The Structure of Mitochondria

The Structure of Mitochondria

E.A. MUNN

Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge, England

Table of Contents

Cover image

Title page

Copyright

Dedication

Preface

Acknowledgements

Chapter 1: Variety in the Structure of Mitochondria in situ

Publisher Summary

Shape of mitochondria in situ

Shape and organization of cristae

Surface area of the mitochondrial membranes

Arrangement of mitochondria in the cell

Mitochondrial inclusions

Chapter 2: Changes in Structure of Mitochondria in situ

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Structural changes during development and differentiation

Structural changes during gametogenesis

Structural changes due to changes in diet

Structural changes due to changes in environment and physiological state

Structural changes in yeast mitochondria

Changes in structure associated with clinical disorders

Chapter 3: The Structure of Isolated Mitochondria

Publisher Summary

Isolation of mitochondria

Structure of intact isolated mitochondria

Changes in mitochondrial structure in relation to changes in osmotic pressure

Changes in mitochondrial structure induced by swelling agents

Reversal of large amplitude swelling

Structures peculiar to isolated mitochondria

Structure of disrupted mitochondria

Process of fragmentation of inner membranes

Chapter 4: Chemical Composition Part 1: Proteins

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Enzymes

Nonhaem iron protein

NADPH-nonhaem iron protein reductase

Electron transferring flavoprotein

Calcium carrier

Structural protein (s) and core protein

Peptide chain elongation factors

Coupling factors

Cytochromes

Chapter 5: Chemical Composition Part 2: Lipids, Metal Ions, Nucleotides and Other Anions

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Lipids

Ubiquinones (coenzymes Q)

Metal ions

Flavins

Nucleotides

Other anions

Coenzyme A

DNA

RNA

Chapter 6: Structure and Functional Organization of the Mitochondrial Membranes

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Structure of the mitochondrial membranes

Role of lipids in the structure and activity of the inner membrane

Mitochondrial structure and the generation of ATP

Long-term stability of mitochondrial structure

Mitochondrial ribosomes

The problem of the assembly of mitochondria

Chapter 7: Structural Aspects of Metabolic Pathways (Compartmentation)

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The enzymes of the tricarboxylic acid cycle

The enzymes involved in amino acid metabolism

The enzymes for hydroxylation of steroids

The enzymes for metabolism of fatty acids

Transport through the mitochondrial inner membrane

Chapter 8: Relationships Between Mitochondrial Structure, Metabolic Control and Ion Transport

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Terminology

Structure in relation to metabolic steady state

Oscillatory changes in mitochondrial structure

Effect of composition of suspending medium on metabolically related structural changes

The effects of ADP, DNP and phosphate on mitochondrial structure

The effects of divalent cations on mitochondrial structure

Metabolically related structural changes of mitochondria in situ

The significance of the changes in the internal organization of isolated mitochondria

Chapter 9: Mitochondria, Chloroplasts and Bacteria

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Mitochondria and chloroplasts

Mitochondria and bacteria

Evolutionary origin of mitochondria

References

Author Index

Subject Index

Copyright

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Dedication

For Patricia D., Deborah B. and Cordelia F.

Preface

The study of the structure of mitochondria has been a topic of great interest for many years. It received its greatest impetus when, in 1952, Palade and Sjöstrand, using the then newly introduced technique of thin sectioning, demonstrated with the electron microscope that the organelles contained organized arrays of membranes. Also at about this time, work on isolated mitochondria had led to the recognition of their fundamental role in the controlled production of energy for the cell. Refinements of earlier procedures and the introduction of new techniques for electron microscopy have since been matched by improvements in the procedures for the fractionation of isolated mitochondria and separation and characterization of their component parts.

When the last extensive account of the structure of mitochondria was published (Novikoff, 1961), the electron microscope had been used to study mitochondria in a wide variety of cells, but it is only more recently that comparative studies of the properties of mitochondria isolated from various sources have been undertaken. The results of these studies have confirmed the contention that there is a basic framework common to all mitochondria and that they possess features characteristic of the particular cell type in which they occur.

In the present account I have set out first of all to illustrate the variety of mitochondrial structure revealed by electron microscopy of intact cells. The structural features made apparent by this technique must be determined by the structure and arrangement of the molecules of which the mitochondria are composed. Some of these are now sufficiently well understood to allow a description of their functional organization within mitochondria and of their role in the interaction of mitochondria with other cell components in vivo.

It is all too easy to lose sight of the fact that some of the properties of isolated mitochondria may result from the act of isolation, nonetheless, they provide good clues as to the way in which the organization of the membranes inside the mitochondria is involved in metabolic control.

By way of conclusion, I have described the interesting similarities of mitochondria, bacteria and chloroplasts and the bearing these have on the speculation about the way in which the relationships between mitochondria and the rest of the eukaryotic cell have evolved.

E.A. MUNN

February, 1974

Acknowledgements

It is a pleasure to acknowledge the help that my wife (and from time to time our daughters) have given me in the preparation of the book. I also wish to thank Mr. D. W. Butcher and his colleagues in the Library and Mr. A. L. Gallup and his colleagues in the Photography Department at Babraham for the excellent facilities they have provided.

