC1 buffer, pH 7.4. nism for this is discussed. Fluorescence Microscopy and Fluorescence Materials and Methods Photobleaching Recovery Fluorescence photomicrographs were taken with a Leitz Ortholux II fluorescence microscope, a standard camera attachment, and Kodacolor I! Fluorescent Lipid Probes 200 ASA daylight film. Measurements of the two-dimensional lateral The following lipid probes were used: 1,1'-di(octadecyl)-3,3,3',3' tetrameth- diffusion of fluorescent lipid probes incorporated into the membranes of ylindocarbocyanine perchlorate (Cis-diI); 1,1'-di (tatradecyl)-3,3,3',3' tetra- parasites were made by the FPR method (2, 23) using the laser-microscope methylindocarbocyanine perchlorate (Cl4-diI); 5-N(octadecanoyl)-amino- combination described by Garland and colleagues (16, 21, 30). In the latter fluorescein (C1s-F1); 5-N (hexadecanoyl)-aminofluorescein (C~-FI); 5-N part of the study we have satisfactorily used a low powered air-cooled argon (tetradeeanoyl)-aminofluoreseein (C~-FI); octadecylrhodamine B chloride ion laser (Lexel model 65; 100 mW all lines maximum power) instead of (Cis-Rh); N-fluorescein N'-phosphatidyletbanolamine thiourea (FI-PE). the more usual water-cooled laser of higher power (e.g., Lexel model 75; All except F1-PE were purchased from Molecular Probe, Inc., Junction 1 W all lines maximum power). A 40X water-immersion objective lens was City, OR. 1, l'-Di(eicosanyl)-3,3,Y,Y tetramethylindoearbocyanine perchlo- used throughout. The 1/e2 radius of the focused laser spot at the sample rate (C20-diI) and 1,1'-di(docosanyl)-3,3,3',3' tetramethylindocarbocyanine was 1-1.2 ~tm, as calculated from measurements of the FPR by two- perchlorate (C2~-diI) were a kind gift from Dr. Michael Edidin. F1-PE was dimensional diffusion of fluoresceinated bovine serum albumin in thin films synthesized using a modification of the method of Fung and Stryer (15). Flu- of glycerol of known viscosity (46). The characteristic times for recovery orescein isothiocyanate (60 mg) was added to a solution of 1,2-dipalmitoyl- of fluorescence after photobleaching were calculated either by the simple sn-glycero-3-phosphatidylethanolaminein 0.75 ml of CHCb and 0.05 ml of 3-point method of Axelrod et al. (2), or by a nonlinear least squares fitting triethylamine. The mixture was stirred gently for 2 h at room temperature, procedure as described by Bevington (3) to fit equation 12 of Axelrod et al. and washed three times with 20 ml of water to remove any unreacted fluores- (2), which is the exact solution for the fluorescence recovery function in the cein isothiocyanate or water-soluble products. The course of the reaction case of a laser spot with a Gaussian transverse energy mode. When the latter was followed by analyzing small samples by thin layer chromatography procedure was used experimental data were collected with a Hewlett- using silica gel plates developed with CHCI3/ MeOH/ I-I20 (65:25:4, Packard Model 85 microcomputer connected with the fluorescence measur- vol/vol/vol). The washed reaction mixture was applied to several 20 × 20 ing electronics (which for FRP measurements were analogue, not photon cm silica gel thin layer chromatography plates (gel thickness = 0.25 mm) counting) by a Microlink interface (Biodata Ltd., Manchester, England). and developed at room temperature with acetone/water (85:15, vol/vol). Vi- The data were stored on disk and subsequently transferred to a Dec 10 main- sualization was by fluorescence under an ultraviolet strip-lamp. In the frame for running the curve-fitting program in Fortran. preparative system the Rf values (i.e., distance migrated by the visualized spot relative to the solvent front) for fluorescein and F1-PE were 1.0 and Fluorescence Quenching 0.45, respectively. The FI-PE band was scraped off the plates, eluted with CHC13-MeOH (2:1, vol/vol), taken to dryness under N2 in a rotary evapo- The depth of insertion of lipid probes in the membrane was determined by rator, redissolved in 0.5 mi CHC13 and taken through the preparative thin measuring the ability of a non-permeant molecule (trypan blue 0.25% layer chromatography procedure three more times. The product was ana- wt/vol) to quench probe fluorescence by Forster resonance energy transfer. lyzed for fluorescein by absorption spectrophotometry and for phospholipid The principles of energy transfer theory have been reviewed by Stryer (43), phosphorus by the method of Raheja et al. (29). The ratio of fluorescein and of depth dependent fluorescent quenching in micelles and membranes to phospholipid phosphorus in the FI-PE was 0.9:1.0. by Blatt and Sawyer (4). We calculated the Ro value (i.e., the distance at which the energy transfer efficiency is 50%) from the absorption spectrum of trypan blue and the emission spectra of the lipid probes. This value was Preparation of Schistosomula and Adult Schistosomes found to be between 3.8 and 4.2 nm for all the lipid probes used. The The life cycle of a Puerto Rican strain of S. mansoni was maintained by pas- fluorescence intensity in these energy transfer experiments was measured sage through laboratory mice strains and Biomphalaria glabrata snails. with a photon-connting variation of the FPR apparatus (30). We also used Adult worms were recovered by porfusion of the hepatic portal system of an additional guard filter (3 mm of Schott BG 18 glass) to cut out the dull infected mice (40), and kept in Eagle's medium (25) containing 10% (wt/vol) far red fluorescence emitted by trypan blue. We obtained independent ex- fetal calf serum or newborn calf serum at 37°C. Experiments were cart.~ed perimental evidence of the calculated Ro value in the case of fluorescein out within 24 h of isolating the worms. Schistosomula were obtained after from a Stern-Volmer plot (4) of the fluorescence quenching of fluorescein- transformation of cereariae either mechanically by passage through a needle conjugated dextran (average molecular weight = 10,500) at varying concen- (9) or by penetration through mouse skin (8). Lung-stage schistosomula trations of trypan blue. The value obtained was 5.0 + L0 nm, and is in rea- were obtained by the procedure of Clegg (7) from the lungs of mice 5-8 d sonable agreement with the theoretical value. Inner filter effects due to the after infection. absorption by trypan blue of either the excitation (514 rim) or emission (>550 nm) wavelengths were removed by measuring front face fluorescence Incorporation of Fluorescent Lipid Probes in 100-I.tm (path length) microcuvettes (Camlab, Cambridge). The various forms of parasite were labeled with fluorescent lipid analogues Electron Microscopy by incubating them for 10 rain at 37°C in 1.0 ml of Eagle's medium to which 5 Isl of ethanol containing 10 I.tg of the probe (other than FI-PE) had been Parasites were prepared for transmission electron microscopy by the method added. The parasites were then washed four times in Eagle's medium lack- of Hockley and McLaren (19) except that schistosomula were fixed in ing calf serum, and either mounted directly for microscopic examination glutaraldehyde for 3 h and adult worms for 4 h. Secondary fixation was for or first subjected to the vesicniation procedure below. When FI-PE was the 2 h at 40°C in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer, pH

