Studies on the fine structure of teleost blood cells

Summary Electron microscopic study of nucleated erythrocytes of the goldfish, Carassius auratus, reveals the microtubular elements comprising the marginal band which encircles the cell. Six to ten units are visible at each pole of the cell, immediately within the plasmalemma. Each tubular unit is composed of an electron dense membrane enclosing a less dense core. Cross-sectional units average 264 Å outer diameter, whereas tubules measured in longitudinal sections average 237 Å.The functions of the microtubules of the marginal bands are analyzed in view of Meves' original interpretation of maintenance of the discoidal form of the nucleated erythrocyte, and the more recent investigations in cell physiology of Trotter and Tilney. It is proposed that the microtubules possess a dual function: the support of the cell which is attributed to the hydroelastic properties of the turgid microtubules resulting from intratubular hydrostatic pressures; and the intracellular transport of materials via the intratubular fluid. The microtubules may, therefore, be considered as a skeletal system and part of an intracellular circulatory system.This project was supported by grants 2 G-895 and 2 G-505 from the United States Public Health Service.     

Mikrotubuli und Filamente in prospektiven Plättchenfeldern der Megakaryocyten

Zusammenfassung Megakaryocyten des Knochenmarkes des Menschen, der Ratte und der Maus wurden nach Fixierung mit Glutaraldehyd und nach Kontrastierung mit Uranylacetat und Bleihydroxyd elektronenmikroskopisch untersucht. Außer den bekannten submikroskopischen Strukturen lassen sich in den prospektiven Plättchenfeldern und in der marginalen Randzone der Megakaryocyten 200–250 Å breite Mikrotubuli und etwa 50 Å breite Filamente nachweisen. Diese gleichen in ihrer Struktur den Mikrotubuli und Filamenten der Thrombocyten des peripheren Blutes. Die Mikrotubuli entstehen im Cytoplasma der Megakaryocyten, ohne zunächst einem bestimmten Plättchenfeld zugeordnet zu sein. In frühen Stadien der Plättchenfelderbildung erkennt man meist nur einige Mikrotubuli innerhalb der Plättchenfelder, die sich zwischen Demarkationsbläschen hindurch in benachbarten Plättchenfeldern fortsetzen. Komplette marginale Bündel von Mikrotubuli sind in den Megakaryocyten nur selten zu beobachten. Die biochemischen und funktionellen Eigenschaften der Mikrotubuli und Filamente werden diskutiert.

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Microtubules and filaments in prospective platelet fields of megakaryocytes

Summary Megakaryocytes of bone marrow of man, rat and mouse were studied electronmicroscopically after fixation with glutaraldehyde and staining the ultrathin sections with uranyl acetate and lead hydroxide. Microtubules measuring 200–250 Å and filaments measuring 50 Å in thickness can be demonstrated in prospective platelet fields and marginal zones of megakaryocytes. These microtubules and filaments resemble those of the platelets of the peripheral blood. The microtubules are formed within the cytoplasm of the megakaryocytes without predilection of certain platelet fields. In early stages of the formation of platelet fields only a few microtubules can be detected within platelet fields which penetrate between demarcation vesicles into neighbouring platelet fields. In megakaryocytes complete marginal bundles of microtubules are rarely observed. Biochemical and functional properties of microtubules and filaments are discussed.

     

Ionophore-induced disassembly of blood platelet microtubules: effect of cyclic AMP and indomethacin

