Observations on tubules derived from the endoplasmic reticulum in leaf glands ofPhaseolus vulgaris

Summary Tubular systems present in bean leaf glands have been studied electron microscopically. Ordered arrays of small tubules (290 Å in diameter) arise from the endoplasmic reticulum in early stages of gland development and remain connected to it. Subsequently larger tubules (560–660 Å in diameter) appear among the smaller tubules and gradually replace many of them. The large tubules are not connected to the endoplasmic reticulum. They contain an electron dense material and their walls exhibit a patterned substructure. In older gland cells the bundles of large tubules run randomly through the cytoplasm. The relationship of the two types of gland tubules to conventional microtubules has been examined morphologically and experimentally. The small tubules have larger diameters and thicker walls than microtubules. Neither type of gland tubule is affected by low temperature or colchicine, or, in thin sections, by pepsin digestion. This suggests that these tubules are not closely related chemically to either cytoplasmic or ciliary microtubules. The two systems of tubules are closely associated with prominent protein vacuoles in the gland cells, but are not directly connected to them.This work was supported in part by grant no. GB-6161 from the National Science Foundation.     

THE NORMAL FINE STRUCTURE OF OPOSSUM TESTICULAR INTERSTITIAL CELLS

The interstitial tissue of the opossum testis includes interstitial or Leydig cells, macrophages, and small cells which morphologically resemble mesenchymal cells. The latter are thought to give rise to mature interstitial cells. The most prominent feature of the interstitial cell cytoplasm is an exceedingly abundant agranular endoplasmic reticulum. This reticulum is generally in the form of a meshwork of interconnected tubules about 300 to 450 A in diameter, but occasionally it assumes the form of flattened, fenestrated cisternae resembling those of pancreatic acinar cells, except for the lack of ribonucleoprotein particles on the surface of the membranes. The interstitial cells vary considerably in their cytoplasmic density. The majority are quite light, but some appear extremely dense, and in addition usually have a more irregular cell surface, with numerous small pseudopodia. These differences may well reflect variations in physiological state. Cytoplasmic structures previously interpreted as "crystalloids" consist of long bundles of minute parallel tubules, each about 180 A in diameter, which seem to be local differentiations of the endoplasmic reticulum. The mitochondria are rod-shaped, and contain a moderately complex internal membrane structure, and also occasional large inclusions that are spherical and homogeneous. The prominent juxtanuclear Golgi complex contains closely packed flattened sacs and small vesicles. The results of the present study, coupled with biochemical evidence from other laboratories, make it seem highly probable that the agranular endoplasmic reticulum is involved in the synthesis of the steroid hormones produced by the interstitial cell. This finding therefore constitutes one of the first functions of the agranular reticulum for which there is good morphological and biochemical evidence.     

Ultrastructural Studies of the Development of Nerves in Hydra

Three types of mature epidermal neurons and several of theirdifferentiating stages aie presented in this ultrastructuralstudy. Each of the three types, neurosensory, neurosecretory,and ganglionic cells, is derived from interstitial cells, (i)Mature neurosensory cells contain elongated nuclei, a well-developedcilium in each cell, and membrane-bounded neurosecretory droplets(700–1300 A in diameter). There may be two or more neuritesin which are numerous microtubules, glycogen particles, ribosomesand many neurosecretory droplets, (ii) Mature neurosecretorycells closely resemble neurosensory cells, except that no ciliumis present. The perikarya contain small, dense nuclei, neurosecretorydroplets (850–1300 A in diameter), mitochondria, glycogenparticles, and microtubules. Active Golgi complexes are presentin both cell types. The nemites are similar to those describedfor neurosensory cells, (iii) Mature ganglionic cells are bipolaror multipolar. The small, dense nuclei are surrounded by a smallamount of cytoplasm. The neurites contain mostly microtubules;a few mitochondria, ribosomes, and glycogen particles are alsopresent, but there are no secretory droplets. To date, only neurosensory and neurosecretory cells have beenobserved in the gastiodermis. They are structurally indistinguishablefrom their epideimal counterparts. A significant finding is that three types of synapses—neuromuscular,neuronematocyte, and interneuronal—are identified in boththe epidermal and gastrodermal neurons.     

