ULTRASTRUCTURE OF THE ENDOPLASMIC FACTOR RESPONSIBLE FOR CYTOPLASMIC STREAMING IN CHARA INTERNODAL CELLS

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1 J. Cell Sci. 36, (1979) I2I Printed in Great Company of Biologists Limited 1979 ULTRASTRUCTURE OF THE ENDOPLASMIC FACTOR RESPONSIBLE FOR CYTOPLASMIC STREAMING IN CHARA INTERNODAL CELLS REIKO NAGAI AND TOMIO HAYAMA Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka, 560 Japan SUMMARY Previous investigators have proposed that cytoplasmic streaming in Chara internodal cells results from the interaction between an endoplasmic factor and fibrils composed of microfilaments in the stationary cortex. Using the internal perfusion technique, we confirmed the observation that organelles which had been attached to the fibrils by decreasing the internal concentration of ATP moved along the fibrils after ATP was introduced. Thin-sectioned specimens revealed that endoplasmic organelles of various shapes were linked to microfuament bundles in the absence of ATP. Linkage was effected by regularly arranged electron-dense materials with a spacing of nm, at definite regions on each organelle. The organelles in question were studied in negatively stained preparations of endoplasm. The organelles had some common features. (1) They were all membrane-limited. (2) Their sizes and configurations varied largely. (3) One or more protuberances were present on them. (4) The protuberances were usually rod- or horn-like. (5) Small globular bodies nm in diameter were found in ordered array with the same spacing as those in thin sections at the surface of the protuberances. (6) Many fine filaments were always attached to the surface of the protuberances. These fine filaments differed from F-actin in diameter (less than 4 nm) and inability to react with heavy meromyosin from rabbit skeletal muscle. The role of such components of the organelles in cytoplasmic streaming is discussed. A paracrystalline array of microfilaments with a transverse periodicity of about 38 nm is presented, together with its optical diffraction pattern. INTRODUCTION It has been generally accepted that rotational cytoplasmic streaming in the characean cell is caused by the active shearing force generated at the interface between the moving endoplasm and the stationary ectoplasm (Kamiya & Kuroda, 1956; Kamiya, 1959). Fibrils running parallel to the direction, of the streaming are located on the inner surface of the chloroplast files in contact with the streaming endoplasm (Kamitsubo, 1966). Each'of the fibrils is a bundle of microfilaments similar in appearance to muscle F-actin (Nagai & Rebhun, 1966). They reversibly bind rabbit skeletal muscle heavy meromyosin to form arrowhead structures (Palevitz, Ash & Hepler, 1974; Williamson, 1974; Palevitz & Hepler, 1975). The arrowheads are oriented opposite to the direction of the streaming (Kersey, Hepler, Palevitz & Wessells, 1976). When the chloroplasts, together with the fibrils, are locally dislodged by strong local irradiation, the streaming endoplasm becomes stagnant or moves slowly, while streaming continues in other, intact areas. However, the streaming returns to normal