Cytoplasmic calcium levels are believed to be important in blood platelet activation. Upon activation, the discrete marginal microtubule band, which maintains the discoid shape of non-activated platelets, becomes disrupted. Present studies demonstrate that the extent of assembly of the marginal microtubule band is related to cytoplasmic calcium levels. The divalent cationophore, A23187, causes platelet aggregation, secretion, and contraction by promoting calcium transport from intraplatelet storage sites into the cytoplasm. A23187 caused disassembly of platelet microtubules. Quantitation of electron micrographs revealed that numbers of microtubules were reduced by approximately 80% after A23187 treatment. Secondly, assembled microtubules in homogenates of platelets, in which microtubules were stabilized prior to homogenization, were decreased in favor of free tubulin in A23187-treated platelets. Thirdly, A23187 increased 14C-colchicine binding by intact platelets; this also indicated a shift in the microtubule subunit equilibrium to favor free, colchicine-binding tubulin subunits. In control experiments, A23187 did not affect the stability of platelet tubulin, the colchicine binding reaction, or the total tubulin content of platelets. Stimulation of colchicine binding depended on A23187 concentration (0.05-0.5 microM) and did not require extracellular calcium. A23187-stimulation of colchicine binding was blocked by dibutyryl cyclic AMP (0.80 mM) and/or 3-isobutyl-1-methylxanthine (50 microM) and by indomethacin (10 microM). Cyclic AMP or indomethacin also interferes with A23187-induced platelet activation, but indomethacin is not likely to completely inhibit the perturbation of intraplatelet calcium gradients by A23187. It is suggested that A23187-induced microtubule disassembly may be an indirect effect of calcium on microtubules.     

A novel microtubule protein in the marginal band of human blood platelets

A characterization is reported of the major cytoskeletal protein, called IEF (isoelectric focusing)-51K, of marginal band microtubule coils from human blood platelets (Kenney, D. M. and Linck, R. W. (1985) J. Cell Sci. 78, 1-22). IEF-51K is a unique biochemical species which is distinguishable from platelet and mammalian neuronal alpha-tubulin and beta-tubulin by 1) its faster mobility on discontinuous sodium dodecyl sulfate electrophoresis corresponding to an apparent Mr 51,000; 2) its more alkaline relative isoelectric point at pH 5.7 compared with that of alpha- and beta-tubulin at pH 5.3 and 5.5, respectively; 3) lack of coincidence in peptide maps prepared with chymotrypsin or Staphylococcus aureus V8 protease; and 4) lack of immunochemical cross-reactivity of polyclonal anti-IEF-51K with alpha- and beta-tubulin and of monoclonal anti-alpha-tubulin and anti-beta-tubulin with IEF-51K. In contrast to its chemical uniqueness, IEF-51K is tubulin-like in some of its properties. IEF-51K is localized in the marginal band of intact platelets by immunofluorescence; it undergoes cycles of microtubule disassembly/reassembly both in vitro and in vivo. Furthermore, IEF-51K was not extracted from isolated Taxol-stabilized marginal band microtubules by elevated NaCl concentrations (to 0.45 M), conditions that do not disrupt the polymeric structure of alpha- and beta-tubulin. These results indicate that IEF-51K together with alpha-tubulin and beta-tubulin are the major structural polypeptides of platelet marginal band microtubules. The unusual subunit composition of the platelet marginal band microtubule may be related to specialization(s) of microtubule structure and function in the marginal band coil of platelets.     

Organization of the cytoskeleton in resting, discoid platelets: preservation of actin filaments by a modified fixation that prevents osmium damage

This study evaluates the structural organization of the cytoskeleton within unactivated, discoid platelets. Previously, such studies have been difficult to interpret because of the ease with which platelets are stimulated, the sensitivity of actin filaments to cell extraction buffers, and the general problem of preserving actin filaments with conventional fixatives, compounded by the density of the cytoplasm in the platelet. In this study we have employed a new fixative containing lysine, which protects actin filaments against damage during fixation and thin-section processing. We used thick (0.25-micron) sections and conventional thin sections of extracted cells (fixed and lysed simultaneously by the addition of 1% Triton X-100 to the initial fixative) as well as thin sections of whole cells to examine three preparations of human platelets: discoid platelets washed by sedimentation; discoid platelets isolated by gel filtration; and circulating platelets collected by dripping blood directly from a vein into fixative. In all of these preparations, long, interwoven actin filaments were observed within the platelet and were particularly concentrated beneath the plasma membrane. These filaments appeared to be linked at irregular intervals to the membrane and to each other via short, approximately 20- to 50-nm-long cross-links of variable width. Although most filaments were outside the circumferential band of microtubules and the cisternae of the open canalicular system, individual filaments dipped down into the cytoplasm and were found between the microtubules and in association with other membranes. The ease with which single actin filaments can be seen in the dense cytoplasm of the human platelet after lysine/aldehyde fixation suggests the great potential of this new fixative for other cells.     

