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We have used 400 kilovolt intermediate voltage electron microscopy (IVEM) to examine thick sections of fibroblasts cultured in collagen gels. In these 3D collagen lattices, the long, narrow pseudopodial extensions that extend out and make contact with the collagen matrix exhibit a complex topography not seen in the processes put out by cells moving on planar substrata. For this reason, sections 1 to 2 microns thick that enclose a whole cell process are more informative of the overall morphology of the interaction between cells and the collagen than are thin sections. To aid the discrimination of topography of cell processes in stereo views of micrographs, some cells were labeled with antibodies and protein A-colloidal gold conjugates. The gold particles provided clear 3D reference points for computer-aided reconstructions of membrane topography from tilt series of IVEM images. Our results confirm that cells that move through collagen lattices lack the well-spread morphology of their counterparts moving on glass. They are generally rather spindly with several long branching anterior pseudopodia. We found that the cell bodies and major pseudopodial processes were cylindrical, as one might expect of cells in a 3D environment, but at the leading edge of advancing pseudopodia there are small flat extensions similar to those seen in cells on glass. This similarity suggests that the lamellipodium is a basic type of protrusive structure used by fibroblasts during locomotion on all types of substratum. The flattened shape of lamellipodia may be part of the mechanism by which cells sense the orientation of fibrillar extracellular matrices during embryonic morphogenesis.

An electron microscopic study of internal and surface-connected membrane systems of leg muscle of the crab shows that there are three kinds of surface-connected membrane systems in addition to an intracellular sarcoplasmic reticulum (SR). One is a system of large infoldings of the sarcolemma referred to as clefts. These are longitudinally-oriented, flattened infoldings of both the plasma membrane and the fibrous sheath of the fiber, and were probably seen earlier with the light microscope. Extending into the fiber both from these clefts and from the free fiber surface are two systems of tubules of much smaller caliber, the Z tubules and the A tubules. The Z tubules are located, as their name indicates, near the Z lines of the myofibrils, and are thought to be attached to them mechanically. The A tubules are found in pairs, near the ends of each A band, and are closely bound to the SR in two-part structures called dyads. Local-activation experiments, like those done earlier by Huxley and Taylor, suggest that the A tubules are involved in excitation-contraction coupling; no such experimental suggestion of function exists for the Z tubules.

The structure of the urinary bladder of the toad Bufo marinus was studied by light and electron microscopy. The epithelium covering the mucosal surface of the bladder is 3 to 10 microns thick and consists of squamous epithelial cells, goblet cells, and a third class of cells containing many mitochondria and possibly representing goblet cells in early stages of their secretory cycle. This epithelium is supported on a lamina propria 30 to several hundred microns thick and containing collagen fibrils, bundles of smooth muscle fibers, and blood vessels. The serosal surface of the bladder is covered by an incomplete mesothelium. The cytoplasm of the squamous epithelial cells, which greatly outnumber the other types of cells, is organized in a way characteristic of epithelial secretory cells. Mitochondria, smooth and rough surfaced endoplasmic reticulum, a Golgi apparatus, "multivesicular bodies," and isolated particles and vesicles are present. Secretion granules are found immediately under the plasma membranes of the free surfaces of the epithelial cells and are seen to fuse with these membranes and release their contents to contribute to a fibrous surface coating found only on the free mucosal surfaces of the cells. Beneath the plasma membranes on these surfaces is an additional, finely granular component. Lateral and basal plasma membranes are heavily plicated and appear ordinary in fine structure. The cells of the epithelium are tightly held together by a terminal bar apparatus and sealed together, with an intervening space of only 0.02 mµ near the bladder lumen, in such a way as to prevent water leakage between the cells. It is demonstrated in in vitro experiments that water traversing the bladder wall passes through the cytoplasm of the epithelial cells and that a vesicle transport mechanism is not involved. In vitro experiments also show that the basal (serosal) surfaces of the epithelial cells are freely permeable to water, while the free (mucosal) surfaces are normally relatively impermeable but become permeable when the serosal surface of the bladder is treated with neurohypophyseal hormones. The permeability barrier found at the mucosal surface may be represented, structurally, either by the filamentous layer lying external to the plasma membrane, by the intracellular, granular component found just under the plasma membrane, or by both of these components of the mucosal surface complex. The polarity of the epithelial sheet is emphasized and related to the physiological role of the urinary bladder in amphibian water balance mechanisms.

The structure of the longitudinal body muscles of Branchiostoma caribaeum has been studied by light and electron microscopy. These muscles are shown to be composed of fibers in the form of flat lamellae about 0.8µ in thickness, more than 100 µ wide, and reaching in length from one intermuscular septum to the next, a distance of about 0.6 mm. Each flat fiber is covered by a plasma membrane and contains a single myofibril consisting of myofilaments packed in the interdigitating hexagonal array characteristic of vertebrate striated muscle. Little or no sarcoplasmic reticulum is present. Mitochondria are found infrequently and have a tubular internal structure. These morphological observations are discussed in relation to a proposed hypothesis of excitation-contraction coupling. It is pointed out that the maximum distance from surface to myofilament in these muscles is about 0.5 µ and that diffusion of an "activating" substance over this distance would essentially be complete in less than 0.5 msec. after its release from the plasma membrane. It is concluded that the flat form of amphioxus muscle substitutes for the specialized mechanisms of excitation-contraction coupling thought possibly to involve the sarcoplasmic reticulum in higher vertebrate muscles.

