Membrane-associated cytoskeleton and coated vesicles in cultured hepatocytes visualized by dry-cleaving

Experimental

Cell Research

MEMBRANE-ASSOCIATED COATED

VESICLES

132 (1981) 169-184

CYTOSKELETON IN CULTURED

VISUALIZED

AND

HEPATOCYTES

BY DRY-CLEAVING

D. A. M. MESLAND,

H. SPIELE and E. ROOS

The Netherlands Cancer Institute, Division of Cell Biology, 1066 CX Amsterdam, The Netherlands

SUMMARY Dry-cleaving is introduced as a new technique for visualization of the cytoplasmic side of adherent membrane in cultured cells. Basically, cells are fixed and critical point-dried in situ and subsequently broken by means of adhesive tape. The plane of cleavage -is dependent on the fixation scheme applied. The method has been used for the study of substrate-adherent membranes in primary cultured hepatocytes, in combination with a variety of scanning- and transmission electron microscopic (SEM and TEM) techniques. It is shown that a two-dimensional fdamentous web is apposed to the entire hepatocytic plasma membrane. Particular patterns within the web can be recognized, among which a ‘spider-web’ pattern appears to be associated with the early stage of coated vesicle development. During successive stages of coated vesicle formation the coat appears to be connected to filaments, directly in its mid-stage and by means of radial spokes in its final stage. The significance of the spider-web pattern with respect to the endocytotic process and the interrelationship of coated vesicles and filamentous structures are discussed.

In many studies actin-containing microfilaments have been found to be associated with the plasma membrane [7, 8, 11, 12, 18, 26, 28, 31, 33, 461. As such, microfilaments can be organized in bundles [16, 23, 44, 451 or as a meshwork of anastomosing filaments apposed to marginal parts of the membrane [16, 451. Some structural investigations suggest the existence of an actin-filament system that may be organized like a web associated with the cytoplasmic side of the plasma membrane during spreading or locomotory activities [8, 11, 441. By means of negative stain electron microscopy of detergent treated fibroblasts Small et al. [43] observed a fine pattern of ‘diagonal microfilaments’ in the plane of the extracted membrane. A microfilamentous web apposed to the membrane and having connections with other cytoskeletal

elements, as is inferred by these studies, may provide a framework for anchorage and for translocational activities of membrane components, i.e. receptor mobility but also endocytosis [7,21, 22,38, 391. In this paper a novel technique is introduced, called dry-cleaving, that allows visualization of the cytoplasmic surface of the adherent membrane without rupturing of the living cells [8, 111. Instead, cells grown on Petri dishes or grids are fixed, dehydrated and critical point-dried in situ according to standard methods. Then the cells are cleaved by tearing them off with a piece of ordinary adhesive tape. The method has been applied to primary hepatocyte cultures and gives very reproducible results in terms of the amount of cells being cleaved and the plane of cleavage. The latter appears to be dependent on the fixa-

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tion scheme applied. Together with a combination of transmission (TEM) and scanning electron microscopy @EM) new observations were made concerning the membrane-associated cytoskeleton of adult rat hepatocytes as well as its relation to endocytosis apparent by coated vesicle formation [17, 391. Our work provide evidence that (1) a rather uniform filamentous web is apposed to the entire cytoplasmic side of the hepatocyte plasma membrane and is continuous with typical cytoskeletal structures such as stress fibres; (2) the site of coated vesicle formation coincides with a typical pattern of the filamentous web; the coated vesicle is connected to filamentous structures during its formation and after its completion.