Morphology of microtubules in rabbit platelets

Summary The morphology of the microtubular wall in rabbit platelets fixed in glutaraldehyde and osmic acid solution and stained with uranyl acetate, or lead hydroxide, or doubly stained, is variable. In cross section, the wall may appear as a uniformly dense annulus, an annulus containing nodular densities about 35 Å in diameter or as a series of contiguous subunits with a circular cross sectional profile about 70 Å wide. The authors relate the varying morphology to the intensity of staining and equate the nodular and circular subunits. Rotational analysis suggests that there are 12 ± 2 subunits in the microtubular wall.We thank Professor A. C. Ritchie for his criticism of the paper and Mrs. M. Lorber and Mrs. M. Mezari for technical assistance. — This work was supported by grants from the Medical Research Council of Canada and the Ontario Heart Foundation.     

Effects of calcium,lanthanum, and temperature on the fluidity of spin-labeled human platelets

Summary Previous platelet studies have shown that calcium plays important roles in stimulus-secretion coupling, aggregation, and other membrane-associated functions. In addition, lanthanum induces platelet aggregation and the platelet release reaction and also influences platelet responsiveness to various stimuli. The spin-label results presented here suggest that one mechanism through which calcium and lanthanum mediate their effects on platelet functions may be by decreasing the lipid fluidity of the surface membrane.The structure of platelet membrane lipids was examined with the spin-label method. Washed human platelets were labeled with the 5-, 12- and 16-nitroxide stearic acid spin probes. Order parameters which measure the fluidity of the lipid environment of the incorporated probe may be calculated from the electron spin resonance (ESR) spectra of 5-nitroxide stearate [I(12,3)]-labeled cells. Evidence is presented which indicates that these spectra principally reflect properties of the platelet surface membrane lipids. The membrane fluidity increased with temperature for the range 17 to 37 °C. Either calcium or lanthanum additions to intact cells increased the rigidity of the platelet membranes at 37 °C, although the La³⁺ effect was larger and occurred at lower concentrations than that of Ca²⁺. For example, addition of 1mm La³⁺ or 4mm Ca²⁺ increased the order parameter of I(12,3)-labeled platelets by 4.3±1.7% or 2.1±0.5%. Preliminary studies conducted on purified platelet plasma membranes labeled with I(12,3) indicated that 1mm LaCl₃ or 4mm CaCl₂ additions similarly decreased the lipid fluidity at 37 °C. The above cation-induced effects on the fluidity of whole platelets were reversed by the use of the divalent cation-chelating agent ethylene glycol-bis-(-aminoethyl ether)-N,N-tetra-acetic acid (EGTA). Lastly, lanthanum (0.2–1mm) caused rapid aggregation of platelets which were suspended in a 50-mm Tris buffer pH 7.4 that did not contain adenosine.     

<Emphasis Type="SmallCaps">l</Emphasis>-carnitine modulates blood platelet oxidative stress

The oxidative stress induced by acute exertion may interfere with blood platelet activation. The beneficial effect of l-carnitine (γ-trimethylamino-β-hydroxybutyric acid) on oxidative stress in blood platelets has not been fully investigated; however, different studies indicate that this compound modulates platelet functions. The aim of our study was to assess the effects of l-carnitine on platelet activation and oxidative/nitrative protein damage (determined by the levels of protein carbonyl groups, thiol groups, and 3-nitrotyrosine residues) in resting blood platelets or platelets treated with peroxynitrite (ONOO⁻, a strong physiological oxidant) in vitro. We also investigated the effects of l-carnitine on the level of platelet glutathione and on the formation of superoxide anion radicals ( O₂ ^{- ·} ) \left( {{\hbox{O}}_2^{ - \bullet }} \right) , lipid peroxidation measured by thiobarbituric acid reactive substances (TBARS) in blood platelets stimulated by thrombin (a strong physiological agonist), and platelet aggregation induced by adenosine diphosphate (a strong physiological stimulator). We have observed that carnitine decreases platelet activation (measured by platelet aggregation, the generation of O₂ ^{- ·} {\hbox{O}}_2^{ - \bullet } , and TBARS production). Moreover, our results in vitro demonstrate that carnitine may protect against oxidation of thiol groups induced by ONOO⁻. Thus, carnitine may have some protectory effects against oxidative changes induced in blood platelets.     

