THE MYOFILAMENT ARRANGEMENT IN THE FEMORAL MUSCLE OF THE COCKROACH, LEUCOPHAEA MADERAE FABRICIUS

The structure of the femoral muscle of the cockroach, Leucophaea maderae, was investigated by light and electron microscopy. The several hundred fibers of either the extensor or flexor muscle are 20 to 40 µ in diameter in transverse sections and are subdivided into closely packed myofibrils. In glutaraldehyde-fixed and epoxy resin-embedded material of stretched fibers, the A band is about 4.5 µ long, the thin filaments are about 2.3 µ in length, the H zone and I band vary with the amount of stretch, and the M band is absent. The transverse sections of the filaments reveal in the area of a single overlap of thick and thin filaments an array of 10 to 12 thin filaments encircling each thick filament; whereas, in the area of double overlap in which the thin filaments interdigitate from opposite ends of the A band, the thin filaments show a twofold increase in number. The thick filament is approximately 205 to 185 A in diameter along most of its length, but at about 0.2 µ from the end it tapers to a point. Furthermore, some well oriented, very thin transverse sections show these filaments to have electron-transparent cores. The diameter of the thin filament is about 70 A. Transverse sections exhibit the sarcolemma invaginating clearly at regular intervals into the lateral regions of the A band. Three distinct types of mitochondria are associated with the muscle: an oval, an elongate, and a type with three processes. It is evident, in this muscle, that the sliding filament hypothesis is valid, and that perhaps the function of the extra thin filaments is to increase the tensile strength of the fiber and to create additional reactive sites between the thick and thin filaments. These sites are probably required for the functioning of the long sarcomeres.     

THE STRUCTURE OF INTERSEGMENTAL MUSCLE FIBERS IN AN INSECT, PERIPLANETA AMERICANA L

The organization of intersegmental muscle fibers associated with the dorsal abdominal sclerites of the cockroach is described. These fibers correspond closely, in the disposition and derivation of the membranes of the transverse tubular system and sarcoplasmic reticulum cisternae, with insect synchronous flight muscle fibers, but differ markedly from these in their fibrillar architecture and mitochondrial content. The mitochondria are small and generally aligned alongside the prominent I bands of the sarcomere, and, in the best-oriented profiles of the A bands, thick filaments are associated with orbitals of twelve thin filaments, a configuration that has also been observed in striated fibers of insect visceral muscle. These structural features of insect muscles are compared and discussed in terms of possible variations in the control of contraction and relaxation, and in the nature of their mechanical role.     

DEGENERATION IN THE EFFERENT NERVE ENDINGS IN THE COCHLEA AFTER AXONAL SECTION

Both roots of the olivo-cochlear nerve bundle to one ear were transected in the brain stems of 12 chinchillas. The animals were sacrificed at times ranging from 2 to 35 days after surgery. The normal olivo-cochlear terminals on the external hair cells in the cochleas of the control ears contained many mitochondria and small vesicles of constant size. The earliest evidence for degeneration was the presence of fine 100 A filaments in the proximal parts of the terminals. These were visible at 2 days. Animals sacrificed at later times showed a greater number of filaments and fewer vesicles, but few mitochondrial changes. After 1 week, disintegration of the terminals was more prominent. A few terminals showed mitochondrial swelling and lysis of the plasma membrane but few or no filaments within the first week. These latter terminals were interpreted as representing a more rapid process of disintegration than those terminals characterized by numerous filaments and seemingly unchanged mitochondria.     

THE DOUBLE ARRAY OF FILAMENTS IN CROSS-STRIATED MUSCLE

The conditions under which one might expect to see the secondary filaments (if they exist) in longitudinal sections of striated muscle, are discussed. It is shown that these conditions were not satisfied in previously published works for the sections were too thick. When suitably thin sections are examined, the secondary filaments can be seen perfectly easily. It is also possible to see clearly other details of the structure, notably the cross-bridges between primary and secondary filaments, and the tapering of the primary filaments at their ends. The arrangement of the filaments and the changes associated with contraction and with stretch are identical to those already deduced from previous observations and described in terms of the interdigitating filament model in previous papers. There are therefore excellent grounds for believing that this model is correct. The alternative models which have been proposed appear to be incompatible both with the present observations and with much of the other available evidence.     