MATERIALS Hepatocyte

isolation

AND

METHODS

and culture

Isolation of adult rat hepatocytes by the collagenasenerfusion method and subsequent culturing on gaspermeable membranes in 50- mm Petripe& diihes (Heraeus) will be described elsewhere [35]. To culture hepatocytes on grids for whole mount transmission electron microscopy (WMTEM), 200 mesh nickel grids, deposited on small pieces (approx. 6x6 mm) of 0.3% Formvar film floatine on doubledistilled water, were picked up with an inverted Petriperrn dish. A number of grids could be sandwiched between the gas-permeable membrane and Formvar film in this manner. Before plating with hepatocytes the dishes were sterilized by ultraviolet irradiation (Philips, 30 W) for 24 h at a distance of 55 cm.

Cell fixation Standard futation scheme. Hepatocyte cultures were rinsed twice with uhosuhate-buffered saline (PBS) 24 h after plating and fixed with 0.1% glutaraldehyde (GA) for 15 min in HMCK buffer (0.1 M KCl, 1 mM CaCl,, 2 mM MgCl,, 10 mM Hepes, pH 6.9 (modified buffer after Kuczmarski & Rosenbaum [23]). The cells were rinsed, treated with 2% tannic acid (Malinckrodt Inc., St Louis, MO) for 30 min, rinsed several times and post-fixed with 2 % 0~0, for 30 min, all in HMCK buffer at room temperature. This procedure stabilized cell structure [3, 401, enhanced fine structural density in WMTEM and increased conErp

Cell

Res 132 (198/J

ductivity in scanning electron microscopy (SEM) [30]. The cells were subsequently rinsed in distilled water, stained with 1% uranyl acetate in distilled water for 30 min. rinsed again and dehvdrated in a graded series of ethanol. From this point on cells were-either critical point-dried from CO, and prepared for drycleaving, SEM or TEM (see below), or embedded in an Epon/Araldite mixture [45] and sectioned parallel or perpendicular to the substrate. Several variants to the standard fixation scheme have been applied. Replacement of 1 mM CaCl, by 2 mM EGTA in the buffer 1431 did not effect cells when fixed in 0.1% GA but considerably disturbed cell morphology when fixed in 2.5 % GA. Fixation in 2.5 % GA instead of 0.1% GA prevented the penetration of tannic acid in hepatocytes and resulted in a coagulated appearance of the microfilaments [ 191. Omission of post-osmication did not influence cell structure but had an adverse effect on dry-cleaving (see below and Results). To remove cell membrane without disturbing cell mornhologv cells were fixed in 0.1% GA for 5 min, rinsed, treated with 1% (v/v) Triton X-100 for 30 min and rinsed again for 10 min (three refreshments) in HMCK buffer. Except for the PBS wash, they were then treated as described in the standard fixation scheme. Experiments with another non-ionic detergent, 30 mM octyl glucoside (Sigma, St Louis, MO), gave similar results [24].

Dry cleaving Hepatocytes, critical point-dried on Petriperm membrane (Petriperm for short) or Formvar-coated nickel grids, were inverted on Scotch tape (ordinary acetate tape (no. 810), or double paper tape (no. 400), as well as other tapes can be applied with similar results), gently pressed against the glue and subsequently removed with a forceps. Dependent on the fixation, cells either cleaved or transferred to tape in their entirety (see Results). Dry-cleaved cells on grids were directly examined in a Philips EM 300 microscope operating at 80 or 100 kV. Stereo micrographs were taken at tilt angles of 6”.

Replicas Dry-cleaved cells on Petriperm were coated with approx. 15 nm carbon at an angle of 90”. The carbon replicas were floated on distilled water, digested on 15 % sodium hypochlorite overnight, washed on water again and picked up on Formvar-coated 100 mesh copper grids. Replicas on grids were rotary shadowed with Pt/Pd at an angle of lo” and examined in a Philips EM 300 microscope operated at 80 kV.

Scanning electron microscopy (SEM) Critical point-dried whole cells or dry-cleaved cells were sputtered with gold/palladium in a Polaron SEM coating unit (2 min, 1.2 kV, 40 mA). Photographs were taken in a Cambridge Stereoscan Electron Microscope Mark IIA operated-at 30 kV.

