Translational diffusion in the plasma membrane of single cells as studied by fluorescence microphotolysis

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MINI-REVIEW TRANSLATIONAL DIFFUSION IN THE PLASMA MEMBRANEOF SINGLE CELLS AS STUDIED BY FLUORESCENCEMICROPHOTOLYSIS

Reiner Peters Max-Planck-Institut fur Biophysik Heinrich-Hoffmann-Strasse 7 6 Frankfurt/Main 71 Federal Republic of Germany 1. Introduction The imagination has been fired by the idea that biological membranes might be Z-dimensional fluid;. Provided, for instance, that membrane viscosity was not much larger than that of the cytoplasm, the reduction of dimensionality would shorten diffusion times and thus enhance reaction rates in membranes over those in the cytoplasm (Adam and Delbriick, 1968). Translational diffusion in membranes could be crucial for the propagation of information in the plane of the membrane, a case which might apply for fertilization and differentiation, and across the membrane as in hormonal regulation (Cuatrecasas, 1974). Furthermore, diffusion in membranes potentially could channel energy, metabolites and electrons through the cell body possibly involving carrier proteins specialized in lateral membrane transport (Skulachev, 1980). Experimentally, translational mobility of proteins in biological membranes from the first has been inferred from cell fusion (Frye and Edidin, 1970), patching and capping phenomena (Taylor et al, 1971) and electron microscopic observations (e.g. Pinto da Silva,

Synonyms for fluorescence microphotolysis (FM) are: fluorescence recovery after photobleaching (Jacobson et al, 1976b) and fluorescence photobleaching recovery (Axelrod et al, 1976a). b

Abbreviations: FM, fluorescence microphotolysis; CFM, continuous fluorescence microphotolysis; IA, irradiated area; diI-Cle(3); 3,3'-dioctadecyl-indo-tri-carbocyanine; diO-C18(3), 3,3'-dioctadecyl-oxa-tricarbocyanine; NBD-PE, N-4-nitro-benz-Z-oxa1,3-diazole phosphatidylethanolamine; DMPC, dimyristoyl-phosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; EPC, egg phosphatidylcholine. 0309-I 651/81/080733-28/$02.00/O

8 1981 Academic

Press Inc. (London)

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1972). The first quantitative data have been provided independently and simultaneously by Peters et al (1974a,b) and by Poo and Cone (1974). Whereas Poo and Cone (1974) exploited particular properties of photo-receptors for measuring diffusion of rhodopsin, Peters et a technique of rather general applicability al (1974b) devised Microphotolysis (FMayb) -and used it to study Fluorescence In retrospect it is amazing that these erythrocyte membranes. initial studies dealt with extremely different membranes. Photo-receptor discs have remained the only biological membranes in which protein diffusion approaches the high rates expected for the 'fluid and Nicolson, 1972), whereas erythrocyte memmosaic model' (Singer branes are characterized by a strong restriction of translational movement. In the meantime, a large body of experimental data on translational in biological membranes has been accumulated by FM meadiffusion The review of these data shows one general aspect: surements. In order to biological membranes are both fluids and solids. appreciate this concept critically, it would appear useful to consider at first the methodological premises (section 2) and then to summarize and evaluate the data (section 3). For preceding of FM measurements, see Elson et al (1976), Webb (1977), summaries Shinitzky and Henkart (1979) and Cherry (1979). 2. Methodological Aspects 2.1 Fluorescence microphotolysis (FM) The FM measurement oroceeds in 3 consecutive steps: 1) A small area (some pm') of a flubrescently labelled cell is irradiated with visible light at a low intensity. Using advanced instrumentation, it is possible to pick up the fluorescence signal originating from the irradiated area (IAb) only a small number of even if fluorophores (e.g. 20,000) is present. IA-fluorescence serves as a relative measure for the number of fluorophores. 2) The light elevated for a short time by a factor of lo4 - lo5 intensity is which induces a rapid irreversible photolysis of fluorochromes and thus partially depletes the IA of fluorescence. 3) Light intensity reduced to the initial low value in order to follow the time is without further photolysis. If course of IA-fluorescence fluorescently-labelled compounds are able to enter the IA by diffusion or by other mechanisms, it will be indicated by a restitution of the IA-fluorescence. The kinetics of fluorescence restitution can be evaluated in terms of transport coefficients. It may be noted that the above scheme is based on some idealizing assumptions: during steps 1 and 3 both light intensity and dose are kept small enough to render photolysis negligible; in step 2 chromophore decomposition is irreversible as well as instantaneous (short as compared to diffusion times).*The range of D-values so far measured by FM is approximately lo2 - 1o-4 pm*/s. The accuracy of FM measurements has been estimated to be, under favourable conditions, about t 20% (Wu et al, 1977, 1978; Wolf et al, 1977).

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The evaluation of FM measurements in terms of transport coefficients in general involves computer curve fitting procedures. Frequently, however, simple methods can be applied. In calculations of Axelrod et al (1976a), diffusion and linear, constant-velocity flow in an infinite plane have been considered. The IA is circular with either a Gaussian or a uniform intensity profile. Photolysis is assumed to be a first-order reaction. Diffusion and flow yield fluorescence recovery curves of a markedly different shape. If the transport type has been established, the transport coefficients can the half-time tI,2 of the fractional be derived by determining fluorescence restitution f and inserting it into eq. (1) or (2), respectively: for

diffusion

D = (wz/4t&D

(1)

for

flow

’ = (w/t,,,hF

(2)

