FREEZE-ETCHED SURFACES OF MEMBRANES AND ORGANELLES IN THE CELLS OF PEA ROOT TIPS

Transcription

1 J. Cell Sci. 3, (1968) I0.0. Printed in Great Britain FREEZE-ETCHED SURFACES OF MEMBRANES AND ORGANELLES IN THE CELLS OF PEA ROOT TIPS D. H. NORTHCOTE AND D. R. LEWIS Department of Biochemistry, University of Cambridge SUMMARY The structure of the inner and outer surfaces of the plasmalemma, the tonoplast and the membranes of the nucleus and endoplasmic reticulum have been investigated. The structure of the plasmalemma probably varies with the metabolic state of the cell, in particular with the synthesis and transport of material for cell-wall formation. The organization of the plasmalemma during the deposition of material in the wall by reverse pinocytosis is shown, and evidence is presented for the possible synthesis of the microfibrillar structure of the wall by synthetic units arranged as particles on and near the plasmalemma surface. A clear indication of substructure along the length of microtubules has been shown, and since views of large surface areas are possible the distribution of the microtubules at the cytoplasmic surface inside the plasmalemma has been revealed; they bound the cell by running around the circumference in one direction only. A definite organization of nuclear pores has been observed and the structure and shape of the pores is described. By the freeze-etch technique it is possible to investigate crystalcontaining bodies, amyloplasts with starch grains and bundles of fibres in addition to mitochondria and Golgi bodies. The method also distinguishes certain spherical organelles by their characteristic surface appearance. INTRODUCTION The technique of freeze-etching (Moor, Miihlethaler, Waldner & Frey-Wyssling, 1961; Moor & Miihlethaler, 1963) allows the surfaces of membranes to be studied, and gives information about the relationship of these surfaces to one another, thus making it possible to suggest functions for the membranes in the synthetic and transport mechanisms of the cell. Cellulose can be recognized by its specific microfibrillar structure and the structural relationship of the plasmalemma and microfibrils can therefore be described in terms of mechanisms for cellulose synthesis. However, there are several alternative interpretations of the images produced by the freeze-etch technique (see Discussion), and this makes any suggestion about the functions of the observed structures very tentative indeed. This paper will report an investigation of the surfaces of the plasmalemma during the period when the cell wall was growing, and will describe the appearance, substructure and distribution of microtubules and the surfaces of various cell organelles. 13 CeU Sci. 3

2 200 D. H. Northcote and D. R. Lewis MATERIALS AND METHODS Peas ('Feltham first') were soaked overnight in water and germinated on cotton wool saturated with water at 8 C for 5 days in the dark. They were then transferred to cotton wool saturated with 20 % glycerol (v/v) and kept at 8 C in the dark. The roots were examined at periods from 1 to 10 days from the incubation with glycerol. The root tip was sliced in half longitudinally under 20 % glycerol and about 2 mm length of the tip used. The tip section was placed on a copper electron microscope grid (3 mm diameter) on the top of a small amount of pressed baker's yeast also moistened with 20 % glycerol. The yeast acted as an adhesive and also served as a constant check of the performance of the subsequent freeze-etch technique. The freeze-etch technique employed was that of Moor & Miihlethaler (1963), using a commercial Balzers (Liechtenstein) freeze-etching apparatus. The specimen on the copper grid was held under Freon at the temperature of liquid nitrogen for 10 sec and then quickly transferred to liquid nitrogen and left till required, when it was placed on the cutting stage of the microtome which was maintained at 150 C and then raised to 100 C before fracture with a knife held at 150 C. The specimen was etched at 100 C at a pressure of 3 x io" 8 torr, and shadowed with Pt/C for 3 sec at an angle of 30. The replica was strengthened by evaporation of carbon on to it for 8 sec. The replica after thawing was floated off the grid under water and then left in sodium hypochlorite solution (10-14% available chlorine) containing 7-5 g NaOH/100 ml for 48 h. It was then washed with water and soaked in 72% (w/w) sulphuric acid for 24 h, washed with water again and taken up on a carbon-coated electron microscope grid and examined with an AEI EM 6 B electron microscope at 60 kv. RESULTS Interpretation of the image produced by the freeze-etch technique The three-dimensional character of the image can be ascertained by a close inspection of the shadows which appear white on the photographs. Organelles which are surface-fractured may be identified either as objects which are sticking out of the principal fracture plane, when their shadow extends beyond the boundary dimension of the organelle, or as objects which have been scooped out of the fracture plane, when their shadow is contained within the boundary of the organelle. Examination of the shadows in this way can indicate clearly the relative positions of the various membranes and organelles in the depth of the replica. A study of the surface images of the membranes of organelles such as the endoplasmic reticulum and nucleus shows that in some specimens the surface image runs continuously into a cross-fractured or transverse-sectioned image (Figs ), in which the membrane appears as a thin line. The fracture of the membrane is seen therefore to pass along the inner or outer external surface of the membrane and the membrane has not been split so that its internal structure is revealed. The interpretation of the freeze-etch image is considered again in the Discussion section of the paper.