Cytoskeleton

Fig. 1. SEM micrograph of primary cultured hepatocytes, spread on a Petriperm dish and showing part of a trabeculum of tightly associated cells. Tilt angle 45”. x1500. Fig. 2. Higher magnification of the area indicated in fig. 1. Note the intertwining of microvilli at the barely vkble borderline between two adjacent cells (arrows).

RESULTS Dry-cleaving Hepatocytes inoculated at a density of 6x lo5 cells/ml spread to form a single cell layer that, after 24 h, shows a pattern of trabeculae with an average thickness of 8 pm. Tightly associated cells within a trabeculum (figs 1, 2) may be organized randomly, or packed like slices in a cake (fig. 3), as can be observed sometimes because of their preferential breakage along intercellular spaces during preparation for SEM [29, 321. The variety of shapes shown by microvilli on the medium-exposed cell surfaces as well as the smooth membrane of the interhepatocyte faces have been described earlier [ 14, 29, 32, 481. Dry-cleaving of the hepatocyte cultures fixed by the standard fixation scheme (Methods) almost quantitatively removes

and coated vesicles in hepatocytes

171

The substrate-facing membrane at this location is slightly up-lifted. Tilt angle 45”. x7600. Fig. 3. ‘Slice-like’ hepatocyte, dissociated from its neighbouring cell in a trabeculum, and showing the smooth intercellular face of its plasma membrane (ZM). Tilt angle 0”. x3 800.

the cell bodies and leaves the remaining adherent cell membranes in a clear mirrorimage pattern, as shown in fig. 4. The original cell positions can be readily observed at higher magnifications (fig. 5). The substrate-facing membrane in the intercellular space has a tendency to remain with the cell bodies (fig. 6), thereby leaving an empty space between the patent adherent membranes (figs 5,7). Although occasionally some embedded mitochondria are left behind, the plane of cleavage appears to be rather invariably just above the adherent plasma membrane. This was confirmed by embedding dry-cleaved material and sectioning it perpendicular to the substrate. Cleavage occurs at a plane between 0 and 150 nm above the membrane. Due to the presence of microvilli underneath, the membrane may be lifted from the substrate

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Figs 4-10. Dry-cleaving. Fig. 4. Low magnification SEM image of hepatocytes fiied by the standard procedure. Dry-cleaved cells glued onto adhesive tape (a). Their respective membranes still attached to the cutten piece of Petriperm membrane (b). Note the clear mirror-images of both preparations. Tilt angle 0”. x 15. Fig. 5. Adherent membranes of dry-cleaved cells. The Exp Cell

Res

132 11981)

original position of the individual cells is clearly shown, as well as the rather uniform thickness of the preparation. Tilt angle 45”. X 1500. Fig. 6. Dry-cleaved cell. View at the cytoplasm of the upside-down cell. Note the membrane remnant (M) located between two adjacent cells. Tilt angle 45”. X3800. Fig. 7. Adherent

membrane of a dry-cleaved

cell.

Cytoskeleton

Table 1. Effect of primary

fixative

and coated vesicles in hepatocytes

and post-osmication

on cleaving

of critical

173

point-dried

hepatocytes Postosmication Fixative

(2 % OSOJ

Cleavage

Figs

O.l%GA

Yes No Yes No

4-10 Not shown Not shown Not shown

No

Above adherent membrane Variable results” Above adherent membraneb Separation of adherent microvilli Variable results?

