Avian erythrocyte development: microtubules and the formation of the disk shape

The development of avian erythrocytes involves a spheroid to discoid transformation in shape. The disk shape of the young erythroid cells is dependent on the presence of microtubules in a marginal bundle in the early stages of postmitotic maturation. Disassembly of microtubules with colchicine, vincristine, sulfate or cold temperature produces the spheroidal shape. Erythrocytes which have acquired the flattened ellipsoidal shape do not alter their shape with disassembly of the microtubules. The number of microtubules decreases as cell maturation occurs. The correlation coefficient for the number of microtubular profiles in one end of erythrocytes and the concentration of ribosomes (cell age) is 0.88. Microtubules of immature erythrocytes disappear more rapidly at 0°C than do microtubules of mature cells.It is concluded that microtubules play little or no role in the maintenance of mature red cell shape; however, they play an important role in the development of the flat discoid shape of avian erythrocytes during maturation.     

Occurrence of microtubules during erythropoiesis in Llama, Lama glama

Studies on the ellipsoid erythroblasts of Llama, L a m gluma, L. during erythropoiesis showed the appearance of the marginal band composed of approximately 29 microtubules. As the maturation of erythrocytes goes on, the number of marginal band microtubules diminishes. No microtubules were found in mature erythrocytes of Llama.     

Development of a differentiated microtubule structure: formation of the chicken erythrocyte marginal band in vivo

The microtubules of mature nucleated erythrocytes are organized into a marginal band that is confined to a single plane at the periphery and that contains essentially the same number of microtubule profiles in each individual cell. Developing erythrocytes can be isolated in homogeneous and synchronously developing populations from chicken embryos. For these reasons, these cells offer a particularly accessible system for study of the pathway leading to a specific microtubule structure in a normal, terminally differentiated animal cell. Along this developmental course, striking changes occur in the properties of the microtubules. Between the postmitotic cell and the formation of the band, a novel arrangement is found: bundles of laterally associated microtubules in each cell, coursing through the cytoplasm but not confined to the periphery. The microtubule organizing centers evident at early stages disappear by the time the band forms. The microtubules in early cells are readily depolymerized by drugs, but that drug sensitivity is lost in the mature cells. The microtubule arrangement of mature cells is faithfully recapitulated after reversible depolymerization, while that of the immature cells is not. Finally, as the band forms, the microtubules and microfilaments increasingly become coaligned. In sum, the microtubules of immature cells have many properties in common with those of cultured cells, but during maturation those properties change. The results suggest that lateral interactions become increasingly important in stabilizing and organizing the microtubules. The properties of marginal band microtubules, and comparable properties of axonal microtubules, may reflect differences between the requirements for cytoskeletal structures of cycling cells and terminally differentiated cells.     

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.     

Molecular organization and in vivo function of the cytoskeleton of amphibian erythrocytes

One prominent cytoskeletal feature of non-mammalian vertebrate erythrocytes is the marginal band (MB), composed of microtubules. However, there have been several reports of MB-associated F-actin. We have further investigated the function of MB-associated F-actin, using newt erythrocytes having large, thick MBs. Confocal microscopy revealed a distinctive band of F-actin colocalizing point- by-point with MB microtubules. Furthermore, the F-actin band was present in isolated elliptical MBs, but absent in membrane skeletons lacking MBs. F-actin depolymerizing agents did not affect F-actin band integrity in isolated MBs, indicating its non-dynamic state. However, exposure to elastase resulted in F-actin removal and MB circularization. These results provide evidence of a strong association of F-actin with MB microtubules in mature ellipsoidal erythrocytes. To assess the true extent of mechanical stress on the cytoskeleton, erythrocytes were observed by video microscopy during flow in vivo. Moving with long axis parallel to flow direction, cells underwent reversible shape distortion as they collided vigorously with other erythrocytes and vessel walls. In addition, cells twisted into figure-8 shapes, a cytoskeletal property that may provide physiological advantages during flow. Our results, together with those of others, yield a consistent picture in which developing erythrocytes undergo transition from spheroids to immature discoids to mature ellipsoids. The causal step in discoid formation is biogenesis of circular MBs with sufficient flexural rigidity to determine cell shape. F-actin binding to MB microtubules then creates a composite system, enhancing flexural rigidity to produce and maintain ellipsoidal shape during the physical challenges of blood flow in vivo.     

Reformation of the marginal band of avian erythrocytes in vitro using calf-brain tubulin: peripheral determinants of microtubule form

The microtubules of nucleated erythrocytes form an extraordinary structure: they are organized into a marginal band at the periphery of the cell. This unusual organelle, recurring in detail in each cell, provides an excellent opportunity to study the determinants of microtubule form. We have been able to reform the marginal band, using detergent-extracted erythrocytes that have been depleted of microtubules in vivo and phosphocellulose-purified tubulin from calf brain. We find that detergent-extracted cytoskeletons incubated under these conditions again have microtubules, and that the pattern of these microtubules recapitulates several features of the intact marginal band. In particular, most of the microtubules after regrowth are located in a band at the periphery of the cell, and curve to form an ellipse. These results support the hypothesis that the specification of microtubule location and shape in these cells is governed by determinants that reside at the periphery of the cell.     