I am indebted to the ninety-seven authors who have given me permission to reproduce their electron micrographs and diagrams. Many of these have been published before and I am most grateful to the publishers for their kind permission to reproduce figures.

Figures 3.3 and 6.14 first appeared in Plant Physiology, published by the American Society of Plant Physiologists; Fig. 8.1, in Intracellular Membranous Structure, published by the Japan Society for Cell Biology; Fig. 2.16 in Journal de Microscopie, published by Societé Française de Microscopie Électronique; Fig. 2.8a in the American Journal of Pathology published by the American Association of Pathologists and Bacteriologists; Fig. 1.53 in the Journal of Submicroscopic Cytology, published by the Instituto di Microscopia Ellectronica, Bologna. Figure 3.8 first appeared in Circulation and is reproduced by permission of the American Heart Association; Fig. 1.30 in The Fungus Spore, published by the Colston Research Society; Figs 2.13 and 2.14 in Acta Tropica, published by Verlag für Recht und Gesellschaft AG., Basel; Figs 4.1, 4.7, 4.8, 4.11a, c, d, e, 4.12, 4.13, 4.18, 4.19 in Journal of Biological Chemistry, published by the American Society of Biological Chemists Inc.; Figs 1.49, 1.50, 3.6 and 9.3 in Handbook of Molecular Cytology, published by ASP Biological and Medical Press; Figs 6.13, 7.7 in FEBS Letters, published by the Federation of European Biochemical Societies; Figs 7.5, 7.6 from European Journal of Biochemistry are reproduced by permission of the Editor-in-Chief. Figure 9.5 is reproduced by permission of the National Research Council of Canada from the Canadian Journal of Microbiology, 11, pp. 935–938 (1965). Figure 2.19 first appeared in Brain, published by Macmillan Journals Ltd.; Figs 1.6, 1.8, 1.10, 1.11, 1.17, 1.19, 1.20, 1.21c, 1.22, 1.24, 1.32, 1.39, 1.40, 1.42, 1.48, 1.52, 1.57, 2.8b, 2.9, 2.11, 2.12, 3.16, 5.6, 6.5, 8.16 and 8.19 in Journal of Cell Biology, published by The Rockefeller University Press; Fig. 1.18 in Journal of Anatomy, Fig. 9.1 in Symposium of the Society of General Microbiology and Fig. 6.11 in Quarterly Review of Biophysics, published by Cambridge University Press; Fig. 2.23 in Journal of Cell Science published for the Company of Biologists Ltd.; Fig. 9.6 in Journal of Bacteriology, published by the American Society for Microbiology. Figure 2.22 is from Phytopath. Zeitschrift, published by Paul Parey. Figures 3.4, 4.6, 5.14, 5.15, 5.16, 5.20, 7.2 and 8.4 which first appeared in Proceedings National Academy of Sciences, U.S.A. are reproduced by permission of the National Academy of Sciences; Figs 4.9 and 4.10 are reprinted with permission from Biochemistry, 5, copyright by the American Chemical Society. Figure 4.16 first appeared in Regulatory Functions of Biological Membranes Figs 4.3, 4.4, 5.11, 5.12, 5.19, 5.21, 6.1, 6.3 in Biochimica Biophysica Acta, published by Elsevier Publishing Company; Fig. 1.36 in Tissue and Cell, published by the Longman Group Ltd.; Fig. 4.2a in Bioploymers, published by John Wiley and Sons, Inc.; Fig. 1.7 in Annals of Botany, published by Oxford University Press; Fig. 2.20 in Acta neuropath., Fig. 1.12 in Archiv für Mikrobiologie and Figs 1.27, 1.28, 1.33, 1.34, 1.44, 1.51, 2.17, 2.18 in Zeitschrift für Zellforschung, all published by Springer Verlag, Berlin–Heidelberg–New York; Fig. 1.21a in Comptes Rendus de l’Academie des Sciences, published by Gauthier-Villars. Figure 4.17 first appeared in Acta Medicinae Okayama; Fig. 1.41 in Journal of Electron Microscopy, published by the Japanese Society of Electron Microscopy; Fig. 1.3 in Protoplasma, published by Springer Verlag, Wien; and Figs 8.5, 8.6, 8.7, 8.10, 8.11, 8.15, 8.17 and 8.18 in Journal of Bioenergetics, published by Plenum Publishing Company Ltd. The scale drawings of molecular models in Chapter 5 are based on drawings in E. DuPraw’s book Cell and Molecular Biology.