Yes

No cleavage

18-20

2.5%

GA

3.5 % Formaldehyde (FA) 0.1% GA followed by 1% Triton X- 100

Not shown

n Cleavage at variable planes through the cell. * Filaments have a coagulated appearance.

which, at its maximum, adds another 100 nm to the total thickness of the preparation (figs 8-10). As shown in table 1, cleaving is dependent on the fixation scheme applied. Omission of post-osmication results in unpredictable cleavage when fixed in 0.1% GA or 3.5 % FA, and in nearly quantitative separation of substrate-adherent microvilli only when fixed in 2.5 % GA. Fixation with 0.1% GA followed by treatment with 1% Triton X-100 before osmication causes no

No membrane is left between the adjacent cells (*, compare with fig. 6, see also fig. 5). Occasionally holes within the membrane can be observed (arrowheads). A complex fibrillar meshwork covers the entire inner surface of the membrane. Sometimes centres from which fibres seem to radiate are distinguishable (arrow). Tilt angle 45”. x3 800. Fig. 8. Dry-cleaved, adherent membrane, embedded in situ and sectioned perpendicular to the substrate. Note the rather constant thickness of the preparation. The absence of membrane in the centre of this image may either reflect space between two neighbouring cells or a hole in the membrane (see fig. 7). Electrondense globules in the background and underneath the membrane probably constitute lipid excretion products [15]. x 16000. Fig. 9. Higher magnification of similar preparation as shown in fig. 8. Distinct filaments running parallel to the membrane and associated with it. ~32000. Fig. 10. Preparation as in fig. 9, showing adherent microvilli (see also fig. 18) and coated vesicles (arrow).

x32000.

cleavage at all, but just leaves inverted whole cells on tape. The details of this result will be dealt with below. 11-14. Membrane-associated skeleton. II. WMTEM of adherent membrane of a hepatocyte fixed by the standard procedure and drycleaved; representative of the majority of cells within a monolayer. A fine fibrous meshwork covers the entire membrane. Locally this meshwork appears more condensed (circles and inset) forming ‘condensations’ or ‘filamentous aggregates’. The strongly electron-dense pattern results from superposition of microvilli underneath the membrane and fdamentous aggregates above it (see figs 9 and 10). The electrondense particles all prove to be coated vesicles at higher magnification (inset, arrows). Note the particular distribution of these vesicles in relation to the dense areas mentioned above. Voltage 80 kV. x7 200; inset, 27500. Fig. 12. Preparation as in fig. 11. Marginal cell in a monolayer showing microfilament bundles organized in two stellate configurations. The cell lacks microvilli. Voltage 100 kV. x 10 350. Fig. 13. Preparation as in fig. 11. Marginal cell with parallel running microfilament bundles. In the lower right comer of this image microvilli, but not filament bundles, can be seen (arrowheads). In stereo view the bundles prove to be extremely flat and directly apposed to the membrane. Many side branches form part of the fine fibrous meshwork in the background. A rare form of coated vesicle is indicated (arrow). Voltage 100 kV. x20000. Fig. 14. Adherent membrane of a hepatocyte fixed by the standard procedure and dry-cleaved; rotaryshadowed replica of its cytoplasmic side. Fine details can be observed of only those structures that have just a small elevation above the surface. The contours of a microfilament bundle that branches out into a fine Wamentous web covering the entire surface can be readily observed (compare with fig. 13). x22750. Figs Fig.

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Cytoskeleton

12-811811

and coated vesicles in hepatocytes

175

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Membrane-associatedfilamentous

web

In the SEM, adherent membranes prepared by standard fixation and dry-cleaving appear to be covered by a dense fibrous network with no particularly striking organization; although sometimes centres can be distinguished from which fibres seem to radiate (fig. 7). Unfortunately, the apparent vulnerability of the Petriperm membrane for the SEM electron beam did not allow for its study at higher magnifications. Drycleaved cells on grids, however, revealed considerable fine structure when studied by stereo TEM [50]. The majority of the cells observed at low magnification show a pattern of electrondense areas, often interconnected, against a background a fine fibrous meshwork (fig. 11). Locally this meshwork appears more condensed in one or more directions, forming a linearly or radially oriented aggregate of thin 5 nm filaments (fig. 11, inset; see ref. [34]). The electron-dense areas result from superposition of such condensations and the system of microvilli underneath the membrane (figs 11, 13, also fig. 10). Quite a number of cells have filament bundles, running some distance along the cell border, or organized in a stellate configuration at the cell edge. At the marginal parts of a trabeculum, cells were encountered in which clear filament bundles or stress fibres were predominant. Those bundles do not have a fixed diameter and are arranged in stellate (fig. 12) or parallel (fig. 13) patterns. The occurrence of microvilli at the substrate side of the membrane and filament bundles at its cytoplasmic side appears to be mutually exclusive. From stereo micrographs it was observed that the filament bundles have a very close apposition to the membrane, and that they invariably form side branches, often perpendicular to