The Journal of Cell Biology, Volume 103, 1986 808 7.4. Thin sections were examined using a Phillips EM 300 electron micro- was continuously monitored from the small area (radius 1.2 scope. lxm) illuminated by the focused (but greatly attenuated) laser beam at the surface under study, in this case the outer mem- brane of the tegument. After establishing a steady reading of Results the fluorescence signal, the average laser beam power was increased by 104 for ~0.1 s before returning to the previous General Form of the Experimental Data power. The high intensity pulse irreversibly bleached almost To illustrate the form of FPR measurements, we show in Fig. a half of the fluorescent molecules in the membrane at the 1 the data obtained from a single experimental measurement focused laser spot. Fluorescence subsequently recovered due of DE for C~rF1 labeling the surface of an adult male to unbleached molecules in the membrane beyond the laser schistosome. Also shown is the computer-generated least spot diffusing laterally into the spot. DE waS calculated from squares best fit for a single value of DE, along with the the kinetics of fluorescence recovery (2). In an idealized residuals for that fit and the autocorrelation function of the bilayer of the fluid mosaic model (37), DL would be ex- residuals. As is usual with such measurements, fluorescence pected to be '~10 -8 era: s -1 and recovery of fluorescence

1.2

4J .p4 o~ E -IJ B E .r-g .6 e-

(J .4 f_ O b_ .2

0 I I I_ I I I I I I 0 50 100 150 200 250 300 350 400 Channel number (125 ~s per channel)

t~ .2 (b} .1

. A.I • 13 • ~.'-'v~ ,.,-.V-~v-'.'lrpV~T . T ,,.v,p-v-- v",ww v, ,( -'"~lrvyv , .t-I 0 ¢/I ~E I I 1 I I I I I I I Figure 1. FPR curve for C~s-FI in the outer 0 50 100 150 200 250 300 350 400 membrane of the body of an adult schisto- Channel number(125 ~s per channel) some. In a, the observed fluorescence in- tensity has been plotted after normalization c to a pre-bleach intensity of 1.0 arbitrary o units. Channel O on the time axis corre- "'-' 05 sponds to the end of the bleaching pulse. The channel width was 0.125 s. The smooth ~4 I (c) .. ,, ,,,...... ~,. A . curve is the nonlinear least squares best fit to the data for a single diffusing species. In r 0 0 b, the residuals (i.e., the difference between U the observed value and fitted value) have O I I I I I .,.., -.05 been plotted. A random scatter of the 0 50 100 150 200 residuals and their (c) autoeorrelation func- tion around zero indicates a satisfactory fit Autocorrelation channel of the calculated curve to the observations.

