COMPLEMENTARY PLASMA MEMBRANE FRACTURE FACES IN FREEZE-ETCH REPLICAS

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1 J. Cell Set. 12, (1973) 445 Printed in Great Britain COMPLEMENTARY PLASMA MEMBRANE FRACTURE FACES IN FREEZE-ETCH REPLICAS N. E. FLOWER Physics and Engineering Laboratory, Department of Scientific and Industrial Research, Private Bag, Lower Hutt, New Zealand SUMMARY Recent studies have all shown that membranes fracture internally during freeze-etching or freeze-cleaving, confirming that 2 internal membrane faces are produced. That these faces are not complementary is one of the main features of freeze-etch replicas still requiring explanation. In the present investigation certain plasma membranes in light organs of the New Zealand glow worm are found to be an exception to this general rule of non-complementarity, as there is a sufficient number of holes present in one membrane face to match the number of particles on the other. This finding suggests that most of the theories put forward to explain noncomplementarity do not apply and that holes are probably lost in most membrane faces by distortion during fracturing. INTRODUCTION Recent studies (Chalcroft & Bullivant, 1970; Sleytr, 1970; Wehrli, Miihlethaler & Moor, 1970) of the 2 replicas obtained by fracturing specimens and replicating both fracture faces have all supported Branton's hypothesis (Branton, 1966) that membranes fracture internally during freeze-etching. If fracturing proceeded without distortion of the faces, and there was no subsequent alteration prior to, or during replication, then the 2 faces produced should be exactly complementary. That this complementarity does not, in fact, exist on the membrane faces produced is one of the main features of freeze-etch replicas still requiring explanation. Small numbers of depressions have been observed on general plasma membrane faces (Bullivant, 1969; Chalcroft & Bullivant, 1970) but never in sufficient numbers even to suggest complementarity of the 2 faces. Only in specialized organelle membranes such as chloroplast lamellae (Branton & Park, 1967) or in specialized regions of plasma membranes such as cell junctions (Chalcroft & Bullivant, 1970; McNutt & Weinstein, 1970; Gilula, Branton & Satir, 1970; Flower, 1970, 1971) are membrane faces seen which approach complementarity. A plasma membrane system is described in this paper in which the 2 fracture faces are almost exactly complementary. This demonstration of a membrane system showing complementarity of the faces is only compatible with one of the suggestions put forward by various authors to explain the lack of complementarity observed in most membranes.

2 446 N. E. Flower MATERIALS AND METHODS Malpighian tubules and the associated light organs were dissected from the larvae of the New Zealand glow worm Arachnocampa luminosa (Skuse) (Dipt: Mycetophilidae). Samples from each of these structures were placed for i h in either 25 % glycerol or 25 % glycerol plus 6 % glutaraldehyde, both solutions buffered to ph 74 with phosphate buffer. Small pieces of tissues cut from these samples were rapidly frozen on Freon 12 at 150 C. Freeze-etching was carried out on a Balzers BA 500 apparatus as described by Moor & Miihlethaler (1963). Some specimens were cut at 100 C and replicated immediately (no etch) while others were etched for 1 min at 100 C before being replicated. The replicas were examined in a Philips EM 200. RESULTS It is proposed to label the 2 internal membrane faces, which are revealed during freeze-etching by the splitting of membranes, as faces A and B (see McNutt & Weinstein, 1970). Face A is the one revealed on the half of the membrane which remains attached to the cytoplasm and so is observed in freeze-etch replicas as though looking from outside the cell towards the cell contents. Similarly, face B is the one revealed on the half of the membrane which is fractured away from the cell and so is observed as though looking from inside the cell. In most membranes A-type faces can be recognized by the large number of randomly distributed 6 ionm particles present on them while B-type faces usually have many fewer associated particles. In replicas showing fractures passing from Malpighian tubule cells into the gland lumen (Fig. 2), microvilli which cover the luminal surface of the cells can be seen. When the 2 types of membrane face revealed on these microvilli are examined, it can be seen that a larger number of particles is present on A-type faces than on B-type faces. Close examination of B-type faces reveals a small number of holes (see inset in Fig. 2) but nowhere near sufficient to match the number of particles on A type faces. Thus, the plasma membranes in these Malpighian tubules show fracture faces very similar in appearance to those observed in other membrane systems. The light organ in A. luminosa is formed by a differentiation of the ends of the Malpighian tubules (Gatenby, 1959). When replicas of fractures through the gland lumen in these light organ regions are examined, it can be seen that the A- and B-type faces are more or less complementary (Fig. 3). Since the Balzers unit at this laboratory is not at present equipped to produce replicas from both fracture surfaces of individual specimens, the number of holes and particles per unit area on exactly the same regions of A and B complementary fracture faces cannot be compared. However, the numbers of holes and particles per unit area on the A face of one microvillus and the B face of a nearby microvillus respectively have been ascertained in a number of replicas. The ratio of holes to particles obtained by these measurements varies between about 0-7 and 1-2. This degree of agreement in numbers is sufficient to strongly suggest that exact complementarity exists. Furthermore, no difference in appearance of the holes in the B faces could be detected between specimens unetched before replication and specimens etched for 1 min. It is noticeable that when one examines other membranes in the light-organ region, they do not show this complementarity of the 2 faces and it

