Biochimica et Biophysica Acta, 457 (1976) 353-384 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 85165

A PORTRAIT OF PLASMA MEMBRANE

SPECIALIZATIONS IN EYE LENS

EPITHELIUM AND FIBERS E. L. BENEDETTI *,a, I. DUN1A~, C. J. BENTZEL**'a, A. J. M.VERMORKEN b, M. KIBBELAARb and H. BLOEMENDALb a lnstitut de Biologie Mol~culaire du C.N.R.S. et de l'Universitd Paris VII, 2, place Jussieu, 75005 Paris (France) and b Department o f Biochemistry, University o f Nijmegen, Geert Grooteplein Noord 21, Nijmegen (The Netherlands)

(Received April 27th, 1976)

CONTENTS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

353

II.

The presence of communicating junctions

355

. . . . . . . . . . . . . . . . . . . .

I11. The control of paracellular routes of permeation across the lens . . . . . . . . . . .

359

IV.

The lens transport model

V.

Protein features of the lens plasma membrane and of the communicating junctions . . .

361

VI.

Development of the communicating junctions in the region of cellular elongation . . . .

372

VII. Concluding remarks

360

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

381

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

382

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

382

Acknowledgements References

. . . . . . . . . . . . . . . . . . . . . . . . . . .

1. INTRODUCTION T h e vertebrate eye lens has been the object o f several investigations on m e t a b o l i c and d e v e l o p m e n t a l aspects. H o w e v e r , o n l y in few instances has at t en t i o n been paid to the biochemical a n d structural characterization o f its membranes.

U n t i l recently

lens m e m b r a n e s were incidentally described as a part o f the so-called " a l b u m i n o i d f r a c t i o n " which remains when the m a j o r part o f the lenticular mass has been dissolved in water or buffer.

A t t e m p t s to characterize the lens m e m b r a n e constituents were

a l m o s t c o m p l e t e l y restricted to the insoluble fractiort which is o b t a i n e d when the ' a l b u m i n o i d ' is t h o r o u g h l y extracted by urea or g u an i d i n e h y d r o c h l o r i d e [1,2]. This

* To whom requests for reprints should be addressed. ** Associated Professor on sabbatical leave from the Department of Medicine, State University of New York at Buffalo.

354

Lensbow

Epithelium

Lenscortex i

~'

Elongahonzone

Fig. 1. Schematic drawing of the structural organization of a vertebrate eye lens. The organ is surrounded by a capsula. The epithelial monolayer covers the anterior surface.

fact is rather surprising since a better knowledge of the organization of the lens membranes could shed some light on the difficult, yet-unsolved problems which are confronting students of the lens functions. The aim of the present review is to outline a number of structural and biochemical features of lens membranes. This non-exhaustive and somewhat arbitrary selection of topics will mainly concern the organization of the lens plasma membranes which have some unique structural and developmental features as compared to the cytomembranes in other cellular systems. The eye lens is an isolated organ without blood vessels and is composed entirely of an epithelial monolayer and of a close package of fiber cells. The fibers arise from epithelial cells and represent the final stage of differentiation [3]. The peripheral (or equatorial) cells of the epithelium have a high rate of mitosis in contrast with those located in the central region of the epithelial monolayer. Each new dividing cell undergoes the process of elongation which will result in the formation of individual fiber cells [4]. The result of this process is an onion-like arrangement of fibers with the eldest cell population in the center of the lens, the nucleus (Fig. 1). During differentiation of the epithelium most of the cellular organelles disappear with the exception of polyribosomes and plasma membranes surrounding each individual fiber. The lens, therefore, is one of the best sources for isolation of these plasma membranes, since the fiber cells have a high surface-to-volume ratio and are devoid almost completely of other membranous constituents which could 'contaminate' the plasma membranes. The latter fraction can be purified by a gentle method of isolation which does not alter the composition and the spatial arrangement of the chemical constituents of the membrane elements [5]. The lens has a rather exceptional chemical composition, since it contains approximately 35% of protein of which about 90~o is represented by the so-called crystallins. Although crystallins form the bulk of the water-soluble material [6-8],

355 a part of these proteins seems to exist in close association with intergral constituents of the plasma membranes. This feature is interesting, since it is related to the more general problem of the definition of specific membrane polypeptides and their interaction with cytoplasmic constituents. Therefore, also in this respect the lens appears to be an appropriate model system.

I1. THE PRESENCE OF COMMUNICATING JUNCTIONS The eye lens has only one function i.e. to form an image by refracting and focusing the light onto the retina in a controlled manner. To fulfil this role, the lens must rely on a number of complex physiological features which are not yet completely understood. As other mural cell layers, the lens separates two different compartments from each other; but it is not clear to what extent other tissues, connected with the lens, might also act as a barrier existing between the aqueous and vitreous h u m o r [9]. It is also unclear to what extent the epithelium, composed of a single anterior cell layer, plays a role in the control of transmural processes [10]. The plasma membranes of the vertebrate eye lens may also represent a useful tool for studying the molecular organization of intercellular junctions, since large areas of the cell surface are connected by extensive junctional complexes [11]. The junctions found in the lens are of the type which usually correlates with short-range communication and the rapid horizontal transfer of ions and active metabolites, directly from one cell to another. Although a general knowledge of the role that these communicating junctions would play on cell growth and differentiation has been acquired during the last ten years, yet very little is known about the chemical nature of these membrane specializations. During differentiation of the epithelium into fiber cells a process of junctional assembly occurs, and therefore the elongation zone can be considered as a favorable system permitting the analysis of junctional formation. Previous electron microscopic observations of lens tissue have revealed the presence of junctional complexes connecting the lens cellular elements [12]; however, these studies did not allow a conclusive identification of the morphological features of the plasma membrane associated with intercellular junctions, since freeze-fracture investigation was still incomplete or lacking. In fact, in a variety of tissues freezefracture has provided more comprehensive information about junctional definition than any other technique of specimen preparation for transmission electron microscopy. By freeze-fracture typical 'gap' junctions* are demonstrated to connect epithelial cells specifically with one another and also the epithelium with fiber cells of the outermost peripheral cortical layer [13]. As in other tissues, the lens 'gap'

* It has been proposed by Simionescu, Simionescu and Palade (1957, Journal of Cell Biol. 67, 863-885) that "gap" junction, otherwise called "nexus", "close junction", "macula" or "fascia occludens", "macular close junction", and "small subunit gap junction", should better be described with the more comprehensive term of "communicating junction" ("macula communicans").