Foley et al. Lipid Probes in the Surface Membrane of S. mansoni 809 would ultimately go to completion (6). In the experiment worm (22). To examine this point further, we measured DE shown, DE was 1.7 X 10-9 cm 2 s -1, which is relatively slow for the probe ClrdiI at the head, ventral sucker, neck, and for a lipid probe. main body of the adult male worm. Fig. 2 shows an adult The final extent (R) to which fluorescence recovery pro- male and female pair with these anatomical regions labeled ceeds to complete recovery is given, in percentage terms, as on the male worm. Table I summarizes the results. In all four R = 100 x (Foo-Fo) / (Fi -/7o); where Foo is the fluores- regions studied DLwas similar, in the range 3.2-5.8 X 10-9 cence signal at infinite time after photobleaching, Fo is the cm2 s -1. The percentage recovery was only 20 % in the body fluorescence signal immediately after photobleaching but region, and highest at the ventral sucker. The gynaecophoric before recovery, and Fi is the fluorescence signal before canal could not be studied because the surrounding worm photobleaching. In practice F~ is either taken as the body was not optically transparent. Nor could adult female fluorescence level beyond which no further recovery appears worms be studied because, even when free or protruding to occur or, better, by computer fitting of the data to a theoret- from the gynaecophoral canal, their high level of autofluo- ical curve. In Fig. 1 the computed value for the percentage rescence interfered with detection of fluorescent probes. recovery was 85%, implying that 15 % of the fluorescent The lateral diffusion of a range of other lipid probes was molecules were unable to diffuse laterally (the so-called im- measured on the body surface of adult male schistosomes. mobile fraction), at least on the time scale of the experiment The results are summarized in Table II. There were marked and over the distance of 1 lma or so set by the radius of the differences between different types of probes. The alkyl- laser spot. substituted indocarbocyanines, CmdiI and C~4-diI, ex- hibited low percentage recoveries (25-34%), as did F1-PE. All the other probes had high percentage recoveries (>75 %) Studies with Adult Schistosomes as shown in Fig. 1 for C18-F1. We were able to obtain ade- Our earlier studies suggested that the surface properties of quate surface labeling with the longer chain length indocar- S. mansoni adults may vary with anatomical position on the bocyanines, C20-diI and C22-diI. Nevertheless, it is clear

Figure 2. A pair of adult male (or) and female (9) Schistosoma mansoni as viewed by scanning electron microscopy. H, head; V, ventral sucker; G, gynaecopic canal; D, dorsal surface of body; O, oral sucker. Bar, 250 Ixm.

The Journal of Cell Biology,Volume 103, 1986 810 Table L Lateral Diffusion* of Cls-diI in the Outer significantly alter the DL value of lipid probes: it remained Membrane of Adult Schistosomes at ~2-6 x 10-9 cm2 s -~, an order of magnitude lower than

Region DL(X 10-9 cm2 s -l) % Recovery the values usually obtained for eucaryotic cell membranes. However, the division of the lipid probes into a group with Ventral sucker 4.7 4- 2.0 (22) 62 4- 12 low recovery and a group of high recovery found in the nor- Head 5.8 4- 1.8 (24) 42 4- 10 mal outer membrane (Table 11) was abolished in the outer Neck 4.9 4- 2.3 (21) 40 + 10 membrane of blebs: all exhibited a high recovery of >80%. Body 3.2 + 2.0 (23) 20 4- 9 Table III summarizes such results with C~s-diI and Cts-F1 as * The values of DL and % recovery are given as mean + standard deviation, representative of the low and high recovery groups, respec- with the number of measurements in parentheses. Approximately 4--6 measure- tively. ments were made on each region for any one worm. Structure of Outer Membrane Blebs from the data of Table II that the length of the alkyl chains Studied by Light Microscopy in the probes did not determine whether a probe fell into the The formation of blebs and vesicles at the surface of schisto- group with low (FI-PE, Cr,-diI, Cis-diI) or high (C~4-FI, somes in response to hypertonic saline treatment (24) can C~6-FI, C~8-Rh) recovery. Statistical analysis was done by readily be followed by phase-contrast microscopy or, if the the Student's t test and differences described in the text are outer membrane has previously been stained with a fluores- significant (p <0.001). DL values for the mobile fractions of cent lipid probe, by fluorescence microscopy. Fig. 3 shows probes were in the range of 3.1 to 4.8 x 10-9 cm2 s -I, with micrographs of views by phase-contrast (Fig. 3, A and B) and the exceptions of C~s-Rh and F1-PE which had DL values fluorescence microscopy (Fig. 3 C) of schistosomes labeled some 10-fold lower. All of the results presented so far refer with Cts-diI. Initially blebs or blisters form (Fig. 3 A) and to worms at 35-37°C. No effects of lower temperature down progress with time to attached (Fig. 3, B and C) and then free to 6°C were found, other than a two- to threefold diminution vesicles. The ring-like distribution of fluorescence in the of the DL value. Percentage recoveries were unaffected at vesicles shows that it is the surface of the vesicle that is la- the lower temperatures. The DE values are rather low for all beled, not the interior. The highly patterned fluorescent probes and C~s-diI and F1-PE have unusually low recoveries staining characteristic of the normal adult surface was lost when compared to lipid analogues in bilayers (6, 14). in the .vesicles and blebs. These observations are similar to Fluorescence microscopy of the worms labeled with the those previously observed with schistosomula (24) and also different probes in all cases revealed the same pattern of body to those obtained using Cls-F1 in place of C~8-diI (data not surface labeling, similar to that described (22) for fluores- shown). cent wheat germ agglutinin or concanavalin A which are presumably non-permeant. It therefore seems very likely Structure of Outer Membrane Blebs that the lipid probes have indeed labeled the surface mem- Studied by Electron Microscopy brane and have not labeled other membranes. Neither long Electron micrographs of thin sections through blebs formed chain alkyl fluoresceins nor carbocyanines fluoresce when on the surface of adult schistomes showed outer membrane they form micelles (36, 38). It therefore follows that the carrying, attached to its inner aspect, many membranous fluorescence which we observe does not arise from micelles structures and small vesicles (Fig. 4 D). The highly indented of the probe adsorbed to the membrane surfaces. and pitted surface of the normal adult became smoothed out The Effect of Bleb Formation on Lipid Probe Diffusion and flattened in the vesicles, in keeping with the loss of sur- face pattern seen by fluorescence microscopy. It appears that After incubating parasites with the lipid probes, membrane the vesicles were formed by detachment of the tegument, car- blebs were formed from parasites by hypertonic saline treat- tying with them the basal membrane (Fig. 4 C). Thus the ment. Peripheral fluorescent staining was observed in blebs vesicles and blebs are bounded by a complex layer, and not with all lipid probes used (listed in Table II) as was the case just a bilayer as in the blebs described by Tank et al. on differ- with several fluorescently labeled lectins (namely, rhoda- ent organisms and cells (44). mine concanavalin A, rhodamine wheat germ agglutinin, We also studied by electron microscopy vesicles formed by and fluorescein peanut agglutin). Bleb formation did not hypertonic saline treatment on the surface of the schistoso-