(D: diffusion coefficient; V: velocity; w: IA-radius, t ' for which f = (F(t) - F(O))/(F(m) - F(0)) = 0.5; F(:{;:F;;;e F(m): IA-fluorescence before, immediately and long after photolysii For a uniform intensity profile yD respectively; yD, yF: constants. for a Gaussian profile yD, yF depend on the = 0.88, yF = 0.81; photolysis degree of photolysis; e.g. y ranges from 1.0 for little to 1.7 for 90% photolysis. Pf a fraction of the labelled molecules is immobile, fluorescence restitution will be incomplete; the mobile fraction is given by: R = (F(m)

- F(O))/(F(-)

- F(0))

(3)

For the evaluation of FM measurements on small cells such as erythrocytes diffusion on the surface of a sphere has been considered (Huang, 1973; Peters et al, 1974b; Koppel, 1980). The effect of an association reaction between mobile and immobile components has been analyzed by Elson et al (1976) and Elson and Reidler (1979). Diffusion in membranes consisting of alternating fluid and rigid stripes has been studied by Owicki and McConnell (1980). Current FM apparatus consist of a fluorescence microscope, a continuous-wave laser (frequently an argon or crypton ion laser), a sensitive light-measuring device and some optical components. Specific instrumental descriptions have been given by: Peters et al, (1974b): 'prototype'; Edidin et al (1976): first laser application; Koppel et al (1976) and Jacobson et al (1976a): giving more details; Smith, B A and McConnell (1978): ruling-like IA; Barisas and Leuther (1979): non-microscopic system for measurement of diffusion in free solution; Koppel (1979), and Peters and Richter (1981): double-beam systems: Peters et al (1981a): continuous fluorescence microphotolysis, (see below). We now

consider

some modifications

of the

basic

FM procedure.

If

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fluorescence is monitored not only in the IA but also in its surthe IA-radius can be directly determined and different roundings, transport types can be easily recognized. An approach in which the beam is scanned along a line through the specimen has measuring been taken by Koppel (1979); applications: Schindler et al, 1980 Sheetz et al, 1980. The IA need not be circular. A slit-like d,b; geometry (Peters et al, 1974b) is advantageous in many instances. An elegant extension of the slit-like geometry is the use of rul1978) and grids (Smith, R A et al, ings (Smith, B A and McConnell, 1979) which, in combination with a photographic recording, is parsuccessful in measurements on large artificial membranes ticularly FM is not res(Rubenstein et al, 1979; Smith, L M et al, 1979a,b). tricted to measuring lateral transport in membranes. Barisas and Leuther (1979) have described a non-microscopic FM-apparatus for in free solution. Employing polarized-light measuring diffusion effects allows, in principle at least, the extension of FM to sublight microscopic spheres (Smith, L M et al, 1981a). 2.2 Continuous Fluorescence Microphotolysis (CFMbi Microscopic membrane studies involve small numbers of molecules. The density of membrane proteins varies from a few molecules per ytn* for rare species such as hormone receptors to about 30,000 molecules per pm2 for crystalline arrays. In FM, photolysis has to It can be easily shown that, when be negligible during measurement. combined, small fluorophore numbers and negligible decomposition measuring signals and thus constitute a lead to extremely small limiting factor for sensitivity and data quality (examples of FM measuring curves: Schlessinger et al, 1976a; Axelrod et al, 1976b; Dragsten et al, 1979; Golan and Veatch, 1980). Such limitations can be overcome if photolysis is permissible during measurement. The experimental procedure of CFM (Peters et al, 1981a,b) area (e.g. of a fluorescently tremely simple: a microscopic belled cell) is irrddiated continuously while fluorescence is The number and distribution of fluorescent particles tored. IA then is the result of two competing processes: depletion reversible photolysis and replenishment by diffusion and/or mechanisms.

is exlamoniin the by irother

If the integral IA-fluorescence is monitored (Peters et al, 1981a), the measuring curve initially displays a steep decay (early time and then, without ever reaching an equilibrium, turns into regime) a slower decay (late time regime). Whereas the early time regime is only determined by the relaxation times of photolysis and diffusthe late time regime, in addition, is determined by the size ion, dnd geometry of the area available for diffusion. By fitting theoretical to experimental decay curves, it is possible to derive values for the reaction rate constants, the diffusion coefficients, and the diffusion area. Optimum conditions for the determination of diffusion coefficients are given if the relaxation times of photolysis and diffusion are about equal. Both parameters can be influenced experimentally: the relaxation time of photolysis by adjust-

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ing the irradiation intensity, the diffusion time IA-radius. Thus, the ra ge of -values accessible 9 1 to that of FM (about 10 - lopmz/s).