2 122 R. Nagai and T. Hayama after the regeneration of the fibrils (Kamitsubo, 19726). These facts indicate that fibrils composed of microfilaments are indispensable for endoplasmic streaming. Factors which interact with microfilament bundles to produce the active shearing have been studied by several investigators. Bradley (1973) discussed the possibility that motive force might be generated by reaction of actin with myosin, which may be anchored at suitable sites on the endoplasmic reticulum. Based on differential treatment of the cell with an SH-reagent, JV-ethylmaleimide, Chen & Kamiya (1975) suggested thatthe factor or the putative myosin is localized in the streaming endoplasm. Williamson (1975), using a vacuolar perfusion technique (Tazawa, 1964), showed that organelles which had been immobilized on the fibrils in the absence of ATP (inactive state) started to move along them after ATP was introduced (reactivation). Williamson's interpretation was that the streaming was caused by interaction between the microfilaments and a myosin-like protein which he supposed was linked to the endoplasmic organelles. The motility required, in addition to ATP, millimolar levels of Mg^ and free Ca a+ at io~ 7 M or less (Williamson, 1975). Tazawa, Kikuyama & Shimmen (1976) succeeded in controlling the cytoplasmic streaming by removing the tonoplast through replacement of the natural cell sap with EGTA-containing solution. They also showed that ATP and Mg^ were indispensable for the cytoplasmic streaming in the tonoplast-free cells. These findings lead us to conclude that the factor which interacts with microfilaments in the presence of ATP and Mg 24 " is localized in the moving endoplasm. As Bradley and Williamson suggested, the factor may be carried by the endoplasmic organelles. The next problem is to identify the organelles in question and to show how and where the factor is arranged on them. The purpose of the present study was to throw light on this problem. Endoplasmic organelles are found to be equipped with manyfinefilaments, apparently different from F-actin and with globular bodies which seem to be the factor interacting with the microfilaments. MATERIALS AND METHODS Cells. Internodal cells of Chara australis were used. The cells were cultured outdoors in a large plastic bucket filled with rainwater with soil at the bottom. In winter, the alga was kept in plastic boxes under artificial illumination in the laboratory. Cells 8-10 cm long were isolated from adjacent cells and kept for at least 1 day before use in pond water. Internal perfusion. Each cell was cut and internally perfused after Ta2awa et al. (1976). The solution for the internal perfusion was composed of 5 mm EGTA, 6 mm MgCl., 290 mm sorbitol and 5 mm Tris-maleate buffer (ph 7, adjusted with KOH). The cell was ligated at both open ends after the natural cell sap had been completely replaced with the artificial medium. Within min after perfusion, the tonoplast disintegrated (tonoplast-free cells, Tazawa et al. 1976). In these tonoplast-free cells active streaming was observed, and they were used as the starting materials for both light and electron microscopy. Light microscopy. As the chloroplasts anchored in the cortical gel layer interfere with the light-microscopic observation of the fibrils, they were dislodged by the 'window technique' of Kamitsubo (19726) before cells were internally perfused. A tonoplast-free cell was then placed on an apparatus similar to that used by Williamson for light-microscopic observation. After replacing the bathing solution (pond water) with 0-3 M sorbitol solution, which is approximately isotonic with the perfusion medium, both ligated ends of the tonoplast-free cell were amputated again for the second and third perfusion. The streaming in the window area was observed under a Zeiss photomicroscope II with differential interference optics.

3 Endoplasmic factor in cytoplasmic streaming 123 Thin sectioning. Tonoplast-free cells were perfused again with the same medium used for the first perfusion. After perfusing the cell interior with an amount of medium 2 to 3 times the cell volume, the cell was ligated at both ends. Endoplasmic flow was not observed after the second perfusion. After confirming the cessation of streaming, the cell was fixed for thin sectioning. Cells in this situation were immersed in 2 % glutaraldehyde containing 6 mm MgClj and 50mM phosphate buffer (ph 7). A few minutes later, the cells were cut into small segments to accelerate fixation, then kept in the fixative for 1 h at room temperature. Cells which had been perfused twice were perfused again with the same medium supplemented with ATP. After restoration of streaming they were fixed with the same medium containing 1 mm ATP to avoid extreme decrease in ATP concentration when the cells were cut into segments in the fixative. All cells were postfixed with 1 % Os0 4 solution containing 6 mm MgCl 2 and 50 mm phosphate buffer for 1 h. The specimens were embedded in Spurr's medium (1969) or Epon 812 (Luft, 1961) after dehydration in an ethanol series. Thin sectioning was done on a LKB-ultratome with a diamond knife. Grids containing sections were stained with uranyl acetate dissolved in methanol and lead citrate before being examined on a JEM-100C electron microscope at 80 or 100 kv. Negative staining. Streaming endoplasm in normal internodal cells was collected by centrifuging the cell at g for 10 min. As the shifted endoplasm always started to move immediately after the cessation of centrifugation, the cells were chilled promptly to prevent movement by replacing the bathing solution in the centrifuge tube with a chilled one. The centrifugal region of the cell was ligated while the cell was chilled. The accumulated endoplasm started rotational streaming in the small segment of the cell when the temperature rose. The ligated endoplasm-enriched segment (volume: o-z 0-5 fil) was placed in a drop (ca. 50 fil) of the perfusion medium containing 3 mm dithiothreitol (DTT) and o-i mm phenylmethyl sulphonyl fluoride (PMSF). The endoplasm was suspended in the solution by cutting the segment. Pieces of cell wall were removed with forceps. After gentle mixing, drops of the endoplasmic suspension were negatively stained with 1 % aqueous uranyl acetate on copper grids coated with Formvar-carbon. RESULTS Cytoplasmic streaming in cells without vacuolar membrane Active streaming in the tonoplast-free cells after first perfusion was probably due to the presence of endogenous ATP still left in sufficient concentration to cause streaming (Shimmen, 1978). When the tonoplast-free cells were perfused again with the perfusion medium without ATP, however, the organelles ceased to move immediately, or within several seconds. Then the fibrils appeared thick due to the attachment of many organelles along their whole length (Fig. IA). The situation corresponds to the inactive state of Williamson. A few minutes after entry into the inactive state, the perfusion medium containing 1 mm ATP was introduced. Organelles anchored on the fibrils promptly started moving along the fibrils. The direction of the movement was the same as before. The number of organelles coming into the observed area diminished with time. The fibrils finally appeared naked and clean (Fig. IB). It is clear from these observations that we could set up the rigor combination between the organelle and the subcortical fibril, and could reactivate the system to produce active movement with Mg-ATP. Using this system, the ultrastructure of the organelle and the linkage between the organelle and the fibril were investigated, as described in the next section.