Table IL Lateral Diffusion* of Various Fluorescent Lipids Table III. Effect of Bleb Formation on the Lateral in the Outer Membrane of the Body of Adult Schistosomes Diffusion* of CmF1 and Cls-dil in the Outer Membrane Lipid DL(X 10-9 cm2 s -l) % Recovery of the Body of Adult Schistosomes

F1-PE 0.42 + 0.09 (11) 29 + 10 State of Lipid membrane DE (X 10-9 cmz s -1) % Recovery C14-diI 3.1 + 2.4 (16) 25 + 15 Cls-diI 4.0 4- 1.0 (68) 34 4- 12 Cls-diI Normal 3.2 + 2.0 (23) 20 + 9 CI4-F1 3.5 4- 2.0 (24) 76 + 8 Cls-diI Bleb 3.9 + 0.8 (21) 83 + 11 CI6-FI 4.8 4- 1.6 (30) 75 4- 10 CI~-FI Normal 3.6 + 1.9 (32) 79 + 19 Cls-Fl 3.6 4- 1.9 (32) 79 4- 19 CtrFl Bleb 2.3 + 0.5 (19) 84 + 8 Cis-Rh 0.40 4- 0.07 (10) 79 4- 15 * The values of DL and % recovery are given as mean + standard deviation, * The values of/)t and % recovery are given as mean -I- standard deviation, with the numbers of measurements in parentheses. Approximately 4-6 mea- with the number of measurements in parentheses. Approximately 3--4 measure- surements were made on any one worm, and were restricted to the body region ments were made on any one worm. or blebs attached to the body region.

Foley et al. Lipid Probes in the Surface Membrane of S. mansoni 811 Figure 3. Light microscopy of blebs induced by hypcrtonic saline treatment of adult schistosomes. A and B are phase-contrast micrographs and C is a fluorescence micrograph. The worms had been stained with Cls-diI before hypertonic saline treatment. Bars, 20 ttm.

The Journal of Cell Biology, Volume 103, 1986 812 Figure 4. Electron micrographs of the outer region of adult schistosomes before and after bleb formation induced by hypertonic saline. (A) The normal tegument is bounded by a basal membrane (bm) internally and a surface membrane (sm) externally, m, muscle; p, pit. (B-D) Progressive stages in bleb formation, sv, small vesicles. Bars, (A, C, and D) 0.5 I~m; (B) 2 ~tm.

Foley et al. Lipid Probes in the Surface Membrane of S. mansoni 813 The Journal of Cell Biology, Volume 103, 1986 814 Table IV. Trypan Blue Quenching* of Fluorescent Lipids in Normal and Vesiculated Outer Membranes of Adult Male Schistosomes c