Blood platelets of the cheetah (Acinonyx jubatus)

The blood platelet count and platelet morphology of 22 adult cheetahs was investigated. The platelet counts of the animals displayed a normal distribution, with a mean count of 344×10⁹/l and a mean platelet volume of 11 fl. Morphological and ultrastructural features of the cheetah platelets revealed the typical platelet morphology of anuclear cells, with granules scattered throughout the cytoplasm. The characteristic surface canalicular system and microtubules were present. True cross‐sections of the platelets had a mean area of 2.146 µm², circumference of 6.805 µm, and mean minimum and maximum projections of 1.000 µm and 2.933 µm, respectively. Zoo Biol 23:263–271, 2004. © 2004 Wiley‐Liss, Inc.     

The fine structure of muscle in a marine turbellarian

Summary The fine structure of the myofibers of Notoplana acticola as studied by electron microscopy indicates that they are composed of thick myofilaments about 200 Å wide with tapering ends and thin myofilaments about 50 Å wide, arranged alongside each other parallel to the long axis of the cell. There is no orderly transverse arrangement of filaments; instead they appear staggered in the fiber. In cross sections 6 to 10 thin filaments form an orbit around one thick filament with possible cross-linkage between the two types of filaments.Dense bodies are associated with the sarcolemma and with the sarcoplasmic reticulum, and appear to serve as attachments for the thin filaments. Dense bodies are compared to elements forming a fragmented Z-disc.Mitochondria, situated in the periphery or the center of fibers, are associated with granules interpreted as glycogen.The sarcoplasmic reticulum consists of: sacs or cisternae in close proximity to the sarcolemma, longitudinal tubular elements between and parallel to the myofilaments, and a tubular network around the filaments. There is no well-defined sarcolemmal-derived transverse tubule system as described in striated muscles. It is hypothesized that in these muscles, the functional equivalent of the T system may be the area of sarcolemma in contact with the cisternae of the sarcoplasmic reticulum.This work was supported by Grant No. GM 10292 from the U. S. Public Health Service to Professor Richard M. Eakin, Department of Zoology at the University of California, Berkeley, USA, where this investigation was conducted during the author's sabbatical leave of absence from the University of Illinois.I wish to thank Professor Eakin for valuable discussions and for his kind hospitality in extending the facilities of his laboratory and the use of the electron microscope to me, and the John Simon Guggenheim Memorial Foundation for the Fellowship which I held during 1964–65.     

Über bandartige Strukturen in den Chromozentren vonLupinus polyphyllus

In the nuclei ofLupinus polyphyllus strongly marked chromocentres occur. In electron micrographs of anther cell nuclei these chromocentres appear either as a homogeneous network or subdivided into two distinct regions, a network-like (NR) and a banded one (GR). In both a 100 Å fiber is the basic unit. The GR is composed of 4 to 6 parallely arranged electron dense bands (240–280 Å wide) and interbands (260–300 Å wide) of low electron density. They appear to correspond to a cylindrical structure with disc-like components and connective sections. These observations are discussed in relation to chromosome structure during interphase and mitosis.

Herrn Prof. Dr. L.Geitler zur Vollendung seines 80. Lebensjahres gewidmet.     

Motor-driven marginal band coiling promotes cell shape change during platelet activation

Platelets float in the blood as discoid particles. Their shape is maintained by microtubules organized in a ring structure, the so-called marginal band (MB), in the periphery of resting platelets. Platelets are activated after vessel injury and undergo a major shape change known as disc to sphere transition. It has been suggested that actomyosin tension induces the contraction of the MB to a smaller ring. In this paper, we show that antagonistic microtubule motors keep the MB in its resting state. During platelet activation, dynein slides microtubules apart, leading to MB extension rather than contraction. The MB then starts to coil, thereby inducing the spherical shape of activating platelets. Newly polymerizing microtubules within the coiled MB will then take a new path to form the smaller microtubule ring, in concerted action with actomyosin tension. These results present a new view of the platelet activation mechanism and reveal principal mechanistic features underlying cellular shape changes.     

