The development of myofibrils in cultured muscle cells: A whole-mount and thin-section electron microscopic study

The development of myofibrils in cultured myotome cells from Xenopus embryos was studied with whole-mount and thin-section electron microscopy. For whole mount, the cells were grown on Formvar-coated grids, fixed, dehydrated, critical-point dried, and examined with a conventional (100 kV) or a high-voltage (1000 kV) electron microscope. Nonstriated bundles of 6- to 8-nm microfilaments, similar to stress fibers in nonmuscle cells, appear prior to nascent myofibrils. These bundles run the whole length of the cell and are inserted into the cell cortex. The transition from striated region to nonstriated region on a single nascent myofibril can be seen in both whole-mount and thin-section images. New sarcomeres appear to be added at the distal end of existing ones. Our data also indicate that these new sarcomeres are formed on a preexisting bundle of thin filaments. This suggests that the bundles of microfilaments are precursors to myofibrils. Evidence for this hypothesis came from the following observations. (1) Nascent myofibrils are anchored to the cell cortex via thin filaments similar to microfilament bundles. (2) Thin filaments in newly formed sarcomeres are often continuous through the middle of the A band. Later they break to form the H zone. (3) Thin filaments appear to be continuous through the developing Z band. Later they interact with the filaments in the Z band to form the staggered appearance.     

AN ELECTRON MICROSCOPE STUDY OF MYOFIBRIL FORMATION IN EMBRYONIC CHICK SKELETAL MUSCLE

The formation of myofibrils in the developing leg muscle of the 12-day chick embryo was studied by electron microscopy. Myofilaments of two varieties, thick (160–170 A in diameter) and thin (60–70 A in diameter), which have been designated myosin and actin filaments, respectively, on the basis of their similarity to natural and synthetic myosin and actin filaments, appear in the cytoplasm of developing muscle cells. There is a greater than 7:1 ratio of thin to thick filaments in these young myofibers. The free myofilaments become aligned in the long axis of the cells, predominantly in subsarcolemmal locations, and aggregate into hexagonally packed arrays of filaments. The presence of Z band material or M band cross-bridges do not appear to be essential for the formation or spacing of these aggregates of filaments. Formation of the Z band lattices occurs coincidentally with the back-to-back apposition of thin filaments. An hypothesis concerning myofibril growth, based on the self-assembly characteristics of the filaments, is presented.     

Aktomyosinartige Filamente im Teilungsmakronukleus des Ciliaten Ichthyophthirius Multifiliis

Just before nuclear division, the chromosomal elements within the large, highly polyploid macronucleus of I. multifiliis carry out rotational movements. Electron micrographs of cells fixed during the rotational movements show islets filled with microfilaments in various states of aggregation. Both thick (80–200 Å) and thin (30–80 Å) filaments occur, either as a highly dense network or as straight, in part parallel, filaments embedded in a filamentous network of lower density. Other islets of the macronucleus contain large and dense aggregates of filaments, sometimes with globular particles measuring 50–60 Å arranged along the thick filaments, occasionally forming cross-bridges with the thinner ones. — After incubation of the cells before fixation in a contractionsolution containing 0.002 M ATP, all nuclear islets show a nearly uniform appear ance of filamentous aggregates: numerous long and thick filaments are arranged in parallel with thin filaments with which they are in some parts connected by bridges. The probable myosinoid and actinoid nature of thick and thin filaments is discussed. It is suggested that the pre-divisional intranuclear rotational movement is a mechanism to avoid aneuploidy by producing a random arrangement of replicated hereditary units prior to division.     