0.8. % Quenching** of fluorescence o Lipid normal worm Blebs ~3 I q- C~s-F1 78 + 27 (40) 95 + 19 (15) Cis-diI 15 + 18 (44) 83 + 28 (8) © C18-Rh 75 + 26 (30) 92 5:21 (8) l F1-PE 40 5:22 (30) 86 5:23 (10) 0J * The values given are mean 5: standard deviation, with the number of mea- 0J surements in parentheses. For the normal worm, 7-10 parasites were examined E o and for blebs one measurement was made on each bleb. Fluorescence was (+- measured by photon-counting from an area '~10 gm in diameter, defined by T.B. suitable focusing of the laser beam. The unquenched fluorescence gave photon 0J o count rates of 20-30 KHz, some 10-fold higher than background due to autofluorescence. The count rates were corrected for autofluorescence, back- ground, dark current, and any residual counts which depended upon worms and was presumably due to chemiluminescence. ** The percentage quenching of fluorescence is equal to 100 × [F(_) - F(+)]/ 14 F(_~, where F(_) and F(.) are fluorescence intensities in the absence and presence, respectively, of trypan blue. In an experiment, 6, 8, or 10 worms Figure 6. Summary of the possible locations of a fluorescent lipid were labeled with a fluorescent lipid. Half were measured for fluorescence im- (black head groups) indicating their relative distance from the ex- mediately, giving F¢-). The other half were added to the trypan blue solution ternal surface of the double surface membrane of S. mansoni. OB, and fluorescence intensity was measured, giving F~+~. outer bilayer; IB, inner bilayer. mula. They differed from those seen in the adult; separation Quenching data are summarized in Table IV. In the normal of the vesicle appeared to leave the basal membrane of the parasite C18-F1 and C~s-Rh are both severely quenched by tegument still attached to the body of the worm, and conse- trypan blue, which would indicate that both lipids are located quently the outer layer of the vesicle was derived from the mainly in the outer monolayer of the outer bilayer. C~8-diI, double outer membrane of the tegument with less membrane however, is deduced to reside in one or more of the remaining structure attached to the inner surface (Fig. 5, A and B). It three monolayers, since its fluorescence was only slightly appeared that the double outer bilayer structure remained in- quenched. On average, just less than half of the F1-PE tact in the blebs. (Fig. 5 C). molecules reside in the outer monolayer of the outer bilayer as judged by this method. Vertical Distribution of Fluorescent The formation of membrane blebs alters the vertical distri- Lipids in the Outer Membrane bution of the lipids in the adult schistosome membrane, since A lipid analogue incorporated into the outer membrane the data show that all fluorescent lipids in blebs are exten- (which, as described above, has two bilayers) could be situ- sively quenched by trypan blue, from which we conclude ated in any one or more of the four constituent monolayers that the fluorescent lipids appear to be located in the outer (i.e., the outer and inner halves of the outer bilayer, and the monolayer of the outer bilayer. These data also indicate that outer and inner halves of the inner bilayer). To explore these the fluorescent lipid is not gaining access to the membranous possible locations we used trypan blue as a non-permeant structures and small vesicles observed within adult blebs. quencher of fluorescein (or rhodamine and di-indocarbocya- (Fig. 4). nine) fluorescence on the external (outer) aspect of the mem- brane. The absorption spectrum of trypan blue overlaps the emission spectra of the fluorescent lipids used, and the Studies with Schistosomula and Lung Forms mechanism of quenching is most probably by Forster reso- Lateral diffusion of two fluorescent lipids, Cls-diI and C~s- nance energy transfer. We calculated that a trypan blue mole- FI was examined on the double outer membrane of schistoso- cule would have a 50 % probability of accepting energy if it mula and lung worms. The results are described in Table V. were 3.8-4.2 nm from an excited fluorescent donor. Because The data illustrate that the D~ values of both lipids are '~10- the fluorophores are hydrophilic, they will be located at the fold lower than for the adult membrane. It is also noted that head group regions of the bilayers. The possible locations are unlike adults, both schistosomula and lung worms show a re- shown schematically in Fig. 6. The relationship between stricted diffusion of C~s-FI. Both skin and mechanically efficiency of energy transfer and increasing separation of do- transformed schistosomula gave very similar FPR results. nor and acceptor is an inverse sixth power law. Accordingly, Quenching experiments reveal that the majority of the only those fluorescent lipids with headgroups on the external fluorescent lipid molecules for both probes are incorporated surface of the outer membrane could be quenched by a non- into the outer monolayer of the outer bilayer (data not permeant energy acceptor such as trypan blue: fluorophores shown). Thus it appears that in both schistosomula and lung located in any of the other headgroup regions are too far re- worms as distinct from adults there is a different vertical dis- moved from trypan blue to be significantly quenched. tribution of fluorescent lipids in the surface membrane.

Figure 5. Electron micrographs of progressive stages in bleb formation in the outer region of schistosomula, induced by hypertonic saline. sm, surface membrane; bm, basal membrane; m, muscle; sp, spine. Bars, (A and B) 1 tma; (C) 0.1 Inn.