356

Fig. 2. Arrays of geometrically packed pits on fracture face B characteristic of communicating junctional domains between epithelial cells of the calf lens. junctions possess geometrically packed arrays of 9-nm inner-membrane particles on fracture face A* and a geometrical lattice of pits associated with the fracture face B of the junctional membrane (Fig. 2). We observed that the gap junctions are localized at the lateral surface of the epithelial cells in the anterior region. Moreover, nexuses have been found along convoluted areas of cell apposition and along penetrating processes between epithelial cells and fibers. The presence of 'gap' junctions, even when they occupy small areas of the cell surface, is a strong indication that the lens epithelial cells are connected by low-resistance pathways (cf. ref. 14). Actually, in several cell types, the electrical and metabolic coupling correlates with the presence of 'gap' regions and in some instances the nexuses are found to be the sole and unique junctional differentiation present between coupled cells [14]. The electrophysiological evidence for electrical coupling between epithelial cells in the lens has not yet been positively reported. It is, however, tempting to correlate the presence o f the extensive network of nexuses in the lens epithelium with cell coupling similar to that observed in other cell systems where the latter phenomenon has been strongly intimated by electrophysiological experiments. * The nomenclature for freeze-fracturing and etching has been revised (Branton, D., et al. (1975) Science 190, 54-56). It has been proposed that for all split biological membranes the half closest to the cytoplasmic matrix should be designed the protoplasmic half (P) and the corresponding fracture face labelled PF (instead of A). The half-membrane closest to extracellular, exoplasmic or endoplasmic spaces should be labeled E and its fracture face EF (instead of B). The true internal or external membrane surfaces, exposed by etching, should be called respectively PS and ES.

357

Fig. 3. a, Extensive junctional domain visualized by freeze-fracture. The junctional particles on fracture face A are closely, but not geometrically, packed. Note on fracture face B the corresponding pitted images, b and c, Tangential and cross-sections of communicating junctions between cortical fibers impregnated by lanthanum hydroxide.

The maintenance of the ionic balance of the total lens would thus depend on the presence of intercellular communication not only between epithelial cells, but also between the fiber cells which form the bulk of the lenticular mass (cf. ref. 15). In fact, extensive areas o f the fiber cell surface in the cortex and deeper in the lens, are occupied by junctions which reside in the larger planar portions of apposing plasma membranes. In freeze-fractured cortical fibers, the junctions are present as a multitude of plaques varying in size and shape. This domain consists of 9.0-nm particles which are visualized at the A fracture faces of the junctional leaflets (Fig. 3). The particulate entities do not appear in an ordered hexagonal arrangement; rather, the

358 particle arrays exhibit a pleomorphism of packing. A similar feature is visible at the junctional fracture face B, where the pattern of the complementary pits is also not hexagonal. These morphological characters of the lens fiber junctions may well be included in the variations of nexus features which have been described in various types of tissues. The variations involve the size, the spacing, the regularity of the packing of the inner membrane particles and of the complementary pits and also the size and shape of the junctional domains [16]. The junctional pleomorphism may correlate with qualitative and quantitative variations of the physiological properties of the nexuses, but the interpretation of the structural features in terms of junctional efficiency cannot, at this time, be positively assessed. For instance, some electrotonic junctions which are impermeable to fluorescein appear identical upon freeze-fracture to the junctional membrane found in nexuses associated both with electrical coupling and with the free passage of fluorescent tracers [l 7]. Recently, it has been claimed that the packing of the particle arrays correlates with changes in junctional permeability. Treatment with uncouplers induces an increase of junctional resistance and, at the same time, a more geometrical and closer packing of the junctional particles [18]. If a less regular packing of the junctional particles correlates with a more efficient coupling, then the junctions connecting lens fibers should be highly permeable. Surprisingly enough, the calculation of the electrical coupling ratio between two adjacent lens cortical fibers yields a value of 0.05 which is not impressive [19-23], compared to the index of communication in other cellular systems [24-26]. However, dye injected into the lens fibers through a microelectrode does not stay in a single fiber, but readily diffuses from cell to cell without leaking into the extracellular spaces [27]. It should be pointed out that lens cells grown in tissue culture do establish rapid cell-to-cell communication and that the communication index of 0.8 [28] is quite high. A variety of data on the size of permeant molecules across the 'gap' junctions [26] and the structural features of the communicating junctions strongly suggest that the low-resistance pathway correlates with an elementary coupling device formed by the association of two distinct 9.0-rim matching inner-membrane particles spanning the entire width of the two junctional membranes (cf. ref. 14). Each particle of the bimodal unit could display a polar surface exposed to the aqueous cytoplasmic environment and another, predominantly non-polar, intercalated into the hydrophilic lipid leaflets. By a proper segregation of other polar residues, the core of the bimodal element could be occupied by a hydrophilic channel connecting the two aqueous cytoplasmic phases directly. The existence of polar transmembrane continuity has been suggested after physico-chemical and freeze-fracture studies of erythrocyte membranes (cf. refs. 69, 78 and 82). The minimum number of the coupling units sufficient for ensuring a lowresistance passageway and whether or not there is a correlation between the cell-tocell permeability and geometry of the reciprocal association of these units remains to be established. Nevertheless, the extensive network of interconnecting 'gap' junctions

