A Freeze-Fracture and Concanavalin A-Binding Study of the Membrane of Cleaving Xenopus Embryos

A Freeze-Fracture and Concanavalin A-Binding Study of the Membrane of Cleaving Xenopus Embryos E. J. SANDERS and R. A. DICAPRIO Department of Physiology, University of Alberta, Edmonton, Alberta, Canada Received May 1976lAccepred Augusl 1976

The freeze-fracture appearance and concanavalin A-binding capacity of the plasma membrane of cells of the cleaving Xenopus embryo have been examined up to the 16-cell stage. It was found that membrane on the outer surface of the embryo, which faces the vitelline membrane and is remote from cleavage furrows, and membrane in the shallow regions of the furrow possessed a high population of intramembranous particles on the PF-face (1 17 1 per pm’). The EF-face of these membranes showed a lower particle population (245 per pm’). By contrast, membrane deep in the furrow and bounding the blastocoel did not display a face with high particle numbers. Both faces of this membrane, which is newly exposed as the furrow grows, were relatively poorly supplied with particles (93 per pm’). Therefore it appears that, in this tissue, newly added membrane possesses fewer intramembranous particles than the pre-existing membrane. Concanavalin A, as detected cytochemically using peroxidase and haemocyanin techniques, bound extensively to both particle-rich and particle-poor membrane. Thus there was no correlation between intramembranous particle frequency and degree of concanavalin A binding.

Introduction It has been demonstrated by a number of investigations that the membrane of the furrow of cleaving amphibian embryos grows by the addition of new membrane. The morphology of the early furrows of Xenopus embryos has been studied in depth and the emergence at the cell surface of new membrane has been considered to be the result of the fusion of Golgi-derived vesicles [28, 30, 36, 371 or possibly due to the acitivity of lamellar bodies which may represent intracellular pools of membrane precursor 12, 3, 351. The possibility of other origins for the new membrane cannot be excluded. Little is known of structural differences that may exist between the newly exposed furrow membrane and the membrane which comprises the outer surface of the embryos, although variations in the lanthanum-binding capacity of the two regions has been demonstrated [361. We have sought to determine whether the new membrane has any structural characteristics which distinguish it from the pre-existing membrane using firstly the freeze-fracture technique, which exposes the hydrophobic core of the membrane and the intramembranous parDifferentiation 7, 13-21 (1976) - 0 by Springer-Verlag 1976

ticles (IMP) which are distributed across the fracture face. These particles can probably be considered to represent integral protein units which span the lipid matrix of the membrane (see [51 for review). Secondly, electron microscope cytochemical techniques have been used which display the binding sites of the lectin concanavalin A (con. A) on the extracellular surface of membrane. This lectin has been shown to bind specifically to the QD-mannopyranoside and a-D-glucopyranoside residues of the membrane glycoproteins 119, 341. In erythrocyte ghost membranes the receptors at the cell surface are linked to the IMP, so that redistribution of the IMP is paralleled by a corresponding change in the distribution of the surface lectin or anionic binding sites 123, 241. This relationship has been found not to be the case in all membranes however, since it has been shown that while con. A receptors are mobile, this may not necessarily be refleced by the IMP distribution [25, 261. Thus the lectin receptors are not always associated with IMP. In the present work we have shown that, while there ar,e gross differences in the numbers of IMP