3 Membrane and organelle surfaces 201 Plasmalemma The surface appearance of this membrane can take several forms. The more typical appearance of the outer surface of the membrane is shown in Figs. 1, io, 12 and 14. The particles on the surface are A in diameter and in places they appear to be organized in rows (Fig. 4). The inner surface of the plasmalemma also carries similar particles (Figs. 2, 4, 12). Pit fields are a conspicuous feature of the surface of the plasmalemma and these are usually organized in definite regions as circular patches, which may contain as few as 8 or as many as 30 plasmadesmata (Figs. 3,4); sometimes a more scattered series of plasmadesmata is found. From the outer surface the group of pores is seen to be raised slightly above the general surface of the plasmalemma, and this is apparent in cross-fractured sections through a pit field. On the fractured outer surface of the plasmalemma the pores have slightly raised edges, and in many instances the centre of the pore contains a small protuberance which may represent the remnants of the pore contents (Fig. 1). The inner surface of the plasmalemma also carries particles and the pores of the pit field are seen as invaginations of the surface. In addition to this more usual appearance, the plasmalemma occasionally presents quite a different image. In these the replica indicates that the surface has been pushed outwards into a series of bulges and folds, and in places an organized distribution of the substructural components of the membrane can be seen (Figs. 5-7). In Fig. 5 both the outer and inner surface of the plasmalemma with these formations can be seen. In many images of cross-fractured cells a large number of vesicles can be seen very close to the cell wall at the plasmalemma (Figs. 17, 26). Relationship of the plasmalemma to microfibril organization The microfibrillar organization and distribution in the cell wall can be seen very clearly in the freeze-etch preparations. The microfibrils are A in diameter and in places they seem to radiate from small point sources within the wall structure (Figs. 2, 11). Amongst the microfibrillar mass, particles of the same size as those at the surface of the plasmalemma are apparent (Figs. 11, 12). The plasmalemma shown in Fig. 12 has been partially stripped and folded back from the underlying wall microfibrils, so that in this preparation the upper and lower surfaces of the same membrane can be seen. In addition, the upper surface of the plasmalemma of a cell fractured at a lower plane is shown. In some preparations the wall fractures at different levels and shows the more randomly oriented microfibrils of the primary wall and the more organized structure of the secondary wall (Fig. 12). During the fracturing process some of the fibrils are quite often splintered and lifted out of the surface of the fracture plane, and these give the appearance of raised fine needle-like projections with long shadows (Fig. 2). Microtubules For the identification of these it was necessary to establish that the structures observed lay on the cytoplasmic side of the plasmalemma and that they were not microfibrils lying along the wall side of the plasmalemma. This was clearly indicated in 13-2