their long axis, which become part of the fine fibrous meshwork. These observations were further substantiated by replicas made of dry-cleaved cells and rotary shadowed at an angle of 10”. Such replicas only reveal line details of structures just above the surface [9, 191. As shown in fig. 14 stress Iibres are readily visible branching into an extensive meshwork of 4-7 nm filaments covering the entire surface-the filamentous web. The rather uniform meshwidths measure about 25-50 nm and although seemingly random, some repeating patterns may be distinFigs 15-23. Filarnentous web. Figs 15-17. Preparation as in fig. 14. Three patterns distinguishable in the filamentous web. All images x45 500. Fig. 15.

Pattern formed by sets of parallel running fdaments (outlined). Fig. 16. Spider-web pattern (squnre). Fip. 17. Hexagonal nattem of coated membrane (ckles). The coats at-the upper left comer are part of a spider-web pattern (centre indicated by arrowhead) as shown by the untrimmed image of this micrograph. Fig. 18. SEM micrograph. Upside-down view of a monolayer of detergent-treated hepatocytes. Cells were fixed, treated with Triton X-100 and critical point-dried as described (Methods). Subsequent adhesion to adhesive tape detaches whole cells from their substrate without cleaving. Cells can be observed with an abundance of microvilli (upper left) or with no microvilli at all (centre and lower right). In the latter a clear outlme‘of stress fibers is apparent (arrowheads). Tilt angle 45”. x 3 400. Fig. 29. Preparation as in fig. 18. Stress fibre endings at the cell border, as shown by tighhtly packed parallel running filaments (FB). Tilt angle 45”. x20000. Fig. 20. Preparation as in fig. lg. Surface architecture showing pattern of fdaments strikingly similar to those of the fdamentous web in figs 15-17. Tilt angle 45”.

x34ooo. Fig. 21.

Upside-up view of detergent-treated hepatocytes. Surface shows similar fiiamentous pattern as in upside-down preparations. I&a the microvillar surface reveals this pattern. Note the clear spider-web pattern in the upper centre of the picture (centre indicated by arrow). Tilt an le 45”. X34OftO. Fig. 22. Thin section of $9ntpn X - lOO-treated hepatocyte. Connected dots at the cell surface represent a cross-sectional view of the filamentous web (arrows). A coated vesicle is indicated (large arrow). x34ooo. Fig. 23.

Grazing section of a surface microvillus vealing the fdamentous web. x 34000.

re-

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Table 2. Relationship

between

Count of vesicles within areas (class B)

Expt 1 Expt 2

coated vesicles andfilamentous

aggregates

1 vesicle diameter of a filamentous aggregate (class A) and of those outside these

Vesicles in class A n

Filament areaa

A-vesicle density

Vesicles in class B n

Void areaa

B-vesicle density

Ratio A : B densities

1 223 1 275

888.1 669.9

1.377 1.903

54 56

602.4 625.4

0.090 0.090

15.3 21.1

a Arbitrary surface units.

guished in favourable areas: (1) patterns formed by crossing two or more sets of parallel-running filaments (fig. 15); (2) spider-web patterns (fig. 16); and (3) hexagonal patterns of coated membrane (fig. 17; see ref. [ 193). When cells prefixed with 0.1% GA for 5 min are treated with 1% Triton X-100 (see Methods), virtually all membrane, except for the inner mitochondrial membrane, is dissolved without other disturbances of cell structure. In an attempt to dry-cleave such cells to visualize the membrane-extracted meshwork, no cleavage occurred but inverted whole cells were glued on tape, ex-