Fine structure of camel erythrocytes in relation to its functions

The fine structure of the red cells in Arabian camels was investigated and certain characteristic features were noted. The plasmalemma of camel erythrocytes are tri-laminar, the inner and outer membranes are of high electron density between which is a zone of lesser electron density. No intracellular organelles were observed with the occasional exception of a small number of mitochondria. In the camel erythrocyte, a marginal band consisting of 30-45 microtubules was observed in many cells. Some of the possible functions of the marginal band in camel erythrocytes are discussed.     

Ultrastructural observations on the erythrocytes and thrombocytes of the tuatara, Sphenodon punctatus (gray)

The erythrocytes of Sphenodon punctatus (Gray) are nucleated, ellipsoidal and flattened, and contain 55--65 microtubules in their marginal band. The thrombocytes are also flattened, ellipsoidal, nucleated cells and in electron-microscopic preparations occurred in aggregrates. The thrombocytes appeared to be 'activated' and possessed many pseudopodia which were devoid of organelles. The latter were concentrated in the perinuclear region and were encircled by a ring of microtubules. The organelles included ribosomes, mitochondria, membrane--bound dense material and numerous actin-like microfilaments. Cytoplasmic vacuoles contained a moderately dense, filamentous material and/or spheroidal electron-dense inclusions, beta-glycogen particles were scattered in the general cytoplasm and were most concentrated in the pseudopodia. The erythrocytes and thrombocytes of S. punctatus are compared with those in other vertebrates.     

Tubulin, but not microtubules, is the substrate for tubulin:tyrosine ligase in mature avian erythrocytes

Chicken erythrocytes, which contain a marginal band of microtubules, were used to study the influence of the aggregation state of tubulin on the post-translational incorporation of tyrosine into the alpha-tubulin subunit. We found that the incorporation of [14C]tyrosine occurs almost exclusively into the nonassembled tubulin pool. The marginal band was practically not labeled. The low incorporation into microtubules was not due to the lack of tubulin with acceptor capacity since after cold-induced disassembly, an additional amount of [14C]tyrosine could be incorporated. 14C-Tyrosinated tubulin of the nonassembled pool could not be incorporated into microtubules of the marginal band after prolonged incubation at 37 degrees C or when the marginal band was regenerated after cold-induced depolymerization. In erythrocytes, tubulin:tyrosine ligase behaved as a soluble entity when the cells were lysed under microtubule-preserving conditions.     

Alterations to the cytoskeleton of erythrocytes infected with frog erythrocytic virus: a fluorescence and electron microscopic study.

Erythrocytes of bullfrogs (Rana catesbeiana) infected with frog erythrocytic virus are spheroid and their nucleus is displaced. In contrast, uninfected cells are ellipsoid and have a centralized nucleus. Fluorescent staining revealed that these changes are correlated with alterations to components of the erythrocyte cytoskeleton. Uninfected erythrocytes contained a broad, continuous marginal band of microtubules, which appeared thinner and interrupted in infected cells. The described disruption of microtubules was associated with an inability to polymerize the tubulin pool with the addition of 12 microM taxol. The arrangement of submembranous microfilaments in uninfected erythrocytes was not significantly altered in infected cells. Vimentin filaments were distributed throughout the cytoplasm and around the nucleus of uninfected cells, and concentrated at the cell and nuclear peripheries. Cytoplasmic pockets that did not contain vimentin filaments were associated with the viral assembly site(s) in infected cells. These data suggest that the distortion of viral-infected erythrocytes could be due, in part, to an irreversible depolymerization of microtubules of the marginal band and a reorganization of the vimentin filament network.     

Erythrocyte microtubule assembly in vitro. Determination of the effects of erythrocyte tau, tubulin isoforms, and tubulin oligomers on erythrocyte tubulin assembly, and comparison with brain microtubule assembly

Two tubulin variants, isolated from chicken brain and erythrocytes and known to have different peptide maps and electrophoretic properties, are demonstrated to exhibit different assembly properties in vitro: 1) erythrocyte tubulin assembles with greater efficiency (lower critical concentration, greater elongation rate) but exhibits a lower nucleation rate than brain tubulin, and 2) erythrocyte tubulin readily forms oligomers whose presence significantly retards the rate of elongation, suggesting that tubulin oligomers may also be important for determining the rate of assembly and the length of microtubules in erythrocytes. Erythrocyte tubulin isolated by cycles of in vitro assembly-disassembly is also demonstrated to contain a 67-kDa tau factor that greatly enhances microtubule nucleation but has little effect on elongation rates or critical concentration. Immunofluorescence microscopy with tau antibody indicates that tau is specifically associated with marginal band microtubules, suggesting that it may be important for determining microtubule function in vivo.     