E. J. Sanders and R. A. DiCaprio

14 in different regions of the embryo, this is not paralleled by changes in the degree of con. A binding sites. Hence in this membrane IMP are not necessarily linked to con. A binding sites. Methods Embryos of Xenopus laevis were obtained by subcutaneous injection of chorionic gonadotrophin, dejellied and washed in Steinberg's physiological salt solution as described previously [361. The material was then immersed in the mixed aldehyde fixative of Kalt and Tandler 1121 which contains 3% glutaraldehyde, 2% formaldehyde, 1% acrolein and 2.5% dimethyl sulphoxide in 0.1 M cacodylate buffer, pH 7.2, at 4OC. Freeze-Fracturing. After aldehyde fixation for 24 h, the embryos at 8- and 16-cell stages were thoroughly washed with buffer and transferred to a 30% glycerol solution. They were stored in this solution, with several changes, at 4' C for up to 3 weeks. No ultrastructural changes were observed to occur during this period of time. The embryos were frozen in Freon 22 at liquid nitrogen temperature prior to fracturing at -100" C, shadowing with platinum and replicating with carbon in a Balzer's Freeze Etch device. The replica was released from the tissue and cleaned by treatment with 20% commercial bleach, followed by washing in distilled water. The replicas were picked up on Formvar-coated copper grids. The distribution frequencies of the IMP on various fracture faces were determined by examination of photographic prints at a magnification of not less than 50,OOO times. Particle counts were made on images of membranes from a large number of different embryos and the mean and standard deviation were calculated for each sample. Concanavalin A-Binding. Two cytochemical techniques were used to determine the site of binding of the lectin, namely the peroxidasediaminobenzidine (PO-DAB) method first described by Bernhard and Avrameas 11I, which is detected by examining thin sections, and the haemocyanin method of Smith and Revel [381, which requires replication of the interblastomeric surface. For the PO-DAB method, after aldehyde fixation, embryos at 2-, 4- and 8-cell stages were split manually, along the plane of cleavage furrows, using a fine tungsten needle in a manner similar to that described previously (291. Following thorough washing in buffer the tissue was immersed in concanavalin A (Sigma Chemical Co.) at a concentration of 100 pg/ml in 0.1 M cacodylate buffer, pH 7.2, for 1 h at room temperature. The split embryos were then washed in buffer and treated with horseradish peroxidase (Type VI, Sigma Chemical Co.) at a concentration of 50 mg/ml in the same buffer for 30 min at room temperature. This solutions was removed by thorough washing in buffer and the tissue was transferred to a saturated solution of 3-3' diaminobenzidine (J. T. Baker Chemical Co.) in 0.5 M TridHCl buffer, pH 7.6, plus 0.01% hydrogen peroxide for 30 min at room temperature. The split embryos were removed from this solution, washed in buffer, and post-fixed in 1% osmium tetroxide in the cacodylate buffer for I h at room temperature. Dehydration was carried out in graded ethanol solutions and propylene oxide, following which the embryos were embedded in araldite, sectioned and examined without further staining. For the controls, a-methyl-D-mannoside (Sigma Chemical Co.) was added to both the con. A and peroxidase solutions to a final concentration of 0.5 M.

For the haemocyanin technique, 2-, 4- and 8-cell embryos were aldehyde-fixed, split and exposed to con. A as described above. After a buffer wash the half-embryos were treated with a 100 mg/ml solution of haemocyanin (keyhole limpet; Calbiochem) for 10min at room temperature. Tissue which was to serve as control was then exposed to 0.5 M a-methyl-D-mannoside. Both samples were then fixed for a further 1-2 h in 2.5% glutaraldehyde in the cacodylate buffer. After a thorough buffer wash the material was dehydrated with graded acetone solutions and dried by the critical point method from carbon dioxide. The interblastomeric surface of the embryos was then shadowed with platinum and replicated with a 250 A layer of carbon. The surface replica was cleaned using 20% commercial bleach, washed in distilled water and picked up on Formvar-coated copper grids.