4 202 D. H. Northcote and D. R. Lewis Fig. 13, where the cytoplasmic surface exposed by the fracture lay directly under the plasmalemma. The plasmalemma in Fig. 13 has faults and folds along its surface and these correspond to similar faults and folds in the underlying surface of the cytoplasm. In this image the microtubules can be seen at this cytoplasmic surface just underneath the plasmalemma. In other fractured preparations the plasmalemma has been stripped away completely from the underlying cytoplasm to give a surface on which the microtubules are clearly distinguished (Figs. 14, 15). The microtubules in groups of 2-4 bound the cytoplasmic surface of the cell and in places a microtubule crossed over and bent from one group to another. There was no overall pattern to the distribution except that the cytoplasm of the cell was bounded by the tubules in one direction round the cell and no tubules running right round the cell at right angles to the general direction were seen. Because of the crossing over of certain tubules from one group to another, in any particular area of the surface it was possible to see tubules running apparently at right angles to one another (Fig. 15). The preparations give a clear indication of the organization and distribution of the tubules at the cell surface and it is apparent that the microtubules ( A in diameter) show a distinct substructure along their length (Figs. 14, 16). Nuclear surfaces The surfaces of both the membranes of the nucleus are visible and the pattern of pore distribution is clearly seen. Like the other membranes of the cell, the membranes of the nuclear envelope carry particles. The outer face of the inner membrane carries more particles than the outer face of the outer membrane. These particles are approximately 130 A in diameter (Fig. 18). The pores are organized in curved interconnecting rows arranged in such a way that nearly circular areas are free of pores (Figs. 19,22,23). Each pore is approximately 1180 A in diameter and appears to be filled with a plug of material which arises within the nucleus and passes through an aperture in the outer membrane and is raised above the fractured surface of this membrane (Figs. 18, 23). When the outer membrane has been stripped away to expose the inner nuclear membrane it can be seen that at the site of the pore the inner membrane is slightly raised at the edges, this probably representing the membrane junction between outer and inner envelopes (Fig. 18). The nuclear pores are visible in cross-fractured sections (Figs. 17, 20), and in the interior of the nucleus, the nucleolus is revealed by a different texture of the replica surface (Fig. 17). Vacuolar membrane The tonoplast is plainly visible as a single membrane in cross-fractured sections (Figs. 20, 25). It is often convoluted at places to give the appearance of material being pinched off into the lumen of the vacuole (Fig. 9). Sometimes, however, in the interior of small vacuoles, ice crystals are present which are not found in the other parts of the cell (Fig. 10). In surface view the vacuolar membrane has a characteristic granular appearance,

5 Membrane and organelle surfaces 203 enabling it to be distinguished from the nuclear membrane and plasmalemma. Like these membranes, however, it carries small particles on both its inner and outer surfaces (Figs. 2, 8, 10). Endoplasmic reticulum The surface membranes of the endoplasmic reticulum are clearly visible and can be seen in close juxtaposition to the nucleus and cell membrane. Like all the other membranes they carry particles ( A in diameter) on both the inside and outside surfaces (Figs ). Examination of Figs allows the individual membranes of the endoplasmic reticulum profile seen in cross-fracture, to be easily related to the surface views. Other organelles Although mitochondria can easily be recognized in cross-fracture when the internal cristae can be seen (Fig. 19), they are much more difficult to distinguish as a surface fracture. A great number of surface-bounded vesicles and organelles are apparent in the replica of the cytoplasm of a freeze-etched cell, and some of these represent fat droplets and other non-etchable material which are not necessarily bounded by a membrane. However, it is possible to distinguish several distinct types of organelle, some of which can be identified by comparison with the images obtained with standard sectioning techniques. The cross-fractured and surface images of Golgi bodies (Fig. 10) have already been described (Branton & Moor, 1964). In addition, organelles such as amyloplasts containing starch grains (Fig. 24), crystal-containing bodies (centre-centre spacing 170 A) (Figs. 27, 29), (see Cronshaw, 1964) and bundles of fibrils (Fig. 8) can readily be identified. Two other distinct organelles can be seen in surface view. One of these is a spherical structure with a distinct pattern of protuberances (450 A in diameter) over its surface (Fig. 25), of which there is about one per cell; the other is a larger body with a distinct patterned substructure in its outer membrane (Figs. 26, 28) which can be seen only infrequently in certain replicas. DISCUSSION It is possible to interpret the surface images of replicas formed from the freezeetch technique in various ways. The alternatives are that: (1) the image could be the upper or lower surface of the membrane, (2) the surface image is the imprint on the underlying material of the original membrane which has been fractured away, or (3) the fracture line has split the membrane to reveal inner surfaces of the membrane not otherwise visible. To decide between these alternative explanations for the image of any one surface seen in a replica it is necessary to have a good knowledge of the normal appearance in thin sections of the particular tissue, to assess the replica image in terms of the shadows it casts and the relative position of known structures within the cell, and to examine enough pictures to be reasonably certain that the alternative interpretations do not better explain the total pictorial evidence. The most serious and difficult alternative to decide upon is the third of these listed above. Branton (1967 a)