Cytokinesis in microspore mother cells of Impatiens sultani

Summary Cytokinesis in Impatiens sultani microspore mother cells is simultaneous. It starts with the formation of small ingrowths of the surrounding callosic wall. Next, an incomplete cell plate is formed by fusion of small dictyosome vesicles. The cell plate consists of a network of anastomosing tubules and sacs. Aggregates of fusing vesicles are associated with bundles of microtubules, which are oriented perpendicular to the plane of the future cell walls. In the sacculate parts of the cell plate, some callose is deposited, while the associated microtubules disappear. The cell walls ultimately develop by enlargement of the previously formed wall ingrowths, which successively incorporate the elements of the cell plate. The enlargement and thickening of the walls is not accompanied by a further fusion and incorporation of dictyosome vesicles.     

Pellicular Microtubules in the Family Trypanosomatidae*

SYNOPSIS. Flagellates of 7 species from 5 genera of the family Trypanosomatidae were prepared for electron microscopy using a combination of surface tension spreading on a simplified Langmuir trough followed by critical point drying. These technics opened the cells and showed the microtubular complex in its 3-dimensional configuration or in a flat sheet with the inside surface facing-up. Tears originated near the flagellar end and proceeded in a posterior direction usually paralleling the tubules. Major differences in the spreading pattern were species-specific. A connection between the flagellum and microtubules was not shown but strongly indicated by their constant association in all degrees of spreading. Most microtubules follow a spiral course from the flagellar end toward the posterior apical area and terminate short of the apex. It is suggested that the concept of antero-posterior orientation in the genus Trypanosoma be reconsidered. Cells of Leptomonas collosoma showed, without exception, a clockwise spiral as the tubules approached the posterior apex. Therefore, both the direction of spiraling and the manner of spreading were consistent. The number of microtubules varied among cells of the same species as well as on different levels of the same cell. Highest counts coincided with the level of greatest circumference. No connections or anastomoses between tubules were found. The microtubules are believed to have limited flexibility. Changes in diameter were interpreted to mean flattening had occurred. An intertubular substance, believed to surround the microtubules in the living cell, was present in all species.     

NUCLEATING SITES FOR THE ASSEMBLY OF CYTOPLASMIC MICROTUBULES IN THE ECTODERMAL CELLS OF BLASTULAE OF ARBACIA PUNCTULATA

In the ectodermal cells of sea urchin blastulae, the microtubules converge and appear to make contact with three distinct cytoplasmic foci or satellites associated with the basal body of the cilium. Beneath the nucleus, which lies in the apical end of the cell, the microtubules are aligned predominantly parallel to the cell's long axis and could thus make contact with the satellites as is directly suggested by observations on sections at or near the planes of the satellites. After the embryos are treated with low temperature (0°C), the microtubules disassemble; however, the satellites can still be recognized. Upon rewarming, the microtubules reappear. In early stages of reformation, when the tubules in the cell consist of short segments, tubules have only been found in the apical part of the cell. One end of each microtubule appears to make contact with its respective satellite, or is aligned so that it could contact one of the satellites, provided serial sections were cut and collected in order. After longer periods of recovery, the microtubules elongate; as before, one end of each makes contact with a satellite or is aligned so that it could attach to a satellite. Segments of microtubules seen in the basal region of the cell are aligned parallel to the long axis of the cell as in the untreated ectodermal cells and are therefore interpreted as extensions of those tubules making contact with one of the satellites. On the basis of these observations, we suggest that assembly of microtubules is initiated at the satellites. These sites, perhaps best referred to as "nucleating sites," thereby could exert considerable control over the distribution of microtubules in cells. It is hoped that this preliminary report will be followed up by a more detailed study using serial sections.     