Foley et al. Lipid Probes in the Surface Membrane of S. mansoni 815 Table V. Lateral Diffusion of Two Fluorescent Lipids in the Differing lipid distribution across membranes has been de- Outer Membrane of Schistosomula and Lung Worms scribed in other biological systems (34, 35), including the *Developmental observation that phosphatidylethanolamine is largely within Lipid stage DE (X 10-9 em 2 s -I) % Recovery the inner leaflet of cell membranes. Our results are in general agreement with Dragsten et al. (11, 12) who, in a study using Cts-diI Schistosomula 0.30 + 0.10 (17) 30 + 13 C~8-diI Lungworm 0.48 + 0.15 (17) 47 + 10 fluorescent lipids on epithelial cells, concluded that 1, l'-di- C~8-FI Schistosomula 0.27 + 0.06 (17) 55 + 11 (hexadecyl)-3, 3, Y, 3'-tetramethylindocarbocyanine perchlorate C~8-F1 Lung worm 0.33 + 0.13 (10) 61 + 16 (C16-diI) can flip-flop and locate itself in the inner mono- layer of those cells whereas C~6-F1 cannot. * Schistosomula transformed by the mechanical process were examined be- Although the presence of one or two hydrophobic chains tween 6 and 24 h after transformation from cercaria when the double outer membrane should be present. Lung worms were used between 2 and 24 h after on the lipid probes appears to correlate with high or low per- perfusion from the lungs of infected mice, cent recovery, respectively, and might lead one to propose The values given are mean + standard deviation, with the number of mea- surements in parentheses. One measurement was made on each worm. that the single-chained lipids do not undergo transmembrane flip-flop whereas the double-chained lipids do, we think this is unlikely unless the chain-length range is restricted to C~4 Discussion upwards. This is because 5-N-(dodecanoyl)-aminofluores- cein (CL~-F1)could label not only the outer surfaces but also FPR and Energy Transfer Studies the body interior when applied externally, showing that it of the Schistosome Membrane could cross many membranes (data not shown), whereas none of the other probes labeled beyond the outer surface. Using the technique of FPR it has been possible to measure The production of membrane blebs from adult worms has the lateral diffusion of various fluorescent lipids inserted into allowed the study of fluorescent lipid diffusion on the outer the outer membrane of S. mansoni. We have assumed membrane detached from the rest of the worm body. This throughout this work, as have many others in this field, that technique of bleb formation has been used in other studies the lipid probes intercalate into the surface lipid bilayers. We to correlate enhanced lateral diffusion in the mem- can exclude from our observations surface aggregates or brane bleb with the disruption of an underlying cytoskeletal micelles of the probe because such assemblies do not network (44). Although cytoskeletal constraints have not flouresce (36, 38). Furthermore, surface adsorption of been invoked to explain the much rarer phenomenon of re- probes by mechanisms other than bilayer intercalation (e.g., stricted lipid diffusion in plasma membranes, we did observe electrostatic interactions with phospholipid head groups) do an analogous release of restrictions of lipid diffusion on adult not necessarily lead to low DE or percent recovery values. blebs. This increase in the fraction of molecules that can An additional point is that the schistosomula surface is diffuse laterally does not seem to occur simply as a result of known to take up host glycolipids, and this has been modeled the reduction of pits and folds of the adult membrane since using transfer of carbocyanin dyes from erythrocyte ghosts Wolf et al. found only slight differences in the diffusion of (5). As revealed in Fig. 2, the surface of this parasite has a a lipid probe due to the effect of surface microvilli (49). In rather complex topography and the membranes cannot be re- addition studies have inferred a physical connection between garded as simply planar bilayers. Although an unusual effect lipids in plasma membrane and the cytoskeleton (31, 47). on lipid lateral diffusion due to surface convolutions cannot The differing distribution of fluorescent lipids found in the be ruled out, several studies both experimental and theoreti- normal adult outer membrane was altered by bleb formation cal, demonstrated only small effects of non-planar topogra- as observed by fluorescence quenching experiments, in phy on lateral diffusion of lipid probes (1, 13, 49). It is proba- which all four fluorescent lipids used were extensively bly the case that the apparent DE values obtained in our quenched when in membrane blebs. The differences, de- study will be reduced by only two- to threefold; however, the scribed in this study, between the lateral diffusion of fluores- observed differences between lipid analogues will still be cent lipids on juvenile stages of the parasite and mature adult valid and the fractional recoveries unaffected. worms, indicate that as the parasite develops a major change One unusual finding is that three fluorescent lipids, Ctr in membrane structure or organization occurs. This has been diI, C~4-diI, and F1-PE give very low mobile fractions suggested previously by a number of workers from indepen- whereas the others did not. In most other biological systems, dent methods (25, 26). Fluorescence quenching studies on lipid probes such as C~8-di/behave as in pure lipid bilayers schistosomula and lung worms indicates that most of the giving a mobile fraction of almost 100% and a high DE molecules of all the fluorescent lipids examined are in the value of t~10-8-10-9 cm 2 s -~. Thus there is restricted lipid outer monolayer of the outer bilayer. Since restricted lipid diffusion in the outer membrane of adult S. mansoni. Lipid diffusion can be observed in these stages it would suggest that domains as detected by restricted diffusion of fluorescent there are gel phase and lipid crystalline phases in this exter- lipids have been observed by other workers (10, 50). Quench- nal monolayer. Hence there is horizontal lipid asymmetry in ing of incorporated lipid fluorescence was used to determine this monolayer in the juvenile stages of the parasite as op- the vertical distribution of several fluorescent lipids parti- posed to vertical asymmetry in the adult. tioned into the membrane. Our results revealed that the majority of molecules of those lipids which were not re- stricted in their lateral diffusion (i.e., C~rF1 and C~8-Rh) Membrane Organization were located in the outer monolayer of the outer bilayer. Extensive tetralayers consisting of two bilayers tightly ap- However, the lipids that exhibited restricted lateral diffusion posed face-to-face over a large area are rare in nature, and (i.e., C~rdiI and F1-PE) were found to reside mainly in one have probably not been studied previously with the methods or more of the remaining monolayers of the adult membrane. used by us. Consequently, we have initially interpreted our