1981

737

by adjusting the by CFM is similar

If experimentally possible, it is of great advantage to monitor the fluorescence distribution within the IA (Peters et al, 1981b). Fluorescence decay, in the centre of the IA, initially will only be determined by the photolysis reaction, thereby producing useful information on its kinetics. Furthermore, different transport mechanisms (isotropic and anisotropic diffusion, flow) yield characteristic patterns of fluorescence distribution in the IA. 2.3 Fluorescent labelling Several lipid-like fluorochromes can be integrated into the bilayer membranes: cyanine dyes with long hydrocarbog phase of biological such as 3,3'-dioctadecyl-indo-tri-carbocyanine (diIC18(3) ) chains 3,3'-dioctadecyl-oxa-tri-carbocyanine and (diO-C18(3)b) (Sondermann, 1971; Sims et al, 1974; Schlessinger, 1977a; Fahey and Wu et al, 1977); fluorescently labelled phospholipids Webb, 1978; such as N-4-nitro-benz-Z-oxa-1,3-diazole phosphatidylethanolamine (NBD-PEb) (Wu et al 1977), and gangliosides substituted with a fluorophore (SchlessiAger et al, 1977b). Membrane proteins may be labelled directly by incubating living cells with reactive dyes such as fluorescein-isothiocyanate (Peters et al, 1974a,b; Edidin et al, 1976; Fowler and Branton, 1977; Schlessinger et al, 1977a; Peters and Richter, 1981), eosine-isothiocyanate (Cherry et al, dichlorotriacinylarnino-fluorescein (Sheetz et al, 1980) and 1976, rhodamine 1981). Direct iodoacctar,lido tetramethyl (Wey et al, labellinq can be highly specific (Cherry et al, 1976; Fowler and labelling methods have Branton, 1977; Wey et al, 1981). Indirect fluorescent derivatives of: native involved the use of concanavalin A and its succinylated (bivalent) (tetravalent) as well as other lectins (e.g. Jacobson et al, 1976a,b; derivative Layyansky and Edidin, 1976; Schlessinger et al, 1976a; Leuther et 1976; Cltlrid~e et al, 1380); inmunoglobulin E (Schlessinger et al , ;11, 1976b), native (divalent) antibodies or their monovalent Fab Schlessirqer et al, 1977c; Johnson and Edidin, ;;Trents (e.g. ( ; Smith, L I'; ct. al, 1979a); cl-bungarotoxin (e.g. Axelrod et al, !'376b); ttorriones such as insulin and epithelial growth factor (e.g. S!iec titer (Levi et al, 1980) and et al. 1378). nerve growth factor tri-iodo-thyronine (riaxfielc! et--al, 1981). . 2.4 }~J.diati:)n-iiI~iuccti
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1979). Membrane proteins showed a differential reactivity, i.e. spectrin was faster cross-linked than the band 3 protein. Cross linking could be completely prevented by glutathione (Z-20 I-IN) and cross-linking depended on the radicystamine (50 mM). Furthermore, ation intensity and dose as well as on the concentration of the fluorescent label (for equal doses at higher intensityless crosslinking). The photolytic decomposition of the fluorescent label also depended on the radiation intensity being more efficient at lower intensities. Thus, at about 10 W/cmz, a dose of about 300 to decompose half of the fluorophores Joules/cm2 was sufficient (Sheetz and Koppel, 1979; Lepock et al, 1978), whereas at 30,000 as typically applied in FM, a dose of about 100,000 w/ Cl112 et al, 1976a). This example Joul es/cm2 was necessary (Schlessinger reveals large differences between model studies and the actual FM A direct investigation of photochemical artifacts in FM situation. measurement (i.e. removal and analysis of the radiation-exposed of the plasma membrane from a single cell) is not feasible. piece However, a number of indirect methods has been worked out: FM measurements have been repeatedly performed at the same membrane area or at a membrane area previously subjected to a prolonged photolysis (Schlessinger et al, 1976a; Jacobson et al, 1978); cells have been labelled with antibodies bearing two different fluorochromes, then the mobility of one of the fluorochromes has been measured whereas the other fluorochrome had or had not been photolzyed beby scannfore hand (Wolf et al, 1980a); the IA has been inspected ing electron microscopy (Jacobson et al, 1978); the impermeability of living cells to trypan blue has been checked after FM (Jacobson et al, 1978); the effect of FM on fertilization and early development of sea urchin eggs has been checked (Peters and Richter, In none of these indirect control experiments has evidence 1981). been disclosed for radiation artifacts. In a few cases it has even been possible to measure diffusion in the same cells by different methods. In the plasma membrane of myoblasts, the diffusion coefficient of diIC18(3) has been determined by FM to 0.9 + 0.4 um2/s and by fluorescence correlation spectrocopy to 0.8 7 0.3 pm2/s (Schlessinger et al, 1977a). In photoreceptor mem6ranes the diffusion coefficient of rhodopsin has been measured by FM as well as by absorption micro-photolysis, as 0.3 - 0.5 um2/s (Poo and Cone, 1974; Wey et al, 1981). Concerning thermal artifacts again, direct measurements have not been possible so far. Calculations by Axelrod (1977) suggest that under favourable conditions, is much the local temperature rise, smaller than 1’C. In artificial lipid membranes, thermal artifacts miyht have shown up as a shift of the phase transition temperature but were not detected (e.g. Wu et al, 1977). In conclusion, it should be recognized that at present no evidence points to radiation artifacts in FM measurements on living cells. However, since it has been demonstrated that irradiation can induce linking, at least in bulk solutions of isolated membranes, cross reservations and concern still remain. In order to remove ambiguit-

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ies completely, we are currently which undergo simple photoreactions Application of such chromophores which lower fluorescence quantum than in FM.