Results

Freeze-Fracturing. The terminology used here to identify the fracture faces is that introduced by Branton et al. [4]. In this nomenclature the fracture face of the halfmembrane closest to the cytoplasm is designated PF and the face closest to the extracellular space is designated EF. Fractured plasma membrane at the surface of the embryo, remote from the furrowing region, displayed IMP of 120-13OA diameter, with a frequency of 1171 k 22 1 particles/pm2 (mean 2 standard deviation) on the PF-face (Figs. 1, 2). This relative abundance of IMP was a consistent finding in all embryos examined, as were all the particle sizes and distribution frequencies to be reported here. The EF-face of the outer membrane showed similar IMP, though these were relatively few in number (245 k 140/pmZ; Fig. 3). The technique employed in this work exposed only small areas of EF-face such as that shown here. The characteristic PF-face frequency of IMP prevailed not only in plasma membrane remote from the furrow but also in the outer region of the furrow itself (Fig. 4). However, then the fracture faces of the furrow membrane were observed deeper within the embryo (Fig. 5), there was a very marked difference, such that the two faces were no longer distinguishable on the basis of their IMP population. The small areas of membrane fractured were typical of those obtained from this preparation. The IMP were also 120-130A in diameter and in this region showed a frequency on all exposed faces of 93 k- 66/pm2 (Fig. 6). The particle frequency on the furrow membrane was therefore only about 8% of that on the PF-face of the outer membrane. In a number of embryos the plasma membrane of several cells lining the blastocoel was fractured. The large fracture faces thus exposed showed identical characteristics with those of the deeper furrow, i.e. they had

Fip. 1. Freeze-fracturereplica of the membrane of the outer surface remote from a cleavagefurrow. The PF-face of the membrane possesses a high density of intramembranous particles. x 46,100 Fig. 2. Higher magnification of PF-face particles. x 116,000 FIB.3. The EF-face of membrane remote from a cleavage furrow, showing the paucity of particles. x 64,800 Fig.4. The shallow regionofacleavagefurrowshowingthefurrowspace(F5')boundcd bymembranehavingparticledistributionsonthePF-faceand EF-face identical to that of membrane outside the furrow. x 55,700

Fig.5.Thedeeper regionofthefurrow showingcloselyapposedmembraneswhichhavefracturefaces(arrows)whichdisplayfewparticles. x 55,700 Fig. 6. A large area of membrane from deep within the furrow. x 55,700 Fig. 7. Vesicles closely associated or fusing with membrane bounding the blastocoel (B). x 55,700 Fig. 8. A Golgi body in the cytoplasm close to the furrow.x 37,100 Fig. 9. A putative lamellar body in the cytoplasm close to the furrow. Fractures through lamellae can be seen (arrows). x 46,100

Fig. 10. A putative lamellar body, with fractures through lamellae (L)and faces showing a structure of subunits (arrows). x 46,100 Fig. 11. Thin section through membrane deep within the furrow showing a dense surface deposit of concanavalin A as revealed by peroxidase and diaminobenzidine. x 37,100 Fig. 12. Membrane remote from the furrow with a continuous layer of reaction product indicating concanavalin A binding. x 27,800 Fig. 13. Membrane from tissue exposed to the inhibitory saccharide showing the negative result obtained in the controls. x 37,100

Fig. 14. A surface replica of the interblastomeric membrane in a cleavage furrow. The rough, shallower, surface ofthe furrow (RS)is separated from the deeper, smoother, surface (SS) by a band of microvilli (MY). x 4,340

Fig. 15. A surface replica of interblastorneric membrane on which the presence of bound concanavalin A is revealed by haemocyanin molecules. When favourably oriented these molecules are seen as rectangular or circular profiles. x 37,100 Fig. 16. A surface replica of interblastomeric membrane treated with concanavalin A, haemocyanin and the inhibitory saccharide. x 36,700

19

Membrane of Cleaving Xenopus Embryos

few particles. There were large numbers of vesicles associated with the membrane bounding the blastocoel (Fig. 7), but it is not possible to distinguish endocytosis from exocytosis in such images. Golgi bodies (Fig. 8) were encountered in the cytoplasm close to the furrow membranes and this supports the suggestion that they may play a role in the synthesis of new membrane 130, 36, 371. Highly characteristic images were repeatedly observed in the cytoplasm close to the course of the furrow membrane (Fig. 9). These showed many jagged, angular fracture faces, indicating that the body cleaved possessed a complex laminar structure. These images were distinctly different from those shown by Golgi bodies. We consider that they probably represent fractured lamellar bodies that have been observed in this region of the cytoplasm by transmission electron microscopy of thin sections and implicated in the formation of new membrane [2, 3, 351. A consistent finding was that one or more fracture faces comprising these lamellar structures possessed an arry of pits 200-250 A in diameter (Fig. 10).