The Journal of Cell Biology, Volume 103, 1986 816 results in terms of the more familiar properties of bilayer Parasitol. 16:133-147. 8. Clegg, J. A., and S. R. Smithers. 1972. The effects of immune rhesus membranes. Our interpretation of these results for the adult monkey serum on schistosomula of Schistosoma mansoni during collection in surface indicates that the outer monolayer of the outer bilayer vitro. Int. J. Parasitol. 2:79-98. allows the greatest freedom for a lipid to diffuse laterally 9. Colley, D. G., and S. K. Wikel. 1974. Schistosoma mansoni: simplified method for the production of Schistosomules. Exp. Parasitol. 35:44-51. (i.e., it behaves as a typical biological membrane with re- 10. Dictus, W. J. A. G., E. J. J. van Zoelen, P. A. T. Tetteroo, L. G. J. spect to lipid lateral diffusion). One or more of the remaining Tertoolen, S. W. de Latt, and J. G. Bluemink. 1984. Lateral mobility of plasma three monolayers however restricts lipid diffusion drasti- membrane lipids in Xenopus eggs: regional differences related to /vegetal polarity become extreme upon fertilization. Dev. Biol. 101:201-211. cally, and in this is atypical of most biological mem- 11. Dragsten, P. R., R. Blumenthal, and J. S. Handler. 1981. Membrane branes. Because the observed restricted lipid diffusion is di- asymmetry in epithelia: is the tight junction a barrier to diffusion in the plasma membrane? Nature (Lond. ). 294:718-722. minished on membrane blebs, it is tempting to speculate that 12. Dragsten, P. R., J. S. Handler, and R. Blumenthal. 1982. Fluorescent it is the parasite cytoskeleton which maintains this lipid re- membrane probes and the mechanism of maintenance of cellular asymmetry in striction in the normal adult. Studies involving the lateral epithelia. Fed. Proc. 41:48-53. 13. Dragsten, P., P. Kenkart, R. Blumenthal, L Weinstein, and J. Schles- diffusion of membrane proteins on adult worms and blebs singer. 1979. Lateral diffusion of surface immunoglobulin, Thy-I antigen, and may clarify this issue. a lipid probe in lymphocyte plasma membranes. Proc..Natl. Acad. Sci. USA. 76:5163-5167. 14. Fahey, P. F., and W. W. Webb. 1978. Lateral diffusion in phospholipid Biological Significance bilayer membranes and multilamellar liquid crystals. Biochemistry. 17:3046- It is possible that the observed immobility of lipid molecules 3053. 15. Fung, B. K-K., and L. Stryer. 1978. Surface Density measurements in in the outer membrane of adult schistosomes may play an im- membranes by fluorescence energy transfer. Biochemistry. 17:5241-5248. portant role in allowing the parasite to resist immune attack. 16. Garland, P. B. 1981. Fluorescence photobleaching recovery: control of laser intensities with an acousto-optic modulator. Biophys. J. 33:481--482. For example, there is a considerable body of evidence which 17. Hockley, D. J. 1970. Ultrastructure of the outer membrane of Schisto- suggests that in order for complement to be fixed and the soma mansoni. J. Parasitol. 56:(4, Pt.2):151. lesion-forming membrane attack complex to integrate into 18. Hockley, D. J., and D. J. McLaren. 1971. The outer membrane of Schistosoma mansoni. Trans. R. Soc. Trop. Med. Hyg. 65:432. the membranes rapid lateral diffusion is required in the 19. Hocidey, D. J., and D. J. McLaren. 1973. Schistosoma mansoni: membrane (28, 45). Thus restricted lipid diffusion may be changes in the outer membrane of the tegument during development from cer- the mechanism used by schistosomes to survive host immune cariae to adult worm. Int. J. Parasitol. 3:13-25. 20. Hocldey, D. J., D. J. McLaren, B. J. Ward, and M. V. Nermnt. 1975. attack, although the relatively "fluid" external monolayer A freeze-fracture study of the segmental membrane of Schistosoma mansoni would presumably provide a suitable environment for com- (platyhelminthes: ). Tissue & Cell. 7:485-496. 21. Johnson, P. J., and P. B. Garland. 1982. Fluorescent probes for measur- plement fixation. ing the rotational diffusion of membrane proteins. Biochem. J. 203:313-321. Restricted lipid diffusion may have a role to play in allow- 22. Johnson, P., P. B. Garland, P. Campbell, andJ. R. Kusel. 1982. Changes ing the schistosomula and the lung worms to evade the im- in the properties of the surface membrane of Schistosoma maasoni during growth as measured by fluorescence recovery after photobleaching. FEBS (Fed. mune response; if this is so then the restriction on lipids in Fur. Biochem. Soc.) Lett. 141:132-136. the young stages differs from that in adults. The outer bilayer 23. Koppel, D. E., D. Axelrod, J. Schlessinger, E. L. Elson, and W. W. behaves as if it has both gel and liquid crystalline phase do- Webb. 1976. Dynamics of fluorescence marker concentration as a probe of mo- bility. Biophys. J. 16:1315-1329. mains (data not shown). This may allow less immune dam- 24. Kusel, J. R., G. Gazzinelli, D. G. Colley, C. P. S. de Souza, and M. N. age to the parasite than an external monolayer which was Cordeiro. 1984. The formation of surface membrane vesicles from schistoso- mula of Schistosoma mansoni. Parasitology. 89:483-494. solely in the liquid crystalline phase by, for example, prevent- 25. Kusel, J. R., L. Stones, and W. Harnett. 1981. The use of membrane- ing lateral diffusion of antigens in the juvenile parasite sur- active compounds to examine the structure of the surface membrane of Schisto- face. If this idea of lipid immobility being involved in evasion soma mansoni. Bioscience Report. 1:253-261. 26. McLaren, D. J. 1980. Schistosome mansoni: the parasite surface in rela- of the immune response is accurate, then it may follow that tion to host immunity. Research Studies Press. John Wiley & Sons, Ltd. parasite membrane blebs, with no observed lipid immobility, Chichester. should be susceptible to host immune damage. We are cur- 27. McLaren, D. J., and D. J. Hockley. 1977. Blood flukes have a double outer membrane. Nature (Lond.). 269:147-149. rently investigating this possibility. 28. Parce, J. W., N. Henry, and H. M. McConnell. 1978. Specific antibody- dependent binding of complement Clq to hapten-sensitized lipid vesicles. Proc. The authors thank John Birmingham, Department of Biochemistry, Univer- Natl. Acad. Sci. USA. 75:1515-1518. sity of Dundee, for his expertise in computer analysis of these results. 29. Raheja, R. K., C. Kaur, A. Singh, and I. S. Bhartia. 1973. New colori- This work was supported by a grant from the Wellcome Trust and the metric method for the quantitative estimation of phospholipids with acid diges- Medical Research Council. tion. J. Lipid. Res. 14:695-697. 30. Rees, A. R., M. Gregoriou, P. Johnson, and P. B. Garland. 1984. 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Hoffman. 1974. to schistosomula of Schistasoma mansoni lyse and fail to transfer membrane Studies on the mechanism by which Cyanine Dyes measure membrane potential components to the parasite. J. Cell Biol. 101:158-166. in red blood cells and phosphatidylcholine vesicles. Biochemistry. 13:3315- 6. Cherry, R. J. 1978. Rotational and lateral diffusion of membrane pro- 3330. teins. Biochim. Biophys. Acta. 559:289-327. 37. Singer, S. J., and G. L. Nicholson. 1972. The fluid mosaic model of the 7. Clegg, J. A. 1965. In vitro cultivation of Schistosoma mansoni. Exp. structure of cell membranes. Science (Wash. DC). 175:720-731.