trying to instead might be yields can

1981

739

employ chromophores of redox processes. restricted to CFM in be tolerated better

3. Results FM measurements so far have had two major objectives: to obtain information about membrane organization, and to elucidate the functional significance of diffusion processes in membranes. We illustrate these objectives by selecting typical examples: the erythrocyte membrane for structural studies (section 3.2), and membranelocalized hormone receptors for functional studies (section 3.3). The basis for both topics is provided by measurements on artificial membranes (section 3.1). The bulk of the experimental material is condensed in Tables I and II (section 3.4). Finally, common features of F?l measurements are summarized in order to arrive at some yeneral conclusions (section 3.5). 3.1 Artificial lipoprotein membranes Oriented multi-bilayers, large multior mono-bilayer vesicles, planar lipid membranes of the MUller-Rudin and the Montal-Mtiller type have been studied. Planar membranes showed exceptionally high D-values and no typical phase transition behaviour, which probably is indicative for a residual solvent content (Fahey and Webb, All other model systems showed similar characteristics; no 1978). differences between mono- and multi-bilayer systems have yet been disclosed. Probes such as diO-C18(3), diI-C18(3) and NBD-PE yielded similar results and thus seem to reflect lipid self-diffusion. In membranes made from dimyristoylphosphatidylcholine (DMPCb), dipalmitoylphosphatidylcholine (DPPCb) or egg phosphatidylcholine temperature (Wu (.EPCb) D s 5 pm*/s at T > T , the phase transition Tt D drops to 1977; At T s et al, 1978). Fah$y and iebb, _s;ill temperature approximately lo-’ pm*/s. Below Tt D is dependent, pm2/s at 22.5'C e.g.Jn DMPC (Tt = 23.5'C) D = 2 x 10 and 0.15 x 10 pm*/s at 9.6'C (Smith, B A and McConnell, 1978). Addition of cholesterol generally reduces D for T > Tt and D for T
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Insertion of typical values (protein radius: 25 fl, viscosity: 1 10 Poise, bilayer thickness: 35 Ii) into the Saffman-Delbriick equation yields D = 0.6 - 6 pmz/s (Cherry, 1979). Experimentally, only the diffusion of relatively small peptides and proteins has been studied so far: gramicidin S (Wu et al, 1978), gramicidin C (Tank et al, 1981), M-13 phage coat protein (Smith, L M et al, apolipoprotein ApoCIII (Vaz et al, 1980) and glycophorin 1979b), Wu et al, 1981). As an example for a typical (Vaz et al, 1981; integral membrane protein we are currently studying bacteriorhodopsin (Peters and Cherry, unpublished results). Let us consider glycophorin (MW 31,000; 60% carbohydrate). It has a transmembrane orientation in the human erythrocyte membrane and can be incorporated into artificial membranes in the same way. Large parts of glycophorin reside outside the lipoprotein bilayer whereas only a small hydrophobic section (2,500 daltons) penetrates the bilayer. In glycophorin-DMPC membranes (protein/lipid i l/1,000 molar) D Q, 2 pm*/s at 24-C for glycophorin; for a lipid probe D = 7 pm*/s (Vaz et al, 1981; Wu et al, 1981). At low temperature D < low2 um*/s for glycophorin and for lipid probes. It appears that the transition from hiyh to low mobility occurs at different temperatures for glycophorin (15'C) and the lipids (23.5.C). High D-values have also been reported for the other lipoprotein membranes mentioned above and thus apparently agree well with the Saffman-Delbruck equation. A membrane made from Escherichia coli phospholipids, lipopolysac cha ides and matrix protein showed interesting characteristics: D< -r; pm*/s for the matrix protein, D s 0.2 pm*/s for a lipid probe 10 independent of matrix protein and concentration, D for the lipopolysaccharide decreased from 0.1 to 0.01 pm*/s for O-60% matrix protein (Schindler et al, 1980b). On the basis of these data, Schindler et al, (1980b) have proposed a polymer model. However, an association of matrix protein and lipopolysaccharide might be sufficient to account for the results (Jahnig, 1981). Diffusion of dextrans (NW 82,000) adsorbed onto planar lipid membranes depends on density: D = 2.1 - 4.5 um*/s for 100 molecules and D = 0.31 - 0.68 for 10,000 molecules/urn* (Wolf et al, /w* 1377). Cross linking of dextran by antibody induced a reduction of t) and, under certain conditions, a 'patching'. D for a lipid probe was not influenced by addition of dextran and its cross-linking. 3.2 Isolated erythrocyte membranes. The erythrocyte membrane is composed of two layers: a 'skeleton' constructed of spectrin and various additional proteins (actin, bands 2.1, 2.2, 2.3, 3a, 4.1, 4.9, 7) and a 'skin' consisting of an asymmetric lipid bilayer into which proteins (e.g. the band 3 protein, ylycophorin) are inserted; a special protein, ankyrin, and possibly other components provide for the coupling of skeleton and skin (review, Lux, 1979). Molecular movement in erythrocyte membranes and its restriction by spectrin were first inferred from

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741

freeze fracture electron microscopic studies. Intra membrane particles probably representing band 3-glycophorin complexes were found to aggregate at conditions of pH, ionic strength and temperature at which precipitation could also be induced in spectrin extracts (Pinto da Silva, 1972; Elgsaeter and Branton, 1974; Elgsaeter et al, 1976). Accordingly, FM measurements on normal human erythrocyte iffusion of integral proteins membranes showed that translational 9 pm*/s at 20-C; Peters et al, is severely restricted (D 5 3 x lo1974b), whereas in erythrocyte variants deficient in spectrin, D is about 50-fold larger than normal (Sheetz et al, 1980). Rotational diffusion of the band 3 protein (Cherry et al, 1976; Nigg and 1980) has two components suggesting that a large fraction Cherry, of band 3 ( B 60%) can freely rotate in the membrane whereas the remaining fraction is tightly linked to the skeleton. It therefore appears that the skeleton restricts translational movement in the lipoprotein skin by both direct linkage and trapping. Applying fusion methods similar to those of Frye and Edidin (1970), Fowler and Branton (1977) observed that the !nbility of integral membrane proteins depends on temperature (D/10 pm*/s: 0.2-0.4 at 37-C, 0.1-0.3 at 30°C, 0.02-0.06 at 23'C, too small to be measured at 0°C) and on the intracellular ATP level. On applying a quantitative version of the fusion technique, Schindler et al (1980a) found that D for integral membrane proteins is increased 4-fold by ATP (12.5 mM) and 2.5-fold by 2,3-phosphoglycerol (12.5 mM) but is reduced more than loo-fold by polyamines (0.6 nf+i neomycin, 0.6 rrlyf spermine). Golan and Veatch (1980) reported that both D and R of the band 3 protein ar strongly and reversibly change -f (increasing from D = 0.4 x 10 = 0.1 to D = 20 x lo- 5! w*/s, R is partially removed from the membrane vm*/s, R = 0.9) if spectrin strength and elevated temperature. Thus, mechanisms by low ionic have been demonstrated by which mobility potentially could be controlled and adjusted according to functional requirements. However, the question of whether these or other mechanisms are effective under physiological conditions is still unresolved. D for lipid probes range from 0.09 pm*/s at 7'C to 0.75 pm*/s at 4O'C (Kapitza and Sackmann, 1980; Thompson and Axelrod, 1980). Slope changes at 12-17-C have been attributed to changes in lipid membranes conformation (Kapitza and Sackmann, 1980). In erythrocyte partially depleted of cholesterol, D was decreased e-fold at -5 to +5'C whereas no changes occurred at higher temperatures (Thompson and Axelrod, 1980). Thus, lipid diffusion coefficients are about lo-fold smaller in erythrocyte membranes than in artificial lipid membranes. Whether and how this restriction is related to the skeleton has yet to be determined. Erythrocytes usually lyse when subjected to a laser pulse in the 500-600 nm range. Apparently, light absorption by hemoglobin leads to a rapid heating, expansion and leakage of the cells. Therefore,