Different aspects of tubulin polymerization in spermatids of cyprinodontidae (fish,teleost)

Our study concerns 10 genera and 26 species of cyprinodontid fish. In the cytoplasm of spermatids tubulin polymerizes in various forms according to species. We have demonstrated the presence of classic microtubules with a diameter of 24 nm and also of tubules of smaller diameter (15 run) and greater diameter (30 to 50 nm). Microtubules are very numerous in the cytoplasm of certain species. They may be arranged without any order or form bundles which may contain several hundred parallel elements. They never form a manchette. In two species (Aplocheilichthys normani and Epiplatys fasciolatus) only spermatids that degenerate show this peculiarity. The microtubules present two kinds of decorations. The first type are small elements composed of MAP which enable two or more microtubules to link up. The second type are curved tubulin elements that give the microtubule that bear them the appearance of an incomplete doublet. Doublets and triplets may also be formed. Cyprinodontidae spermatocytes and spermatids probably synthesize a very large quantity of tubulin which polymerizes in certain species.     

THE FINE STRUCTURE OF TESTICULAR INTERSTITIAL CELLS IN GUINEA PIGS

In guinea pig testes perfused with either glutaraldehyde or osmium tetroxide fixative, the cytoplasm of the interstitial cells contains an exceptionally abundant agranular endoplasmic reticulum. The reticulum in central regions of the cell is a network of interconnected tubules, but in extensive peripheral areas the reticulum is commonly organized into closely packed, flattened cisternae which are fenestrated. Occasional small patches of the granular reticulum occur in the cytoplasm and connect freely with the agranular reticulum. The mitochondria have a dense matrix and contain cristae and some tubules. The Golgi complex is disperse and shows no evidence of secretory material. The cytoplasm also contains lipid droplets. Lipofuscin pigment granules are probably polymorphic residual bodies and contain three components: (1) a dense material which at high magnification shows a 75-A periodicity; (2) a medium-sized lipid droplet; and (3) a cap-like structure. In glutaraldehyde-perfused testis the interstitial cell cytoplasm appears to have the same density from cell to cell, and the agranular reticulum is tubular or cisternal but not in the form of empty vesicles. Thus the "dark" and "light" cells and the vesicular agranular reticulum sometimes encountered in other fixations may be artifacts. Biochemical results from other laboratories, correlated with the present findings, indicate that the membranes of the agranular endoplasmic reticulum in guinea pig interstitial cells are the site of at least two enzymes of androgen biosynthesis, the 17-hydroxylase and the 17-desmolase.     

Coalescence of Golgi fragments in microtubule-deprived living cells

The process of stack coalescence, an important mechanism of Golgi recovery from mitosis, was examined using novel experimental paradigms. In living cells with disrupted (by nocodazole) microtubules, galactosyl transferase-GFP-labelled Golgi fragments constantly appeared, grew, sometimes moved with a speed of 1-2 microns/min, coalesced or gradually diminished and disappeared. The rate of Golgi fragment turnover and coalescence was highly balanced to maintain a constant number of Golgi units per cell. Moreover some Golgi islands appear and some received new GalTase-GFP after photobleaching of cell cytoplasm. Short tubules extending from the rims of scattered Golgi fragments frequently formed bridges between ministacks, inducing their coalescence. The frequency of coalescence could also be inhibited by disruption of actin microfilaments. After the Golgi redistribution into endoplasmic reticulum induced by brefeldin A, either the growth of small Golgi fragments or their coalescence leads to compartmentalized stack formation without the participation of microtubules. These results demonstrate that this coalescence between isolated Golgi stacks is microtubule-independent and could thus be mediated by membranous tubules.     