4 I2 4 R. Nagai and T. Hayama Linkage between fibrils and organelles Thin sections of cells which had been fixed in the inactive state were made along the long axis of the chloroplast files. Fig. 2 shows one of these sections. Rows of bundles (mf), some of which are associated with chloroplasts (chl), and several other organelles (arrows) anchored to the bundles, can be seen. The arrangement of the bundles and their spacing coincide well with that observed previously (Nagai & Rebhun, Fig. 1. Fibrils in the' windowed region' of a Chara cell, A,fibrilsappear thick due to the anchoring of many endoplasmic organelles in the absence of ATP. B, fibrils appearing naked and clean after the restoration of movement of organelles by introduction of 1 mm ATP. x ). It is thus reasonable to conclude that the bundles are identical with subcortical fibrils composed of F-actin (Palevitz & Hepler, 1975). Most of the endoplasmic contents were dispersed due to the lack of tonoplast. The organelles shown in the figure can reasonably be assumed to be the same as those seen tightly bound to the fibrils observed under the light microscope, since such organelles were not observed

5 Endoplasmic factor in cytoplasmic streaming 12 in cells fixed after the restoration of cytoplasmic flow produced by introduction of ATP, as will be described later. The organelles are linked to the microfilaments by means of electron-dense structures projecting from their surface. These are arranged in periodic order along the surface of the organelles. Thicker sections were prepared to find whether or not the appearance in Fig. 2 represents the configuration of the organelles as a whole. In sections of around 0-15 /tm Fig 2. A longitudinal thin section of a tonoplast-free cell which is ATP-defkient. Rows of microfilament bundles {mf), chloroplasts (cht) and several organelles (arrowheads) anchored to the bundles are seen. Notice electron-dense materials projecting from the surface of each organelle. They are arrayed regularly at the surface of the organelles. x CEL 36

6 R. Nagai and T. Hayama 3 A \ - /",. B Fig. 3. A balloon-shaped organelle with 3 long protuberances (A), and with a single protuberance (B) observed in a thicker section (about 0-15 fim) of ATP-deficient tonoplast-free cells. Both are linked to microfilament bundles through electron-dense materials. The electron-dense materials in (B) are arrayed at intervals of ioo, 100, 100 and 110 respectively from the right (arrows), A, x 51000; B, X

7 Endoplasmic factor in cytoplasmic streaming 127 we obtained more extensive views of their configuration, as can be seen in Fig. 3A and B. The organelle shown in Fig. 3 A resembles a balloon with 3 long protuberances; 2 are closely associated with a bundle of the microfilaments over their whole length and the third bends partly but finally comes into contact with the microfilaments at its tip. Notice that there are regularly arranged bridges between the protuberances and microfilaments. Fig. 3B shows a balloon with a single protuberance. Here also electron-dense bridges (arrows) are noticeable. The spacings between the bridges shown are 100, 100, 100 and 110 nm, respectively from the right. We also observed an organelle attached to 2 adjacent microfilament bundles simultaneously at 2 sites. Based on the distinctive morphology of the 2 organelles shown above, the organelles in Fig. 2 may show part of organelles linked to the microfilaments. A B r Spacing, nm X 10"' Spacing, nm X 10"' Fig. 4. Histograms showing the frequency distribution of spacing A, of the electrondense materials on sectioned specimens (n = 84), and B, of the globular bodies on negatively stained specimens (n = 124). These electron micrographs revealed that (1) there must be large variations in organelle size and morphology; (2) the organelles are linked, in the absence of ATP, to the microfilament bundles through special region(s), the protuberance(s), which are functionally and morphologically differentiated in each organelle; (3) in these structures, electron-dense bridges are regularly arrayed; (4) 64% of the electrondense bridges are located at intervals of nm. Variation of spacing is shown in Fig. 4A. The average value (with S.D.) is calculated to be 111 ± 15 nm (n = 84). When, the cell in the inactive state was perfused again with the perfusion medium suplemented with 1 mm ATP, the cytoplasmic streaming was restored due to restoral of an adequate ATP concentration (Shimmen, 1978). In sections of such cells, organelles linked to the microfilament bundles were not observed. This may be 9-2

