Microtubules and macrotubules in fish meiosis

During meiosis in the male of a cyprinodontid fish, Aphyosemion splendopleure, and during the organization of the spindle of division, the spindle is made of two types of tubules: microtubules (20-25 nm) and macrotubules (30-50 nm). The macrotubules are associated only with the polar region of the meiotic apparatus and are located outside the spindle of microtubules. At the end of meiosis, the spindle microtubules depolymerize whereas the macrotubules remain. One can find them throughout the entire process of spermiogenesis; later, they disappear only at the end of spermatid maturation. We have studied four populations from Cameroon, three of them with macrotubules.     

Vinblastine-induced paracrystals and unusually large microtubules (macrotubules) in rat renal cells

Summary Vinblastine sulfate was administered to adult rats by intravenous injections. Kidney cortex was fixed after 1, 2, or 5 hours of treatment and studied by routine transmission electron microscopy.In control animals, cells of distal convoluted tubules possessed numerous microtubules with an average diameter of 280 Å. In treated animals, the microtubules of these cells were reduced in number, and paracrystalline inclusions characteristic of vinblastine treatment were common. Macrotubules (570 Å average diameter) were also present and often were seen close to, or in apparent continuity with, paracrystals. Since the work of others indicates that vinblastine-induced paracrystals contain microtubular protein (tubulin), observation of continuities between paracrystals and macrotubules is interpreted as evidence that macrotubules are also composed of tubulin and that macrotubules may become incorporated into paracrystals.Unlike the ordinary microtubules of cells of the distal tubules, vinblastine-induced macrotubules exhibited cross-striations in longitudinal view and subunit structure in cross section.Macrotubules and paracrystals were also observed in cells of the proximal convoluted tubule, mesangium, glomerular endothelium, parietal epithelium of Bowman's capsule, and visceral epithelium of Bowman's capsule. Continuities between macrotubules and paracrystals, although relatively common in occurrence in distal tubule cells, were only rarely seen in the other kinds of cells examined. Acknowledgements. The authors gratefully acknowledge the technical help of Mrs. Dawn Bockus, Miss Judy Groombridge, Mrs. Jeri Hunter, Mrs. Jolan Pinter, Miss Franque Remington, Miss Mary Stewart, Miss Louise Young, Mr. Reginald Pickering, and Mr. W. J. Masten. This research was supported by N.I.H. grants AM 16 236, GM 00 100, and HE 03 174, by Institutional Cancer Grant IN-26L from the American Cancer Society, and by the Graduate School Research Fund of the University of Washington.     

Nucleation of macrotubules on double-walled microtubule seeds

It is known that histone H1 is able to cause the formation of double-walled microtubules from microtubule protein. Now, we demonstrate that in dependence on the mass ratio H1/microtubule protein upon addition of tubulin to short pieces of double-walled microtubules either their inner or their outer wall elongates resulting in normal microtubules or in macrotubules, respectively. Because of their genesis we suggest that macrotubules like double-walled microtubules (see Unger et al., Eur. J. Cell Biol. 46, 98-104 (1988)) expose those sides of tubulin dimers at their surface which usually form the lumen face of microtubules.     

Hyperosmotic stress induces formation of tubulin macrotubules in root-tip cells of Triticum turgidum: their probable involvement in protoplast volume control

Treatment of root-tip cells of Triticum turgidum with 1 M mannitol solution for 30 min induces microtubule (Mt) disintegration in the plasmolyzed protoplasts. Interphase plasmolyzed cells possess many cortical, perinuclear and endoplasmic macrotubules, 35 nm in mean diameter, forming prominent arrays. In dividing cells macrotubules assemble into aberrant mitotic and cytokinetic apparatuses resulting in the disturbance of cell division. Putative tubulin paracrystals were occasionally observed in plasmolyzed cells. The quantity of polymeric tubulin in plasmolyzed cells exceeds that in control cells. Root-tip cells exposed for 2-8 h to plasmolyticum recover partially, although the volume of the plasmolyzed protoplast does not change detectably. Among other events, the macrotubules are replaced by Mts, chromatin assumes its typical appearance and the cells undergo typical cell divisions. Additionally, polysaccharidic material is found in the periplasmic space. Oryzalin and colchicine treatment induced macrotubule disintegration and a significant reduction of protoplast volume in every plasmolyzed cell type examined, whereas cytochalasin B had only minor effects restricted to differentiated cells. These results suggest that Mt destruction by hyperosmotic stress, and their replacement by tubulin macrotubules and putative tubulin paracrystals is a common feature among angiosperms and that macrotubules are involved in the mechanism of protoplast volume regulation.     