E.A.M

1

Variety in the Structure of Mitochondria in situ

Publisher Summary

This chapter discusses the variety in the structure of mitochondria in situ. Cells in different tissues can be distinguished by their different, and often characteristic, shape and organization and, as has long been apparent to light microscopists, by the numbers, shapes, sizes, and distributions of the mitochondria which they contain. The techniques of electron microscopy that have provided most information on mitochondrial structure are thin sectioning and negative staining, and the more recently introduced technique of freeze-etching. The apparent structure of mitochondria in situ revealed by thin sectioning depends on the type of cell in which the mitochondria occur, the physiological state of the cell, and, not least, on the procedure to which the cell is subjected in its preparation for examination in the electron microscope. The chapter also explains the shape of mitochondria in situ, shape and organization of cristae, surface area of the mitochondrial membranes, and arrangement of mitochondria in the cell. The chapter further explains mitochondrial inclusions. A large number of mitochondrial inclusions have been described; nearly all have been seen in sectioned material and one or two in negatively stained preparations. Most inclusions occur in the matrix, but a few have been observed in the intracristal and peripheral spaces, and some are derived from modifications of the cristae. The inclusions may be broadly classified as granular, tubular or filamentous; they commonly form crystalline or paracrystalline arrays.

Cells in different tissues can be distinguished by their different, and often characteristic, shape and organization and, as has long been apparent to light microscopists, by the numbers, shapes, sizes and distributions of the mitochondria which they contain. The application of electron microscopy to the study of the structure of cells and their mitochondria has brought out the details of their differences but, at the same time, has emphasized the number of common features which they share.

The techniques of electron microscopy which have provided most information on mitochondrial structure are thin sectioning and negative staining, and the more recently introduced technique of freeze-etching. In the most commonly employed procedure for sectioning, specimens are fixed, dehydrated and then embedded in a supporting medium. Reagents widely used as fixatives include osmium tetroxide, glutaraldehyde and potassium permanganate. Frequently combinations of these reagents, either sequentially or together, are employed. In the early work on mitochondria, fixed, dehydrated specimens were embedded in methacrylates, but these have been largely superseded by epoxy resins and to a lesser extent by polyester resins. A limited amount of work has been done on the structure of mitochondria in sections of tissues preserved by freeze-drying or by freeze-substitution.

Because biological specimens lack inherent electron density, they are usually stained with electron-dense molecules. Staining is carried out during fixation with osmium tetroxide or potassium permanganate, or subsequently by treating the fixed specimen with a solution of heavy metal salt (phosphotungstic acid, uranyl acetate, lead hydroxide, tartrate or citrate). These stains enhance the contrast between some parts of the specimen, thus distinguishing them from each other. In cytochemical studies specific staining is sought at the sites of certain chemically reactive groups or enzymes.

In negative staining the specimen is surrounded by an aqueous solution of an electron-dense salt (e.g. potassium phosphotungstate, ammonium molybdate) which is allowed to dry in a thin film around the specimen and is thought to preserve it in a hydrated state, or, at least, one resembling the hydrated state (Bangham and Horne, 1964). The negative stain penetrates more or less into the hydrophilic parts of specimens, and the resulting image in the electron microscope reveals the unpenetrated regions as relatively electron-transparent structures against an electron-dense background. The specimen may be treated with a fixative before negative staining. In the freeze-etching technique the specimen is rapidly frozen to about −190°C; under vacuum the frozen material, now at about −100°C, is fractured to expose a surface, which may be freeze-dried to a depth of a few hundred ångströms (etching), and is then shadowed with platinum-carbon to form a replica which can be examined in the electron microscope. Shadowing is also used in the study of mitochondrial components, particularly DNA (see Chapter 5).

The apparent structure of mitochondria in situ revealed by thin sectioning depends on the type of cell in which the mitochondria occur, the physiological state of the cell, and, not least, on the procedure to which the cell is subjected in its preparation for examination in the electron microscope. The apparent structure of isolated mitochondria further depends on their treatment during isolation and prior to their preparation for sectioning. Within these limits all mitochondria can be referred to a general basic pattern as first deduced by Palade (1952, 1953), Sjöstrand (1953) and Sjöstrand and Rhodin (1953) (Figs 1.1 and 1.2). Each mitochondrion consists of a limiting or outer membrane within which is a peripheral inner membrane which in turn encloses an inner space called the matrix. Lying in the matrix are a variable number of membranous structures called cristae which appear either to lie free, or to be associated with the inner membrane. The name cristae (more fully, cristae mitochondriales) was originally used by Palade (1952) to describe the shelf-like lamellae seen in rat liver mitochondria, but the term is used here, and more generally, for all forms of arrangement of these internal membranes. Sometimes the cristae contain a space, the intracristal space, and there is also a space between the outer and inner membranes, the peripheral space. Occasionally the inner membrane and the cristae membranes are seen to be continuous so that there is continuity between the intracristal and peripheral spaces. These spaces have also been termed the intermembrane space (Ernster and Kuylenstierna, 1969) or membrane space (Parsons, 1965); use of the latter term may lead to confusion with the space occupied by the membranes themselves. Since the peripheral inner membrane and the internal or cristae membranes are continuous, the term inner membrane is frequently used to include both parts.