359 throughout the lens has led to the postulate that the anterior epithelial layer containing most of the ATPase is not only responsible for trafismural movement of ions and water, but also maintains the internal milieu of fiber cells throughout the lens, as a sort of single membrane syncitium. Ill. THE CONTROL OF PARACELLULAR ROUTES OF PERMEATION ACROSS THE LENS Electrophysiological observations have provided compelling evidence that the flow of water and ions across different types of mural epithelia, displaying variable values of transepithelial electrical resistance, is essentially associated with paracellular routes of permeation, rather than with variation of the specific conductance of the epithelial plasma membranes [29]. Actually, the main low-resistance pathway by-passes individual cells and runs longitudinally through the intercellular spaces. The paracellular gateways are controlled by a specific type of plasma membrane specialization called a tight junction forming a continuous closing belt around each cell. Freeze-fracturing has revealed that the structural features of these occluding junctions are quite different from those of the gap junctions [30,31 ]. In fact, when the fracture passes through the "zonula occludens" or tight junction, the two membranes in tight apposition are simultaneously split and a complicated meshwork of particulate ridges and complementary linear furrows is exposed. Tentatively, the efficiency of the sealing device, evaluated by the electrical and permeability properties of the paracellular shunt and by the penetration of electron-dense tracers across the region of membrane apposition, has been correlated with the extension and complexity of the interconnected series of ridges and furrows [32]. However, the latter conclusion has been recently questioned [33]. The electrical conductance of the extracellular space, if assumed to contain physiological concentrations of electrolyte, would be determined by the area-tolength ratio of the extracellular space and by the resistance of the occluding junctions. For epithelia transporting large amounts of sodium and water isoosmotically such as jejunum and proximal kidney tubule, the extracellular conductance path is considerably larger than individual cell membrane conductance ("leaky" epithelia of Fr0mter and Diamond [29]). A leaky epithelium is also considered to be self-shortcircuiting, i.e. it supports only a small transmural potential difference and usually has a resistance of 300 c2 - cm z or less. In contrast, epithelia with low osmotic permeability may transport ions anisotonically and have large resistances associated with well developed occluding junctions. The lens, with a translenticular potential of about --25 mV with respect to the anterior surface and a resistance of about 1000 -Q • cm 2 [20-23], would be classified as an intermediate epithelium. It is surprising that occluding zones (tight junctions) are neither seen in the epithelial layer nor between cortical fibers. Most, if not all, reabsorbing epithelia are characterized by occluding junctions where ridge-groove structures are seen in freezefractured samples [32,33].

360 What allows the lens to maintain this rather high resistance and translenticular potential difference without tight junctions? In the absence of studies on isolated lens epithelia the contribution either of the extracellular space or of the intracellular compartment, to the total translenticular resistance is not easily discerned. If the resistance path is primarily extracellular, then it is reasonable to expect some contribution of the extracellular space to this total resistance. This postulate is supported by the observation that the extracellular space occupies less than 3 ~o of the lens, and fiber cells are both tightly apposed and highly interdigitated [12,15]. Moreover, it cannot be completely ruled out that the elaborate and very extensive junctional domains connecting lens fibers by low-resistance pathways might also control the overall translenticular permeability by significantly restricting the free movement of water and ions along the narrow extracellular environment circumventing each interlocking junctional unit (a paracellular vertical gateway working in association with a horizontal communicating gateway). The latter postulate does not necessarily imply that the paracellular pathways are completely sealed. The extracellular extension of the communicating junction may present an additional restriction to the translenticular flow over that offered by close plasma membrane apposition. As in gap junctions found in other tissues, the array of interlocking junctional units in lens fibers are longitudinally permeated by the extracellular flow of tracers, such as colloidal lanthanum hydroxide (compare Fig. 3).

IV. THE LENS TRANSPORT MODEL The presence of communicating junctions between lens epithelial cells where the cation 'pump' (ATPase) is mainly located [10] and between fiber cells, suggests that maintenance of electrochemical gradients across the entire lens can be interpreted in the framework of a single-cell syncitial model. In this model, the source and means of energy production would lie in the epithelium and through the extensive communicating network, energy would be supplied both for transport and to maintain the internal milieu of each individual cell. As proposed for frog skin [34] the fiber cells, though interconnected among themselves and to the epithelium by low-resistance pathways, would maintain their normal high intercellular potassium environment through a Na+/K + exchange mechanism across individual cell membranes independent of the transport function of the epithelium. This interpretation is given additional support from Rae's microelectrode measurements of two domains of potential in the deeper layers of the lens; one, probably a fiber cell membrane potential of about --65 to --75 mV and the other an extracellular potential of about --25 mV [21-23]. The sign of the latter potential is consistent with the direction of net Na ÷ transport, that is from the lens interior to the anterior surface, a secretory direction. At least one other type of epithelium primarily concerned with the secretion of ions, the salt gland of elasmobranchs and marine catfish, has not been observed to have

361 typical occluding junctions [35]. It should, however, be re-emphasized that unless the freeze-fracture technique is used, confusion can arise as to which type or types of junction are characteristic of an epithelium. The ion-secreting epithelium usually transports ions from the blood luminal side (inside to outside) through the cell via an extensive array of plasma membrane foldings [34]. According to present concepts, the transepithelial potential is maintained through the action of an active cellular transport mechanism requiring some degree of ion restriction in the intercellular space. If ion restriction exists, it correlates with the presence of occluding junctions. Since in the lens no occluding junctions have been found so far, we have to conclude that the organization of the intercellular space characterized by large numbers of communicating junctions must act as a partial barrier to flow as mentioned before. One might anticipate that the lens communicating junction system, especially near the cortical border, would have either spatial and/or electrical properties which restrict, at least partially, back-flow of cations in a direction opposite to active transport. It is tempting to speculate that these junctions would also be comparatively non-restrictive to osmotic flow of water, so that osmotic forces could not be generated across the epithelium. In this way the anisotonic 'secretory' function of the epithelium could be accomplished without a tight occluding zonula with its resulting large unfavorable electrical potential gradient opposing active cation transport.

V. PROTEIN FEATURES OF THE LENS PLASMA M E M B R A N E A N D OF THE COMM U N I C A T I N G JUNCTIONS

It is evident that some basic knowledge of lens protein is required in order to enable the analysis of polypeptides associated or adsorbed to the plasma membranes of the lens fibers. The water-soluble lens proteins, the so-called crystallins, may be fractionated on the basis of their size, electrophoretic mobility and immunochemical properties [8,36,37]. By gel-filtration (on Sephadex G-200, Biogel P-300, Ultrogel AcA 34 or Biogel A5m) four rather well-defined fractions are identified, namely a-, fin-, ilL- and y-crystallin (Fig. 4). a-Crystallin consists of closely related proteins of differing size classes (average molecular weight 800 000) which, however, are composed of the same sort of polypeptides held together by hydrophobic linkages and hydrogen bonds [7]. Part of a-crystallin exists as a very high-molecular-weight component (HM crystallin) (cf. ref. 8). The naturally-occuring a-crystallin is composed of four kinds of polypeptide chains, namely etA1, aA2, aB~ and aB2, which migrate with different electrophoretic mobilities in polyacrylamide gels containing 7 M urea at alkaline pH [6,38, 39] (Fig. 5). Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis the four a-crystallin polypeptides are separated only into two ]~ands with apparent molecular weights of 19 500 and 22 500, respectively, since both aA1 and aA2 as well as aBj

362 E280nm

1.8 1.6 1.4

1.2 1.0 0.8 0.6 0.4 0.2

I9; /~L 20

40

60

80 100 Fraction number

Fig. 4. Separation of the water-soluble lens proteins by gel-filtration on a Sephadex G-200 column (The peak beyond the 7 fraction contains nucleotides).