THE ORIGIN OF PROTEIN AND FATTY YOLK IN RANA PIPIENS : I. Phase Microscopy

Study of living frog oocytes with the phase microscope has shown that the early yolk appears in two forms. One of these, the protein yolk, consists of thin, dense, plate-like bodies which in face view are almost always regular hexagons. The other form, the fatty yolk, occurs as clusters of globules of varying sizes. The plate-like bodies occur both singly and in clusters. As the oocytes mature these plate-like bodies grow in size while retaining their hexagonal outline. Mitochondria have been observed to increase in length and numbers as the oocytes mature; they are rods or filaments at all stages of growth up to an oocyte diameter of 300 microns. The oocyte cytoplasm gradually becomes packed with long mitochondria, plate-like bodies, and clusters of globules.     

THE STRUCTURE AND FORMATION OF CILIA AND FILAMENTS IN RUMEN PROTOZOA

The large oligotrich rumen protozoa Diplodinium ecaudatum and Ophryoscolex caudatus have been studied by electron microscopy during interphase and division. The structure of mature cilia is contrasted with that seen during their formation particularly in a tuft where development lags and is arrested. Here the shaft is only a few micra long and is composed of filaments that have circular cross-sections not in the typical circular arrangement. In their diameter and appearance the filaments are similar to filaments associated with the nuclei during division. The macronucleus has within it randomly directed filaments, while the micronucleus contains well aligned filaments and other arrangements typical of an intranuclear mitotic process. An extranuclear filament system is also present and is elaborated during division. The infraciliary filament system is particularly elaborate in these organisms. Filaments ranging from 14 to 22 mµ have been observed with some tendency for a bimodal distribution in diameters of 15 and 21 mµ. Formation of such filaments has been observed and consists of an initial orientation of very fine elements followed by filament formation. The observations are discussed in relation to filament involvements in cell movements. The concepts are discussed that filaments are metastable structures and that the transitions from one state to another are functionally significant.     

THE ORGANIZATION OF FLIGHT MUSCLE FIBERS IN THE ODONATA

The cytological organization of flight muscle fibers of Odonata has been investigated. These fibers, in representatives of the Zygoptera and Anisoptera, have been compared and found to be similar, except that, in the former, pairs of lamellar fibrils, rather than single fibrils, alternate with the mitochondria. In each instance, in these synchronous muscles, the actin filaments of the myofibrils are found to lie opposite to and midway between pairs of myosin filaments—a configuration previously reported in asynchronous flight muscle fibers. The disposition of the T system and sarcoplasmic reticulum membranes in glutaraldehyde-fixed anisopteran muscle is described in detail: the T system tubules are shown to be radially continuous across the fiber, and are derived as openmouthed invaginations from the surface cell-membrane. The detailed organization of the dyad junctions between these tubules and the adjoining cisternae of the sarcoplasmic reticulum is described. The accessibility of the T system interior to diffusion exchange with the general extracellular milieu has been investigated by studies on the penetration of ferritin into the fiber: molecules of this marker have been found to diffuse solely along the T system tubules, and their presence in the tubule extremities adjoining the centrally placed nuclei confirms the morphological evidence suggesting that these tubules provide open diffusion channels extending across the radius of the fiber. The possible physiological role of these membrane components and their distribution in synchronous muscles of insects and vertebrates and in asynchronous insect flight muscle are discussed.     

THE ORGANIZATION OF CONTRACTILE FILAMENTS IN A MAMMALIAN SMOOTH MUSCLE

Ordered arrays of thin filaments (65 A diameter) along with other apparently random arrangements of thin and thick filaments (100–200 A diameter) are observed in contracted guinea pig taenia coli rapidly fixed in glutaraldehyde. The thin-filament arrays vary from a few to more than 100 filaments in each array. The arrays are scattered among isolated thin and thick filaments. Some arrays are regular such as hexagonal; other arrays tend to be circular. However, few examples of rosettes with regular arrangements of thin filaments surrounding thick filaments are seen. Optical transforms of electron micrographs of thin-filament arrays give a nearest-neighbor spacing of the thin filaments in agreement with the "actin" filament spacing from x-ray diffraction experiments. Many thick filaments are closely associated with thin-filament arrays. Some thick filaments are hollow circles, although triangular shapes are also found. Thin-filament arrays and thick filaments extend into the cell for distances of at least a micron. Partially relaxed taenia coli shows thin-filament arrays but few thick filaments. The suggestion that thick filaments aggregate prior to contraction and disaggregate during relaxation is promoted by these observations. The results suggest that a sliding filament mechanism operates in smooth muscle as well as in striated muscle.     