In 1902, Veratti published a description of a fine reticular network pervading striated muscle fibers that could be revealed by infiltration with osmium and silver, a procedure developed by Golgi and referred to as his black reaction (Veratti, 1902, 1961). Much later it was shown by electron microscopy that Veratti’s “reticulum” corresponds to a network of transversely oriented tubules (T tubules) that can be visualized by electron microscopy (Porter, 1961; Smith, 1961; Peachey, 1965). T tubules are invaginations of the plasma membrane at the surface of the muscle fiber, and their content is continuous with the extracellular space. Thus Veratti used the osmium-silver technique to infiltrate extensions of the extracellular space, rather than to infiltrate the cell’s interior, as is believed to be the case when the same method is used to study neurons (Ramon y Cajal, 1972). We have slightly modified the original black reaction of Golgi in a way that results in a superior preservation of fine structural details for light and electron microscopy. Our results show that this procedure is ideally suited for the visualization of small and large invaginations of the extracellular space into cells, both by light microscopy and by electron microscopy in the range of 100-1000 kV. Test objects used in this study were a variety of types of muscle fibers whose surface topography is greatly complicated by the presence of clefts and various tubular invaginations. The highlights of our results are as follows

The disposition of surface invaginations (clefts, Z and T tubules) and of the sarcoplasmic reticulum has been examined by electron microscopy at three accelerating voltages (100, 200 and 1000 kV) and by phase-contrast light microscopy in crustacean muscles infiltrated by the "Golgi stain." In long-sarcomere, tonic type fibers, an extensive system of invaginating clefts has been observed, along with both Z and T tubules. Z and T tubules form interconnections with each other, but only T tubules form specific contacts with the sarcoplasmic reticulum, which in these fibers forms an extended and continuously fenestrated network. In short-sarcomere, phasic type fibers, a ladder-like disposition of an abundant T network is found. Z tubules are absent in these fibers. The sarcoplasmic reticulum forms more frequent junctions with flattened areas of T tubules and with clefts, but has less extensive free surfaces than in the long-sarcomere fibers.

Publisher Summary This chapter focuses on three-dimensional structure of the T-system of skeletal muscle cells. The T-system or transverse tubular system, of a striated muscle cell is a predominantly transversely oriented network of interconnected tubules invaginating into the fiber from many points on its surface. These tubules are believed to provide a route for electrical excitation to spread into the fiber interior to activate contraction. The morphological studies through 1975 defined the T-system as a series of planar, continuous, transverse networks crossing the fiber at regular intervals in registration with the sarcomere banding. More recent morphological studies have revealed several ways in which the T-system deviates from this pattern, either by having a longitudinal component or with some form of interruption in its planar arrangement. This chapter reviews and summarizes these newer findings. Most of the results described in the chapter are based on studies using selective stains to enhance the density of the T-system and high voltage electron microscopy to examine thick preparations, often stereoscopically.

The sarcoplasmic reticulum of the frog's sartorius muscle was examined by electron microscopy following sequential fixation in glutaraldehyde and osmium tetroxide and embedding in Epon. The earlier results of Porter and Palade on Ambystoma muscle were confirmed in the sartorius. In addition, the transverse tubules were observed to be continuous across the width of the fiber, a set of flat intermediate cisternae was seen to connect the terminal cisternae to the longitudinal tubules in the A band, and the continuous reticulum collar at the center of the A band was found to be perforated by circular and elongated pores (the fenestrated collar). The transverse tubules have a volume about 0.3 per cent of the fiber volume, and a surface area about 7 times the outer cylindrical surface area for a fiber 100 µ in diameter. The terminal cisternae, the intermediate cisternae, and the longitudinal tubules together with the fenestrated collar each have a volume of 4 to 5 per cent of the fiber volume and a surface area 40 to 50 times the outer surface area of a fiber 100 µ in diameter. Some evidence for continuity of the transverse tubules with the fiber surface is presented, but this is thought to be not so convincing as evidence presented by others. The results are discussed in terms of a possible mechanism for a role of the transverse tubules and sarcoplasmic reticulum in excitation-contraction coupling, as suggested by their morphology and a variety of physiological studies. In this scheme, the transverse tubules are thought to be electrically coupled to the terminal cisternae, so that depolarization of the fiber surface spreads inward along the transverse tubules and to the terminal cisternae, initiating the release of a contraction-activating substance.

A hypothesis, suggested previously by morphological studies, for impulse conduction from the sarcolemma to the contractile material via the sarcoplasmic reticulum is discussed. The relation of reticulum morphology and cell size to speed of contraction in smooth and striated muscle agrees with the hypothesis and thus supports it. Additional support comes from evidence concerning an unusual morphological relationship between the sarcolemma and contractile fibrils in striated muscle of amphioxus.

Cat extraocular muscles consist of two regions: orbital, and global. The orbital region contains predominantly small diameter fibers, while the global region contains a variety of fibers of different diameters. The differences in ultrastructural features among these muscle fibers indicate that the extraocular muscles of cats contain at least five structurally distinguishable types of fibers.Superior rectus muscles were studied by light and electron microscopy, mapping the distribution of each fiber type with its distinctive features. A mixture of 4% paraformaldehyde and 4% glutaraldehyde was perfused through the carotid arteries of anesthetized adult cats and applied locally to exposed superior rectus muscles during the perfusion.