6 204 D. H. Northcote and D. R. Lewis has suggested that nearly all the surface views of freeze-etched material represent pictures of the internal structure of the membrane and he has detailed the arguments in favour of this interpretation (Branton, 1967a, b; Branton & Park, 1967). The results of the work reported in this paper do not rule out the possibility that fractures of the type envisaged by Branton might occur. But a study of the images produced from the pea root tissue indicated that they were best interpreted as replicas of the external surfaces of the membranes or of the underlying surfaces after the membranes had been fractured away. The studies showed that the plasmalemma did not have a constant appearance and that its surface and substructural organization varied during growth and the accompanying changes in metabolism of the cell. The bulges seen in some images of the plasmalemma could represent the discharge into the wall of material contained in the Golgi vesicles; this is known to occur in actively growing cells (Northcote & Pickett- Heaps, 1966). The structure of the plasmalemma not only showed the apposition of the internal vesicles upon it, but indicated that changes in its own structure had occurred to accommodate these vesicles by the reverse pinocytotic process, since the image of the plasmalemma showed that the surface substructure was not homogeneous. Patches of organized material were visible which could represent different functions and altered permeability of the membrane during this active metabolic condition. Another possible function of the plasmalemma is its role in cellulose synthesis. A recent report of freeze-etch studies on Chlorella (Staehelin, 1966) indicated that the particles seen at the surface of the plasmalemma were concerned with microfibrillar synthesis. The work reported here would support the view that a similar process occurred in higher plants, since the microfibrils of cellulose were seen in some instances to arise and radiate from a point centre, and these centres were occasionally located near the plasmalemma. Particles were present on the plasmalemma surface and occasionally these were organized in rows suggestive of microfibrillar synthesis. In addition, the particles were seen within the microfibrillar structure and Staehelin (1966) suggested that in Chlorella these particles not only gave rise to microfibrils but that they migrated from the plasmalemma into the wall structure to sites of microfibrillar synthesis. However, suggestive as this evidence is, it is by no means conclusive since the particles seen on the plasmalemma and within the wall were ubiquitous, and were found on all membranes. They were even found on the freeze-etched surface of cross-fractured starch grains (Fig. 24). It may be that they represent units concerned with synthesis, but it may also be that they are artefacts of the freeze-etch technique. The observations of the microtubules by the freeze-etch technique showed their distribution and gave some indication of their substructure. Subunits were visible along their length but the resolution of the micrographs did not permit any detailed analysis of the structure. When the distribution of the pores on the nuclear membrane was examined on large areas of the surface a distinct organization became apparent and they were seen to occur in short rows which encircled patches of membrane where no pores occurred. The outer membrane of the nucleus could be fractured away from the inner envelope