I 60

60

100

1 120

I 140

Vesicle diameter (nm) Fig. 24. Coated vesicles measured from WMTEM of hepatocytes fixed by the standard procedure and dry-cleaved. Exp Cell

Res

132 (1981)

posing their substrate sides (fig. 18). SEM observation of these cells disclosed a surface architecture strikingly similar to the filamentous web described above (fig. 20). Obviously the filaments observed were thicker due to a gold/palladium coat of about 15-20 nm, but still the same overall meshwidths and same patterns were apparent. In fact, despite lower resolution, a better image of the web was obtained, since no other structures masked its observation compared with the inside-view in normally dry-cleaved preparations. Even the presence of filament bundles could be inferred from the parallel arrangement and close apposition of filaments in parts of the upside-down cell aggregate (fig. 19). In addition, all surfaces of the hepatocyte, including those of the intercellular face and including the microvilli, exposed the filamentous web (fig. 21). In thin sections of embedded material the web could be inferred from the repetition of interconnected dots at the cell surface (fig. 22). It could be actually seen in grazing sections of microvilli (fig. 23). Coated-vesicle occurrence and development

An interesting in standardly the abundance heights above

feature most readily visible dry-cleaved preparations is of coated vesicles at variable the adherent membrane (figs

Cytoskeleton

and coated vesicles

in hepatocytes

179

Fig.

25. WMTEM of adherent membrane of hepatocytes fixed by the standard procedure and drycleaved. Distribution of coated vesicles parallel to filament bundles. Virtually all vesicles appear to be

connected to fdaments. No overlapping of filament bundles and coated vesicles occurs. Voltage 100 kV. x5oooo.

11, 12, 13). Hepatocytes perform considerable endocytotic activity [5, 17, 20, 21, 29, 38, 471, part of which being reflected apparently by the formation of coated vesicles at the cytoplasmic side of the membrane. As shown in fig. 24 the vesicle diameter was normally variable with an

average diameter of 82 nm. Observation of dry-cleaved cells such as shown in fig. 11 suggested the distribution of coated vesicles to be non-random and restricted, laterally, to filamentous aggregates (see above). To evaluate this suggestion we counted the vesicles located within one vesicle diameter E.rp Cell

Res 132 (IYRl)

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Cytoskeleton of an aggregate (A) and those located outside these areas (B). Then we measured the respective area surfaces and calculated the vesicle density of both classes. Table 2 lists the distribution densities found in two experiments and shows an average A/B ratio of 18 : 1. In cells with filament bundles coated vesicles rarely occupy space overlapping the bundles, but frequently are found associated with side branches or bundle termini. As such, series of coated vesicles can be found laterally aligned with stress fibres (fig. 25). According to Heuser [19], stereo micrographs made it possible to deduce developmental stages in coated-vesicle formation, based on the extent of curvature shown by their coats. Shown in fig. 26 are three stages that were distinguished: (1) Earlystage, presented by a flat (fig. 26a) or slightly curved (fig. 266) coat, highly integrated in the filamentous web (see also fig. 17). The coat seems to result from additional linkages within a spider-web pattern of the filamentous web. In upside-down preparations of Triton X-100 treated cells for SEM (see above) pits could be observed suggesting a developing coated vesicle at the cell’s interior [2, 17, 191, and being the very centre of a spider-web pattern (fig. 27). (2) Mid-stage, typified by a progressive curvature of the coat. At this stage of development the coat delimits part of a con-