Fig. 1.1 Electron micrograph of a section through a mitochondrion (in a piglet intestinal cell). Fixed in glutaraldehyde, treated with osmium tetroxide, embedded in Araldite and the section stained with uranyl acetate and lead citrate. The membranes are distinct only where they are perpendicular to the plane of the section. When the peripheral membranes lie in the plane of the section, the separation of the mitochondrial contents from the cytoplasm is no longer apparent.

Fig. 1.2 Diagrammatic representation of a section through a mitochondrion, based on Fig. 1.1.

The matrix appears more or less electron dense (two extremes are shown in Fig. 1.3) and finely granular, and it may contain discrete electron dense granular, crystalline or fibrous inclusions (see p. 43).

Fig. 1.3 Electron micrograph of section of Amoeba proteus showing mitochondria with electron dense (D), relatively electron transparent (L) and intermediate density (I) matrices. The cristae of these mitochondria are tubular. Fixed with glutaraldehyde and formaldehyde. From Flickinger (1968).

The structure of mitochondria in situ deduced from the examination of thin sections is confirmed by freeze-etching and such negative staining as can be applied to whole cells.

Shape of mitochondria in situ

Electron microscopy has confirmed the early observations of light microscopists that mitochondria have a variety of shapes, ranging from spheres to branched filamentous forms. In any one cell type the mitochondria may all have the same shape and this is particularly true of mature spermatozoa. In mammalian spermatozoa for instance, all the mitochondria, save one or two at the proximal end of the mid-piece, are short crescentic rods wrapped helically around the axoneme (Fig. 1.4). In the mid-piece of earthworm spermatozoa, the mitochondria are longitudinally arranged rods which are triangular in cross-section (Fig. 1.5). Many other types of cells contain mixtures of mitochondria of different shapes, but nonetheless it is often possible to relate the shape of the mitochondria to the cell type in a general way.

Fig. 1.4 Electron micrographs of mitochondria in mid-piece of mammalian spermatozoa. (a) Section of boar spermatozoon (fixed glutaraldehyde, osmium); (b) frozen etched rat spermatozoon (generously provided by Dr. J. K. Koehler, see Koehler, 1966); (c) bull spermatozoon negatively stained with ammonium molybdate.

Fig. 1.5 Diagram to show the characteristic shape and arrangement of mitochondria in mid-piece of earthworm, Lumbricus terrestris, spermatozoa. The mitochondria are encased by the plasma membrane and lie between the nucleus and the flagellum. See Anderson et al. (1967).

In rat liver, the tissue whose mitochondria have been most extensively studied, the mitochondria appear to be either small spheres or rods of various lengths, and both forms occur together (Fig. 1.38). In the proximal tubule of the kidney, both these shapes are again present in the same cell, but the distal part contains only elongated mitochondria (see Fig. 1.15).

Fig. 1.15 Electron micrographs of sections of mitochondria (a) at the base of a kidney proximal tubule cell; (b) in a rat liver cell.

Fig. 1.38 Electron micrographs of section of rat liver showing mitochondria containing electron-dense granules 250–500 Å across in the matrix. Electron micrograph by C. Rouiller, R. Pictet and L. Orci, generously provided by Professor C. Rouiller.

Hagopian (1967) has described three distinct populations of mitochondria in the femoral muscle of the cockroach, Leucophaea mederae, that are recognizable by their shape, size and position in the cell. (a) Just beneath the plasma membrane are oval mitochondria about 2 µm long and 0·6 µm maximum diameter. (b) The second type are commonly 3 µm long, but may be up to 25 µm long; they are about 0·5 µm wide. They lie between, and parallel to, the myofibrils and they have characteristic surface indentations which accommodate elements of the sarcoplasmic reticulum and limbs of the third type of mitochondria. These (c) are Y-shaped, nearly 3 µm in overall length and are wrapped around the myofibrils. One of the arms of the Y lies next to, and in the plane of, the Z-disc, and the other two arms lie either side of the Z-disc. Y-shaped mitochondria, and mitochondria with several branches, also occur frequently in rat diaphragm where they may form rings or braces around the I-bands (Palade, 1953).

Annular mitochondria are found in logarithmically growing Tetrahymena pyriformis (Elliot and Bak, 1964) (Fig. 1.6), in the mid-piece of some sea urchin spermatozoa (Longo and Anderson, 1969), in Papilio osmeterium (Crossley and Waterhouse, 1969) and in the pathogenic yeast-like organism Cryptococcus neoformans (Edwards et al., 1967). Annular mitochondria also encircle the basal body of the uniflagellate zoospores of the aquatic fungus Blastocladiella emersonii (Cantino and Truesdell, 1971) (see Fig. 1.30).

Fig. 1.6 Electron micrographs of mitochondria in sections of logarithmically growing Tetrahymena pyriformis. The sections are in planes at right angles to each other. From Elliot and Bak (1964).

Fig. 1.30 Electron micrographs of sections through zoospores of Blastocladiella emersonii. (a) Showing the zoospore in longitudinal section; (b) transverse section through the mitochondrion. From Fuller (1966).