Convanavalin A-Binding. The PO-DAB technique showed that there was a dense reaction product on the surface of membrane deep within the furrow (Fig. 1l), and also on the outer membrane of the embryos (Fig. 12). The deposit was not uniformly thick in all regions. The reaction product in the deep furrow was most often discontinuous and clumped, similar to the manner in which lanthanum nitrate deposits [31, 361. Measurements indicated that approximately 75% of the membrane of the deep furrow was covered by a detectable deposit. Deposits on the outer surface and shallower regions of the furrow appeared as a uniform continuous layer providing 100% coverage of the membrane. Control tissue from both regions that had been exposed to the inhibitory saccharide showed no surface precipitate in any region (Fig. 13). Examination of replicas of the interblastomeric surface of half-embryos which had not been treated with con. A allowed the identification of the various types of membrane topography within the furrow (Fig. 14). This could be directly compared with the topography observed using scanning electron microscopy on these surfaces [3, 361. Thus it was possible to identify (Fig. 14) the rough surface in the shallower region of the furrow which was due to .the presence of membrane protuberances and the band of microvilli which separates this rough surface from the smoother surface deep in the furrow. These changing zones of surface architecture are in complete agreement with the results obtained by scanning electron microscopy.

When such surface replicas of haemocyanin-treated tissues were examined, the presence of these molecules was disclosed on the deep furrow membrane (Fig. 15), on the rough shallower furrow and outer membranes. No difference in the frequency of occurrence of the marker was seen in the deeper or shallower regions of the furrow. Replicas of control tissue indicated that far fewer molecules were bound, and large areas of surface were entirely free of the marker (Fig. 16). As described by Smith and Revel [381, the cylindrical haemocyanin molecules appear as either rectangular or circular profiles, depending on the aspect from which they are viewed. The longest dimension of the molecule measured in replicas prepared by us was approximately 340 A, which compares favourably with the molecular size of gastropod haemocyanin determined by negative staining techniques 161.

Discussion The results presented here show that the IMP population in the membrane of the outer surface and the shallow region of the cleavage furrow is considerably greater than the population in membranes deep in the furrow and bounding the blastocoel. Furthermore, there is not a comparable difference in the binding of the lectin con. A to the surface of these forms of membrane as judged by the cytochemical techniques used here. It therefore appears that membrane synthesized in the furrow during cleavage has a low number of particles when compared with the membrane existing prior to the cleavage process. This finding parallels that of Pfenninger and Bunge [211, who demonstrated that newly inserted plasma membrane at the growth cone of nerve fibres is relatively particle-poor and that this membrane apparently “matures” with time and acquires a higher population of particles. The question of a “maturation” process by the membranes of the Xenopus embryo requires further examination by study of the membranes within the embryo at successive developmental stages. The significance of the low particle population is unclear, although the current view is that this indicates that the membrane is low in proteins which span the lipid component 151. There has been discussion in the literature concerning the electrical properties of. the new membrane in the furrow of cleaving Xenopus embryos. It has been proposed that this membrane displays a higher ionic permeability than the pre-existing membrane [ 131, although this view is not necessarily consistent with all the data available [7-91. The present results indicate that the new membrane has fewer IMP, which