ELECTRON MICROSCOPE AND EXPERIMENTAL INVESTIGATIONS OF THE NEUROFILAMENTOUS NETWORK IN DEITERS'' NEURONS : Relationship with the Cell Surface and Nuclear Pores

The assembly of filamentous elements and their relations to the plasma membrane and to the nuclear pores have been studied in Deiters' neurons of rabbit brain. Electron microscopy of thin sections and of ectoplasm spread preparations have been integrated with physicochemical experiments and differential interference microscopy of freshly isolated cells. A neurofilamentous network extends as a continuous, three-dimensional, semilattice structure throughout the ectoplasm, the "plasma roads," and the perinuclear zone of the perikaryon. This space network consists of ~90-Å wide neurofilaments arranged in fascicles which are interconnected by an exchange of neurofilaments. The neurofilaments consist of intercoiled ~20-Å wide unit-filaments and are associated through cross-associating filaments with other neurofilaments of the fascicle and with microfilaments. The ~20–50-Å wide microfilaments display intimate associations with the plasma membrane and with the nuclear pores. Electron microscopy of thin sections from glycerinated and heavy meromyosin-treated Deiters' neurons shows that actin-like filaments are present in the pre- and postsynaptic regions of synapses terminating on these neurons. It is proposed that the neurofilamentous space network serves a transducing function by linking plasma membrane activities with the genetic machinery of the neuron.     

The effects of cytochalasin D on differentiating muscle in culture

The spontaneous contractility of myotubes is enhanced by cytochalasin D (CD), which also causes myotobes to constrict and retract. Within an hour, large accumulations of filament masses appear in the cortical cytoplasm; they are later sequestered by fusion of sarcoplasmic membranes. More prolonged treatment induces the formation of many leptomerie bodies. The myofibrils of differentiating myotubes are disoriented by CD, but stacking of thick and thin filaments is not affected. In developed myotubes the orientation of myofibrils is maintained. Small pulsating myotubes can dcvelop in the continued presence of CD. After short exposure, effects of CD are rapidly reversible but after long exposure recovery is delayed or incomplete.     

ACTION OF CYTOCHALASIN D ON CELLS OF ESTABLISHED LINES : I. Early Events

HeLa, Vero, L, HEp2, and MDBK cells respond immediately to 0.2–0.5 µg/ml cytochalasin D (CD) with sustained contraction (contracture), loss of microvilli, expression of endoplasmic contents (zeiosis), nuclear protrusion, and extension of cytoplasmic processes. The development of these changes is depicted, and the dose-response patterns in these cell lines are described. MDBK is generally most resistant and HeLa most sensitive to these effects of CD. Cells in G₁ are most sensitive to CD; responsiveness decreases progressively during early S and is least in mid S through G₂. CD inhibits transport of [¹⁴C]deoxyglucose in HeLa by about 45% but has no significant effect on hexose uptake in Vero and MDBK; sugar transport is thus apparently unrelated to any morphologic effect of CD. Although spreading and attachment are impeded, CD does not decrease and may even enhance the adhesiveness of established monolayers. Contraction appears to be a primary early effect of CD, upon which other visible changes follow. It is prevented by some inhibitors of energy metabolism (deoxyglucose and dinitrophenol) and does not occur in glycerinated models without ATP. The possible bases of the contractile response to CD are discussed. Although direct or indirect action of CD on some microfilaments may occur, a generalized structural disruption of contractile filaments by CD is considered unlikely.     

THE DISTRIBUTION, ULTRASTRUCTURE, AND CHEMISTRY OF MICROFILAMENTS IN CULTURED CHICK EMBRYO FIBROBLASTS

The distribution, ultrastructure, and chemistry of microfilaments in cultured chick embryo fibroblasts were studied by thin sectioning of flat-embedded untreated and glycerol-extracted cells, histochemical and immunological electron microscopic procedures, and the negative staining of cells cultured on electron microscopic grids. In these cultured cells, the microfilaments are arranged into thick bundles that are disposed longitudinally and in looser arrangements in the fusiform-shaped cells. In the latter case, they are concentrated along the margins of the flattened cell, on the dorsal surface, and particularly at the ends of the cell and its ventral surface, where contact is made with the plastic dish or with other cells. Extracellular filaments, presumably originating from within the cell, are found at these points of contact. The microfilaments are composed in part of an actin-like protein. These filaments are between 70 and 90 Å in diameter, they are stable in 50% glycerol, they have an endogenous ATPase (myosin-like?) associated with them, they bind rabbit muscle heavy meromyosin, and they specifically bind antibody directed against isolated actin-like protein. In the cultured chick embryo fibroblasts, the microfilaments are essential for the establishment and maintenance of form, and they are probably critical elements for adhesion and motility. The microfilaments might also serve as stabilizers of intramembranous particle fluidity.     