Immunocytochemical identification of cytoskeletal linkages to smooth muscle cell nuclei and mitochondria

In avian smooth muscle cells, desmin-containing intermediate filaments (IFs) are a prominent component of the cytoskeleton and are readily seen in several domains, including the axial intermediate filament bundle (IFB). Both the nucleus and some of the mitochondria are partly surrounded by elements of the IFB. By using anti-desmin and protein-A-colloidal gold labeling, we have identified intermediate filaments that form linkages with the nuclear envelope and with mitochondria. These linkage regions seem to occupy a proportionately greater part of the mitochondrial surface than of the nuclear envelope. The existence of these linkages in smooth muscle cells is consistent with results that support similar linkages to mitochondria and other cellular structures in various cells that contain either vimentin or keratin IFs. These linkages could functionally restrain or assist in homeostatically restoring organelles to their normal position after the rearrangement that accompanies the substantial shortening of smooth muscle cells.     

OBSERVATIONS ON THE FINE STRUCTURE OF THE DEVELOPING SPERMATID IN THE DOMESTIC CHICKEN

The kinetic apparatus, the acrosome and associated structures, and the manchette of the spermatid of the domestic chicken have been studied with the electron microscope. The basic structural features of the two centrioles do not change during spermiogenesis, but there is a change in orientation and length. The proximal centriole is situated in a groove at the edge of the nucleus and oriented normal to the long axis of the nucleus and at right angles to the elongate distal centriole. The tail filaments appear to originate from the distal centriole. The plasma membrane is invaginated along the tail filaments. A dense structure which appears at the deep reflection of the plasma membrane is identified as the ring. The fine structure of the ring has no resemblance to that of a centriole and there is no evidence that it is derived from or related to the centrioles. The tail of the spermatid contains nine peripheral pairs and one central pair of tubular filaments. The two members of each pair of peripheral filaments differ in density and in shape: one is dense and circular, and the other is light and semilunar in cross-section. The dense filaments have processes. A manchette consisting of fine tubules appears in the cytoplasm of the older spermatid along the nucleus, neck region, and proximal segment of the tail. The acrosome is spherical in young spermatids and becomes crescentic and, finally, U-shaped as spermiogenesis proceeds. A dense granule is observed in the cytoplasm between acrosome and nucleus. This granule later becomes a dense rod which is interpreted as the perforatorium.     

VARIATIONS IN THE STRUCTURE OF MITOCHONDRIA

A characteristic internal structure, consisting of a double-layered outer wall enclosing a matrix-filled space through which pass double-layered membranous folds, would appear to comprise as satisfactory a definition of mitochondria for electron microscopy as their intravital affinity for Janus green affords for light microscopy. Relying for identification upon this characteristic internal structure, mitochondria appear to be pleomorphic structures which vary in size, shape, complexity, and density. They are labile also in that their number may increase or decrease under controlled conditions. The possibility therefore exists that these organelles are constantly being formed and destroyed, perhaps by their participation in metabolic processes. The problem of the origin of mitochondria is in an unsatisfactory state. New organelles unquestionably are formed in particular physiological states. The possibility that new bodies are produced by fission of ones already present does not seem adequate. On the other hand, the possible fabrication of new mitochondria out of intracellular membranes, although an attractive hypothesis, has not been adequately substantiated.     

MEMBRANE JUNCTIONS IN THE INTERMEMBRANE SPACE OF MITOCHONDRIA FROM MAMMALIAN TISSUES

There have been several reports describing paracrystalline arrays in the intermembrane space of mitochondria. On closer inspection these structures appear to be junctions of two adjoining membranes. There are two types. They can be formed between the outer and inner mitochondrial membranes (designated outer-inner membrane junctions) or between two cristal membranes (intercristal membrane junctions). In rat heart, adjoining membranes appeared associated via a central dense midline approximately 30 Å wide. In rat kidney, the junction had a ladder-like appearance with electron-dense "bridges" approximately 80 Å wide, spaced 130 Å apart, connecting the adjoining membranes. We have investigated the conditions which favor the visualization of such structures in mitochondria. Heart mitochondria isolated rapidly from fresh tissue (within 30 min of death) contain membrane junctions in approximately 10–15% of the cross sections. This would indicate that the percentage of membrane junctions in the entire mitochondrion is far greater. Mitochondria isolated from heart tissue which was stored for 1 h at 0°–4°C showed an increased number of membrane junctions, so that 80% of the mitochondrial cross sections show membrane junctions. No membrane junctions are observed in mitochondria in rapidly fixed fresh tissue or in mitochondria isolated from tissue disrupted in fixative. Thus, the visualization of junctions in the intermembrane space of mitochondria appears to be dependent upon the storage of tissue after death. Membrane junctions can also be observed in mitochondria from other stored tissues such as skeletal muscle, kidney, and interstitial cells from large and small intestine. In each case, no such junctions are observed in these tissues when they are fixed immediately after removal from the animal. It would appear that most studies in the literature in which isolated mitochondria from tissues such as heart or kidney were used were carried out on mitochondria which contained membrane junctions. The presence of such structures does not significantly affect normal mitochondrial function in terms of respiratory control and oxidative phosphorylation.     