On macrotubule structure.

The number of protofilament pairs in macrotubules was calculated as a function of macrotubule diameter, protofilament angle to the long axis, and pair width. A comparison of these calculations with published observations suggests utilizasion of all 13 microtubule protofilaments in the formation of macrotubules in vivo.     

Influence of vincristine on the Golgi complex of leukaemic lymphoblasts

In vitro and in vivo effects of vincristine on the Golgi complex of leukaemic lymphoblasts were studied. The cells incubated in vitro for 4 hours with vincristine of 1.25 x 10(-5) M concentration lacked microtubules, but regularly contained paracrystals and parallel arrays of macrotubules associated with ribosomes. The Golgi complex in control lymphoblasts was represented by 1-3 dictyosomes (stacks of cisternae) grouped in one area. After exposure to vincristine the dictyosomes lay at a considerable distance from each other. In many of them the cisternae were shorter than in controls and distended or transformed into large vacuoles. In cells incubated in vitro with lower concentrations of vincristine (1.25 x 10(-6) and 1.25 x 10(-7) M) and in cells obtained after the second therapeutic dose of vincristine (in the course of normal clinical treatment) neither changes in the Golgi complex nor formation of paracrystals and macrotubules were observed.     

Some Observations on the Structure of the Peristomial Membranelle of Spirostomum ambiguum

SYNOPSIS. Electron-microscopic observations of Spirostomum ambiguum have demonstrated additional details of superficial and deep tubular connections with peristomial and somatic kinetosomes. The superficial peristomial tubules appear to connect adjacent rows of kinetosomes. Anatomically, they course distally from the proximal kinetosomal plate. The deep tubules run proximally from the kinetosomal plate. Those in the somatic region appear to enter the endoplasm; those in the peristomial region leave the kinetosome as bundles of either 10 or 11 tubules which steadily converge to form 2 compact rows of 10 tubular bundles. These tubules connect to 2 of the 3 rows of 10 cilia each, the rows of 3 being separated by membranous folds protruding perpendicular to the peristomial groove. The rows of bundles converge further, enter the endoplasm and fan out again into tubular sheets, some of which appear to course in an antero-posterior direction. Another set of tubules arises from each of the kinetosomes in the 3rd row of 10 kinetosomes and courses proximally at a different angle from those arising from the 2 other kinetosome rows. Terminations have not been observed for the deep somatic or peristomial tubules. Their possible role in producing the forceful longitudinal contraction of Spirostomum is discussed.     

The effects of anaerobiosis and uncouplers of oxidative phosphorylation on mitotic timing and the induction of intranuclear macrotubules in the slime mold,Physarum polycephalum