CYTOPLASMIC FILAMENTS OF AMOEBA PROTEUS : I. The Role of Filaments in Consistency Changes and Movement

The role of filaments in consistency changes and movement in a motile cytoplasmic extract of Amoeba proteus was investigated by correlating light and electron microscopic observations with viscosity measurements. The extract is prepared by the method of Thompson and Wolpert (1963). At 0°C, this extract is nonmotile and similar in structure to ameba cytoplasm, consisting of groundplasm, vesicles, mitochondria, and a few 160 A filaments. The extract undergoes striking ATP-stimulated streaming when warmed to 22°C. Two phases of movement are distinguished. During the first phase, the apparent viscosity usually increases and numerous 50–70 A filaments appear in samples of the extract prepared for electron microscopy, suggesting that the increase in viscosity in caused, at least in part, by the formation of these thin filaments. During this initial phase of ATP-stimulated movement, these thin filaments are not detectable by phase-contrast or polarization microscopy, but later, in the second phase of movement, 70 A filaments aggregate to form birefringent microscopic fibrils. A preparation of pure groundplasm with no 160 A filaments or membranous organelles exhibits little or no ATP-stimulated movement, but 50–70 A filaments form and aggregate into birefringent fibrils. This observation and the structural relationship of the 70 A and the 160 A filaments in the motile extract suggest that both types of filaments may be required for movement. These two types of filaments, 50–70 A and 160 A, are also present in the cytoplasm of intact amebas. Fixed cells could not be used to study the distribution of these filaments during natural ameboid movement because of difficulties in preserving the normal structure of the ameba during preparation for electron microscopy.     

Effects of mechanical tension on protrusive activity and microfilament and intermediate filament organization in an epidermal epithelium moving in culture

Mechanical tension influences tissue morphogenesis and the synthetic, mitotic, and motile behavior of cells. To determine the effects of tension on epithelial motility and cytoskeletal organization, small, motile clusters of epidermal cells were artificially extended with a micromanipulated needle. Protrusive activity perpendicular to the axis of tension was dramatically suppressed. To determine the ultrastructural basis for this phenomenon, cells whose exact locomotive behavior was recorded cinemicrographically were examined by transmission electron microscopy. In untensed, forward-moving lamellar protrusions, microfilaments appear disorganized and anisotropically oriented. But in cytoplasm held under tension by micromanipulation or by the locomotive activity of other cells within the epithelium, microfilaments are aligned parallel to the tension. In non-spreading regions of the epithelial margin, microfilaments lie in tight bundles parallel to apparent lines of tension. Thus, it appears that tension causes alignment of microfilaments. In contrast, intermediate filaments are excluded from motile protrusions, being confined to the thicker, more central part of the cell. They roughly follow the contours of the cell, but are not aligned relative to tension even when microfilaments in the same cell are. This suggests that the organization of intermediate filaments is relatively resistant to physical distortion and the intermediate filaments may act as passive structural support within the cell. The alignment of microfilaments under tension suggests a mechanism by which tension suppresses protrusive activity: microfilaments aligned by forces exerted through filament-surface or filament-filament interconnections cannot reorient against such force and so cannot easily extend protrusions in directions not parallel to tension.     

