The fine structural distribution of acid phosphatase in the digestive gland ofArion hortensis (Fer.)

Summary Acid phosphatase activity has been found in association with the extensive brush border of the excretory cells and also within the lysosomal vacuoles of actively phagocytosing digestive cells. A release of enzyme into the cytoplasm occurs in some cells of the digestive epithelium. This appears to be linked with cellular lysis and may be a prelude to cell replacement.We would like to thank Professor D.Bellamy for providing facilities and encouragement.     

Fine structure and absorption of ferritin in the digestive organs of Loligo vulgaris and L. Forbesi (Cephalopoda,Teuthoidea)

The digestive organs possibly involved in food absorption in Loligo vulgaris and L. forbesi are the caecum, the intestine, the digestive gland, and the digestive duct appendages. The histology and the fine structure showed that the ciliated organ, the caecal sac, and the intestine are lined with a ciliated epithelium. The ciliary rootlets are particularly well developed in the ciliated organ, apparently in relation to its function of particle collection. Mucous cells are present in the ciliated organ and the intestine. Histologically, the digestive gland appears rather different from that of other cephalopods. However, the fine structure of individual types of squid digestive cell is actually similar to that of comparable organs in other species, and the squid cells undergo the same stages of activity. Digestive cells have a brush border of microvilli, and numerous vacuoles, which sometimes contain “brown bodies.” However, no “boules” (conspicuous protein inclusions of digestive cells in other species) could be identified in their cytoplasm; instead only secretory granules are present. In the digestive duct appendages, numerous membrane infoldings associated with mitochondria are characteristic features of the epithelial cells in all cephalopods. Two unusual features were observed in Loligo: first, the large size of the lipid inclusions in the digestive gland, in the caecal sac, and in the digestive duct appendages; and second, the large number of conspicuous mitochondria with well-developed tubular cristae. When injected into the caecal sac, ferritin molecules can reach the digestive gland and the digestive duct appendages via the digestive ducts, and they are taken up by endocytosis in the digestive cells. Thus, it appears that the digestive gland of Loligo can act as an absorptive organ as it does in other cephalopods.     

Ultrastructural study of the regressing prothoracic glands of blattarian insects

Summary The prothoracic glands, source of the molting hormone ecdysone, regress within a few days after the final molt, a process which was analyzed with electron microscopic methods in the cockroaches Leucophaea and Blaberus. This strictly timed event is accompanied by drastic alterations in cellular fine structure. Early signs of breakdown appear in groups of nuclei whose substance becomes segregated into patches of contrasting electron density characteristic of pyknosis.The most conspicuous change in the cytoplasm of parenchymal cells concerns the appearance of large, heterogeneous inclusion bodies in which various cellular elements become segregated. These compartments seem to represent autophagic vacuoles within which the gradual degradation of much of their contents takes place, presumably under the influence of lysosomal enzymes. Undigested swirls of membranous character may remain sequestered within these packets for some time.At advanced stages of cellular atrophy, plasma membranes and nuclear envelopes have gradually disappeared, and masses of protoplasm undergoing autolysis become invaded by a greater number of hemocytes than are present in nymphal glands. These phagocytic elements appear to engulf debris of parenchymal cells as well as some degenerating connective tissue elements. After the completion of the regressive process, the axial band of musculature characteristic of the nymphal gland persists on its own. Whether or not some parenchymal cells (or possibly their precursors) capable of reactivation persist in the proximity of this muscle is unknown.The resorption of the prothoracic gland in the newly emerged insect is the result of physiological autolysis and seems to be aided by the activity of phagocytic hemocytes.Dedicated to Professor W. Bargmann on his 60th birthday in friendship and admiration.This study was supported by Research Grants AM-03984, NB-02145 and NB-05219 from the U.S.P.H.S.I wish to express my thanks to Mrs. S. Wurzelmann, Mrs. C. Jones, Mrs. C. Grubman, and Mr. S. Brown for their excellent technical assistance.     