8 128 R. Nagai and T. Hayama interpreted to mean that the organelles in question could not remain linked to the microfilaments during fixation because of the presence of internal and external ATP. Structures of organelles revealed by negative staining When characean internodes are centrifuged at moderate force, the streaming endoplasm collects at the centrifugal end of the cell. The shifted endoplasm always starts to move immediately after centrifugation along the chloroplast files, in the same direction as before centrifugation (Kamiya & Kuroda, 1956; Hayashi, 1963). Therefore, the endoplasm collected by centrifugation was expected to contain many of the organelles in question. In negatively stained specimens, we observed many organelles of characteristic appearances, as expected from the sectioned specimens. Fig. 5A-D shows examples of organelles of various sizes and shapes. They had features in common with those in sectioned specimens. Further it was found that: (1) one or more protuberances were present on their bodies; (2) protuberances were usually rod- or horn-like; (3) small globular bodies were attached in ordered array on the surface of the protuberances (arrows); and (4) many fine filaments were always attached to and frequently frayed out from the surface of the protuberances (to be shown later). The organelle in Fig. 5 A has the same configuration as that in Fig. 3B. The organelle in Fig. 5D is similar to that in Fig. 3 A. The globular bodies on the horn-like protuberance are shown in Fig. 6 under higher magnification. One globule is always found at the pointed end of the horn, (arrow 1). Except for the tip area, sets composed of 2 globules (arrows 2-4), perpendicular to the axis of the horn, are arrayed periodically at 120 run on average. Similar globular bodies, nm in diameter, arranged along the surface of the rod-like protuberance are shown in Fig. 7. The spacing of the globules agrees well with that of the electron-dense material in sectioned specimens. About 85% of the globular bodies lie within nm apart. The variation of spacing is presented in Fig. 43. The average value (with SD) was calculated to be 120 ±9 nm (n = 124). Notice a filament (Fig. 7, small arrows) originating from the globular body and other long filaments running parallel to the long axis of the rod. Some of the long filaments (large arrows) spread out from the area where the globular body is absent. It is not clear whether thesefilamentsare identical or not. The diameter of an individual filament is less than 4 nm. They did not react with heavy meromyosin from rabbit skeletal muscle, indicating that they differ from F-actin (data not shown). The endoplasmic organelles rarely had globular bodies along the whole length of the rod-like protuberance. However, one globule was usually located in the region of the tip, even when globules were rare or absent elsewhere. It is not clear whether the globules are detached during preparation from the surface of the rod-like protuberances or are intrinsically rare or absent. Fig. 8 shows part of a large mat-like organelle. Globular bodies (arrows) of constant spacing on the organelle surface are noticeable and fine filaments are also numerous. In some instances, the protuberance is composed of some smaller vesicles, which

9 Fig. 5. Various kinds of organelles in negatively stained specimens of endoplasm-rich suspension. O n e (A)o r more (B,c,D) protuberances are present on each organelle. T h e protuberances are of rod-like (A,B) o r horn-like (B,c,D) appearance. Small globular bodies (arrowhead) are arranged at the surface of the protuberance in periodic order with a constant ; x 180~0. spacing. A, x w ; B, x 23000; C, x ~ ) D, Y N