ELECTRON MICROSCOPIC STUDIES ON THE INDIRECT FLIGHT MUSCLES OF DROSOPHILA MELANOGASTER : I. Structure of the Myofibrils

The myofibrils in Drosophila have thick and thin types of myofilaments arranged in the hexagonal pattern described for Calliphora by Huxley and Hanson (15). The thick filaments, along most of their length in the A band, seem to be binary in structure, consisting of a dense cortex and a lighter medulla. In the H zone, however, they show more uniform density; lateral projections (bridges) also appear to be absent in this region. The M band has a varying number of granules (probably of glycogen) distributed between the myofilaments. The myofilaments on reaching the Z region appear to change their hexagonal arrangement and become connected to one another by Z filaments. The regular arrangement of the filaments found in most regions of the fibrils is not seen in the terminal sarcomeres of some flight muscles; the two types of filaments appear to be intermingled in an irregular pattern in these parts of the fibrils. The attachment of myofibrils to the cuticle through the epidermal cells is described.     

Morphological evidence for the existence of a more complex cytoskeleton in Amoeba proteus

Summary Various stabilization and extraction procedures were tested to demonstrate the ultrastructural organization of the cytoskeleton in normal, locomoting Amoeba proteus. Most reliable results were obtained after careful fixation in glutaraldehyde/lysine followed by prolonged extraction in a polyethylene glycol/Triton X-100 solution. Before dehydration in a graded series of ethanol and critical-point drying, the amoebae were split by the sandwich-technique, i.e., by mechanical cleavage of cells mounted between two poly-L-lysine-coated glass slides. Platinum-carbon replicas as well as thin sections prepared from such cell fragments revealed a cytoskeleton composed of at least four different types of filaments: (1) 5–7-nm filaments organized as a more or less ordered cortical network at the internal face of the plasma membrane and probably representing F-actin; (2) 10–12-nm filaments running separately or slightly aggregated through the cytoplasm and probably representing intermediate filaments; (3) 24–26-nm filaments forming a loose network and probably representing microtubules; and (4) 2–4-nm filaments as connecting elements between the other cytoskeleton constituents. Whereas microfilaments are responsible for protoplasmic streaming and other motile phenomena, the function of intermediate filaments and cytoplasmic microtubules in amoebae is still obscure.     

SARCOPLASMIC RETICULUM OF AN UNUSUALLY FAST-ACTING CRUSTACEAN MUSCLE

The fast-acting, synchronous "remotor" muscle of the lobster second antenna was examined by light and electron microscopy and was found to have a more profuse sarcoplasmic reticulum (SR) than any other muscle known. Myofibrils are widely separated from one another and occupy only about one-fourth of the volume of the muscle; most of the remaining volume is taken up by the SR, which resembles the smooth-surfaced reticulum of steroid-secreting cells. Dense granules (0.03–0.1 µ in diameter) are scattered through the reticulum. T-tubules penetrate into the fibers and form dyads along the A bands of myofibrils; however, ferritin-labeling experiments show that the volume of the T-system is very small compared with that of the SR. Myofibrils are ~0.5 µ x 1.0 µ in cross section and consist of thick filaments, which appear tubular except at the M region, and thin filaments, which are situated midway between neighboring thick filaments. The ratio of thin to thick filaments is 3:1. The extreme development of the SR in this muscle is discussed in relation to the exceedingly short duration of the contraction-relaxation cycle.     

FORMATION OF ARROWHEAD COMPLEXES WITH HEAVY MEROMYOSIN IN A VARIETY OF CELL TYPES

Heavy meromyosin (HMM) forms characteristic arrowhead complexes with actin filaments in situ. These complexes are readily visualized in sectioned muscle. Following HMM treatment similar complexes appear in sectioned fibroblasts, chondrogenic cells, nerve cells, and several types of epithelial cells. Thin filaments freshly isolated from chondrogenic cells also bind HMM and form arrowhead structures in negatively stained preparations. HMM-filament complexes are prominent in the cortex of a variety of normal metaphase and Colcemid-arrested metaphase cells. There is no detectable binding of HMM with other cellular components such as microtubules, 100-A filaments, tonofilaments, membranes, nuclei, or collagen fibrils. The significance of HMM-filament binding is discussed in view of the finding that arrowhead complexes form in types of cells not usually thought to contain actin filaments.     