Apparent annuli would result from cross-sectioning cup-shaped mitochondria. Mitochondria of this shape, first described by André (1958, 1959) in the spermatocyte of the scorpion Euscorpius flavicaudis (Fig. 2.3, see p. 73 for details) and called by him culichondries, have also been observed in tissues from other animals, plants and fungi. They have been described in rat testicular interstitial cells (Christensen and Chapman, 1959), in hamster adrenocortex (De Robertis and Sabatini, 1958), in a few cells of an apparently normal rat liver (Stephens and Bils, 1965), in gills of the crab Callinectes sapidus (Copeland and Fitzjarrell, 1968) (see p. 38), in antimycin-treated guinea pig pancreatic exocrine cells (Jamieson and Palade, 1968), in archegonia of the fern Pteridium aquilinum (Bell and Mühlethaler, 1964), in meristoderm cells of the brown alga Egregia menziesii (Bisalputra and Bisalputra, 1969) and in Neurospora crassa hyphae (Weiss, 1965). Mitochondria in tissue freshly excised from dormant tubers of the Jerusalem artichoke characteristically contain large numbers of mitochondria shaped like cups with thick sides and rims (containing numerous cristae), and thin, crista-free bases (Fig. 1.7a). The mitochondria which are frequently sectioned to reveal annular profiles (Fig. 1.7b) apparently develop in cultured cells into more bell-shaped structures with thin walls (Fig. 1.7c) (Bagshaw et al., 1969).

Fig. 1.7 Electron micrographs of mitochondria in sections of vacuolated parenchyma cells of Jerusalem artichoke tubers. From Bagshaw et al. (1969).

Senger and Saacke (1970) have reported a related unusual morphological type of mitochondrion abundant in oocytes from mature bovine Graafian follicles. The mitochondria have a characteristic distribution of cristae and matrix, and at one end the peripheral membranes enclose only a small thickness of matrix and are folded to give a hooded appearance (Fig. 1.8). The pleomorphic mitochondria in cat ventricular papillary muscle often possess slender processes in which the peripheral membranes are closely apposed and which contain only one, or are free of, cristae (Fawcett and McNutt, 1969). Disc-shaped mitochondria with flattened centres consisting of closely apposed outer membranes have been reported as a characteristic feature of neonatal rat liver (Stempak, 1967) and in the myoepithelium of rat submaxillary gland (Tamarin, 1966). Similar mitochondria in which the closely apposed outer membranes form a sandwich with the fused inner membranes, have been described in columnar cells of the midgut of Hyalophora cecropia (Anderson and Harvey, 1966) and in the salt absorbing cells of crab gills (Copeland and Fitzjarrell, 1968).

Fig. 1.8 Drawing to show appearance of hooded mitochondria in electron micrographs of mature bovine Graafian Follicles. Characteristically, each hood encloses a single cisterna of the endoplasmic reticulum (see Fleming and Saacke, 1972). From Senger and Saacke (1970).

By comparison with some of their mammalian counterparts, the mitochondria in insect flight muscles are of comparatively simple shape, but they tend to be large. In the flight muscle of the wasp Polistes the mitochondria are cylindrical and only about 1·6 µm long and 1·2 µm wide. The mitochondria of the asynchronous flight muscles of the blowfly Calliphora are more generally spheroidal in shape and some 3–6 µm long by 1 µm wide (Smith, 1963), while the mitochondria of the synchronous flight muscles of the dragon fly Aeschna are slab-like structures about 9 µm by 9 µm and 1 µm thick (Smith, 1961).

Large mitochondria 1·5–3·2 µm in diameter occur frequently in the large groups which lie in the peripheral sarcoplasm of the red fibres in rat soft palate muscle (Leeson and Leeson, 1969), and large mitochondria (up to 10 µm long and 1·5 µm wide) are numerous in the supracoracoideus and pectoralis muscles of the hummingbird, Archilochus colubris (Grinyer and George, 1969). So-called giant mitochondria, spheres with diameters of 2–3 µm, have been found occasionally in the infranuclear region of mink endometrial gland cells (Enders et al., 1963), and mitochondria up to 7 µm diameter containing numerous cristae are present in the human uterus in the second half of the menstrual cycle (Merker et al., 1968). The incidence of giant mitochondria in certain human disorders is considered later (p. 102).

Shape and organization of cristae

On the basis of their appearance in mitochondria fixed in situ, cristae may be broadly classified as either lamellar, tubular (villous), or tubulo-saccular. The lamellar form is the commonest and a variety of shapes, sizes and arrangements of lamellar cristae have been described (see below). Tubular cristae may be circular in cross-section and of uniform internal diameter, or they may have irregular dimensions, or more rarely, they are triangular (prismatic) or square in cross-section. Forms intermediate in appearance between lamelliform and tubular cristae are found. It is possible that cristae do not have a fixed shape in vivo (André, 1959, 1962; Rouiller, 1960); certainly considerable changes in their appearance can be induced in vitro.