LIGHT AND ELECTRON MICROSCOPE STUDIES OF MORPHOLOGICAL CHANGES OF MITOCHONDRIA DURING SPERMATOGENESIS IN THE GRASSHOPPER

1. Observations on the morphological changes of mitochondria preparatory to the formation of the nebenkern, as well as changes within the nebenkern, are reported. 2. Mitochondria enlarge and divide during the meiotic divisions. 3. The mitochondria of the spermatid elongate, become filamentous, form a crescent, and partially encircle the nucleus. 4. Nodes which develop on either end of the crescent become entwined as they move toward each other. 5. The two nodes coalesce to form a filamentous or early type nebenkern which is described by others as chromophilic. 6. Internal rearrangement and partial dissolution of the filaments result in the development of the late or chromophobic nebenkern which separates into two distinct bodies. 7. The nebenkern moieties send out processes toward the centrosome, and after making contact, elongate and occupy part of the space between the tail filaments and sheath of the spermatozoon.     

Giant mitochondria in the A cells of the pancreas of a lizard: Varanus niloticus

Pancreatic A cells of the lizard Varanus niloticus are characterized by the presence of two types of mitochondria: (a) normal, small mitochondria (about 0.4 X 1 micron), and (b) giant mitochondria, measuring up to 9 micron in length and 1 micron in diameter. Giant mitochondria show various shapes. Their matrix is filled with tubules, filaments, and dense granules. Transverse sections of tubules are polygonal in shape and about 20 nm in diameter. They are grouped in bundles. The filaments, about 9-10 nm in diameter, are arranged in parallel layers crossing each other at a 57 degree angle. In a closely related species, Varanus exanthematicus, pancreatic A cells do not show these peculiar features.     

THE STRUCTURE OF THE CENTRAL REGION IN THE SYNAPTONEMAL COMPLEXES OF HAMSTER AND CRICKET SPERMATOCYTES

The fine structure of bivalents from golden hamster and house cricket spermatocytes has been studied with a whole mount surface-spreading method combined with negative staining. The elements of the synaptonemal complex show detail of structure which is absent in other preparative procedures. The transverse filaments found in the central region of the synaptonemal complex from both species are straight and have a similar width, 1 6–1 8 nm These filaments occur mainly in bundles The central element differs in architecture in the two species In hamster bivalents it is formed of longitudinal stretches of filaments 1.6–1 8 nm wide and a small amount of an amorphous material similar to that of the lateral elements In the cricket, the central element contains transverse fibrils which are continuous with the transverse filaments of the central region, and an amorphous material lying mainly along the sides of the central element All of the components of the central region of the synaptonemal complex are resistant to pancreatic DNase. The overlapping ends of the transverse filaments, together with additional protein material, make up the central element The widespread occurrence and close morphological and histochemical interspecies similarities of the transverse filaments indicate that they serve an essential role, probably one concerned with holding synapsed bivalents together via the lateral elements. Restrictions placed by the observations reported here on current models of the synaptonemal complex are discussed.     

ON THE ROLE OF MICROTUBULES IN MOVEMENT AND ALIGNMENT OF NUCLEI IN VIRUS-INDUCED SYNCYTIA

Infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes rapid and extensive cell fusion. Time-lapse cinematography shows that when cells fuse, their nuclei migrate straight to the center of the syncytium at rates of 1–2 µ/min. Nuclei are often arranged in long, tightly packed, parallel rows in syncytia derived from the fibroblastic BHK21-F cells. Polarization microscopy shows birefringent material between and parallel to these rows of nuclei, and electron microscopy shows bundles of cytoplasmic microtubules, ~250 A in diameter, and filaments, ~80 A in diameter, parallel to and between the rows of nuclei. Colchicine treatment causes disappearance of microtubules from BHK21-F cells and an apparent increase in the number of 80-A filaments. Although colchicine-treated, SV5-infected cells fuse, their nuclei do not migrate or form rows but remain randomly scattered through the syncytial cytoplasm. Incubation at 4°C does not disrupt microtubules in BHK21-F cells. Rows of nuclei have been isolated from SV5-induced syncytia, and the nuclei in them have been found to be intimately associated with microtubules but not with other cytoplasmic structures. These results suggest that microtubules demarcate cytoplasmic channels through which nuclei migrate and that they may also be involved in the mechanism of nuclear movement.     