Human leukocytes as viewed by stereo high-voltage electronmicroscopy

Whole-mount preparations of adherent leukocytes were investigated by stereo high-voltage electronmicroscopy (HVEM) to determine the organization of the cytoplast in unstimulated, motile, and phagocytosing cells. A highly ordered structured cytoplast is revealed. All cytoplasmic organelles are held within an intricate network of fine strands, termed the microtrabecular lattice (MTL), which appears more complex in neutrophils than eosinophils or monocytes. In neutrophils, the tendency of the MTL to expand and contract during cell movement and the responding deformability of the granules appear to influence granule shape. This pleomorphism in granule shape is particularly prominent in exceptionally elongated neutrophils that have not established directionality and demonstrate the appearance of having two leading lamellipodia. Results suggest that the morphology of neutrophil granules is influenced by cell motility, and may account for the pleomorphic populations of granules observed by standard transmission EM. Examination of the cytoskeleton of these elongated cells after detergent extraction reveals separation of the centrosome into two solitary centrioles, with each centriole surrounded by an aster of microtubules. A complex network of microfilaments, intermediate filaments, and microtubules is integrated within a thin area of cytoplasm separating the two cell bodies. Interaction between the MTL, microfilaments, intermediate filaments, and microtubules probably influences granule translocation in these elongated cells. Phagocytosis stimulates a reorganization of the cytoplast; all organelles are found in more central areas of the cytoplasm, bordered by a thin area of hyaloplasm. The MTL appears to limit cytoplasmic granules to a compartment around phagocytic vacuoles, which probably provides the framework for efficient phagolysosome fusion.     

Microfilaments in epidermal cancer cells

The occurrence and structure of microfilaments in epidermal cancers induced in mice by treatment with 3,4-benzpyrene were investigated with the electron microscope. With malignant change, pleomorphic, undifferentiated cells with a cortical zone of microfilaments became increasingly abundant. The microfilaments were 40 Å in diameter and occupied the cortex of the cells beneath the plasma membrane, extended into cell processes, and were situated in the cores of microvilli. At high magnification, the filamentous areas were formed by an interconnected meshwork of filaments which in favorable planes had a polygonal arrangement. When exposed to high concentrations of cytochalasin B, the microfilaments became clumped and moderately disrupted. At the same time, the processes and microvilli of the cells were blunted. The structure of these filaments and their sensitivity to cytochalasin B place them in a class of microfilaments believed to be related to cell motility. Their presence in malignant cells may be correlated with the motile, invasive properties of these cells.     

Actin-organelle interaction: association with chloroplast in arabidopsis leaf mesophyll cells.

The role of the cytoskeleton in the regulation of chloroplast motility and positioning has been investigated by studying: (1) structural relationship of actin microfilaments, microtubules, and chloroplasts in cryofixed and freeze-substituted leaf cells of Arabidopsis; and (2) the effects of anti-actin (Latrunculin B; LAT-B) and anti-microtubule (Oryzalin) drugs on intracellular distribution of chloroplasts. Immunolabeling of leaf cells with two plant-actin specific antibodies, which react equivalently with all the expressed Arabidopsis actins, revealed two arrangements of actin microfilaments: longitudinal arrays of thick actin bundles and randomly oriented thin actin filaments that extended from the bundles. Chloroplasts were either aligned along the actin bundles or closely associated with the fine filaments. Baskets of actin microfilaments were also observed around the chloroplasts. The leaf cells labeled with an anti-tubulin antibody showed dense transverse arrays of cortical microtubules that exhibited no apparent association with chloroplasts. The application of LAT-B severely disrupted actin filaments and their association with chloroplasts. In addition, LAT-B induced aberrant aggregation of chloroplasts in the mesophyll and bundle sheath cells. Double labeling of LAT-B treated cells with anti-actin and anti-tubulin antibodies revealed that the microtubules in these cells were unaffected. Moreover, depolymerization of microtubules with Oryzalin did not affect the distribution of chloroplasts. These results provide evidence for the involvement of actin, but not tubulin, in the movement and positioning of chloroplasts in leaf cells. We propose that using motor molecules, some chloroplasts migrate along the actin cables directly, while others are pulled along the cables by the fine actin filaments. The baskets of microfilaments may anchor the chloroplasts during streaming and allow control over proper three-dimensional orientation to light.     