E. J. Sanders and R. A. DiCaprio:

20 can be taken to mean that this membrane has a higher lipid-to-protein ratio. This finding may be pertinent to the discussion of new membrane permeability, since it may be shown that the specific resistance of pure lipid bilayers is orders of magnitude greater than that of biological membranes [ 141. Thus, it might be expected that the new membrane would display a lower ionic permeability than the pre-existing membrane, instead of the converse. However, this interpretation is rendered speculative by the fact that the nature of the proteins responsible for the IMP is unknown. A further similarity between the present results and those of Pfenninger and Bunge I211 and Pfenninger and Maylit-Pfenninger 1221 is that the density of lectin binding does not parallel the distribution density of intramembranous particles. Thus in the work described here, con. A bound both to the surface of particle-rich and particle-poor membrane to a degree that did not correlate with particle frequency. Results obtained with growing neuronal processes [221 indicate a similar inconsistency between lectin binding and particle frequency. These results raise questions regarding the nature of the carbohydrate-carrying moiety within the membrane, since this has often been considered to be a protein which penetrates the lipid bilayer and should therefore appear as an intramembranous particle [51. Pfenninger and Bunge 12 1 I have speculated that the carbohydrate components are carried by proteins which are not integral, or by glycolipids. The observation made here that the membrane of the shallower furrow region is identical with the membrane of the outer surface of the embryo and different to that of the deep furrow is readily understandable when reference is made to the mechanism of furrow initiation. This process is by cortical contraction which causes a furrowing of the surface membrane 121. Membrane growth then ensues to complete the cleavage. Thus the shallower furrow membrane is derived directly from existing membrane and does not consist of any newly incorporated membrane. The particle population on the fracture faces on the outer surface of the early Xenopus embryo has been briefly alluded to previously [151. The contention expressed in this earlier report, that the particle distribution density is approximately equal on both PF- and EFfaces, is not in accordance with the findings described here. We find that the particle number on the EF-face is only 21% of that of the PF-face. Cytochemical observations on the binding of con. A to early Xenopus embryos using fluorescein-conjugated lectin [201 indicated that, during cleavage, binding occurs predominantly along or near the cleavage furrows.

Furthermore, autoradiography showed that “C-con. A bound to the blastomere surfaces within the furrow, as we have found in the present study using different techniques. It appears to have been established that migratory cells during early sea urchin embryogenesis display a different distribution of con. A receptor sites from the less migratory cells in the same embryo. Thus, the migratory micromeres, while not binding more lectin, have von. A receptors which are more mobile than those on the macromeres and mesomeres. It is argued that this greater mobility renders the micromeres more readily agglutinable by the lectin 118, 271. The possibility of differential lectin affinity among cells in the early amphibian embryo is still open. While it has been reported that the motile cells during gastrulation in Xenopus bind con. A more avidly than the non-motile ones [201, this result was not obtained using Rana species, where no differences have been observed [lo].The present results did not indicate any major variation in binding capacity among the cells during Xenopus cleavage. It has been shown that binding of con. A to the surface of embryonic amphibian cells inhibits their normal morphogenetic migrations by a mechanism unknown [ 16, 171.

The fracture faces of membrane bounding the blastocoel cavity, showing activity which may suggest vesicle fusion, lend support to the proposal that such activity is of considerable significance in the growth and development of the blastocoel 11 ll. Vesicle fusion has also been proposed as a contributing factor in furrow advancement 1361, but little evidence of this has thus far been accumulated using the freeze-fracture technique, due undoubtedly to the difficulty in finding fracture faces in the region where fusion is most apparent. The close association with the furrow of Golgi bodies and putative lamellar bodies seen by freeze-fracturing supports the view that these are involved in new membrane formation. The significance of the pits revealed within the lamellar bodies is unclear. Acknowledgements: This work was supported by the Medical Research Council of Canada in a grant to E.J.S. and a studentship to R.A.D. We thank Dr. S. K. Malhotra for the use of the freeze-etchig equipment and Dr. J. C. Tu for assistance.

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