Fig. 26. Stereo micrographs, WMTEM as in fig. 25. Coated vesicle formation as distinguished in three stages: (1) early-stage, presented by a flat (a) or slightly-curved coat (b); the coats are integrated in a spider-web pattern of the fdamentous web (centres indicated by arrows), (2) mid-stage, progressive curvature of the coat (c, d); the coats are connected to branches of a filament bundle; (3) final-stage, coated vesicles with radial suokes (e, f, arrowheads) connected to filament bundles (FB) or fdamentous aghalo’s gregates (FA). Note the electron-translucent formed by the fnamentous aggregates (e). Voltage (a, e,f) 100 kV; (b, c, d) 80 kV. x80000.

and coated vesicles in hepatocytes

181

Fig. 27. SEM preparation as in fig. 21. Pit in cell surface being the centre of a spider-web pattern in the fdamentous web (arrow). Tilt angle 45”. x20000.

densed area of the filamentous web or is directly connected with a branch of a filament bundle (fig. 26c, d). (3) Final-stage, coated vesicle in its strict sense, linked by radial spokes to a filamentous aggregate or filament bundle (figs 25, 26e, f). The arrangement of the filamentous aggregate at some distance around the vesicle often creates an electron translucent halo, in which the radial spokes are clearly visible (fig. 26~). In favorable cases as many as 10 spokes can be counted per vesicle. DISCUSSION The technique of dry-cleaving introduced in this paper has been shown to offer considerable potential for the study of membrane-associated structures. The introduction of artefact by this technique is limited by that of standard electron microscopic fixation and dehydration techniques and therefore well documented. This is an advantage over those techniques using a buffer stream to blow off attached cells [8, 111, which gives comparable preparations of adherent cell membrane, but at the cost of considerable shearing forces applied Exp Cd

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to the living cell. Furthermore, dry-cleaving basically effects all cells which become attached to the adhesive tape similarly, and therefore produces a reliable image of the in situ situation of cultured cells. The usefulness of dry-cleaving for other cell types than primary cultured hepatocytes has yet to be established. However, the application of adhesive tape on dry cells opens up a variety of preparative possibilities as long as the conditions chosen are such that fixed-cell cohesiveness is weaker than cellsubstrate and cell-glue adhesions. It has been shown in this study that the combination of dry-cleaving and WMTEM allows visualization of structures at both sides of the single-fold adherent membrane and facilitates the making of topographical correlations. Interpretation difficulties due to superposition were overcome by the use of stereo micrographs [50] and by rotaryshadowed replicas [9, 191, which produced clear images of the cytoplasmic side of the adherent membrane. Moreover, extraction of membrane by Triton X-100 after brief fixation with 0.1% GA prevented cleavage, but enabled us to obtain an outside view of the subplasmalemmal structure system by means of SEM. The combination of these techniques has produced strong evidence for the existence of a filamentous web apposed to the cytoplasmic side of the hepatocytic membrane. Since the same distinct configurations of this web in cleaved preparations can be observed at the entire cell surface in detergent-treated preparations, we propose that in both cases the same web is visualized from two different sides. Especially the outside view of the web reveals a strikingly regular system of criss-crossing filaments, possibly similar to the ‘diagonal filaments’ described by Small [43], spider-web patterns and filament bundles, all of which Exp

Cell

RES 132 f/98/)

constitute a two-dimensional filamentous system [51]. The inside view of the web shows its continuity with filament bundles or stress fibers and less organized filamentous constellations described as filamentous aggregates or condensations. The whole system can be considered to be a twodimensional framework supporting the plasma membrane and stratified by both vectorily and focally organized filament complexes. The web itself may be analogous to the spectrin-actin membrane skeleton as described in erythrocytes [38, 511. No direct biochemical data concerning the web are yet available, but actin associated with the medium-facing side of the membrane has been shown in primary hepatocyte cultures [27, 42, 491. Also preliminary experiments indicate the web’s sensitivity to cytochalasin B (our unpublished SEM observations, see ref. [25]). Moreover, the filament diameter of the web measures about 4-7 nm and therefore the presence of actin as one of its constituents may well be expected. Obviously, with respect to the importance of the web, many speculations can be made referring to structural support and functional control of membrane phenomena. However, we like to point out that the very presence of the web subdivides the membrane into small domains of about 1000-2 000 nm2, bordered by filaments which may have associations with membrane proteins [4, 7, 231, and thus may influence the molecular composition and kinetic behaviour of a particular domain [6,

101.