3 Complementary freeze-etch fracture faces 447 appears that only the cell surface abutting the gland lumen, through which the products necessary to produce the luminosity must pass, shows this structure. DISCUSSION Several suggestions have been made to account for the lack of complementarity in membrane faces revealed by freeze-etching. These include (a) plastic deformation of membrane components during fracture; (b) melting of the edges of holes during etching; (c)fillingin of holes by contamination; (d) localized heating during shadowing leading to melting of the edges of holes; and (e) filling in of the holes by shadowing material (Branton, 1969; Bullivant, 1969, 1970; Clark & Branton, 1968; Chalcroft & Bullivant, 1970; McNutt & Weinstein, 1970). During the present investigation identical techniques have been used during freeze-etching of both the Malpighian tubules and light organs. However, the appearance of the B-type membrane faces in the 2 tissues are completely different. As has been reported for most plasma membranes, few holes are present on B-type plasma membrane faces in the Malpighian tubule specimens. In the light-organ regions, however, the B-type plasma membrane faces are covered with sufficient holes for them to be complementary to the particle arrays on the A-type faces. Furthermore, in these specimens there was no detectable difference between no etch and 1 -min etch in the appearance of the holes on the B-type faces. This eliminates the possibility that the holes are caused by the etching process. Such effects can sometimes be observed in some membranes but only after deep etching. These results therefore strongly suggest that the preparation procedures used do not affect the presence or otherwise of holes on B-type membrane faces and so are only consistent with the suggestion that some deformation of the membrane components must occur during fracturing. The complementary replicas observed in the light-organ region can only be explained if the positions of particles and fracture plane are such that the fracture plane meets and passes around the particles as in Fig. IA. No deformation of either face is necessary to explain the observed fracture faces. The more usual type of A and B faces as observed in the Malpighian tubules can be explained in 2 different ways, both involving distortion of one membrane face after fracturing. One possibility is that the fracture takes place as in the light-organ regions but that distortion, involving the material underlying or surrounding the holes either springing up or being pulled up, occurs during fracturing so as to level out the membrane face. The second possibility requires some asymmetry in the membranes as well as some distortion of the membrane faces. If the position of the particles or fracture plane is so asymmetrical that the particles do not penetrate the B-type faces (Figs. 1B, c) no holes would be expected in these faces. However, for particles to occur on the corresponding A-type faces it would be necessary for the particles to be stretched or pulled out of the A face during fracture so that they stand proud of the face. The present results cannot differentiate between these 2 possibilities. However, several recent publications have a bearing on these 2 suggestions. In chloroplast lamellae, Park & Pfeifhofer (1969) have suggested that an asymmetric fracture plane

4 448 N. E. Flower Fig. i. Schematic diagram showing 3 possible membrane models with respect to both the position of particles within membranes and the level of the fracture plane which could explain the faces observed in freeze-etch replicas. If the fracture plane is centrally located and passes around particles (A) then complementary faces are produced. Non-complementary faces could be produced from this model by a distortion of face Bfillingin the holes. Non-complementary fracture faces can also be produced if the fracture plane is asymmetrically placed (B) or if the particles are asymmetrically located (c). In both cases membrane faces as observed would only be produced if the particles were pulled out of the A faces during fracturing. could account for the relative ease with which large ragged holes can be etched into the B-type faces. A similar effect has been seen in both sarcoplasmic reticulum in muscle and endoplasmic reticulum in yeast (W. S. Bertaud, personal communication). This would seem to indicate that an asymmetric fracture plane could lead to the observed membrane faces in cell organelles. However, prolonged etching of plasma membranes does not appear to have this effect. Furthermore, Flower (1971) has shown that protuberances are present on the true outer surface of flagellar membranes as revealed by deep etching. These protuberances occur exactly over particles on the underlying A-type faces. Similar protrusions have been shown to occur over particle aggregates on the true outer surface of erythrocyte ghosts by Pinto da Silva, Douglas & Branton (1971). These results indicate that the outer half of each membrane was disturbed by the presence of particles on the A face. The B face therefore cannot have been flat before fracturing and yet in both membranes few holes are normally seen on