10 R. Nagai and T. Hayama are interconnected by many fine filaments (Fig. 9). The fine filaments unite to form thicker curly filament bundles. Bundles of microfilaments In negatively stained preparations of the endoplasm, microfilament bundles were frequently seen. They may come from the ectoplasmic layer of the endoplasmenriched cell segment. The bundles were associated with large dense structures, which were probably chloroplasts. Some bundles were associated with large membranous structures and others lay free on the grids. Organelles linked to microfilaments were rarely observed, perhaps because the ATP concentration of the endoplasmic 4 r 3 r 2 r 1 Fig. 6. Enlargement of the tip area of a horn-like protuberance. One globule is always found at the pointed end of the horn (arrow 1). Except for the tip area, sets composed of 2 globules (arrows 2-4), lying perpendicular to the axis of the horn, are arrayed periodically, x suspension was not extremely low, i.e. 2-5 /tm. This value was estimated from the ratio of the volume of an endoplasm-enriched segment to a drop of the medium in which the endoplasm was suspended (cf. Methods), and the cytoplasmic ATP concentration, which is around 0-5 ram (Hatano & Nakajima, 1963). Fig. IOA shows a microfilament bundle embedded in a membranous structure. Microfilaments in the bundle are parallel to each other and a transverse periodicity of about 38 nm is obvious. Fig. IOB shows another example of a bundle in which the twisted and beaded nature of each microfilament is more obvious. The transverse periodicity is also nm. These filament bundles closely resemble a paracrystal formed in vitro from purified F-actin or formed from actin, tropomyosin and troponin in the presence of Mg 2+ (Moore, Huxley & DeRosier, 1970; Spudich, Huxley & Finch, 1972; Ohtsuki & Wakabayashi, 1972; Gillis & O'Brien, 1975; Wakabayashi, Huxley, Amos & Klug, 1975). In addition, naturally occurring actin paracrystals have been reported in the acrosomal process of Limulus sperm (Tilney, 19750,6). Fig. 10c and D shows the

11 Endoplasmic factor in cytoplasmic streaming i i Fig. 7. Enlargement of a rod-shaped protuberance. Notice globular bodies arranged periodically, fine filaments originating from the globule (small arrowheads) and long filaments running parallel to the long axis of the protuberance. Some of the long filaments spread out from the area where the globular body is absent (large arrowheads), x Fig. 8. Part of a large mat-like organelle equipped with globular bodies (arrowheads) and many fine filaments, x

12 I 32 R. Nagai and T. Hayama B Fig. 9. Protuberance composed of small vesicles interconnected by manyfinefilaments. Fine filaments unite to form thicker curly filament bundles, x Fig. 10. Microfilament bundles observed in endoplasm-rich suspension, A, a bundle of microfilaments embedded in a membranous structure. Microfilaments in the bundle lie parallel to each other with transverse periodicity of about 38 nm. The twisted and beaded nature of each microfilament is more obvious in B. C, optical diffraction pattern from (A); I-O cm corresponds to 9-17 nm. D, optical diffraction pattern from B; i*o cm corresponds to 7-47 nm. A, X ; B, x