Über schlauchförmige Ausstülpungen an der Zellmembran der Makrogametocyten von Eimeria perforans

Summary The surface of the gametocytes and gametes of Eimeria perforans reveals tube-like extrusions which have not been discovered so far in coccidia. These slender tubes are 650 Å in width and at least 1,3 in length. Probably they represent resorbing organelles. The tubes occur only at the surface of older female gametocytes and gametes and have not been observed in merozoites, schizonts, microgametocytes and male gametes. In transversal sections the tubes look like vesicles and are bordered by a membrane the layers of which are not sharply defined. The interior of the tubes seems to be empty after electron microscope observations. In longitudinal sections the membrane of the tubes is striated. The repeating unit of the dark and light bands amounts to about 165 Å. This appearance cannot be explained so far.

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Für beratende Hilfe sind wir Herrn Prof. Dr. R. Danneel und Herrn Prof. Dr. K. E. Wohlfarth-Bottermann zu Dank verpflichtet, für technische Unterstützung Fräulein cand. rer. nat. B. Volkmann. Herrn Dr. D. Spiecker von der Forschungsstelle für Jagdkunde und Wildschadenverhütung, Beuel, danken wir für die Durchführung der Infektionen. Die Mittel für die Untersuchungen stellte uns die Deutsche Forschungsgemeinschaft zur Verfügung.     

Effects of tensile stress on the α1 nicotinic acetylcholine receptor expression in maxillofacial skeletal myocytes

The present study was to test the hypothesis that 11,12-epoxyeicosatrienoic acid (11,12-EET), a metabolic product of arachidonic acid by cytochrome P450 epoxygenase, regulates nitric oxide (NO) generation of the l-arginine/NO synthase (NOS) pathway in human platelets. Human platelets were incubated in the presence or absence of different concentrations of 11,12-EET for 2 h at 37°C, followed by measurements of activities of the l-arginine/NOS pathway. Incubation with 11,12-EET increased the platelet NOS activity, nitrite production, cGMP content, and the platelet uptake of l-[³H]arginine in a concentration-dependent manner. In addition, 11,12-EET attenuated intracellular free Ca²⁺ accumulation stimulated by collagen, which was at least partly mediated by EET-activated l-arginine/NOS pathway. It is suggested that 11,12-EET regulates platelet function through up-regulating the activity of the l-arginine/NOS/NO pathway.     

Geitler's “Plattenband” in the diatomSynedra cf.ulna in the light of TEM investigations

The ultrastructure of the diatomSynedra cf.ulna was examined paying special attention to the Plattenband (platelet band). This structure was first described byGeitler in 1948 on the basis of LM observations and denotes a linear array of dictyosomes along the apical axis of the cell. The present investigation confirmsGeitler's observations in all essential details and demonstrates that the dictyosomes are arranged along polarized nuclear extensions running towards the cell poles. Laterally the extensions are accompanied by a number of microtubules. In large cells the total length of the nucleus thus may reach 400 µm and more. Since only the central part of the nucleus is DNA-positive with DAPI and acridine orange, the nuclear nature of the backbone of the Plattenband cannot be recognized by LM techniques. TEM investigation of serial apical and transapical sections, however, prove unambiguously the identity with extended parts of the nucleus.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.     

A lineage-restricted and divergent beta-tubulin isoform is essential for the biogenesis, structure and function of blood platelets