Summary Mitotically synchronous plasmodia of the slime moldPhysarum polycephalum were subjected to brief exposures of either pure atmospheres of carbon dioxide or nitrogen gases or to pulsetreatments with respiratory poisons (sodium azide, sodium arsenate, or 2,4-dinitrophenol, DNP) at many different phases of the mitotic cycle to assess their effects on the mechanism(s) controlling the timing of mitosis. Plasmodia were fully viable after a pulse of CO₂ lasting up to 90 minutes or after a N₂-pulse of 30 minutes in duration. Upon return to normal aeration, all treated plasmodia entered a fully synchronous mitosis with a variable excess mitotic delay, which was dependent on the duration of the pulse and time of application in the mitotic cycle. Likewise, plasmodia exposed to 15-minute-pulses of a sublethal dose of sodium arsenate (0.1 mM), sodium azide 0.05 mM) and 2,4-DNP (0.2 mM) yield characteristic patterns of excess mitotic delay upon returnal to normal culture conditions. Two different types of phase response curves (PRC) were generated by these treatments. This suggests that at least two distinct respiratory-linked physiological mechanisms are involved in control of mitosis onset and regulation of mitotic timing inPhysarum.Electron microscope observations of CO₂-treated plasmodia reveal the induction of intranuclear 40–60 nm diameter macrotubules at all stages of the G₂ phase up to and including prometaphase. Both anoxia and sodium azide treatments are effective in macrotubule induction, and both reversibly disrupt the normal tubular cristae organization of mitochondria. In early G₂, macrotubules polymerize in association with both the inner membrane of the nuclear envelope and the nucleolus, while the tubule-organizer region, TOR, serves as the only nucleating site for macrotubules in late G₂ nuclei, coincident with the onset of mitosis and TOR formation.     

Associations between microbodies and a system of cytoplasmic tubules in oil-body cells of Marchantia

Summary Preliminary observations on differentiating oil-body cells of Marchantia sp. revealed that the cytoplasm possesses conventional cytoplasmic microtubules as well as a greater number of other tubules, which average 35 nm in diameter and appear in cytoplasmic regions rich in endoplasmic reticulum. These tubules, at a stage of active synthesis of oil, increase in number, may form well-organized bundles traversing the cytoplasm close to the vacuole, and show a preferential spatial relationship to elongated microbodies. One layer of densely arrayed cytoplasmic tubules surrounds the microbodies partially or totally. Fine links bridge the tubules to one another or some of them to the microbody bounding membrane.     

STRUCTURE OF THE MITOTIC SPINDLE IN L STRAIN FIBROBLASTS

The mitotic spindle of L strain fibroblasts, fixed with glutaraldehyde followed by osmium tetroxide, contains many 150- to 180-A tubules. They appear first in the cytoplasm. They extend from the centrospheres to the kinetochores, and from one centrosphere to the other. Only occasionally can points of continuity between the spindle tubules and the tubules of the centrioles be observed. The chromosomal insertion is by a means of a thin dense plate of the kinetochore. The total number of continuous spindle tubules is between 500 and 600. Occasionally, tubules appear paired. At anaphase, short lengths of individual spindle tubules possess a coating of a substance of high density midway between the poles. These parts of the spindle tubules aggregate to form irregular groups, comprising the stem-body, and, by becoming aligned into a plate, they form the mid-body.     

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.     

Giant sperm cells with accessory macrotubules in a neuropteran insect

The flagellar axoneme of the atypical spermatozoa (paraspermatozoa) of Mantispa perla (Neuroptera, Planipennia) contains accessory microtubules or rather macrotubules that are 55 nm in diameter and that has a wall consisting of about 40 protofilaments. The sperm tail further contains two giant mitochondrial derivatives, which during spermiogenesis store an electron dense material. The mature spermatozoon has a flattened acrosome and a elliptical nucleus. These giant spermatozoa may furnish nutrients to the functional spermatozoa (euspermatozoa) when they reach the female genital tracts or/and they function in sperm competition filling the spermatheca.     

The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast

The dynamin-related GTPase Dnm1 controls mitochondrial morphology in yeast. Here we show that dnm1 mutations convert the mitochondrial compartment into a planar 'net' of interconnected tubules. We propose that this net morphology results from a defect in mitochondrial fission. Immunogold labelling localizes Dnm1 to the cytoplasmic face of constricted mitochondrial tubules that appear to be dividing and to the ends of mitochondrial tubules that appear to have recently completed division. The activity of Dnm1 is epistatic to that of Fzo1, a GTPase in the outer mitochondrial membrane that regulates mitochondrial fusion. dnm1 mutations prevent mitochondrial fragmentation in fzo1 mutant strains. These findings indicate that Dnm1 regulates mitochondrial fission, assembling on the cytoplasmic face of mitochondrial tubules at sites at which division will occur.     