There are some difficulties in distinguishing lamelliform and tubular cristae in single sections as may be seen by reference to the drawings in Fig. 1.9.Figure 1.9a shows the face-view of a discoidal, lamelliform crista lying in the plane of the paper. The crista is connected through short tubes with the inner membrane at two places. The appearance of cross-sections of this crista at various levels are shown in Fig. 1.9b. Figure 1.9c and d show the corresponding views of two tubular cristae lying in the plane of the paper. Clearly, although only the tubular cristae will give the profile seen in Fig. 1.9d (i), one cannot distinguish Fig. 1.9b (i), d (iii); b (ii) or d (ii).

Fig. 1.9 Drawings to compare the appearance of sections through lamelliform and tubular cristae. See text for details.

The intracristal and peripheral spaces usually appear to be empty, that is, they do not contain any material which reacts with the electron-dense stains (see however section on inclusions). Possibly in situ the peripheral space contains some material connecting inner and outer membranes, since the two often follow an essentially parallel course through numerous changes in direction (see Fig. 1.12 for instance). The width of the peripheral space in sections of mitochondria in situ varies, and the exact width (from 0–500 Å) depends on the fixative employed, as well as the cell the mitochondria are in. The width of the intracristal space (of lamellar cristae) is considerably more variable, but again is influenced by the fixation procedure. With a given fixation procedure, however, the width of the intracristal space varies from cell type to cell type and even within a given cell type. There has been considerable discussion as to the extent to which the intracristal spaces, and the space between the inner and outer membranes, are continuous. In sections of some mitochondria there appear to be numerous connections between cristae and inner peripheral membranes, e.g. in blowfly flight muscle mitochondria (Smith, 1963) (Fig. 1.10) and in Neurospora (Luck, 1965) (Fig. 1.11); whilst in others there are apparently no connections (Potswald, 1967). Daems and Wisse (1966) concluded from studies by the serial sectioning technique, that in mitochondria in mouse liver fixed with OsO4 the cristae are attached to the inner membrane through tubes (called pediculi cristae) of varying length and approximately 300 Å in diameter. It follows that cristae can be expected to reveal continuity with the inner membrane only when they are sectioned through a pediculus crista (see Fig. 1.9) and, since the pediculus forms only a very limited part of the periphery of a crista, that when there are only a few pediculi per crista continuity will be seen infrequently. Examination of Daems and Wisse’s electron micrographs of mitochondria sectioned tangentially through the region of pediculi cristae suggest that in mouse hepatic mitochondria many cristae have at least four pediculi, and the authors also show portions of cristae lying in the plane of the section with three pediculi joining them to the inner membrane. Daems and Wisse have also observed pediculi cristae in the mitochondria of mouse Kupffer cells and thrombocytes, and Drosophila germinal cells. The clearest examples of pediculi are seen in the sections of Euglena spirogyra examined by Leedale et al. (1965) (see Fig. 1.12). Andersson-Cedergren (1959), in a study of serial sections of mouse skeletal muscle mitochondria, had earlier observed that of seventeen clear contacts between cristae membranes and inner peripheral membrane only two showed continuity of the intracristal and peripheral spaces. The appearance of sectioned mitochondria in other cells (see, e.g. Miller and Palade, 1964, Figs 1, 11) are also in accord with the pediculi cristae model (Daems and Wisse, 1966). Study of negatively stained fixed preparations enabled Whittaker (1963) to demonstrate that the intracristal spaces of mitochondria isolated from brain tissue, connected with the peripheral space through narrow orifices. The connections are prominent in many isolated mitochondria (see Chapter 3) in which the intracristal spaces are enlarged by suspension of the mitochondria in 0·25M-sucrose media.

Fig. 1.10 Electron micrographs of parts of mitochondria in sections of (a) blowfly (Calliphora erythrocephala) and (b) housefly (Musca domestica) flight muscle. The mitochondria in these tissue are characterized by their numerous cristae which contain fenestrations (*), and which show frequent connections to the peripheral inner membrane. The arrowheads point to matrix between cristae, the arrows point to intracristal spaces. In (a) the section passes through the cristae transversely at 1, and obliquely at 2; the cristae are in the plane of the section at 3. In (b) the cristae membranes are sectioned transversely. From Smith (1963b) and Smith et al. (1970).

Fig. 1.11 Electron micrographs of mitochondria in section of Neurospora crassa. Connections of the cristae membranes with the peripheral inner membranes are common and in most cases the intracristal space is continuous with the peripheral space. This implies that the cristae are attached to the inner membrane over a relatively long length. From Luck (1965).

Fig. 1.12 Electron micrograph of mitochondria in sections of Euglena spirogyra, showing the characteristic form of the cristae and their attachment by pediculi to the peripheral inner membrane. From Leedale et al. (1965).