7 Membrane and organelle surfaces 205 and pictures of replicas of these preparations showed a plug of material which arose from inside the nucleus and passed out through the apertures in the membrane. Some of the profiles of the holes in the membrane, although not clearly octahedral as demonstrated by the negative-staining technique used by Gall (1967), showed a polyhedral outline in which some straight sides and angles between these sides were distinguished. When the fracture line passed through an organelle to give a cross-fracture the image was similar to that seen in sectioned material and it was easy to recognize organelles such as mitochondria and Golgi bodies which have distinctive features in cross-section. Surface views were more difficult to recognize. However, some surfaces of the organelles seen in the replicas were distinctive, and it was possible to recognize an organelle in the meristematic root cell which had regular protuberances over its surface and one in which there was an ordered substructure to the outer membrane. It is possible that the technique offers a method of distinguishing between spherical organelles which occur in the cell and which in section would look very similar to one another. We wish to thank the Science Research Council for grants for the purchase of the freeze-etch apparatus and the electron microscope. REFERENCES BRANTON, D. (1967a). Fracture faces of frozen membranes. Proc. natn. Acad. Sci. U.S.A. 55, BRANTON, D. (19676). Fracture faces of frozen myelin. Expl Cell Res. 45, BRANTON, D. & MOOR, H. (1964). Fine structure in freeze-etched Allium cepa L. root tips. J. Ultrastruct. Res. n, BRANTON, D. & PARK, R. B. (1967). Subunits in chloroplast lamellae. J. Ultrastruct. Res. 19, CRONSHAW, J. (1964). Crystal containing bodies of plant cells. Protoplasma 59, 318. GALL, J. G. (1967). Octagonal nuclear pores. J. Cell Biol. 32, 3 QI MOOR, H. & MUHLETHALER, K. (1963). The fine structure in frozen-etched yeast cells. J. Cell Biol. 17, MOOR, H., MUHLETHALER, K., WALDNER, H. & FREY-WYSSLING, A. (1961). A new freezing ultramicrotome. J. biophys. biochem. Cytol. 10, NORTHCOTE, D. H. & PICKETT-HEAPS, J. D. (1966). A function of the Golgi apparatus in polysaccharide synthesis and transport in the root cap cells of wheat. Biochem. J. 98, STAEHELIN, A. (1966). Die Ultrastruktur der Zellwand und des Chloroplasten von Chlorella. Z. Zellforsch. mikrosk. Anat. 74, (Received 27 September 1967)

8 206 D. H. Northcote and D. R. Lewis BBR #^ / g i inm m n 0 EVIATIONS ON PLATES direction of shadowing by platinum bundle of fibrous material Golgi body inner surface of endoplasmic reticulum inner nuclear membrane mitochondria nucleus outer surface of endoplasmic reticulum onm pi po S t(o) V w outer nuclear membrane inner surface of plasmalemma outer surface of plasmalemma spherical body with protuberances tonoplast (outer surface) vacuole wall Fig. i. Outer plasmalemma surface carrying particles and showing structure of the plasmadesmata. x Fig. 2. Fracture through a cell showing inner surfaces of the tonoplast and plasmalemma. Numerous microfibrils have been raised above the fracture plane of the wall; in places the microfibrils can be seen to radiate from point sources, x Fig. 3. Outer plasmalemma surface at a pit field composed of about 30 plasmadesmata. x Fig. 4. Inner and outer plasmalemma surfaces. The contents of the cell at the left have been fractured away, so that the inner plasmalemma surface has been exposed. A portion of the wall between the upper and lower cells is visible. The particles on the upper plasmalemma surface are in places arranged in rows (arrows). Pit fields composed of about eight plasmadesmata are present, x

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10 Fig. 5. Outer and inner surfaces of the plasmalemma. The cell at the right has been fractured away so that the inner plasmalemma surface has been exposed. A portion of the wall between upper and lower cells is visible. Vesicles are apposed to the plasmalemma and the folds around them are apparent, x Fig. 6. Outer surface of the plasmalemma at which vesicles are apposed. Longitudinal patches of an organized substructure of the membrane can be seen in relation to the apposed vesicles, x Fig. 7. Outer surface of the plasmalemma at which vesicles are apposed. Some of the vesicles have been cross-fractured and the structure around the vesicles can be seen, x