The subcellular localisation of certain crop-juice esterases in the digestive gland ofCepaea (Mollusca: Helicidae)

Summary Three methods have been used to localise specific crop-juice esterases within the cells of the digestive gland ofCepaea nemoralis andC. hortensis. A comparison withHelix aspersa has also been made. Autolysis experiments showed that Est. 1 and Est. 9 were very resistant to denaturation and might therefore be of lysosomal origin. Ultracentrifugation of digestive gland homogenates suggests that these same esterases are within vacuoles and this is confirmed by histochemical studies at the electron microscope level using thiolacetic acid as a substrate. It is shown electrophoretically that only esterases within set 1 (Oxford, 1977), which includes Est. 1 and Est. 9, hydrolyse this substrate to any marked extent. Thiolacetic acid esterase activity is found within the phagolysosomes and endoplasmic reticulum of digestive cells. It is suggested that at least some of the digestive enzymes present in the crop juice originate within phagolysosomes and are specifically released from digestive cells.     

Fine structure of the retina in Haliotis discus

Summary The fine structure of the retina of Haliotis discus has been studied by means of electron microscopy. The visual cell has an apical projection which protrudes beyond the retinal surface into the ocellar cavity. The surface of this projection shows elaborate in- and outfoldings. Besides, two apposed laminae of the folded plasma membrane contact each other to form a quintuple-layered compound membrane. The analogy of this compound membrane system to the rhabdomere of the visual cell of Cephalopoda has been discussed. Subsurface endoplasmic reticulum and giant multivesicular bodies are also notable structures in Haliotis visual cells. The observations include the fine structure of supporting cells, plexiform layer, optic nerve and transitional zone of retina to epidermis.The author wishes to express his appreciation for the encouragement and suggestions given by Dr. Toshi Yuki Yamamoto, Professor of Anatomy, Tohoku University School of Medicine, through all stages of this work.     

Immunocytochemical localization of cathepsin D in lysosomes of cortical collecting tubule cells of the rat kidney

Immunocytochemical localization of cathepsin D in rat renal tubules was investigated by means of indirect immunoenzyme and protein A--gold techniques. By light microscopy, fine granular staining was seen in the mesangial cells of glomeruli. Heavy reaction deposits were present in the cortical tubular segments and some of the medullary collecting tubules. The proximal tubules contained a few positive granules. Other segments were negative for cathepsin D. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were present in cytoplasmic granules and multivesicular bodies of the segment of the cortical collecting tubule. These cytoplasmic granules were presumed to be digestive vacuoles (secondary lysosomes) from their morphological profile. The proximal tubule cells contained the very weakly labeled secondary lysosomes. No specific labeling was noted in other segments of the nephron. Control experiments confirmed the specificity of the immunostaining. Quantitative analysis of the labeling density in each subcellular compartment also confirmed that the main subcellular sites for cathepsin D are the secondary lysosomes and multivesicular bodies. The labeling density in these granules of the lysosomal system varied widely with the individual granules, suggesting that there is a considerable heterogeneity of enzyme content among the granules of the lysosomal system. The prominent presence of cathepsin D in the cortical collecting tubule suggests a certain segment-specific function of this proteinase.     

Cellular basis for polarized light perception in the spider crab,Libinia

Summary Differential increases in the numbers of pinocytotic vesicles, multivesicular bodies and total complex bodies occurred in the cytoplasm of specific photoreceptor cells in the compound eye of the crab Libinia exposed for six hours to polarized light with various e-vector orientations. These data coupled with previous results on the same species proved that the seven retinular cells in each ommatidium formed two functional groups selectively light adaptable by e-vectors oriented 90° apart. One group (Channel I, comprising Cells 1, 4 and 5) was more affected by horizontal polarization; the other (Channel II, comprising Cells 2, 3, 6 and 7) was more affected by vertical polarization.This confirmed by a quite independent technique the conclusion reached from electrophysiological experiments on the crab Cardisoma that decapod compound eyes have two orthogonal polarization analyzer channels. In addition the present data showed that both channels occur in each ommatidium as hypothesized on previous electron microscopic evidence and that the axes of maximum absoprtion in the two retinal channels were parallel to the long axes of their cells' rhabdom microvilli, horizontal in Channel I and vertical in Channel II. The latter relations in turn supported the hypothesis that the dichroism of rhodopsin was fundamental to the analyzer mechanism.This research has been supported by U. S. Air Force Grant AFOSR 1064 and NASA Grant NGR 07-004-055. The authors wish to thank Professor Joseph G. Gall for generously sharing his electron microscope facilities.     