Messenger RNA synthesis in the testis of immature rats: effect of gonadotropins and cyclic AMP.

Two hours after the intratesticular injection of FSH, hCG or cyclic AMP, the incorporation of labeled uridine into poly(A)-rich RNA was increased. Pretreatment with actinomycin D inhibited the incorporation of uridine into mRNA. After the seminiferous tubules and interstitial cells were separated by treatment with collagenase, FSH treatment increased mRNA synthesis only in the tubules whereas hCG stimulated mRNA synthesis only in the interstitial cells. Cyclic AMP increased the synthesis of mRNA in both interstitial cells and seminiferous tubules. These results suggest a differential action of the two gonadotropic hormones in the cells of the testis; both effects appear to be mediated by cyclic AMP.     

Evidence for a species-specific barrier to sperm transport within the vagina of the chicken hen

Following their insemination into the vagina of chicken hens, turkey spermatozoa did not appear to reach the ovum within the upper magnum or infundibulum and were only occasionally found within the sperm storage tubules at the uterovaginal junction. Turkey spermatozoa were able to populate chicken uterovaginal sperm storage tubules as (or more) efficiently as fowl spermatozoa in uterovaginal junction tissue in vitro. They also populated uterovaginal junction sperm storage tubules in vivo after insemination directly into the uterovaginal region. Thus, a barrier to foreign spermatozoa appears to exist within the vagina of the chicken and not at the level of the uterovaginal junction sperm storage tubules. The nature of this barrier is not known; however it can be shown that while chicken and turkey spermatozoa have similar morphological features and motility characteristics, they have distinct surface antigenicity. Recognition of surface antigenicity by a localised immunological mechanism may be the basis of sperm selection within the hens vagina.     

Electron microscopy of Japanaut and Tilligerry viruses: two proposed members of the orbivirus group.

Japanaut and Tilligerry viruses were studied by thin-section and negative-contrast electron microscopy, and from their morphology and morphogenesis appear to be typical orbiviruses. Characteristic intracellular structures are associated with the development of the two viruses. These structures include coated tubules and one or more types of structure which appear to be composed of a number of parallel sheets of moderately electron-dense material.     

Ultrastructural changes in isolated plastids

Summary Etioplasts were isolated from maize leaves and the changes in their ultrastructure were followed in light and in darkness for several hours. It has been shown that the regular crystalline structures of prolamellar bodies, present after the isolation in darkness, disappear after 30 to 60 minutes of illumination, and long straight tubules appear within prolamellar bodies. Their appearance is influenced by the molarity of the isolation medium used, by light intensity, duration of illumination and by the temperature at which the isolates are kept. Long tubules appear, however, also in isolated etioplasts incubated for several hours in complete darkness.In isolates illuminated for 2–3 hours long tubules disappear again, and prolamellar bodies produced eventually consist of irregularly connected short tubules. In prolamellar bodies, regions with regular and very dense arrangement of tubules sometimes develop at this stage. The thylakoids (usually perforated) are now arranged concentrically in the plastids. True grana or poly-thylakoids can never be found in isolated etioplasts, not even when the etioplasts have been illuminated for 6 hours or more (up to 24).The present investigations have indicated that in isolated etioplasts in light, tubular elements, which build up the prolamellar bodies, cannot normally be transformed into thylakoids as is the case with intact tissue.The survival of isolated etioplasts is limited at present, and for this reason changes in their fine structure could be followed successfully for as long as 6 hours (in light at 15 °C), although a certain percentage of plastids survive up to 24 hours.     