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the FM studies discussed above have been performed on isolated erythrocyte membranes. CFM, however, can be performed on intact erythrocytes without inducing lysis (D N 0.5 pmz/s for diO-C18(3) at Peters et al, unpublished results) which will permit the ZO'C; study of membrane diffusion processes under physiological conditions. 3.3 Plembrane-localized hormone receptors. High specificity and rapid control found in systems involving memare difficult to understand on brane-localized hormone receptors the basis of a sirnple reversible bimolecular reaction between horIt is very likely that the biological response mone and receptor. is the result of a series of reactions into which feedback mechanisms are integrated. One such scheme is the 'mobile-receptor' hypoFor instance, in the case of systems thesis (Cuatrecasas, 1976). involving adenylate cyclase it is assumed that receptor and cyclase are discrete and separate structures which are mobile in the plane of the membrane forming complexes only after the receptor has been rnodified by hormone. Formation of the hormone-receptor-cyclase comoff the activation of cyclase and induces further plex triggers events leading to the biological response. The low density of hormone receptors in the plasma membrane is a serious challenge for FM measurements (see 2.2). In some cases this problem can be solved by synthesis of highly fluorescent hormone derivatives. Insulin and epithelial growth factor have been coupled to lact-albumin substituted with rhodamine at a molar ratio of 7 (Shechter et al, 1978), nerve growth factor has been directly labelled with rhodamine at a ratio of 8-10 (Levi et al, 1980). Receptors for insulin, epidermal and nerve growth factor were found to be diffusely distributed in the plasma membrane of cultured cells. D-values for the hormone-receptor complex are around 5 x 10m2 pm*/s with mobile fractions ranging from 0.4 to 0.8 (Schlessinger et al, Levi et al, 1980). However, at 23'C and faster at 37-C, the 1978a; hormone-receptor complexes form clusters which are visible in the light microscope. Formation of clusters can be inhibited by various amines, inhibitors of transglutaminase and sulfhydryl reagents (Schlessinger, 1980). At 37-C, the clusters are quickly removed from the cell surface by endocytosis, transferred to lyosomes and degraded. Clustering and endocytosis are regarded (Schlessinger, 1980) as a potential mechanism of 'down regulation', the gradual of membrane-localized receptors in the presence of disappearance elevated hormone concentrations. Preceding to the appearance of large clusters the formation of oligomers has to be assumed. Oligomerization is a pre-requisite for transmission of the hormonal into the cell (Shechter et al, 1979) and may be the central signal event in signal amplification (DeLisi, 1981). 3.4 Other bM measurements not discussed in preceding sectionsare Table I (lipid probes) and Table II (membrane proteins).

compiled

in

diI-C18(3)

diI-C18(3)

diI-C18(3)

Rat peritoneal mast cell

L-6 rat embryo myoblast

Chicken myotube

diI-Cl8(3)

diI-C18(3)

Rat myotube

Mouse egg cell

embryo

Probe

Cell

1.91+0.27 0.3 0.01

0.25+0.15 0.2-0.6

0.7+0.1 -

0.12+0.03 -

O-85-1.0

1.0 o-3-0.7

1.0

R

0.6+0-l -

-1

0.15+0.03 -

O-2-0.6

0.15 0.15

0.9-Fo.4 -

0.8

D/pm's

25-30

-

-

12-31

23

T/OC et al

and remarks (1976b)

Johnson and Edidin (1978) : in unfertilized egg. : paraformaldehyde fixed. : about 3h after fertilization

Axelrod et al (1978a) : in membrane areas where acetylcholine receptors are diffusely distributed : in receptor patches. diI-density same in patches and other areas.

Axelrod et al (197813) Enrichment of plasma membrane with unsaturated fatty acid chains: no effect.