Microanatomy of the digestive tract and accessory organs of the Japanese flathead (Inegocia japonica Cuvier, 1829) (Scorpaeniformes,Platycephalidae)

The Japanese flathead, Inegocia japonica Cuvier, 1829 is a commercially important fish in small-scale coastal fisheries in Thailand; however, an explanation of its digestive biology is missing. This study describes the digestive tract and accessory organs of I. japonica, using morphological and histological methods. The fish (10 individual fish, 24.5 ± 0.98 cm in total length) were obtained from Libong Island, Thailand. Integrated morphological and histological data showed that the digestive tract was composed of oesophagus, stomach, pyloric caeca and intestine, with accessory organs. All digestive tracts consisted of four layers, including mucosa, submucosa, muscularis and serosa. Two stomach regions were identified (cardiac and pyloric stomachs). Several clusters of gastric glands were identified in the cardiac stomach. Each gland was a unicellular structure. The apical surface of this gland contained the vacuolar cell. The intestine was lined with a simple columnar structure with goblet cells that was similar to pyloric caecum. Goblet cells were rare in the anterior intestine, in contrast to the posterior intestine where goblet cells were abundant. The numerous of hepatocyte was mostly observed in the liver, whereas an exocrine acinar cell of pancreas was also identified. The results of our observations provided the first information of the digestive tract of I. japonica and can be applied to advanced study, such as physiology and histopathology.     

Factors influencing the degradation of photoreceptor membrane in the crayfish,Procambarus clarkii

Summary Light-induced degradation of photoreceptor membrane in the crayfish was studied by quantitative light and electron microscopy. The production of lysosomal organelles within the photoreceptor cells was enhanced by presenting the light stimulus intermittently (i.e., flicker) or by doubling its intensity. The enhancement was seen primarily as an increase in the number and size of multivesicular bodies. As these stimulus conditions are likely to facilitate intracellular Ca⁺⁺ fluxes, the results are compatibl with recent speculations that Ca⁺⁺ ions may regulate membrane degradation. To test the possibility that Ca⁺⁺ acts as a signal coupling receptor stimulation with membrane loss, retinas were incubated in the dark with the ionophore A23187 in the presence or absence of external Ca⁺⁺. The results demonstrate that A23187 produces a Ca⁺⁺-dependent increase in lysosomal organelles, predominantly multivesicular bodies. These data are consistent with a role for intracellular Ca⁺⁺ in the degradative process; however, the exact locus of the effect is unclear.Supported by a grant (BNS 8004587) from the National Science Foundation to G.S.H. The authors gratefully acknowledge the helpful discussions and expert technical assistance of Thomas R. Tokarski     

Electron microscopic observations of lung alveolar epithelial cells of normal young mice,with special reference to formation and secretion of osmiophilic lamellar bodies

Summary Using the electron microscopy and electron microscopic histochemistry the authors studied the lung alveolar epithelial cell of normal young mice.Type II cell of the alveolar epithelium has characteristically numerous osmiophilic lamellar bodies. The lamellar boies are formed in the cytoplasmic vesicle, and never originate from the mitochondrion. These bodies have abundant acid phosphatase activity in their limiting membrane therefore it is considered to be lysosomal origin, but the mitochondria have no such enzyme activity.The body which is newly formed in the cytoplasmic vesicle grows up to the large lamellar body as a result of an accumulation of the fibrous dense substance, migrates to the free margin of the type II cell of alveolar epithelium, and then is discharged into the alveolar lumen as a merocrine type secretion.Acknowledgement is given to Professor Dr. Y. Sano and Professor Dr. H. Fujita, Department of Anatomy, and Assistant Professor Dr. S. Fujita, Department of Pathology, for their kind advice and criticism.     