In Euglena spirogyra (Leedale, 1967) and crithidia of Trypanosoma (Steinert, 1960) the cristae are circular discs with a diameter somewhat less than half that of the mitochondria. They are arranged along the periphery of the organelle so that there is a clear central channel (see Fig. 1.12). Similar discoidal cristae are seen in the mitochondria of Blastocladiella emersonii zoospores (Fuller, 1966; Reichle and Fuller, 1967). Somewhat larger discoidal cristae occur in the mitochondria of Hydra interstitial cells (Slautterback, 1963). In general, lamelliform cristae run transversely and more or less completely across elongated mitochondria. They are sometimes very regularly arranged as seen in mitochondria of the mid-piece of Aeschna spermatozoa (Fig. 1.13) (Kessel, 1966a), or in Cypridopsis spermatozoa (Reger and Florendo, 1969a, b) or in many brown adipose tissue mitochondria (Fig. 1.14), but usually are somewhat less regularly arranged as in the elongated mitochondria at the base of proximal tubule cells (Fig. 1.15a) or elongated hepatic mitochondria (Fig. 1.15b). In some cells the cristae are orientated primarily in a direction parallel to the long axis, e.g. in many neurons, and motor end plates (De Harven and Coers, 1959), in parafollicular cells of rat thyroid gland (Ekholm and Ericson, 1968) and in Helix aspersa ovotestis (Powers et al., 1956). Mitochondria in the proximal tubular cells of the frog Rana pipiens, nephrons, examined during the summer when the frogs are feeding, have fairly regularly orientated, transverse cristae; on starvation the cristae of many of the mitochondria become arranged longitudinally (Fig. 2.13). This structural change is correlated with a loss of cytochrome oxidase activity (Karnovsky and Himmelhoch, 1961; Karnovsky, 1962, 1963 and see p. 87).

Fig. 1.13 Electron micrograph of section of dragonfly spermatozoa showing the regular arrangement of the cristae. From Kessel (1966a).

Fig. 1.14 Electron micrograph of section of rat brown adipocyte tissue, showing the regular arrangement of the cristae the majority of which extend the full width of the mitochondrion; they are probably connected to the peripheral inner membrane through very narrow pediculi.

In many cells the cristae show no preferred orientation and this seems particularly true of plant mitochondria (Figs. 1.7, 1.16), although mitochondria in the cotyledons of the pea Pisum sativum have been reported to contain regularly orientated cristae (Warner and Schidlovsky, 1963).

Fig. 1.16 Electron micrographs of sections of (a) part of a transfer cell from young pine Pinus pinea; (b) part of a developing phloem sieve element from sycamore. Acer pseudoplatanus. Generously provided by Dr. F. B. P. Wooding.

Where there are numerous lamelliform cristae in a mitochondrion these usually are more or less parallel to one another, and frequently they are orientated predominantly transversely to the long axis of the mitochondrion. In several instances, however, while retaining their parallel arrangement, the cristae in one part of a mitochondrion are orientated in a different direction to those in another part. The mitochondria in late spermatids of the teleost Oligocottus maculosus, for instance, contain cristae which are in parallel array in stacks of about six to eight; the membranes of adjacent stacks being perpendicular to one another (Stanley, 1969). The curving cristae of mitochondria in the basalar flight muscle of Achalarus lyciades are arranged in groups of three or four which show no orientation related to the shape of the mitochondria (Reger and Cooper, 1967). Similarly the cristae of Aeschnaflight muscle mitochondria are organized in groups consisting of sub-parallel arrays of curved membranes (Smith, 1961). In the retina of the tree shrew Tupaia glis, which contains numerous large mitochondria, the cristae are arranged in ordered rows of whorls, each whorl containing four or five cristae (Fig. 1.17) (Samorajski et al., 1966). The mitochondria in blowfly flight muscle contain occasional whorls of inner membranes (Smith, 1963), and cristae arranged in whorls are common in cardiac mitochondria in the quail, Colinus virginiahus (Slautterback, 1965), where they are frequently aligned in adjacent mitochondria.

Fig. 1.17 Electron micrographs of mitochondria in the retina of the tree shrew Tupaia glis, at the junction of the inner and outer segments of the cone, (from the area enclosed in the rectangle in the phase contrast micrograph in the inset. The ellipsoids (*) are packed with mitochondria). From Samorajski et al. (1966).

In some cells the cristae may branch and interconnect. Particularly marked examples of anastomosing branched cristae may be seen in the sperm of the polychaete Spirorbis (Potswald, 1967) and the scorpion spermatocyte (Fig. 2.3) (André, 1959).

The lamelliform cristae of Calliphora flight muscle mitochondria contain numerous fenestrations about 600 Å across which are precisely aligned in successive cristae (Fig. 1.10) (Smith, 1963). Fenestrated cristae have also been observed in cardiac mitochondria of the shrew, Blarina brevicauda (Slautterback, 1961), the canary, Serinus canarius (Slautterback, 1965) and the rat (Porter and Palade, 1957).

Concentrically arranged cristae have been observed in cardiac muscle (Moore and Ruska, 1957), in invertebrate and mammalian spermatids (Grassé et al., 1956; Fawcett, 1958, 1959), in the outer fascicular zone of the mouse adrenal (Zelander, 1964) and in the smaller mitochondria in the peripheral sarcoplasm of the red fibres of rat soft palate muscle (Fig. 1.18) (Leeson and Leeson, 1969). In some of the latter the cristae membranes show a wavy outline with abrupt, sharp angles (Fig. 1.18b).