SYNAPSES IN THE CENTRAL NERVOUS SYSTEM

A number of different synapses have been described in the medulla, cerebellar cortex, and cerebral cortex of the rat. All of these possess the same fundamental fine structure as follows: 1. Close apposition of the limiting membranes of presynaptic and postsynaptic cells without any protoplasmic continuity across the synapse. The two apposed membranes are separated by a cleft about 200 A wide, and display localized regions of thickening and increased density. 2. The presynaptic expansion of the axon, the end-foot or bouton terminal, contains a collection of mitochondria and clusters of small vesicles about 200 to 650 A in diameter. Although the significance of these structures in the physiology of the synapse is still unknown, two suggestions are made: that the mitochondria, by means of the relation between their enzymatic activity and ion transport, participate in the electrical phenomena about the synapse; and that the small synaptic vesicles provide the morphological representation of the prejunctional, subcellular units of neurohumoral discharge at the synapse demanded by physiological evidence.     

THE FINE STRUCTURE OF THE CILIA FROM CTENOPHORE SWIMMING-PLATES

The ctenophore swimming-plate has been examined with the electron microscope. It has been recognized as an association of long cilia in tight hexagonal packing. One of the directions of the hexagonal packing is parallel to the long edge of the swimming-plate and is perpendicular to the direction of the ciliary beat. All the cilia in the swimming-plate are identically oriented. The effective beat in the movement of the swimming-plate is directed towards the aboral pole of the animal, and this is also the side of the unpaired peripheral filament in all the cilia. The direction of the ciliary beat is fixed in relation to the position of the filaments of the cilia. The swimming-plate cilium differs from other types of cilia and flagella in having a filament arrangement that can be described as 9 + 3 as opposed to the conventional 9 + 2 pattern. The central filaments appear in a group of two "tubular" filaments and an associated compact filament. The compact filament might have a supporting function. It has been called "midfilament." Two of the peripheral nine filaments (Fig. 1, Nos. 3 and 8) are joined to the ciliary membrane by means of slender lamellae, which divide the cilium into two unequal compartments. These lamellae have been called "compartmenting lamellae." Some observations of the arrangement of the compartmenting lamelae indicate that they function by cementing the cilia together in lateral rows. The cilia of the rows meet at a short distance from each other, leaving a gap of 30 A only. The meeting points are close to the termini of the compartmenting ridges. An electron-dense substance is sometimes seen bridging the gap. Some irregularities are noted with regard to the arrangement of the compartmenting lamellae particularly at the peripheral rows of cilia. In many cilia in these rows there are small vesicles beneath the ciliary membrane.     

THE FATE OF MITOCHONDRIA DURING AGING IN TETRAHYMENA PYRIFORMIS

During the growth cycle of Tetrahymena pyriformis the mitochondria undergo changes in position, number, and structure. Ciliates in the logarithmic growth phase possess elongated mitochondria which are aligned along the plasma membrane and are closely associated with the kinetosomes and kinetodesmata. Mitochondria appear to divide across the long axis at this time, resulting in two or more products. Throughout this phase of growth mitochondrial divisions keep pace with cytokinesis so that the population of mitochondria remains at essentially the minimal level. As the ciliates enter the stationary growth phase the mitochondria increase in number, become oval to spherical in shape, and some migrate into the cytoplasm. Intramitochondrial masses of various configurations appear at this time. Some of the mitochondria lying in the cytoplasm become incorporated into vacuoles. Within these vacuoles either a single mitochondrion appears or several mitochondria may be seen along with other cytoplasmic structures. Later in the stationary growth phase the contained mitochondria are dense and the tubules are more compact than normal. Various stages in disorganization of the mitochondria are observed in a single large vacuole. Cytochemical tests reveal the presence of acid phosphatase, suggesting that hydrolysis of the vacuolar contents occurs. Lipid droplets increase in number during the middle and late stationary phase of growth. These events are interpreted as being associated with the normal process of aging in T. pyriformis.