Schlessinger et al (1977a) D = 0.8+0.3 pm2/s as measured by fluorescence correlation spectroscopy. Con A, Con A + anti-Con A: no effect. : glutaraldehyde fixed : Con A + anti-Con A + glutaraldehyde

Schlessinger

Reference

TABLE I. Summary of FM measurements on the mobility of lipid probes in the plasma membrane of living cells. ( Only measurements not discussed in 3.1 - 3.3 )

diI-C18(3) fluoresceinganglioside

diI-C18(3)

fluoresceinganglioside

diI-C18(3)

Chicken embryo fibroblast

Chicken embryo fibroblast Chicken embryo muscle cell

Mouse neuroblastoma cell

Mouse neurobastoma cell

diI-C18(3) NBD-PE

diI-C18(3)

Human polymorphonuclear leukocyte

Human lymphocyte

fluoresceinganglioside

Probe

Cell

-1

0.18+0.02 0.38TO.02 0.4850.03 0.61~0.004 0.31+0.03 0.42TO.03 0.56TO.02 O-62+0.03 1.7+0.8 2.3yO.5 o-92+0.44 -

0.57-0.65 0.71-0.99

o-15-0.3

0.3

o-9+0.3 0.042+0.003 -

D/km"s

-

-

R

1.0

1.0 1.0

0.85

20-24

21-22

T/OC

TABLE I (cont.) and remarks

and yguerabide

( 1979)

et al

M early Gl late Gl mid S Petty

: : : :

(1980)

de Laat et al (1980) (cell cycle :M : early Gl : late Gl : mid S

phase)

de Laat et al (1979) : in perikaryon. : in neurites. Differentiation up to 48h:no

2-3

difference

: maximum of D = 0.3 bm7/s occured days after plating.

Elson

Schlessinger et al (1977b) No significant changes of D,R by binding of CSP (cell surface protein) to cell surface.

Reference

diO-C18(3)

egg

egg

Sea urchin

Sea urchin

NBD-PC

diI-C16(3)

diI-Cl8(3)

fluoresceinganglioside

diI-C18(3)

3T3 mouse fibroblast

3T3 mouse fibroblast

Human lymphocytes

Mouse spleen lymphocytes

diI-Cl8(3)

diI-C18(3)

Rabbit

lymphocyte

Probe

Cell

-1

0.93+0.09 0.8OTO.15 0.78+0.18 0.6770.15 0.55Yo.13 0.3270.15 -

0.59+0.26 0.31To.13 0.20+0.11 0.48TO.39 -

0.82+0.14 -

0.6+0.3 -

O-56+0.12 0.27TO.12 -

o-95+0.07 0.95+0.08 0.92+0.10 0.89+0.08 -

0.95+0.04 0.91To.09 -

o-95+0.22 0.8650.21 0.90+0.08 0.8770.13 0.50+0.07 0.4250.13 -

0.95

0.9

R

and remarks with Con A: no changes.

transformed.

15

: : : :

unfertilized fertilized unfertilized fertilized

Wolf et al (1981) Also measured: diIs with ClO,C12,C14

fertilization

(1981)

Peters and Richter : unfertilized : 15-25 min after

cell. virus

: normal : Simian

transformed.

(1980)

20-23

et al cell. virus

Eldridge : normal : Simian

Dragsten et al (1979) Different subgroups: no significant differences of D; on capped regions: D = 1.1 pm2/s, R = 0.75 : flat cells ( no microvilli ). : round cells ( normal microvilli ).

Leuther et al (1979) : T cells. Incubation : B cells. significant

Reference

Wolf et al (1980b) No significant changes of D,R by binding of dextran to cell surface.

)

37

37

T/OC

I ( cont.

1.1-l-8

1.89-2.00 1.43-1.67

D/m2 s

Table

myoblast

Human embryo fibroblast

L-6 rat

line

fWGAC

fConA fsConA

fConAC fsConA

Mouse embryo fibroblast, primary culture

Transformed fibroblast, Cl Id

fsCon AC

receptors

Label

3T3 mouse fibroblast

1. Lectin

Cell

0.8

0.29+0.08 0.42TO.08 -

0.1-l

D/10-2km's

-'

0.8

0.65 0.65

0.4

R

25

23

22

22

T/OC

and remarks

Jacobson No effect and time

et al (1976a) of WGA-dosis after labeling

(2.6-26 on D.

yg/ml

)

Schlessinger et al (1976a) Values pertain to 2 I.Lg/ml ConA, measured 15min after labeling. D decreases with increasing ConA-dosis (0.5-lObg/ml) with increasing time after labeling (15215min), with decreasing temperature, 37-ooc. No effect of 1OmMNaN3, l-100 ClM colchicine. lOpg/ml cytochalasin B: D reduced to l/3 of control.

Zagyansky and Edidin (1976) R = 0 in transformed cells using fConA or fsConA. Small R in primary culture cells using fConA up to lh after labeling. Slightly increased R using sfConA. R decreases with time after labeling.

Jacobson et al (1976b) No significant differences between normal and Simian virus-transformed cells.

Reference

TABLE II. Summary of FM measurements on the mobility of membrane proteins. ( Only measurements not discussed in 3.1 - 3.3 )

rConA

Rabbit

rConA rsConA

trans-

Simian formed

virus 3T3

rConA rsConA

3T3 mouse fibroblast

Mouse embryo fibroblast

fsConA

fWGA

NIL-8M

lymphocyte

rsConA

l-26+0.83 3.68TO.42 -

1.29+0.86 5.1051.66 -

NO.1 mo.01

1.6-0.65

o-4+0.05 -

0.4 0.09

0.39+0.11 -

rsConAC

Chicken embryo fibroblast

0.15+0.03 (0.06

rConAC,20fig/ml lOOpg/ml

3T3 mouse fibroblast

D/10-2&

Label

Cell s -l

(cont.)

0.07+0.05 0.4570.19 -

0.17+0.12 0.73To.17 -

-

0.5

-

R

TABLE II

-

22-23

37

25

T/OC

et al

(1978)

et al (197713)

Eldridge

et al

(1980)

Smith, B.A. et al (1979) :parallel to stress fibres :perpendicular to stress fibres Isotropic diffusion in regions out stress fibres.

with-

Leuther et al (1979) Within 4 hr after labeling D is reduced 2-fold; 24 hr after labeling D is restored to the initial value. 20mM azide or IpM cynide reverses the initial reduction of D. Similar effect of cytochalasin B and colchitine.