Foley et ai. Lipid Probes in the Surface Membrane of S. mansoni 817 38. Sldar, L. A., M. C. Doody, A. M. Gotto, Jr., and H. H. Pownall. 1980. 45. Taylor, P. W. 1983. Bactericidal and bacteriolytic activity of serum Serum lipoprotein structure: resonance energy transfer localization of fluores- against Grin negative bacteria. Microbiol. Rev. 47:46-83. cent lipid probes. Biochemistry. 19:1294-1301. 46. Thompson, N. L., T. P. Burghardt, and D. Axelrod. 1980. Reduced 39. Smithers, S. R., D. J. McLaren, and F. J. Ramalho-Pinto. 1977. Immu- lateral mobility of a fluorescent lipid probe in cholesterol-depleted erythrocyte nity to schistosomes: the target. Am. J. Trop. Med. Hyg. 26:11-17. membranes. Biochim. Biophys. Acta. 597:155-165. 40. Smithers, S. R., and R. J. Terry. 1965. Infection of laboratory hosts with 47. Utsumi, K., E. Okimasu, Y. M. Morimoto, Y. Hishihara, and M. Miya- cercariae of Schistosoma mansoni and the recovery of adult worms. Parasitol- hara. 1982. Selective interaction of cytoskeletal proteins with liposomes. FEBS ogy. 55:695-700. (Fed. Fur. Biochem. Soc.) Lett. 141:176-180. 41. Smithers, S. R., and R. J. Terry. 1967. Resistance to experimental infec- 48. Webb, W. W. 1981. Molecular mobility on the cell surface. Biochem. tion with Schistosoma mansoni in rhesus monkeys induced by the transfer of Soc. Symp. 46:191-205. adult worms. Trans. R. Soc. Trop. Med. Hyg. 61:517-533. 49. Wolf, D. E., A. H. Handyside, and M. Edidin. 1982. Effect of microvilli 42. Smithers, S. R., and R. J. Terry. 1969. The immunology of Schistosomi- on lateral diffusion measurements made by the fluorescence photobleaching asis. Adv. Parasitol. 7:41-93. recovery method. Biophys. J. 38:295-297. 43. Stryer, L. 1978. Fluorescence energy transfer as a sprectoscopic ruler. 50. Wolf, D. E., W. Kinsey, W. Lennerz, and M. Edidin. 1981. Changes Annu. Rev. Biochem. 47:819-846. in the organization of the sea urchin egg plasma membrane upon fertilization: 44. Tank, D. W., E-S. Wu, and W. W. Webb. 1982. Enhanced molecular indications from the lateral diffusion rates of lipid-soluble fluorescent dyes. diffusability in muscle membrane blebs: release of lateral constraints. J. Cell Dev. Biol. 81:133-138. Biol. 92:207-212.

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