13 Endoplasmic factor in cytoplasmic streaming 133 optical diffraction patterns of the bundles in Fig. IOA and B, respectively. Both patterns show features characteristic of F-actin bundles: the layer line at a spacing of about 37 nm arises from the double-stranded nature of F-actin and one with a spacing of about 5-8 nm is the reflexion from the basic helix. The spacings of these layer lines are 578 and 37-0 nm in Fig. 10c and 5-84 and 37-8 nm in Fig. IOD. The spacing ratio of the 2 layer lines is 6-40 in Fig. 10c and 6-47 in Fig. IOD. These values indicate that the basic helix of F-actin in our preparation has 28 subunits per 13 turns. The nature of F-actin in the microfilament was also confirmed with specimens prepared by squeezing out the cytoplasm of the perfused cells. The microfilaments were decorated with heavy meromyosin from rabbit skeletal muscle to form arrowheads (data not shown) which were shown previously by Palevitz et al. (1974). DISCUSSION It has been suggested (Palevitz & Hepler, 1975) that microfilaments are packed in vivo in a paracrystalline array in characean internodes. A paracrystalline array of microfilaments with a transverse periodicity of about 38 nm is clearly shown in the present paper. The paracrystal of microfilaments was probably not formed in the perfusion medium supplemented with DTT and PMSF, since the Mg 2+ concentration in the medium is lower (6 mm) than that necessary for paracrystal formation from actin in vitro (25-50 misi). DeRosier et al. (1977) showed that paracrystals consisted of hexagonal arrays of actin filaments cross-linked by a second protein having a molecular weight of about Bradley (1973) observed bridges connecting each microfilament in Nitella. Allen & Condeelis (unpublished) found a Dalton component in Nitella extracts. It may be possible that a protein of Daltons has the role of maintaining the paracrystalline array of the microfilaments in vivo. The endoplasmic organelles which we observed here were quite different from other endoplasmic organelles from the structural point of view. First, they bore characteristic protuberance. Secondly, the organelles were equipped with globular bodies arrayed in periodic order on the surface of their protuberances and were equipped also with fine filaments attached to the surface of the protuberances. We mentioned above that in the inactive state produced by the absence of ATP, in which actin and myosin would be in rigor combination, the organelles were linked to the microfilaments by electron-dense material. This must correspond to the globular bodies seen in negatively stained specimens, because of the similarity in size and spacing on the surface of the organelles (cf. Fig. 4A, B). We can reasonably suppose that the globular bodies act as functional units when the endoplasmic organelles slide along the microfilaments. Myosin-like protein has been extracted iromnitella and studied(kato & Tonomura, 1977). The protein formed bipolar aggregates resembling those of myosin from rabbit skeletal muscle and other sources. Although we have no direct evidence, such as immunological identification, the globular bodies may be composed at least of the

14 134 R- Nagai and T. Hayama functional head of myosin or myosin aggregates, possibly with some other unknown protein. Their periodic array itself suggests that the period might be determined by the tail portion of the myosin molecule, or other unknown protein(s). Fine filaments were also seen usually on the surface of the protuberances or on a certain other region of the organelles. In this connexion, it is interesting to touch upon the endoplasmic filaments in Nitella revealed by scanning electron microscopy (Allen & Reinhart, 1976; Allen, 1977). They are thinner than subcortical fibrils and interwind to form loose networks. Some of them seem to originate from granular cytoplasmic structures much smaller than the chloroplast (fig. 2 in Allen, 1977). Since the fine filaments observed in the present study often fray out from the organelle and have a property of uniting to form thicker curly filament bundles, they morphologically look like the endoplasmic filaments of Allen. He has suggested that the endoplasmic filaments are composed, in part, of F-actin. Our fine filaments, however, are apparently different from F-actin in their inability to react with HMM from rabbit skeletal muscle and in diameter (less than 4 nm). It is unlikely that these properties come from damage to F-actin during preparation of the endoplasm, since in the same preparation actin filaments usually keep their native appearances and exist in the form of bundles (Fig. IOA, B, and at upper left corner of Fig. 8). The role of the fine filaments in the movement of the organelles must be important, although it is unlikely that they play the leading role in the sliding mechanism. They may (1) stiffen the protuberances by attaching to them; (2) provide chemical sites for the myosin-like molecules to locate properly, with a distinct polarity; or (3) help propel viscous endoplasm when frayed out into the endoplasm. Also, we cannot exclude the possibility that'the rod parts of myosin lacking functional heads might assemble themselves longitudinally to form fine filaments. In some specimens in which the direction of the streaming had been confirmed before fixation, the tips of the protuberances pointed downstream, in the same direction as the streaming, and the body part upstream. This appears very effective for carrying viscous endoplasm together with the sliding organelles. Several protuberances around a single'organelle, as shown in Fig. 5D, probably point in the same direction and interact with a single bundle of the microfilaments, as in Fig. 3A. They may also interact with a few bundles simultaneously. When larger mat-like organelles move along the microfilaments, the endoplasm could be more effectively dragged. Cytoplasmic streaming could be explained solely by this mechanism. The idea of 'undulating endoplasmic filaments' as a cause of streaming (Allen, 1974) does not seem to be pertinent. The ultrastructural basis for rotational cytoplasmic streaming now appears to have been established. It remains to discover the molecular mechanism for the generation of the sliding force. We wish to express our sincere thanks to Professor N. Kamiya of National Institute of Basic Biology for continuous interest and valuable criticism throughout the period of this study, and also to Dr Y. Nonomura for profitable suggestions. We are indebted to Dr R. Kamiya for taking the photographs of the optical diffraction pattern, and also to Professor Y. Tonomura

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