Fig. 5. Electrophoretic separation on polyacrylamide gels containing 6 M urea of the lens protein fractions obtained from the Sephadex G-200 column (compare Fig. 4). By this technique the subunit structure of the individual crystallin chains is visualized.

363

Fig. 6. Electrophoretic separation, on polyacrylamide gels containing 0.1 ~ sodium dodecyl sulfate, of the fractions obtained from the Sephadex G-200 column (compare Fig. 4).

and aB2 coincide [40]. ft, and flL crystallins have also a subunit structure, with the major polypeptide chain flBp of about 24 000 daltons in common [36] (compare Figs. 5 and 6). in contrast to a- and fl-crystallins, y-crystallin seems to consist of at least four polypeptides which occur in a monomeric form. The molecular weight of the main constituent is about 20 000 [41]. An improved separation of the total population of eye-lens polypeptides has recently been achieved by two-dimensional polyacrylamide gel electrophoresis [42]. Isolated lens fiber plasma membranes (see Fig. 7) have a characteristic protein pattern. In sodium dodecyl sulfate gel electrophoresis (Fig. 8) a multitude of polypeptide bands are seen varying in molecular weight from roughly 18 000 to approx.

364

Fig. 7. Isolated lens fiber plasma membranes characterized by extensive junctional complexes and vesicular profiles of various size and shape.

100000 [11].* A very significant fact is that crystallin polypeptides - especially a crystallin subunits - remain detectable in the lens-membrane protein pattern (bands ranging from approx, from 18 000 to approx. 31 000 daltons. These crystallin polypeptides are not eluted by repeated washing in low-ionic-strength buffer and, even, resist mild proteolytic digestion applied to the isolated plasma membranes. Moreover, when the isolated membranes are eluted with 6 M urea, the crystallin polypeptides are only partially solubilized, in particular a-crystallin [5]. That the bands in the 20 000-dalton region represent crystallin chains can be ascertained from the fact that u p o n isolation and removal of the detergent they form a similar aggregate as native a-crystallin polypeptides. This finding probably reflects the close association to the plasma membranes o f ' s o l u b l e ' crystallins which should be regarded, at least partially, as integral membrane constituents. It is also clear from those observations that the a m o u n t and the type of protein which can be removed from the lens plasma membranes varies depending upon the elution procedure, and that the definition of the protein components, which should be called either "'intrinsic" or "peripheral" to the membrane leaflet is essentially operational (cf. ref. 43).

* In order to facilitate discussion between the several groups interested in lens membrane proteins we recently proposed a more rational nomenclature than used hitherto. Since at this moment the only reasonably we[[ defined parameter for lens membrane proteins is their molecular weight, this data ( × 10-3) is used as designation behind the two capitals M (membrane) P (protein). For instance, the two major components are indicated as MP26 and MP34, respectively [41a].

365

A

B

C

D

E

•

F

G

Fig. 8. Sodium dodecyl sulfate gel electrophoresis of lens plasma membrane proteins: A, structural lens proteins (for comparison); B, proteins from purified membranes; C, proteins from membranes purified after trypsin treatment; D, proteins from junctions purified after treatment with trypsin, hyaluronidase and collagenase, followed by deoxycholate treatment; E, as D but instead of deoxycholate, sarkosyl was used; F, occasionally,instead of the result depicted under E the pattern shown here was observed; G, same as F but trypsin only was used as enzyme (the numbers on the bands indicate the approximate molecular weights of the corresponding polypeptides x 10-3).

It should also n o t be u n d e r e s t i m a t e d that a consistent a m o u n t o f m e m b r a n e a n d j u n c t i o n a l fragments remains in the urea-soluble s u p e r n a t a n t where they can be detected by electron microscopy. This pitfall is due to the fact that the high urea c o n c e n t r a t i o n forms a g r a d i e n t u p o n centrifugation preventing the s e d i m e n t a t i o n o f some m e m b r a n e fragments. This p h e n o m e n o n c a n n o t be circumvented by dialysis p r i o r to centrifugation, since reassociation o f solubilized m e m b r a n e c o m p o n e n t s m a y occur when the urea is removed.

366

Fig. 9. Isolated lens fiber plasma membranes thoroughly extracted by 6 M urea. Note that the junctional and membrane profiles are mixed with densely packed amorphous material.

The urea-insoluble fraction of the isolated plasma membranes (Fig. 9) is characterized by a number of bands which are originally present in the protein profile of the plasma membranes untreated by the perturbant (compare Fig. 10a with Fig. 8B). Two major components of the urea-insoluble fraction are the 34 000- and 26 000-dalton polypeptides, in some experiments urea treatment of the membranes results in a polypeptide of approximately 22 000 daltons instead of the 26 000-dalton component while the 34 000-dalton band is unaffected (Fig. 10b). Electron microscopic observations show that the urea-insoluble fraction from the total lens homogenate represents essentially fiber plasma membranes and a very large number of junctions (Fig. 11). The protein pattern of this fraction (Fig. 12c) is very similar to that of the urea-washed isolated plasma membranes (compare Fig. 10a). Moreover Fig. 12b clearly demonstrates that the 34000-dalton component cannot be extracted with urea (note that all fractions contain a certain amount of a-crystallin polypeptides). The question arises: which among these membrane polypeptides is the major constituent of the junctional architecture? It has been claimed that gap junctions, regardless of the tissue from which the%, have been isolated, are built up from a unique type of protein or at least by a very small number of related proteins. This attractive hypothesis gains support from the observation that the communicating junction is composed of identical 9.0-nm particulate entities which form geometrically packed arrays on freeze-fracture [13,14]. As far as the chemistry of the inner membrane particulate entities, which are visualized by freeze-fracturing, is concerned, earlier observations using labeling

367 a

b ~c

ii~!~iil ;;

iiiii i!I

iii~iUiiiii!~ili!i

~!~i!~ ~iiii!i!!iiiil ~

iiiiiiiiii!!!!

m

/

Fig. 10. Sodium dodecyl sulfate gel electrophoresis of lens plasma membrane proteins: a, insoluble in urea; b, insoluble in urea (this pattern is found occasionally); c, total lens proteins (for comparison). (The numbers indicate the molecular weights of the corresponding polypeptides × 10-3).