This consideration becomes the more important by the observations of coated vesicle formation, a process involved in receptor-mediated endocytosis in many cell types and believed to function in hepa-

Cytoskeleton tocytes for the uptake and secretion of serum components and liver products respectively [5, 17, 201. Early-stages of vesicle development, defined by flat coats, seem to coincide with so-called spider-web patterns of the filamentous web. In addition, cell surface pits considered to be the site of accumulation for material to be endocytosed [2, 17, 191, are observed to be the center of spider-web patterns in SEM preparations. This indicates that the spider-web may constitute a specific configuration of the filamentous web, possibly involved in the aggregation of membrane proteins [2, 17, 21, 361. Concerning the occurrence of spider webs two alternatives can be entertained: (1) spider-web patterns form as a joint event during accumulation of coat material; (2) spider-web patterns preexist and coated vesicle formation occurs preferentially at these locations. So far, our micrographs provide evidence for the second possibility, since spider-web patterns do occur in which the presence of coat material is at least doubtful. On the other hand, aggregation of membrane proteins may in general involve spider-web pattern formation, while not all of these events precede the development of coated vesicles [5, 17, 391. Both possibilities are equally intriguing. What is important is the obvious logic of the pattern, if one considers filaments to support some centripetal translocation of membrane components [ 10, 361. The next two stages distinguished in the development of coated vesicles, mid-stage and final-stage, both proceed with connections to a filamentous aggregate or filament bundle. Mid-stage coated vesicles appear to be directly attached to a small bundle of filaments or condensation of the fdamentous web. In the final stage the coated vesicle contains spokes radiating from its sur-

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face and connected to a nearby fdamentous aggregate that, by its particular location, often creates a translucent halo around the vesicle. This arrangement suggests a kind of interaction between the vesicle and the filamentous aggregate. Associations between vesicle coats and filaments have been observed [ 13, 191, as well as an equal distribution of stress fibres and coated vesicles on the light microscopic level [l]. Drycleaving readily revealed the coated vesicle distribution on the electron microscopic level and we were able to show that coated vesicles are almost exclusively distributed along areas with filamentous aggregates (see also [41]). Two patterns could be distinguished: (1) a more or less random distribution in membrane areas provided with microvilli; (2) a linearly distribution along stress fibres. No overlapping of coated vesicles and stress fibres was found, although it cannot be excluded that overlapping may occur in a plane above the plane of cleavage. This possibility however is not supported by cross section data (not shown). It is tempting to speculate that the peculiar connection by means of radial spokes to filaments and the position along these filaments are significant for selective transport of the vesicle to either lysosomes, Golgi apparatus or nucleus [ 17, 391. As remarked by Heuser [19] it cannot be excluded that connections observed are artefactually induced by GA fixation. However, we believe that the spatial interrelationship between vesicles and filamentous aggregates strongly argue for a functional dependency irrespective of the fact whether the connections seen are real. In conclusion, coated vesicle formation appears to be intimately associated with filaments. The particular constellation of these filaments becomes more complicated at progressive stages of vesicle development. Presumably Exp Call

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they may lend support for transportation the coated vesicle to its final destination.

of

We acknowledge the hospitality of the Laboratory for Electron Microscopy of the University of Amsterdam for use of their SEM facilities and the expert technical assistance of Cor Bakker. We are grateful to Dr C. A. Feltkamp and Dr 0. P. Middelkoop for stimulating discussions, and to Mr I. V. van de Pave& Mr N. Ong and Mrs M. A. van Halem for technical and administrative support.

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