The intranuclear mitosis of the ciliates Paracineta limbata and Ichthyophtirius multifiliis

Electron microscope studies on the premetaphase stages of micronuclear divisions of Paracineta limbata and Ichthyophtirius multifiliis showed that spindle material also exists during interphase. In the case of I. multifiliis scattered microtubule fragments persist in the nuclear space; in P. limbata the micronuclei contain a small paracrystalloid which is suggested to be microtubular protein. Wide microtubules, varying in diameter from 300 to 400 Å develop during intranuclear prophase near the nuclear envelope in both cases. There are good reasons to assume that they function as a kind of stem body during the enlargement of the surface area of the nuclear envelope. Later micronuclear prophase stages of both species show a some-what different development. In I. multifiliis, there are scattered groups of short microtubular segments, partly in parallel array, whereas in P. limbata the wide tubules are transformed into normal microtubules of 180–200 Å diameter. The nuclei of both species are similar at late prophase and prometaphase stages. Bundles of interpolar microtubules run between the chromosomes, and single microtubules, presumably induced by the chromosomes, cross them at different angles. The chromosome-induced microtubules appear a short time after the interpolars. At prometaphase stage all microtubules show a highly parallel arrangement and therefore it is suggested that chromosomal tubules reach their final polar orientation by the action of cross-bridges.     

Effects of various treatments on microtubules and axial units of lung-fluke spermatozoa

Summary Sperm of the frog lung-fluke, Haematoloechus medioplexus, were treated in various ways and their microtubules and axial units were subsequently studied in sectioned and negatively-stained material. Microtubules and axial units were generally unaffected by exposure to colchicine, cold, and KCl, although with KCl certain lateral projections from doublet tubule A appeared more prominent in negatively-stained preparations. Both mercaptoethanol and urea have a dissociative effect on doublet tubules and microtubules, with doublet tubules being the more sensitive. Pepsin-HCl initially digests the dense region associated with the A tubule of a doublet pair and the core of the axial unit. Microtubules and B tubules of doublet units are later digested; in microtubules, there appears to be a proteinaceous material in the lucent central region which is digested before disappearance of the wall of the microtubule. Further evidence is presented indicating that the characteristically helical wall of the microtubules is made up of spherical subunits about 50 Å in diameter, with about 8 subunits in one turn of the helix. Under certain conditions, the helical structure may be altered to form a wall comprised of longitudinal filaments. It is emphasized that not all microtubules are structurally and chemically equivalent, and it follows that all microtubules do not share a common function.This research was supported by U.S. Public Health Service Grant AI-06448 and an institutional grant from the American Cancer Society.     

The fine structure of differentiating interstitial cells in Hydra

Summary Interstitial cells of hydra are small undifferentiated cells containing an abundance of free ribosomes and few other cytoplasmic organelles. They are capable of differentiating into epitheliomuscular, digestive, glandular, nerve cells, and cnidoblasts. Developing epitheliomuscular and digestive cells acquire bundles of filaments, 50 Å in diameter, which later are incorporated into the muscular processes. Early gland cells develop an elaborate rough-surfaced endoplasmic reticulum and one or more Golgi apparatus. Secretory granules originate in the Golgi region eventually filling the apex of the cell. Neurons are recognized first by the presence of an elaborate Golgi apparatus, absence of a well-developed endoplasmic reticulum, and later the appearance of cytoplasmic processes. The most striking feature of nematocyst formation by cnidoblasts is the presence of a complex distribution system between protein synthesizing rough-surfaced endoplasmic reticulum and the nematocyst. This system consists of connections between cisternae of the endoplasmic reticulum with smooth Golgi vesicles which in turn are connected to minute tubules, 200 Å in diameter. The tubules extend from the Golgi region around the nematocyst finally entering the limiting membrane of the nematocyst. It is suggested that the interstitial cells of hydra represent a model system for the investigation of many aspects of cell differentiation.This work was supported by grants from the National Cancer Institute (TlCA-5055) and from the National Institute of Arthritis and Metabolic Diseases (AM-03688), National Institutes of Health, Department of Health, Education and Welfare.The author is indebted to Dr. Russell J. Barrnett for his guidance and interest throughout this investigation.     