Polarity of actin filaments at the initial stage of myofibril assembly in myogenic cells in vitro

The polarity of thin filaments in relation to thick filaments in developing muscle cells in vitro was investigated. The majority of thin filaments exhibited the right polarity and spatial position similar to that seen in mature myofibrils. It appears that the interaction between thick and thin filaments exists in the initial phases of myofibrillogenesis. Cortical microfilaments are found to have their polarities arranged randomly.     

Organization of the actin cytoskeleton during pollen development inGasteria verrucosa (Mill.) H. Duval visualized with rhodamine-phalloidin

The three-dimensional organization of the microfilamental cytoskeleton of developingGasteria pollen was investigated by light microscopy using whole cells and fluorescently labelled phalloidin. Cells were not fixed chemically but their walls were permeabilized with dimethylsulphoxide and Nonidet P-40 at premicrospore stages or with dimethylsulphoxide, Nonidet P-40 and 4-methylmorpholinoxide-monohydrate at free-microspore and pollen stages to dissolve the intine.Four strikingly different microfilamentous configurations were distinguished. (i) Actin filaments were observed in the central cytoplasm throughout the successive stages of pollen development. The network was commonly composed of thin bundles ramifying throughout the cytoplasm at interphase stages but as thick bundles encaging the nucleus prior to the first and second meiotic division. (ii) In released microspores and pollen, F-actin filaments formed remarkably parallel arrays in the peripheral cytoplasm. (iii) In the first and second meiotic spindles there was an apparent localization of massive arrays of phalloidin-reactive material. Fluorescently labelled F-actin was present in kinetochore fibers and pole-to-pole fibers during metaphase and anaphase. (iv) At telophase, microfilaments radiated from the nuclear envelopes and after karyokinesis in the second meiotic division, F-actin was observed in phragmoplasts.We did not observe rhodamine-phalloidin-labelled filaments in the cytoplasm after cytochalasin-B treatment whereas F-actin persisted in the spindle. Incubation at 4° C did not influence the existence of cytoplasmic microfilaments whereas spindle filaments disappeared. This points to a close interdependence of spindle microfilaments and spindle tubules.Based on present data and earlier observations on the configuration of microtubules during pollen development in the same species (Van Lammeren et al., 1985, Planta165, 1-11) there appear to be apparent codistributions of F-actin and microtubules during various stages of male meiosis inGasteria verrucosa.Abbreviation DMSO dimethylsulfoxide     

ULTRASTRUCTURAL STUDIES ON THE CONTRACTILE MECHANISM OF SMOOTH MUSCLE

Fresh taenia coli and chicken gizzard smooth muscle were studied in the contracted and relaxed states. Thick and thin filaments were observed in certain (but not all) cells fixed in contraction. Relaxed smooth muscle contained only thin filaments. Several other morphological differences were observed between contracted and relaxed smooth muscle. The nuclear chromatin is clumped in contraction and evenly dispersed in the relaxed state. The sarcolemma is more highly vesiculated in contraction than in relaxation. In contraction, the sarcoplasm also appears more electron opaque. Over-all morphological differences between cells fixed in isometric and in unloaded contraction were also noticeable. The results suggest a sliding filament mechanism of smooth muscle contraction; however, in smooth muscle, unlike striated muscle, the thick filaments appear to be in a highly labile condition in the contractile process. The relation between contraction and a possible change in pH is also discussed.     

Association of actin filaments with axonal microtubule tracts

Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 µm with some at least as long as 3.5 µm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.