Jacobson

CSP

Schlessinger

(1977c)

with

et al

and remarks

: CSP-poor areas : CSP-rich areas Cells prelabeled

Schlessinger

Reference

D/10-2~m2s

r-anti-Ig

Mouse splenic lymphocyte

r-anti-Thy

rFab anti-Ig

rFab(RaP388)

3T3 mouse fibroblast

rIgE

1

to.oi

2.6+1.7


0.1-3 (0.01 2.8+0.06

2.6+0.8 1.9To.5 0.156+0.05 0.131To.055 0.36+0.11 0.625.1

<0.06

0.3-0.9 o-o.3 o-73+0.17 0.16+0.15 0.58TO.25 0.1870.12

dO.1

0.3-0.8 0.5-0.7

1.5 +0.5 1.25TO.65 -

R

0.5-0.8 0.6-0.8

-1

1.65+0.3 1.2 TO.6 -

and immune receptors.

fIgE,

antigens

Label

Rat peritoneal mast cell

2. Surface

Cell

TABLE II

-

T/OC

(cont.) and remarks

:after

al

(1977c)

cross-linking

with

with

Dragsten

anti-IgG

anti-Fab

et al(1979)

+ colcemid platelets platelets + colcemid

cross-linking

low r-anti-Ig high r-anti-Ig :after

:at :at

Schlessinger et : control : 20Dg/ml ConA :lOO@g/ml ConA :lOO~g/ml ConA :ConA-labelled :ConA-labelled

Schlessinger et al (197623) After labelling with 1:l mixture of fIgE and rIglJ: : fIgE (diffuse staining) : rIgE (diffuse staining) + horse anti-fluorescein: : fIgE (microaggregates) : rIgE (diffuse staining) + rabbit anti-horse serum: : fIgE (patches) : rIgE (diffuse staining) D not changed. Azide, colchicine: Cytochalasin B: D = 0.34 x 10-2pm2s-1 R = 0.2-0.5

Reference

Chicken embryo fibroblast 3T3 fibroblast Transformed 3T3

fFab of antibody against G protein of vesicular stomatitis virus

rFab(RaP388)

3T3 mouse fibroblast

BHK cell

rFab(RaE14)

Mouse neuroblastoma cell

fFab xenoantibody

Mouse egg cell

rFab(RaE14)

fFab anti-C3breceptor fFab anti-HIA

Human leukocyte

Mouse neuroblastoma cell

Label

Cell

0.65+0.09 0.68+0.16 0.23TO.21 -

5.421-l 6.7+1.9 -

o-74+0.13 0.7450.13 -

0.47+0.18 0.3650.17 -

0.3

0.3-0.4

4.5+1.3 6.2T2.2 7.2+1.6 -

2.4+0.54 l-25+0.46 -

1.3+0.2 3.1To.3 2.750.3 1.870.1 -

1.8-2.1 3.5+0.5 -

16 and 145 (0.1 and 50

37

39

31 39

-

20-24

-

phase)

Eldridge et al (1980) normal cell Simian virus transformed Reidler et al (1981) Multiplicity of infection and postinfection time had no effect on D,R. Temperature sensitive mutants with defective M protein: at restrictive temperatures no chnages of D,R; at permissive temperatures D not changed, but R reduced to about 0.5.

de Laat et al (1980) :M (cell cycle :early Gl :late Gl :mid S

changes of D during up to 24 h.

and Edidin (1978)

leukocyte Johnson

(1979)

fertilization

de Daat et al :in prikaryon :in neurite No significant differentiation

:unfertilized :3h after

:on polymorphonuclear :on lymphocyte

(1980)

and remarks

et al

Reference

1.5+0.5 6.974.8 25-30

T/OC Petty

0.24-0.60 0.08-0.28

R

(cont.)


D/10-2pm2 s -'

TABLE II

r-o.bungarotoxin

: ConA, concanavalin r, rhodamine-labeled;

'Abbreviations

Iodoacetamidotetramethylrhodamine

Rat myotube

4. Other

Frog photoreceptors

FITC

FITC

3T3 mouse fibroblast

egg

FITC

Rat myoblast

Sea urchin

D/10-2pn2 s -l

-

0.40+0.12 -

0.92+0.06 0.81+0.07 -

O-62+0.1 -

0.25

0.6

R

22,35

22 35

22

20-23

37

23

22

T/OC

(cont.) and remarks

et al

(1976a)

2h after labeling

no

limited

et al (1978a)

-

membrane area,

: in membrane areas Axelrod : where acetylcholine-receptors diffusely distributed. patches. : in receptor

Wey et al (1981) If corrected for R 0.66-O-77

Peters and Richter (1981) : unfertilized : 15-25min after fertilization

Wolf et al (1980b) No significant changes of D,R by binduq of dextran to cell surface.

Schlessinger

Edidin et al (1976) Values are for measurements labeling; immediately after fluorescence restitution.

Reference

f, fluorescein-labeled; A; WGA, wheat germ agglutinine; s, succinyl; FITC, fluorescein isothiocyanate; CSP, cell surface protein

0.01

0.5+0.2 1.6TO.3 -

30-53

3.5+0.7 0.7T;O.i5 -

7+3 -

2.2+1-o -

2.6+1.6 -

membrane proteins.

FITCc

labeled

Label

Transformed mouse fibroblast Cl Id

3. Directly

Cell

TABLE II.