Fig. I 1. Total urea-insoluble fraction from lens cortex showing that the membranous and junctional elements are contaminated by heterogeneous, non-membranous material.

368

ii;i !!i!iil~ Fig. 12. Electrophoretic separation on polyacrylamide gels containing 0.1 ~ sodium dodecyl sulfate

of different fractions from lens cortex extract: a, water-soluble fraction; b, water-insoluble but ureasoluble fraction; c, urea-insoluble but sodium dodecyl sulfate-solublefraction; d, the sodium dodecyl sulfate-soluble fraction treated with deoxycholate; e, the total water-insoluble fraction. techniques for exposed receptors have provided indirect evidence for the association of inner membrane particles with (glyco-)proteins [43,43a)]. Direct proof has been given by incorporation of amphipatic protein into the lipid hydrophobic domain. Pure lipid vesicles quenched from above the phase transition temperature have smooth fracture faces. Globular particles arise on the fracture surfaces of frozen lipid-protein complexes only when amphipatic proteins or their hydrophobic polypeptide segments become associated with the lipid bilayer [44-47]. The appearance of the particles and their density seem to be dependent on a "critical multimeric concentration" of the protein [47]. One can, thus, visualize the inner membrane particles as consisting of protein and the ~mooth areas essentially of lipid. However, it should not be concluded that, in bilayer regions corresponding to the smooth fracture faces, the proteins are rigorously excluded. These regions could

369 still comprise a small amount of proteins - below critical multimeric concentration which would not extend appreciably across the hydrophobic lipid core [43,48]. In most plasma membranes of animal cells the size of the intramembranous particles is heterogeneous. It may vary even within the same plasma membrane type between 4.0 and 15.0 rim. The most probable explanation of this difference is that presumably the particles are associated with heterogeneous classes of polypeptides. If a single protein component or a few equivalently related polypeptides become predominant, then the particle size tends to be uniform (cf. ref. 14) as it occurs in the junctions. The existence of common proteins forming the communicating junctions could account for the fact that metabolic cooperation, electrical coupling and passage of tracers are not exclusively between cells of the same origin and may even exist between cells that are genetically different. The existence of some common molecular feature among nexuses is also supported by the discovery that gap junctions are more resistent to solubilization by detergents than the non-junctional plasma membranes [49,50]. The same properties can be ascribed to the lens fiber junctions which can be isolated from the rest of the plasma membrane since they are resistent to the action of deoxycholate and sarkosyl [13]. When sodium dodecyl sulfate gel electrophoresis is applied to the detergent insoluble junctional fraction, the major component coincides with the 34 000-dalton region (Fig. 8). In most experiments probably consistent with variation of the purity of the junctional preparation, another polypeptide of 26 000 daltons, which appears to be a major component of the isolated plasma membrane and of the urea insoluble membrane fraction, also seems to characterize the protein pattern of the junction-rich fraction. The 26 000-dalton polypeptide is particularly abundant if deoxycholate solubilization has not been preceded by proteolytic digestion (Fig. 9). It can therefore be postulated that the junctional core, in contrast with the general plasma membranes, consists of a very limited number of intrinsic components which may well form the intramembranous junctional particles. In fact, freeze-fracture of detergent-isolated lens junctions shows that the membranes are built up by arrays of 9.0-nm particle (Figs. 13 and 14). From our experiments, however, it can not be completely ruled out that some other constituents of the junctional membrane could be either eluted or degraded, since the isolation of these specialized areas involves a mild proteolytic digestion and eventually hyaluronidase and collagenase treatment prior to detergent solubilization. For instance, the presence of an additional diffuse band in the 13 000dalton region occasionally found in the sodium dodecyl sulfate gel electrophoretic pattern of the detergent-insoluble junctions, could be due to degradation of other junctional polypeptides of higher molecular weights. Former experiments of Goodenough [51 ] provided some evidence that a 34 000-dalton polypeptide, found in gap junctions isolated from mouse liver, can be split into smaller molecular-weight fragments in the presence of reducing agents. In this connection, it should be mentioned that in all our experiments using a similar isolation technique, this condition has been fulfilled, nevertheless we do not observe a significant disassembly either of the

370

Fig. 13. Fiber junctions purified by deoxycholate solubilization of the isolated plasma membrane and sucrose density gradient centrifugation (final concentration 1.0 % deoxycholate). The structural features of the junctional membranes are preserved, a and c, thin sections stained with uranyl acetate - lead citrate; b, Replica of freeze-fractured isolated junctions.

34 000-dalton or o f the 26 000-dalton polypeptides. N o t a b l y , the electrophoretic behavior o f isolated a-crystallin used as internal m a r k e r for m e m b r a n e experiments, proves that no disulfide bridge f o r m a t i o n occurs under o u r conditions since only the m o n o m e r i c aB chain is observed. In a later paper, G o o d e n o u g h [52] claims t h a t the mouse liver gap j u n c t i o n s consist of a single protein of 18 000 d a l t o n s which can be reduced to a 9000-dalton c o m p o n e n t . It should be stressed that the isolation procedure applied by this a u t h o r involves a very long and drastic trypsin digestion of the isolated p l a s m a membranes. On the other hand, G i l u l a has postulated that gap

371

~:,t i

Fig. 14. Fiber junctions purified by sarkosyl solubilization and sucrose density gradient centrifugation (final concentration 0.5 ~ sarkosyl NL97). The junctions are mostly fragmented in short segments• a and b, thin sections stained with uranyl acetate - lead citrate; c, particulate organization of membrane fragments visualized by freeze-fracturing of isolated junctions•

junctions from rat liver are characterized by two low-molecular-weight components of 10 000 and 20 000 daltons [53]. Unfortunately, it is not clear whether or not these polypeptides are also characteristic of the isolated plasma membrane protein profile. The varying statements concerning the proper molecular weight of plasma membrane and isolated junctional proteins demonstrate the difficulties inherent in the physicochemical characterization of this kind of proteins. It should also be taken into consideration that so far most of the methods used for the isolation of communicating junctions are based on a rather drastic pre-treatment of the crude pre-