Golgi associated acid phosphatase in muscle and nerve cells fromArion ater (L.)

Summary Golgi associated acid phosphatase has been demonstrated within muscle and nerve cells from the sub-epithelial layers of the crop, intestine, and digestive gland ofArion ater. Enzyme activity was detected in the saccules, vacuoles, and vesicles of the Golgi apparatus of both nerve ganglia and muscle cells. Other vacuolar sources of acid phosphatase could also be distinguished within the cytoplasm of these cells.     

Anatomical and histochemical features of the digestive system of Octopus vulgaris Cuvier, 1797 with a special focus on secretory cells

This study reports a detailed anatomical and histological study of the digestive system of Octopus vulgaris. Emphasis was placed on characterising the glands and glandular cells and their distribution throughout the digestive tract. The use of classic histological and histochemical techniques revealed two morphological types of glandular cells: granular and mucous. Moreover, the histochemical analysis indicated specialisation of mucous glandular cells in the buccal mass, the submandibular gland and the caecum for secreting acid and neutral glycoconjugates. The cells of the anterior salivary glands are specialised for secreting neutral glycoproteins, and those of the posterior salivary glands are specialised for granular and mucous secretion. The oesophagus, crop and stomach lack glandular cells, but both granular and mucous glandular cells are found in the intestine. An unusual structure resembling the typhlosole of bivalves is described for the first time in the intestine of O. vulgaris. The highly ciliated epithelium and location of the structure in the anterior part of the intestine suggest a possible role in bypassing the caecum, stomach and intestine. We discuss how these cells and organs contribute to the process of digestion in the light of the present histological and histochemical data and of previously published information on the morphology and physiology of digestion in the octopus.     

Electronmicroscopical observations on yolk and yolk formation in Ophryotrocha labronica LaGreca and Bacci

Summary In Ophryotrocha labronica LaGreca & Bacci mature yolk granules are found only in the ovocyte. Other typical yolk elements are lipid droplets, small vesicular bodies, multivesicular bodies and dense bodies. The two last-mentioned also appear in the accompanying nurse cell and from there obviously pass over unchanged into the ovocyte through a specific intercellular bridge, the fusome.The mature yolk granules are considered as aggregates of mitochondrial, endoplasmic and Golgi material, to which also is added pinocytotically incorporated external material. Mitochondria apparently play a fundamental role in the process, as the multivesicular bodies, most likely the direct precursors to the yolk granules, in all probability represent transformed mitochondria.Labelling with ³H-thymidine during vitellogenesis reveals presence of DNA in the yolk granules. From the labelling pattern, which shows DNA-synthesis both in the ovocyte and the nurse cell nucleus, it is concluded that the labelled material present in the cytoplasm of both cells — most of it in yolk granules and dense bodies — is of nuclear origin. The possible mitochondrial nature of yolk granule DNA is discussed.The author is indebted to Dr. Bertil Åkesson, Zoological Institute, Lund, for kindly supplying the initital material for the Ophryotrocha cultures. The excellent technical assistance of Mrs. Mariann Carleson is gratefully acknowledged. My thanks are also due to Mrs. Siv Nilsson for skilful assistance with the photography. This work has been supported by the Swedish Natural Science Research Council and Kungliga Fysiografiska Sällskapet, Lund.     

The fine structure of the cells of the mouse peritoneum

Summary The cells of the peritoneum of the mouse have been examined with the electron microscope both by studying the gastro-splenic omentum and by washing the cells out of the peritoneal cavity. They comprise mesothelial cells, mast cells, lymphocytes and macrophages. The mesothelial cells were probably nearly all degenerate. The mast cells released granules which were phagocytosed by the other cells. The lymphocytes were either classical small lymphocytes, or rather larger cells similar to previously described immunoblasts. The macrophages varied considerably in size. Some were probably derived from the covering cells of the milk spots. They contained varying numbers of dense bodies, with the structure of lysosomes. A series of appearances was seen which suggested that these were synthesized in the granular endoplasmic reticulum. A gradation of structure was seen between lymphocytes and small macrophages.The gastro-splenic omentum consisted of two layers of mesothelium, in places fenestrated. The milk spots which were scattered throughout this structure were covered by cells similar to macrophages, and had a core of lymphoid cells in which ran a small blood vessel. The most notable difference between the mesothelial cells and the macrophages was the presence of many small caveolae at the surface of the mesothelial cells, and of larger vacuoles and indentations at the surface of the macrophages. Acknowledgements. I am grateful to Professor R. Barer for much advice and criticism, to Dr. G. A. Meek for guidance on electron microscopy, and to Miss M. Tune and Mr. M. Turton for photographic assistance.This work was supported by a grant from the Medical Research Council and by grants to the Department from the S.R.C., Nuffield Foundation and Unilever Limited.     