Excurrent duct system of the male turtle Chrysemys picta

The epididymis and efferent duct system of the turtle Chrysemys picta were examined. Seminiferous tubules are drained by a series of ducts that form a rete exterior to the tunica albuginea. The rete is located lateral to the testis and consists of anastamosing tubules of varying diameters, lined by a simple epithelium consisting of squamous to cuboidal cells. The rete is highly vascularized. A series of tubules (efferent ductules) connect the rete to the epididymis proper. The efferent ductules are highly convoluted, running between the epididymal tubules and are of varying diameters. The simple columnar epithelium lining these tubules possesses tight junctions, with every third or fourth cell possessing long cilia that protrude into the lumen. The cytoplasm of these epithelial cells contains abundant mitochondria. In the central portion of the efferent ductule, epithelial cells possess granules that appear to be secreted into the lumen by an apocrine process. The epididymis proper is a single, long, highly convoluted tubule that receives efferent ductules along its entire length. It is lined by a pseudostratified epithelium containing several cell types. The most abundant cell (vesicular cell) lacks cilia, but has a darkly staining apical border due to numerous small vesicles immediately beneath the luminal membrane. The small vesicles appear to fuse with each other basally to form larger vesicles. These cells appear to have an absorptive function, and occasionally sperm are embedded in their cytoplasm. The second-most abundant cell is a basal cell found along the basement membrane. The number of these cells fluctuates throughout the year, being most abundant in late summer and early fall. A small narrow cell with an oval nucleus and darkly staining cytoplasm, extending from the basement membrane to the apical surface, is present in small numbers, particularly in the caudal regions of the epididymis. This cell is frequently found in association with another narrow cell having a rounded nucleus and abundant mitochondria in its cytoplasm.     

Excretion in the house cricket Acheta domesticus: Effects of diuretics on the structure of the mid-tubule

Most structural studies of insect Malpighian tubules focus on freshly dissected tissue, which is in fact stimulated by diuretic factors. In this study, we examine tubules from the house cricket Acheta domesticus in four discrete secretory states: control (freshly dissected); unstimulated (held in vitro for 90 min prior to fixation); corpus cardiacum-stimulated (held in vitro for 60 min, then stimulated with corpus cardiacum homogenates for 30 min prior to fixation); and cAMP-stimulated (held in vitro for 60 min, then stimulated with dibutyryl cAMP for 30 min prior to fixation). In unstimulated tubules, we see a reduction in vacuolization and a near-complete collapse of the basolateral infolds. Stimulated tubules show several major structural shifts: mitochondria are darkly stained with well-defined cristae, there is extensive vacuolization of the tissue and expansion of the basolateral spaces, and the CaPO₄ spherites appear to be ejected into the lumen. cAMP-stimulated tubules showed the most pronounced structural changes, including the presence of a newly reported ultrastructural feature for A. domesticus Malpighian tubules, referred to here as paracrystalline arrays, which appear as stacks of membrane localized in the perinuclear region. © 1996 Wiley-Liss, Inc.     

The structure of the Golgi apparatus: a sperm’s eye view in principal epithelial cells of the rat epididymis

 The Golgi apparatus of epididymal principal cells shares many structural features with other cell types. Saccular regions are arranged in a cis-Golgi network, eight flattened saccules, and several trans-Golgi networks (TGNs). Dilated tubules form intersaccular connecting regions which joint together saccules at the same or different levels between adjacent stacks. Wells exist as large perforations in register with the four cis-most saccules and serve as areas of vesicular interactions. TGNs are variable and can appear to peel off the stack or to be detached from it in the form of an anastomotic tubular network with pale dilated areas corresponding to prosecretory granules connected by short narrow bridges. Elongated or discoid dilated cisternae of endoplasmic reticulum (ER) (sparsely granulated) lie over the cis face of the stack, from which they are separated by an intermediate compartment filled with vesicles and tubules. The ER is also closely juxtaposed to the TGNs and the eighth saccule but interconnections are never seen between them. Vesicles of the COP variety reside at all levels of the stack and appear to bud off the cis-located ER and the edges of the saccules, while clathrin-coated vesicles appear mainly on the trans face of the stack and next to lysosomes. In the supranuclear cytoplasm, clusters of vesicles and tubules, at times budding off enveloping ER, appear to radiate toward the Golgi stacks where they fuse with cis Golgi elements. Taken together, these observations suggest dynamic functions and interactions for the various Golgi elements, associated vesicles, ER, and vesicular tubular clusters. Accepted: 29 January 1998