372 paration with proteolytic and other hydrolytic enzymes, whose action upon the molecular architecture of the membrane components is not easily predictable. Even small variations of the incubation conditions may result in major changes in the protein pattern though the electron-microscopic features of the subunit pattern of the plasma membranes and of the junctions seem to remain unaffected. A group of authors [54,55], who used the urea-insoluble fraction from the crude total lens homogenate, have claimed that the 26 000-dalton component is the major constituent (approximately 50/0) °/ of this membrane-rich fraction. However, these results must be evaluated with great care because the urea-insoluble fraction did not undergo sucrose gradient centrifugation, which is consistently applied in our isolation procedure of the lens plasma membranes. Therefore highly insoluble proteins, for instance some fl-crystallin polypeptides which even resist urea solubilization, may contaminate and/or overshadow the protein profile in the region of 24 000-26 000 daltons, known to be the molecular weight range of the major fl-chains (Figs. 9 and 12). Experiments in progress in our laboratories show that the membrane 26 13130dalton polypeptide is not identical to flBp, a question recently raised by the group of Maisel [55]. If the molecular weight is estimated by sodium dodecyl sulfate gel electrophoresis it should be borne in mind that the values are not exact. A very illustrative example is the two polypeptide chains of a-crystallin the molecular weights of which, according to the recently elucidated primary structure, are 19 830 for the A-chain and 20 900 for the B-chain [56]. Nevertheless, there is a clear-cut separation in the sodium dodecyl sulfate gel electrophoresis pattern of both chains with calculated molecular weights 19 500 and 22 500 daltons, respectively. In view of this and similar findings reported in the literature, differences in molecular weights estimated by sodium dodecyl sulfate gel electrophoresis may be misleading, as long as more stringent chemical information is not available.

Vl. D E V E L O P M E N T O F T H E C O M M U N I C A T I N G CELLULAR E L O N G A T I O N

JUNCTIONS

IN T H E R E G I O N

OF

The onset and the mode of formation of the intercellular junctions have been the object of many interesting observations carried out on embryonic and developing tissues (cf. ref. 39). From some of these experiments, however, it is not readily apparent whether the plasma membrane features revealed by freeze-fracturing represent the assembly of the junctions or steps in their breakdown and modulation during organogenesis and tissue differentiation. The advantage of the lens compared to other systems, resides in the fact that this tissue is made of only one cell type, the anterior epithelium, which during the entire life of the animal differentiates into lens fibers. The lens fibers are formed continuously and in one direction even in the adult lens in a quite similar fashion, although slower, than during the embryonic development [4]. As mentioned before the fibers, generation after generation, are progressively packed and stored within the lenticular core or nucleus. Therefore, the progress

373

Fig. 15. Developing junctional domains found in the region of cellular elongation. The 9.0-nm junctional particles visualized on fracture face A form either linear rows or loosely packed polygonal arrays. Note on fracture face B the rows of pits corresponding to the particle array on fracture face A. of the regional specialization of the plasma membrane can be followed, step by step, in the same evolving lerLticular tissue. During elongation the amount of surface membranes of the fibers as compared to the epithelial cuboid cells increases almost a thousand times, and this process involves the accumulation of newly synthesized plasma membrane lipid and protein [53,57]. In the elongation zone, the sequences of the membrane differentiation involving the assembly of intercellular junction(s) are most remarkable. The onset of the junctional assembly seems to correlate with the appearance of a new class of intramembranous particles visualized primarily on the fracture face A of elongating cell plasma membranes. These particulate entities are homogeneous in size (9.0 nm in diameter) and exhibit the tendency to form either short linear rows or small clusters (Figs. 15 and 16). It is remarkable t h a t the linear arrays or the small clusters of 9.0-nm particles generally reside in or appear to be sequestrated into a region of the fracture face A void of other usual types of heterogeneous particles (Fig. 16). The presence of a smooth halo around junctional particles seems to be a general feature of their assembly since it has been reported also by Decker and Friend in the gap junction formation during amphibian nerulation [58] and by Revel in regenerating liver [59]. At this stage of junctional development, it is not possible to establish if the 9.0-nm particles penetrate through the membrane leaflet to form complementary

374

Fig. 16. In the elongation zone: 9.0-nm particles forming linear rows or small clusters reside in a region of the fracture face A void of other usual type of heterogeneous particles.

depressions on the corresponding outer half of the bilayer and thus match with complementary 9.0-nm particles on the opposite plasma membrane. As cell elongation proceeds, longer and more elaborated arrays and clusters of particles are visualized on the fracture face A (Fig. 17), complementary arrays of pits are clearly visible on the fracture face B - in close proximity to membrane regions where the intercellular space seems to be abruptly reduced. The particulate linear arrays converge and intermingle. Conceivably, the rows of particulate entities are the structural device holding two neighboring plasma membranes in close proximity. The rows of matching pairs of 9.0-nm particles spanning the entire width of the two adjoining plasma membranes may already represent preferential and initial sites of cell-to-cell communication. It is likely that the linear arrays and clusters of 9.0-nm particles also provide an inner membrane frame which may favor further particle assembly concomitant with the progression of junctional efficiency [60,61]. The most advanced step of the junctional development is characterized by the accumulation of packed particle clusters (Fig. 17) enclosed within the polygonal areas limited by the pre-existing particulate rows. Similar particle arrays acting as nucleation sites have also been observed during the junctional development in other types of tissue [58,62]. Arrays of large particles (10.0 nm) have also been found where the assembly of gap junctions could be observed [59]. It is not yet fully understood whether these 10.0-nm

375

Fig. 17. Multiple junctional domains of various sizes between elongating fibers. Probably during the junctional assembly smaller clusters merge into more extensive particle arrays.

particles are precursors of the smaller particles (9.0 nm) forming the gap junction, or represent the membranous determinant site for initial cell recognition [59]. In our observations, 9.0-nm particulate entities seem to make up both the primitive rows or strains characteristic of junctional onset as well as the large aggregates of close-packed particles which represent the developed junctions connecting the lens fibers. It may therefore be postulated that lens-fiber junctions arise from a multitude of linear arrays or small clusters consisting of particulate entities, rather than from a unique center of particle accumulation. A similar process has also been outlined during the assembly of tight junctions. The remarkable difference from gap junction formation