5 Complementary freeze-etch fracture faces 449 the B-type faces after freeze-etching. This would seem to indicate that in plasma membranes distortion of the outer half of the membrane, leading to a rilling in of holes, accounts for the observed membrane faces. A similar fiuing-in of holes on the B-type faces could also account for the appearance of the membrane faces in cell organelles if the etching effect was caused by some weakness in the membrane allowing it to disrupt as water is evaporated from the cytoplasm underlying the membrane. Thus the present results suggest that in most plasma membranes, and possibly in cell organelle membranes, holes in B-type faces are lost by membrane distortion during fracturing and that the plasma membranes in the light-organ regions are sufficiently different in structure so that this distortion does not occur. Recently, the present author has found another plasma membrane exhibiting complementarity in its 2 freeze-etch fracture faces. This membrane is the luminal surface of the goblet cells found in the mid-gut epithelium of the Mediterranean flour moth, Anagasta kuhniella. It is interesting that these goblet cell membranes are believed to be actively engaged in secretion (Anderson & Harvey, 1966) as is the light organ of the glow worm and that in both cases only the plasma membranes abutting the gland lumen show this fracturing property. Thus it is possible that the difference in structure leading to complementary fracture faces is related to a secretory function in which large numbers of small molecules are being actively passed through the membrane and out of the cell. The interesting question, which of course cannot be answered by electron microscopy, is what is this difference that causes the variation in fracture properties of membranes. I would like to thank Mrs S. M. O'Kane and Mr L. M. Adamson for expert technical assistance and Messrs W. S. Bertaud and D. M. Hall for helpful discussions. REFERENCES ANDERSON, E. & HARVEY, W. R. (1966). Active transport by the Cecropia midgut. II. Fine structure of the midgut epithelium, jf. Cell Biol. 31, BRANTON, D. (1966). Fracture faces of frozen membranes. Proc. natn. Acad. Set. U.S.A. 55, BRANTON, D. (1969). Membrane structure. A. Rev. PI. Physiol. ao, BRANTON, D. & PARK, R. B. (1967). Subunits in chloroplast lamellae. J. Ultrastruct. Res. 19, BULLIVANT, S. (1969). Freeze fracturing in biological materials. Micron 1, BULLIVANT, S. (1970). Present status of freezing techniques. In Some Biological Techniques in Electron Microscopy (ed. D. F. Parsons), pp New York and London: Academic Press. CHALCROFT, J. P. & BULLIVANT, S. (1970). An interpretation of liver cell membrane and junction structure based on observation of freeze-fracture replicas of both sides of the fracture. J. Cell Biol. 47, CLARK, A. W. & BRANTON, D. (1968). Fracture faces in frozen outer segments from the guinea pig retina. Z. Zellforsch. mikrosk. Anat. 91, FLOWER, N. E. (1970). Frozen etched septate junctions. Protoplasma 70, FLOWER, N. E. (1971). Septate and gap junctions between the epithelial cells of an invertebrate, the mollusc Cominella maculosa. J. Ultrastruct. Res. 37, GATENBY, L. N. (1959). An investigation of the structure of the New Zealand glow worm. Trans. R. Soc. N.Z. 87,

6 450 N. E. Flower GILULA, N. B., BRANTON, D. & SATIR, P. (1970). The septate junction: A structural basis for intercellular coupling. Proc. natn. Acad. Set. U.S.A. 67, MCNUTT, N. S. & WEINSTEIN, R. S. (1970). The ultrastructure of the nexus. A correlated thin section and freeze-cleave study. J. Cell Biol. 47, MOOR, H. & MOHLETHALER, K. (1963). Fine structure in frozen etched yeast cells. J. Cell Biol. 17, PARK, P. B. & PFEIFHOFER, A. O. J. (1969). Ultrastructural observations on deep-etched thylakoids. J. Cell Set. 5, PINTO DA SILVA, P., DOUGLAS, S. D. & BRANTON, D. (1971). Localization of A antigen sites on human erythrocyte ghosts. Nature, Lond. Z32, SLEYTR, U. (1970). Die Gefrieratzung korrespondierender Bruchhalften: Ein neuer Weg zur Aufklarung von Membranstrukturen. Protoplasma 70, WEHRLI, E., MOHLETHALER, K. & MOOR, H. (1970). Membrane structure as seen with a double replica method for freeze fracturing. Expl Cell Res. 59, (Received 10 July 1972)

7 Complementary freeze-etch fracture faces The encircled arrows in the figures indicate the direction of shadowing. Fig. 2. Freeze-etch replica showing microvilli at the Iuminal surface of Malpighian tubule cells in the New Zealand glow-worm, A. luminosa. A-type fracture faces bear many more particles per unit area than do B-type faces, x Inset is a highermagnification view of the area marked showing one of the few holes (arrowed) present on the B-type faces in this micrograph, x

8 452 N. E. Flower Fig. 3. Freeze-etch replica of the microvilli at the luminal surface of cells in the light-organ region of A. luminosa. The large number of particles per unit area present on A-type membrane faces is matched by the large number of holes per unit area on the B-type faces, x