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12 Fig. 8. Fractured cell in which the tonoplast and a bundle of fibres are visible, x Fig. 9. Cross-fractured section through a vacuole; a vesicle is apparently being pinched off into the vacuole contents, x Fig. 10. Cross-fracture through a cell. The vacuole contains small ice crystals. The outer surface of the plasmalemma and tonoplast and the membrane surfaces of the Golgi cisternae are visible, x Fig. 11. Freeze-etched portion of a cell wall in which the microfibrils can be clearly seen. The microfibrils appear to radiate from definite loci within the wall structure, x Fig. 12. Outer and inner surfaces of the plasmalemma and the relationship of the plasmalemma to the cell wall. The contents of the cell on the left have been fractured away, the inside surface of the plasmalemma has been exposed, and at the edge of the cell it has been folded back to reveal the outside surface of the membrane and the underlying microfibrils. Particles are present on the plasmalemma surfaces and within the microfibrillar mass, x

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14 Fig. 13. Plasmalemma outer surface and the underlying cytoplasm. Microtubules are visible immediately under the plasmalemma. x Fig. 14. Distribution of microtubules immediately under the plasmalemma at the surface of the cytoplasm. The microtubules run in one direction with reference to the cell axis. A substructure along the length of the microtubules is apparent, x Fig. 15. Fractured cells which show the distribution of microtubules at the cytoplasmic surface. The cell at the upper right has been fractured at a much deeper level in the cytoplasm. In this cell the inner surface of the plasmalemma is visible, x Fig. 16. High magnification of microtubules to show the subunits (arrows) along their length, x

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16 Fig. 17. Cross-fracture of two cells. The nucleus, nucleolus and cross-fractures of the nuclear membranes are visible. Small vesicles close to the cell wall at the plasmalemma are present, x Fig. 18. Surface-fracture of a nucleus. The outside surfaces of the outer and inner membranes of the nucleus are shown. Both surfaces carry particles, but the inner membrane is more densely covered than the outer. The structure of the pores is visible and the cross-fractured images of these structures have a polyhedral rather than a circular profile (arrows), x Fig. 19. Surface-fracture of the outside surface of the outer membrane of a nucleus to show the organization of the pores. The mitochondrion on the left of the picture has been cross-fractured and the cristae are visible, x Fig. 20. Cross-fracture through a vacuole and nucleus. The structures in the vacuole are due to invaginations of the tonoplast (compare with Fig. 9). The nucleus is at the extreme left-hand corner of the picture; the double nuclear envelope and crossfractured nuclear pores are shown, x

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18 Fig. 21. Surface- and cross-fractured images of the endoplasmic reticulum. Inside and outside surfaces of the endoplasmic reticulum are shown. The surfaces and membranes can easily be followed from the cross-fractured to the surface images, so that the identification of the structure which has been fractured is apparent, x Fig. 22. Surface- and cross-fractured images of the endoplasmic reticulum and surfacefractured image of the nucleus. The surface- and cross-fractured images of the endoplasmic reticulum are continuous. Both the inner and outer membranes of the nucleus are visible, x Fig. 23. Surface- and cross-fractured images of the endoplasmic reticulum and surfacefractured image of the nucleus. Both the inside and outside surfaces of the endoplasmic reticulum membranes carry particles, x

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20 Fig. 24. Cross-fractured image of an amyloplast carrying starch grains. The fractured surfaces of the starch grains carry particles, x Fig. 25. Surface-fracture of a spherical organelle which has small protuberances distributed over its outer surface, x Fig. 26. Surface-fracture of a spherical organelle. The substructure of the outer membrane is arranged in an ordered manner (see Fig. 28). Vesicles are closely applied to the plasmalemma at the wall, x Fig. 27. Cross-fracture of a crystal-containing body (see Fig. 29). x Fig. 28. High magnification micrograph of the ordered membrane structure shown in Fig. 26. x Fig. 29. High magnification micrograph of the crystalline pattern shown in Fig. 27. x

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