376 is that the particulate entities forming the linear array in developing tight junctions tend to fuse one another and/or to be coated; from these processes the smooth ridges of the occluding network emerge [63-65]. The morphological sequence characterizing junctional assembly during cell elongation does not provide any direct information on the molecular mechanism underlining the structural rearrangement of the plasma membrane. Primarily, it would be interesting to know if the 'nascent' or pre-existing junctional intramembranous particles visualized on the freeze-fractured inner membrane faces are also associated with entities exposed at the true cell surfaces (receptors, antigenic and recognition substances, glycosyl-transferases, sugar acceptors, etc.). The reciprocal interaction of these exposed entities could, during elongation, enhance specific adhesion, guide movements and trigger close cell-to-cell contact and communication. Unfortunately, many controversial results have been presented concerning the association of the intramembranous particles with other membrane components exposed either at the outer or at the inner true plasma membrane surfaces [66-68]. Only in a very few instances, the rearrangement of exposed receptors correlated with or was concomitant to the change in the distribution of intramembranous particles [69]. We have reported that cross-linking of the exposed receptors with concanavalin A induced intramembranous particle aggregation in mouse plasmocytoma agglutinated plasma membranes [70]. However, this event seems to be restricted only to a few types of cell since concanavalin A on normal and transformed 3T3 cells and on lymphocytes [67,68], was unable to change the long-range particle distribution which remained random. Similar negative results have been also reported during the agglutination of Entameba histolytica [66]. It can only be reported that many membrane-mediated functions, such as cell fusion, endo-exocytosis and cell contact, which should be dependent upon membrane surface interactions and transmembranous control, are characterized by the rearrangement of the protein particles located in the width of the plasma membrane. In all these instances membrane-to-membrane recognition is associated with the formation of apposed matching particle arrays in the partner membrane [71], indicating that some specialized membrane functions require an ordered spatial arrangement rather than randomness and variation of the membrane intercalated protein particles. Permanence of membrane differentiation is needed [71] in Tetrahymena and in Paramecium during the secretory process in vesicles which has a tight schedule of events; arrays of intramembranous particles (annulus and rosette) are found respectively in the plasma membrane and mucocyst or tricocyst membranes. Thus, at least temporarily, there is a stable and ordered position, permissive or stringent, for the vesicle and plasma membrane 'rendez-vous'. The increasing particle distance and the final centrifugal disassembly of the array during the final step of fusion may indicate the developing free lateral mobility of the particulate entities. The mobility of inner membrane particles within the 'fluid' lipid domain seems in fact to be de-

377 pendent upon or regulated by a variety of parameters. Restriction in selected areas of the plasma membrane of the free lateral mobility of protein may depend upon local accumulation of more highly saturated fatty acids in the chains of membrane phospholipids; restriction may also come from high concentration of cholesterol which is thought to impair the mobility of phospholipid fatty acid chains, thus provoking an 'intermediate fluid condition', rather close to the gel phase [72]. These circumstances would favor the segregation of preexisting or newly inserted proteins from the rather rigid lipid domain and enhance protein-to-protein interaction. It has been reported that restriction of the random incorporation of hydrophobic protein into Escherichia coli membranes correlateswith the relative amount of ordered paraffin chains of the phospholipids [73]. Other experiments have clearly shown that in Acholeplasma laidlawii cells grown at 37 °C and enriched with relatively straight chain fatty acids, the intramembranous particle distribution tends to be clustered rather than random even when the samples are quenched for freezefracturing experiments, from above the phase transition temperature. The particle aggregation is dramatically increased when the cells are slowly brought to the phase transition temperature and then rapidly frozen [74]. From all these observations, it could be postulated that the assembly of the 9.0-nm inner-membrane particles in ordered arrays, and progressively into segregated junctional domains, could reflect the restraints of lateral mobility of the proteins within a rather rigid lipid domain. The latter view is strongly supported by the fact that the differentiation of the epithelium in lens fibers correlates with remarkable changes in the lipid features of the developing fiber membranes. In particular, the ratio of cholesterol to phospholipid increases during the elongation and aging of the fiber, and sphingomyelins are preponderant and become the major class of membrane lipids [57,75]. It is also remarkable that during the process of cellular elongation in the lens the sphingomyelin fatty acids show an increase in main chain length and monounsaturation (nevronic acid, C24 : 1) or full saturation (palmitic acid, C1 x : 0) [75]. From these properties of the membrane lipids, it can be expected that inner-membrane particle aggregation would be due to the slow lateral diffusion of lipid which would prevent a random equilibrium distribution of the protein. Furthermore, the assembly of an ordered lattice of identical or equivalently related protein subunits will restrict by itself the translational diffusion and favor growing particle accumulation. The halo or the aisles, void of particles, that are found to border the arrays and clusters of junctional particles could reflect the accumulation of those lipids which have a specific affinity with the same membrane proteins, i.e. the junctional proteins. It has been thoroughly demonstrated that certain membrane lipids have preferential binding properties with the proteins and that the activity of the latter largely depends upon the convenient choice of the lipid close environment ("lipid annulus" [76] or "strongly bound lipid" [77]). The observation that the 9.0-nm junctional particles tend to be aggregated whereas the other classes of inner membrane particles remain randomly distributed in the general plasma membrane, may be interpreted in the

378

Fig. 18. Isolated fiber plasma membrane, negatively stained with uranyl formate, after repeated washing in low ionic strength buffer. The plasma membrane sheets appear to be associated with microfilaments 'decorated' by molecules having common features with 'water-soluble' crystallin polypeptides(~-crystallin?), a, arrow points to crystallin molecules.

light of the selective association of protein and lipid domains. This association seems to be strongly dependent upon the proper conformation of the hydrophobic polypeptides; hence certain classes of proteins (glycophorin, bleached rhodopsin) are not excluded even from the S-phase lipid, whereas other polypeptides (unbleached rhodopsin, (Mg2+ + Ca2÷)-ATPase) are rapidly segregated when the lipid phase becomes ordered [46]. The free lateral diffusion o f intrinsic membrane proteins within the lipid core seems also to be controlled by the interaction of the inner