The fine structure of photoreceptors in a marine flatworm

Summary The ocelli or eyes of the marine polyclad turbellarian Notoplana acticola are clustered on the paired dorsal nuchal tentacles and in two longitudinal bands lateral to the cerebral ganglion. The ocelli, studied by electron microscopy, were characterized as rhabdomeric and non-ciliary in origin. There are 60 to 80 ocelli per animal each enclosed in a fibrous capsule to which muscle fibers may attach. An ocellus consists of a pigmented eyecup into which 30 to 50 photoreceptor cells send dendritic processes through interruptions in or among pigment cell projections across the eyecup opening. The dendritic processes terminate in numerous long intertwined microvilli which fill the eyecup. The nucleated cell body of each photoreceptor cell lies outside the eyecup and projects an axonal process to the cerebral mass. Within the dendritic processes are observed mitochondria, ribosomes, neurotubules, multivesicular bodies, vesicles and vacuoles. The cell body contains smaller mitochondria, endoplasmic reticulum, ribosomes, vesicles and prominent Golgi complexes.After dark adaptation, there are some structural alterations in terms of swelling of microvilli, increased numbers of vacuoles associated with the microvilli and dendritic processes, and changes in the pigment cell projections.This work was supported by Grant No. GM 10292 from the U.S. Public Health Service to Professor Richard M. Eakin, Department of Zoology at the University of California, Berkeley, U.S.A., where this investigation was conducted during the author's sabbatical leave of absence from the University of Illinois, and by Grant No. 1 SO 1 FR 5369 from the U.S. Public Health Service to the University of Illinois at the Medical Center.I express appreciation to Professor Eakin for interesting discussions and generous hospitality to me as a guest in his laboratory, and to the John Simon Guggenheim Memorial Foundation for the Fellowship which I held during 1964–65. I thank Dr. John P. Marbarger, Director of the Aeromedical Laboratory for the electron microscope facilities used at the University of Illinois.     

Phosphatase activity in the hepatopancreas of Helix aspersa

• 1.1. Phosphatase acid (PhA) activity in the digestive gland (hepatopancreas) of the common garden snail Helix aspersa has been investigated using cytochemical methods.
• 2.2. All the cells composing this gland show PhA activity, the distribution pattern differing according to the cell type.
• 3.3. The digestive cells show the most widely distributed reaction product (brush border, phagolysosomes, multivesicular bodies and autophagic vacuoles).
• 4.4. In the excretory cells this activity appears in large sacs, while in the calcium cells the reaction product is abundant in the calcium granules.
• 5.5. Cellular digestion processes performed by each of these cell types is discussed together with their role in the detoxification of heavy elements derived from the environment.

     

Photoreceptor membrane breakdown in the spider Dinopis: The fate of rhabdomere products