Cell Biology

International

Reports,

Vol. 5, No. 8, August

7981

751

3.5 General aspects Plasma membranes of animal cells display apparently contradicting The maintenance, for instance, of characteristic surproperties. face geometries such as microvilli, of specialized membrane regions such as gap junctions, and of the highly polar organization of epithelial cell surfaces seem to require a rigid matrix. Translational and rotational mobility of certain membrane components, on the other hand, imply the membrane to be fluid. Apparently, this duality manifests itself in the FM measurements and we will try to incorporate it into a model of membrane structure. The summary of FM measurements has shown that translational diffusion of both lipids and proteins is restricted in cellular as compared to artificial membranes. Whereas DN 5 pmz/s for lipid probes in artificial lipid membranes at T z Tt, Dw 0.1 - 1.0 pm*/s for lipid probes in cell membranes. DN 0.5 - 5.0 pm*/s for proteins in artificial lipoprotein membranes. Membrane proteins, to a considerable fraction, are immobile whereas the mobile fractions typically have D-values of (0.1 - 10) x 10m2 pmz/s.

The restriction of lipid mobility in cell membranes may have simple reasons. High protein concentrations, for instance, may reduce lipid mobility by geometrical hindrance and lipoprotein association. In artificial membranes of bacteriorhodopsin and DMPC D of di0 -C18(3) is reduced from 5 pmZ/s at 0 wt% bacteriorhodapsin to 0.7 pmz/s at 60 wt% bacteriorhodopsin (30-C; Peters and Cherry, unpubAdditional components of cell membranes such as lished results). cholesterol further impede lipid diffusion. It is still unresolved, as to why a considerable variability of lipid mobility is however, found among different cell membranes (cf. erythrocytes and lymphocytes) and why the manipulation of membrane viscosity by changes of lipid composition does not influence the mobility of lipid probes (Axelrod et al, 1978b). The strong restriction of protein mobility in cell membranes cannot easily be understood in terms of bilayer viscosity. Therefore, various other mechanisms have been considered (summary: Elson and Reidler, 1979). Some evidence points to the involvement of cytoskeleton-membrane interaction (e.g. anisotropic diffusion of concanavalin A receptors in membrane areas above stress fibres; Smith, B A et al, 1979). However, experiments with microtubuli-and microfilament-disrupting agents do not support this hypothesis (e.g. colchitine had no effect on protein mobility, cytochalasin B induced even a 2 to lo-fold decrease; Schlessinger et al, 1976b, 1977a,c). Extensive cross-linking of membrane proteins by lectins and antibodies (Schlessinger et al, 1976a, b) can reduce and even abolish mobility. External protein coats, as far as studied (Schlessinger et

752

al,

Cell Biology 1977b),

did

not

international

Reports,

impede the mobility

Vol. 5, No. 8, August

of integral

1981

membrane pro-

teins.

In order to interpret the main features of FM data, we would like to turn to a different approach. As summarized in section 3.2, the an example for a cell membrane in erythrocyte membrane provides which translational mobility and its restriction is understood to a considerable extent at the molecular level. It is also obvious that a membrane model consisting of two tightly coupled layers, a protein skeleton and a fluid lipoprotein skin, could, in general, account for the dual aspects of membrane dynamics discussed above. In the following, we therefore collect evidence for the occurrence of membrane skeletons and consider potential mechanisms of skin-skeleton interaction. Patching and capping phenomena of surface receptors, the modulation of these phenomena by concanavalin A as well as FM measurements have led Edelman and co-workers to postulate that lymphocytes and many other eukaryotic cells contain a so-called surface-modulating assembly (see e.g. McClain and Edelman, 1979; Schlessinger et al, 1977c). Cytoskeletal elements such as microtubules are an essential part of the surface-modulating assembly. However, in order to account for the coupling of cytoskeleton to the plasma membrane an actin-containing layer located at the inner surface of the lipoprotein bilayer of the plasma membrane has been assumed which, in our may be classified as a membrane skeleton. Direct evidence concept, for membrane skeletons in murine tumour cells, lymphoid cells, and possibly human lymphocytes has been reported recently (Mescher et al, 1981). These membrane skeletons consist of actin and four additional proteins not identical with known cytoskeletal proteins and also different from components of the erythrocyte membrane skeleton. As a basic version of the 'skin and skeleton' model, a structure may be considered consisting of a static protein network as skeleton and a fluid lipoprotein bilayer as skin. Effects of an association reaction involving a mobile and an immobile component have been analyzed by Elson and Reidler (1979): roughly, high-affinity binding sites immobilize a fraction of the otherwise mobile component whereas low-affinity binding sites reduce apparent diffusion rates. Therefore, the occurrence of immobile fractions together with small diffusion coefficients of the mobile fractions could be accounted for by assuming that the skeleton has two classes of binding sites for skin proteins with high and low affinity, respecSince skin proteins can have large sections residing outtively. side the lipoprotein bilayer (e.g. glycophorin), an alternative or additional mechanism of immobilization may be their trapping in meshes of the skeleton. However, if such mechanisms are considered, the dynamic properties of the skeleton should be taken into account. Cell shape changes, rheological properties and certain pathological conditions 1979) apparently require that links of bx,

Cell Biology

International

Reports,

Vol. 5, No. 8, August

1981

753

the skeleton can be solved and rearranged. As a model for this situation, diffusion within a dynamic two-dimensional network can be considered (Passow, personal communication). A realistic membrane model, therefore, may have to account for both chemical and physical properties and interactions of skeleton and skin. Acknowledgements: I should like to thank Professor H Passow for suggesting this review. Furthermore, I wish to thank him for his continuous support without which my recent work would not have been possible. Mrs C Raine masterly prepared the typescript adding various linguistic spices. Support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged. References of dimensionality in Adam, G. and DelbrUck, M. (1968) Reduction biological diffusion processes. In: Structural chemistry and molecular biology. (Rich, A. and Davidson, N., eds) W.H. Freeman and and London. co., San Francisco Axelrod, bleaching Axelrod,

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