Microtubular Structures in Macronuclei of Synchronously Dividing Tetrahymena pyriformis

SYNOPSIS. When heat-synchronized cultures of Tetrahymena pyriformis , amicronucleate strain GL, were examined by electron microscopy, intramacronuclear microtubules were observed in dividing cells. These tubules have a diameter of 180–230 A and occur either singly or packed together in bundles. They are predominantly associated with outpocketings and invaginations of the nucleus. Sections as well as negatively stained preparations of isolated macronuclear envelopes indicate that the microtubules are inserted at the inner nuclear membrane.
The findings suggest that microtubules of the spindle type participate in the process of macronuclear division.     

MICROTUBULES AND EARLY STAGES OF CELL-PLATE FORMATION IN THE ENDOSPERM OF HAEMANTHUS KATHERINAE BAKER

A fine structure study of the phragmoplast and developing cell plate has been made on glutaraldehyde-osmium tetroxide-fixed, dividing, cultured cells of the liquid endosperm of Haemanthus katherinae Baker. The phragmoplast arises between the telophase nuclei, usually in association with a remnant strand of spindle elements, and consists of an accumulation of microtubules oriented at right angles to the plane of the future cell plate. The microtubules, which are 200–240 A in diameter, occur in small clusters spaced at approximately 0.2–0.3 µ intervals along the plate. Short interconnections interpreted as "cross-bridges" have been observed between individual microtubules. Within each cluster there is an electron-opaque zone about 0.3 µ in width which can be attributed in part to an overlap of microtubules from both sides of the plate and in part to a local accumulation of an amorphous electron-opaque material. During development these dense zones become aligned in a plane which itself defines the plane of the plate. Vesicles, commonly observed in long files, are derived from a cytoplasmic matrix rich in elements of the endoplasmic reticulum and sparse in dictyosomes. They aggregate between the clusters of microtubules and eventually coalesce to form the cell plate.     

ELECTRON MICROSCOPIC OBSERVATIONS ON TUBULAR STRUCTURES INVOLVED IN CRYSTAL FORMATION IN THE RED ALGA PLEONOSPORIUM SQUARRULOSUM (HARV.) ABBOTT1

Hexagonal or angular crystalline inclusions in Pleonosporium (Naeg.) Hauck vegetative cells were examined using electron microscopy. Ultrastructural analysis reveals that the inclusions initially contain tubular elements resembling microtubules but, with continued differentiation, are transformed into rod containing crystals. The tubular structures initially measure 25 nm in diameter. Scattered tubules become arranged in a parallel and alternate pattern and undergo subsequent enlargement to approximately 29 nm. Following enlargement, each tubule apparently disaggregates into rods that form a crystal having hexagonally arranged rod-like subunits. It is suggested that these tubules may represent microtubules and the resultant crystals are composed of tubulin.     

THE ULTRASTRUCTURE OF THE CAT MYOCARDIUM : II. Atrial Muscle

The ultrastructure of the cells specialized for contraction in the atrium and ventricle of young adult cats are compared. The cells specialized for conduction are not included. In addition to possessing distinctive atrial granules, the cells of the atrium are smaller in diameter (5–6 µ) than ventricular cells (10–12 µ) and have strikingly fewer T tubules. These latter differences are discussed in terms of their possible significance for the rate of conduction of the action potential. It is suggested that the very small number of T tubules in atrial cells may compensate for the small cell diameter, and thus permit rapid conduction of the action potential across the surface of the atrium. Coated dense vesicles found in association with the sarcoplasmic reticulum at the level of the Z line in ventricular muscle are more evident in atrial cells. In the virtual absence of T tubules in atrial cells, the sub-sarcolemmal cisternae of the sarcoplasmic reticulum are almost exclusively at the cell periphery. The ends of the cells and their processes in ventricular muscle are rectilinear with the interdigitated portions of the intercalated discs oriented transversely, whereas those of the atrium are often oblique to the myofilament axis. This difference may be related to the lower mechanical tension on atrial cells.     