379

Fig. 19. High magnification of the 'water-soluble' crystallin-microfilament complex visualized by negative staining with uranyl formate, a, arrow points to crystallin molecules. membrane entities with cytoplasmic peripheral components such as microfilaments or microtubules [69,72,78,79]. In freeze-fractured plasma membranes the partition coefficient of the inner membrane particles, which adhere more to the cytoplasmic fracture face A, than to the complementary extracellular fracture face B, probably reflects this interaction [80]. On the other hand, freeze-fracture experiments indicate that accretion and clustering of membrane particles are enhanced when the plasma membrane is 'destabilized' by extraction of the extrinsic protein (i.e. spectrin) [81,82]. Most of the junctional particles in the lens plasma membrane remain strongly attached to the fracture face closer to the cytoplasm, and in negatively stained preparations of isolated lens plasma membrane microfilaments are seen to end at the membrane inner surface (Figs. 18 and 19). In this connection, it should be mentioned that metabolic interaction between cells growing in vitro, which correlates with the assembly of communicating junctions, is prevented or affected by cytochalasin B [83]. But the

380 relevance of these interesting observations with the hypothesis that the junctional assembly is controlled by a direct plasma membrane-microfilaments interaction is still unclear. So far the most conclusive indication of the association of inner membrane particle array with cytoplasmic components has been provided by Satir and Gilula studying the "microtubules-membrane complex" in ciliary necklace formation [84]. It remains to be discussed whether the assembly of junctional devices during lens-fiber elongation relies on newly synthesized material or is mainly dependent on lateral displacement of pre-existing junctional particles. Evidence derived from other rapidly growing cells in vitro or from reaggregation experiments [60,61] shows that the times of coupling onset are rather short (1-40 min). Yet the establishment of low-resistance pathways monitored by the increasing junctional conductance is a progressive event reflecting a gradual accretion of junctional units [60,61]. It is probable that these rather rapid events at the plasma membrane level are not associated with a program involving new biosynthesis and insertion of junctional constituents. Hence junctional assembly could rely primarily upon the lateral displacement of pre-existing randomly distributed 9.0-nm particles which subsequently become reorganized and polygonally packed. Conversely in other cellular systems the establishment of communicating junctions seems to be dependent on metabolic events which tightly control the biosynthesis and the assembly of new junctional constituents [85,86]. In the lens, during elongation, there is an obvious increase of the plasma membranes. The latter process must involve the accumulation of newly synthesized plasma membrane lipids and proteins. Incorporation experiments using specific lipid precursors show that the highest incorporation of lipids is in the elongation zone [57]. Since most of the lipids are associated with membranes these experiments indicate that fiber differentiation is accompanied by the formation of new membrane constituents. As far as protein biosynthesis is concerned the few available results show that [~4C]leucine is incorporated to the greatest extent by the epithelium and by the outer cortex [83,87]. These results, however, cannot give a direct indication of whether or not membrane specific proteins have been synthesized de novo. Vermorken et al. [88] have recently shown that polysomes derived from the cortical part of calf lens are able to direct the biosynthesis of specific membrane proteins even in a heterologous cell-free system. Hence one may conclude that also protein constituents of the plasma membrane are newly synthesized during lens cell differentiation. The assembly of intercellular junctions should thus coincide with the biosynthesis of membrane protein and lipid, and we have already postulated that the accretion and extension of the segregated junctional domain are probably favored by the intrinsic features of the lipid bilayer. It will be a matter of further interest to investigate whether the difference observed between the structural organization of gap junctions in lens epithelium and the developed fiber junctions reflects the appearance or the selection of a specific protein pattern during elongation.

381 VII. C O N C L U D I N G R E M A R K S

The morphological and biochemical data presented in this review are not in contradiction with the current molecular model for biological membrane. According to this model, the architecture of biological membranes relies on the association of at least two main components: a lipid phase primarily occurring as an asymmetrical bilayer, and intercalated protein entities. From a purely biophysical or physicochemical point of view, biological membranes may be regarded as a viscous heterogeneous two-dimensional fluid lacking a well developed order (cf. ref. 44). There is, however, increasing data supporting the postulate that an ordered spatial arrangement of the membrane constituents is needed, at least temporarily, to fulfil many essential membrane functions. The plasma membrane in the eye lens provides a good example of the structural complexity of a specialized membrane type. Functionally also this type of plasma membrane may be regarded as a receiving-transmitting assembly, where the regulatory mechanisms are probably inherent consequences of oligomeric or even polymeric associations of protein and lipid active membrane components, held together, both accurately and economically, by non-covalent bond interactions. The experimental data, which we have outlined, demonstrate the existence of local highly specialized forms of supramolecular organization of the membrane proteins within the lipid domain. The membrane proteins are an integral part of the plasma membrane backbone and form a rather 'solid' framework preventing randomness or variation of the specialized membrane sites. Polymeric association of protein subunits, either forming linear arrays or bidimensional lattices of repeating subunits are the common features of lens plasma membrane differentiation at points of mutual contact of cells. These specializations of the surface cell membrane seem to be associated with the regulation of short-range intercellular communication permitting rapid horizontal transfer of ions and active metabolites directly from one cell to another. We have also proposed that these communicating junctions might somehow control the flow of water, ions and metabolites along the paracellular routes of permeation. On the basis of available evidence it seems that the permanence of the differentiation of the plasma membrane may also code for reciprocal membrane-to-membrane recognition and probably represents a template favoring the stereospecific apposition of newly formed membrane protein and lipid. Our gel electrophoretic experiments and a direct visualization by negative staining of isolated plasma membrane preparations, tend to indicate that 'watersoluble' lens proteins, in particular a-crystallin, form with other integral plasma membrane constituents and cytoplasmic microfilaments a rather stable association. It will be the subject of further study whether a-crystallin or other membrane polypeptides are the favored binding protein for microfilaments.

382 ACKNOWLEDGEMENTS

The authors gratefully acknowledge the help of Miss M. C. Potjens in the preparation of the manuscript. The work has been supported by the D616gation G6n6rale &la Recherche Scientifique et Technique (contrat No 74.7.0173) and in part by the Netherlands Foundation for Chemical Research (S.O.N.) through financial aid from the Netherlands Organization for Pure Research (Z.W.O.).

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