BACKGROUND: Mammalian megakaryocytes release blood platelets through a remarkable process of cytoplasmic fragmentation and de novo assembly of a marginal microtubule band. Cell-specific components of this process include the divergent beta-tubulin isoform beta1 that is expressed exclusively, and is the predominant isoform, in platelets and megakaryocytes. The functional significance of this restricted expression, and indeed of the surprisingly large repertoire of metazoan tubulin genes, is unclear. Fungal tubulin isoforms appear to be functionally redundant, and all mammalian beta-tubulins can assemble in a variety of microtubules, whereas selected fly and worm beta-tubulins are essential in spermatogenesis and neurogenesis. To address the essential role of beta1-tubulin in its natural context, we generated mice with targeted gene disruption. RESULTS: beta1-tubulin(-/-) mice have thrombocytopenia resulting from a defect in generating proplatelets, the immediate precursors of blood platelets. Circulating platelets lack the characteristic discoid shape and have defective marginal bands with reduced microtubule coilings. beta1-tubulin(-/-) mice also have a prolonged bleeding time, and their platelets show an attenuated response to thrombin. Two alternative tubulin isoforms, beta2 and beta5, are overexpressed, and the total beta-tubulin content of beta1-tubulin(-/-) megakaryocytes is normal. However, these isoforms assemble much less efficiently into platelet microtubules and are thus unable to compensate completely for the absence of beta1-tubulin. CONCLUSIONS: This is the first genetic study to address the essential functions of a mammalian tubulin isoform in vivo. The results establish a specialized role for beta1-tubulin in platelet synthesis, structure, and function.     

A neurofibrillar body in the perikaryon of nervus terminalis ganglion cells in teleosts

Summary In three species of Teleosts (Tinea tinea L., Leuciscus cephalus cabeda Risso, Epinephelus guaza L.) a round strongly argentophilic body of considerable size occurs in the cytoplasm of the nervus terminalis ganglion cells. In Tinea, surgical interruption of functional connections of the ganglion cells does not produce any apparent change either in the number or in the size of these cytoplasmic bodies.Electron microscopical observations show that the neurofibrillar body is made up of densely packed and irregularly arranged bundles. In cross section, each of these bundles appears to be composed of neurofilaments (100 Å in thickness) and neurotubules (diameter: 300 Å). Each tubule is surrounded by 9–10 filaments equi-distant from one another, and at a distance of 30–40 Å from the central tubule.The authors are indebted to Prof. G. Palladini for helpful histochemical advice, to Prof. B. Bertolini for electron micrographs and to Mr. D. Scorsini for skilful technical assistance.     

Megakaryocyte fragments and the microtubule coil

We have examined megakaryocyte process fragments that migrate out of bone marrow explants after a short period of incubation and assume a beaded form, consisting of 2 or more putative platelets. The fragmentation appears to occur in vivo and supports the proposal that platelet liberation does not always occur in a sequential manner from the distal ends of megakaryocyte processes. Transmission electron microscopy revealed that microtubules were generally oriented longitudinally in the process fragments. Rarely, a microtubule coil was found in a terminally located putative platelet. The observations favour the view that the marginal coil of microtubules, which is a characteristic of circulating platelets, does not usually form until after platelets have been liberated.     

Observations on the ultrastructure of nucleated erythrocytes and thrombocytes,with particular reference to the structural basis of their discoidal shape

Summary The marginal band of nucleated erythrocytes in the toadfish is found, in electron micrographs, to be composed of about twenty-five microtubules approximately 200 Å in diameter. These form a bundle that encircles the erythrocyte just beneath the plasma membrane. These observations support the interpretation of Meves 1904, that this relatively stiff equatorial band may contribute to the maintenance of the discoid shape of nucleated erythrocytes in fish, amphibians, reptiles and birds.Similar microtubules form an annular bundle encircling the nucleus in fish thrombocytes. The number of tubular elements involved here is in excess of one hundred and they are located deep to the ectoplasmic layer instead of immediately beneath the plasmalemma. The term endoplasmic ring is therefore proposed for this structure.Comparative observations on nucleated erythrocytes of various species are presented showing that the density and fine structure of the material occupying the interchromosomal areas of the nucleus, always matches the cytoplasm and is related to the hemoglobin concentration of the species. These ultrastructural observations are consistent with the optical absorption and biochemical findings of other investigators indicating the presence of intranuclear hemoglobin in nucleated erythrocytes. Crystalline order is occasionally found in electron micrographs of the hemoglobin rich areas of the nucleus in toadfish erythrocytes but is not found in the cytoplasm.This research was supported by grant G-12916 of the National Science Foundation.