Fine Structure, Reconstruction and Possible Functions of Components of the Cortex of Tetrahymena pyriformis

SYNOPSIS. Improved methods of preparing cells for electron microscopic study have permitted a more complete examination of the tubular and fibrous elements already reported in the cortex of Tetrahymena pyriformis. Three of these structures, a kinetodesmal fiber, a band of postciliary microtubules and a band of transverse microtubules, are all associated at one of their ends with the proximal end of a basal body-cilium complex. From this position they radiate out as tho from one corner of a tetrahedron, each passing separately to one of the other 3 corners. Available evidence is presented and discussed for a structural function for these elements. A firm bonding of these fibers and bands to the basal body at one of their ends and to the amorphous material under the pellicle at their other ends is thought to provide for this support. Connecting the proximal ends of the left side of the basal bodies of a kinety is another, previously undescribed, set of tubules. Their diameter, 24 mμ, and cross sectional structure are similar to those of the other microtubules. However, their more sinuous longitudinal appearance, small number of tubules per kinety and different reaction to fixatives suggest a different function. Because of their location it is proposed that they may be a communication line between basal body-cilium complexes of a kinety. A 3-dimensional drawing shows the positioning of the above structures in the cortex. Bodies with an internal tubular structure appear anterior to the proximal end of some basal bodies. They are referred to as probasal bodies due to their resemblance to procentrioles; they may be immature basal bodies. Smooth-membraned cisternae which have bristle-coated pits and bear a resemblance to the outer pellicular membrane appear in the cytoplasm. Their origin is discussed.     

LH and FSH stimulation of cyclic AMP in specific cell types isolated from the testes.

The direct effect of LH and FSH on cyclic AMP levels in specific cell types, isolated from the rat testes, was investigated in vitro. LH significantly stimulated cyclic AMP production in isolated interstitial cells and had only a slight effect on the isolated germ cells. FSH significantly stimulated cyclic AMP production in isolated seminiferous tubules, organ cultures of testes explants, and isolated Sertoli cells, with only a small response elicited in the germ cells. FSH had no effect on the cyclic AMP levels in interstitial cells and either freshly isolated or cultured peritubular cells. These data indicate that the Sertoli cells and interstitial cells are the main cell types in the testes which respond to FSH and LH respectively with increased cyclic AMP production. A possible slight effect of either hormone on the cyclic AMP level in the germ cells has not be ruled out.     

An observation of fine tubules within the endoplasmic reticulum in a dog liver

Summary During a survey of liver tissue from 100 dogs, fine tubules were observed within the cisternae of the endoplasmic reticulum of hepatocytes in one dog. The tubules were 300 Å in diameter with electron dense walls of 100 Å thickness. Many cells contained the tubules but their significance is unknown. The tubules varied from other reported microtubules in their location, size and characteristics of fixation and staining.     

Microtubules,protoplasts and plant cell shape

Indirect immunofluorescence has been used to study the function of cytoplasmic microtubules in controlling the shape of elongated carrot cells in culture. Using a purified wall-degrading preparation, the elongated cells are converted to spherical protoplasts and the transverse hoops of bundled microtubules are disorganised but not depolymerised in the process. Since microtubules remain attached to fragments of protoplast membrane adhering to coverslips and are still seen to be organised laterally in bundles, it would appear that re-orientation of the transverse bundles is due to loss of cell wall and not to the cleavage of microtubule bridges. After 24 h treatment in 10^-3 M colchicine, microtubules are depolymerised in elongated cells but, at this time, the cells retain their elongated shape. This suggests that wall which was organised in the presence of transverse microtubule bundles can retain asymmetric shape for short periods in the absence of those tubules. However, after longer periods of time the cells become spherical in colchicine. Neither wall nor tubules therefore exert individual control on continued cellular elongation and so we emphasize the fundamental nature of wall/microtubule interactions in shape control. It is concluded that the observations are best explained by a model in which hooped bundles of microtubules—which are directly or indirectly associated with molecules involved with cellulose biosynthesis at the cell surface—act as an essential template or scaffolding for the orientated deposition of cellulose.