Fig. 1.18 Electron micrographs of mitochondria in the peripheral cytoplasm of red fibres in rat soft palate muscle. From Leeson and Leeson (1969).

Revel et al. (1963) first drew attention to the presence of clearly defined angles in the course of the cristae membranes of mitochondria from a wide variety of animals and tissues (bat cricothyroid muscle and pancreas, frog gastric mucosa, fish chloride cells and leech neurones).

These angulated cristae have also been observed in rat heart muscle, rat corpus striatum (Mugnaini and Walberg, 1964), embryonic chick breast muscle in culture (Ishikawa, 1968), guinea pig pancreatic exocrine cells (Jamieson and Palade, 1968) and red fibres of pigeon breast muscle (Ashhurst, 1969). In its most common form one membrane of a crista diverges slightly from the other, then forms an angle of about 90° that brings it back close to the other membrane, which then forms a similar sharp angle a short distance further along the crista on the opposite side, giving a zigzag course to the crista. In some tissues, leech neurone for example, one membrane of a crista may have two or more successive angles with no corresponding reflections of the other membrane, so that the crista is curled upon itself. Although the zigzag form of the cristae is normally found in only a few mitochondria in a given cell, Luft et al. (1962) described this as a common feature of the interfibrillar mitochondria in the sartorius muscle of a patient suffering from hypermetabolism (see p. 98).

In 0·1 to 1·0% of the mitochondria in root-tip cells of Phaseolus vulgaris (and some other plant cells) up to four of the cristae, which otherwise are irregular in shape and with wide intracristal spaces, are in the form of longitudinal plates formed by fusion of the cristae membranes. The matrix-facing surfaces of the cristae membranes are thickened and show a series of oblique bands 40–50 Å in diameter and 400–500 Å long (Fig. 1.19), and Newcomb et al. (1968) liken the structure of the whole modified crista to that of a tight junction between animal cells.

Fig. 1.19 Electron micrograph of mitochondrion in a section of root tip cell from Phaseolus vulgaris. From Newcomb et al. (1968).

The cristae of the mitochondria of bat cricothyroid muscle are abundant (20 per µm) and usually take the form of closely packed parallel septa that extend the full width of the organelle and are perforated by occasional circular fenestrations. Many of the mitochondria also contain prismatic tubular cristae which appear to be continuous with the more common lamelliform cristae. The sides of the prismatic cristae are about 600 Å long and the cristae are often arranged radially in groups of six, so that their sides are parallel. The fascicles usually run straight but may bend abruptly through approximately 90° (Revel et al., 1963). Prismatic tubules are also encountered in mitochondria in mouse diaphragm, astrocytes of the hamster brain (Fig. 1.20) (Blinzinger et al., 1965) and of the cat (Mugnaini and Walberg, 1964), and associated with filamentous inclusions in the matrix of some rat kidney mitochondria (Suzuki and Mostofi, 1967, and see p. 51) and the rows of ovoid granules in the kidney mitochondria of ammocoetes of Petromyzon marinus (Fig. 1.40) (Youson, 1971).

Fig. 1.20 Electron micrograph of mitochondrion in a section of an astrocyte from hamster brain. From Blinzinger et al. (1965).

Fig. 1.40 Electron micrographs of sections of the posterior tip of Petromyzon kidney, showing mitochondria with orderly arrays of ovoid granules between the prismatic cristae. Compare the arrangement of the cristae with those in Fig. 1.20. From Youson (1971).

The prismatic tubular cristae in the hamster brain mitochondria form a hexagonal lattice with a centre to centre spacing of 470–480 Å. The sides of the triangles seen in cross-sections are 250–330 Å long and occasionally they are slightly concave. The tubules are arrayed longitudinally in the mitochondria, but occasionally bend outwards to become continuous with the inner membrane (Fig. 1.20).

Crayton and Mirolli (1968) reported the occurrence of two types of mitochondria in neurones of the buccal ganglia of the opisthobranch mollusc Navana, namely, conventional mitochondria, and other very large mitochondria up to 14 µm long and 3 µm wide which have long, longitudinally orientated cristae which in cross-section appear as square profiles in orthogonal arrays. These mitochondria are present in all the neurones, but not in the glia or in the cells of the connective tissue sheath; they are found only in the peripheral region of the cell where they are closely associated with lysosome-like bodies, glycogen and rough endoplasmic reticulum.

Fain-Maurel (1968) demonstrated the occurrence of square-section cristae in mitochondria of salivary gland mucocytes of the gastropod Limnaea stagnalis associated with refeeding of the starved animals. The cristae became aligned in parallel rows, 700 Å centre to centre, the sides of the cristae being 300–500 Å (Fig. 1.21). These show a marked similarity to structures seen in brain neuroglia of the lizard Lacerta viridis (Gray, 1960). The cristae, in hexagonal