Summary Photoreceptor membrane breakdown at dawn in the posterior median eyes of the spider Dinopis is described. Coated and smooth vesicles are shed into the receptor cytoplasm and are assembled into multivesicular bodies of two kinds: (i) Coated vesicles form loosely-assembled multivesicular bodies (mvbs) whose bounding membranes are derived from endoplasmic reticulum. (ii) Smooth vesicles generated by the mass disintegration of membrane aggregate to yield tightly-assembled multivesicular bodies which are not membrane-bound. Both types are either lysed in the inter-rhabdomeral cytoplasm, or degrade via multi-lamellar bodies to residual bodies (rbs) while they are being transported to the intermediate segments. Two systems are associated with lysis. Nebenkerne produced by the rapid differentiation of GERL in the intermediate segments fuse with membrane-bound mvbs or rbs and may inject them with hydrolases. Partially-differentiated rigid tubules (Blest et al., 1978) travel to the receptive segments together with RER from the intermediate segments and also fuse with or engulf mvbs. Both systems may contain pro-enzymes which are activated at their target sites. No evidence of a close or necessary geometrical relationship between GERL and Golgi bodies has been seen, and there is no clear demarcation between RER, smooth ER and GERL which is entering into continuity with or engulfing mvbs. The implications of these findings for hypotheses about the origins of isolation membranes and autolytic systems in invertebrate systems are briefly discussed.The authors thank Professor D.T. Anderson, F.R.S., for our use of field facilities at the Crommelin Biological Field Station of Sydney University at Warrah, Pearl Beach, N.S.W., Andrew & Sally Austin and Sally Stowe for help in the field, and Joanne Maples for technical assistance. Professor T.H. Waterman and Dr. V.B. Meyer-Rochow kindly gave us access to certain of their results prior to publication. We are indebted to Rod Whitty and the Electron Microscopy Unit for advice and support throughout these studies     

Multiple sources of esterase enzymes in the crop juice ofCepaea (Mollusca: Helicidae)

Summary Previous workers have (a) compared pulmonate crop juice and digestive gland extracts and found a close similarity in the enzymic complements from these two sources, and (b) located specific enzymes within the various cell types of the digestive gland. The digestive gland seems to be the major source of extracellular enzymes but what is not clear is which of the enzymes associated with particular intracellular structures are actively secreted into the crop juice. The present study has used polyacrylamide disc gel electrophoresis to investigate the digestive gland and crop juice esterases ofCepaea nemoralis andC. hortensis. It appears that only some of the digestive gland esterases are specifically secreted. The variation shown in crop juice esterases suggests three independent sources in the digestive gland. Less detailed studies ofHelix aspersa andArianta arbustorum also indicate multiple sources of extracellular esterases.     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Summary Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed and increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and -N-acetyl-d-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and -glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the German Research Foundation (Sfb 174)     

The sources of acid hydrolases for photoreceptor membrane degradation in a grapsid crab

Summary Dawn photoreceptor breakdown in the crab Leptograpsus variegatus is analysed at the ultrastructural level. Coated vesicles derived from microvilli are assembled as multivesicular bodies (mvbs), which degrade to multilamellar bodies (mls) and are lysed. Cytochemical markers for hydrolases were a fluorideinhibited -glycerophosphatase and a fluoride-insensitive p-nitrophenyl phosphatase, with indistinguishable distributions when localised at pH 5.0. These enzymes are injected into the secondary lysosomes from two sources: (i) Immediately after dawn Golgi bodies are highly active, and differentiate a transtubular network, from which tubules and vesicles detach, and can be seen fusing with mvbs and mlbs. (ii) Saccules derived from the rough endoplasmic reticulum (RER) provide a second source and are most often seen in association with late mlbs. Both kinds of primary lysosome rarely give AcPh-positive responses when free in the cytosol, but are seen to do so as they make contact with their secondary lysosomal targets. Lipid droplets and lipofuscin bodies are interpreted as the residual products of breakdown. These results are discussed in relation to previous findings on photoreceptor membrane breakdown in a dinopid spider. Attention is drawn to the implied diversity of organisation of lysosomal compartments in receptors which internalise membranes of similar compositions.The authors thank Professor T.H. Waterman for hospitality extended to A.D. Blest at Yale University, for allowing them to see results prior to publication, and for enthusiastic encouragement of at present project during a visit to this University in 1979. We also thank Dr. Gary Hafner and Dr. D.R. Nassel for sending us results prior to publication, and others, especially Dr. Dean Bok, for discussions in correspondence. Bruce Ham helped to collect crabs. Rod Whitty and the staff of the Electron Microscope Unit provided advice and support throughout these studies. Chris Snoek prepared Fig. 1 a, b