Fine structure of developing hagfish erythrocytes with particular reference to the cytoplasmic organelles

Cytological changes accompanying the maturation of erythrocytes in the “Pacific hagfish” (Eptatretus stoutii) were studied. Great numbers of immature and mitotically dividing red blood cells in the peripheral circulation of the hagfish appear to indicate that extensive differentiation and proliferation occurs in the blood stream of this animal. The immature erythrocytes contained mitochondria, Golgi membranes, centrioles, microtubules and a high density of ribosomes in the cytoplasm. Intermediate stages revealed lysosomes in the cytoplasm. With progressive differentiation the hagfish erythrocytes accumulate hemoglobin and lose most of their cytoplasmic organelles. The various cytoplasmic organelles are apparently lost through a degradation process brought about by lysosomal autolysis. The undigested products of degradation such as mitochondrial and other intercellular membranes are apparently extruded by way of the plasma membrane. The plasma membrane of young as well as mature erythrocytes display evidence of intense pinocytotic activity. The nucleolus undergoes a reduction in size with progressive maturation. The cytoplasm of mature erythrocytes consists predominantly of hemoglobin. An equatorial microtubular marginal band is identifiable in differentiating erythrocytes.     

Altering microtubule stability affects microtubule clearance and nuclear extrusion during erythropoiesis

Mammalian erythrocytes are highly specialized cells that have adapted to lose their nuclei and cellular components during maturation to ensure oxygen delivery. Nuclear extrusion, the most critical event during erythropoiesis, represents an extreme case of asymmetric partitioning that requires a dramatic reorganization of the cytoskeleton. However, the precise role of the microtubule cytoskeleton in the enucleation process remains controversial. In this study, we show that microtubule reorganization is critical for microtubule clearance and nuclear extrusion during erythropoiesis. Using a rodent anemia model, we found that microtubules were present in erythroblasts and reticulocytes but were undetectable in erythrocytes. Further analysis demonstrated that microtubules became disordered in reticulocytes and revealed that microtubule stabilization was critical for tubulin degradation. Disruption of microtubule dynamics using the microtubule-stabilizing agent paclitaxel or the microtubule-destabilizing agent nocodazole did not affect the efficiency of erythroblast enucleation. However, paclitaxel treatment resulted in the retention of tubulin in mature erythrocytes, and nocodazole treatment led to a defect in pyrenocyte morphology. Taken together, our data reveals a critical role for microtubules in erythrocyte development. Our findings also implicate the disruption of microtubule dynamics in the pathogenesis of anemia-associated diseases, providing new insight into the pathogenesis of the microtubule-targeted agent-associated anemia frequently observed during cancer chemotherapy.     

The cytoskeletal system of mammalian primitive erythrocytes: studies in developing marsupials

Seeking to resolve conflicting literature on cytoskeletal structure in mammalian "primitive" generation erythrocytes, we have utilized the circulating blood of developing marsupials. In young of the Tammar Wallaby (Macropus eugenii) and the Gray Short-tailed Opossum (Monodelphis domestica), relatively large, nucleated primitive erythrocytes constituted nearly 100% of the circulating population at birth (= day 0) and in fetuses (Tammar) several days before birth. These cells were discoidal or elliptical, and flattened except for a nuclear bulge. Their cytoskeletal system, consisting of a marginal band of microtubules enclosed within a cell surface-associated network (membrane skeleton), closely resembled that of non-mammalian vertebrate erythrocytes. By day 2 or 3, much smaller anucleate erythrocytes of "definitive" morphology, lacking marginal bands, appeared in abundance. These accounted for greater than 90% of the circulating population of both species by day 6-8. Non-nucleated erythrocytes of a different type, constituting 1-6% of the cells in most blood samples up to day 7, were identified as anucleate primitives on the basis of size, shape, and presence of a marginal band. Thus, loss of erythrocyte nuclei in mammals appears to begin earlier than generally recognized, i.e., in the primitive generation. Counts of these anucleate primitives in young of various ages implicated nucleated primitives as their probable source. Pointed erythrocytes, occasionally found in younger neonates of both species, occurred in greatest number in fetuses (Tammar) prior to birth. This is in accord with previous work on non-mammalian vertebrates suggesting that such cells are morphogenetic intermediates. The results confirm the long-suspected similarity between mammalian primitive erythrocytes and the nucleated erythrocytes of all non-mammalian vertebrates.     

Dynamics of microtubules from erythrocyte marginal bands.

Microtubules can adjust their length by the mechanism of dynamic instability, that is by switching between phases of growth and shrinkage. Thus far this phenomenon has been studied with microtubules that contain several components, that is, a mixture of tubulin isoforms, with or without a mixture of microtubule-associated proteins (MAPs), which can act as regulators of dynamic instability. Here we concentrate on the influence of the tubulin component. We have studied MAP-free microtubules from the marginal band of avian erythrocytes and compared them with mammalian brain microtubules. The erythrocyte system was selected because it represents a naturally stable aggregate of microtubules; second, the tubulin is largely homogeneous, in contrast to brain tubulin. Qualitatively, erythrocyte microtubules show similar features as brain microtubules, but they were found to be much less dynamic. The critical concentration of elongation, and the rates of association and dissociation of tubulin are all lower than with brain microtubules. Catastrophes are rare, rescues frequent, and shrinkage slow. This means that dynamic instability can be controlled by the tubulin isotype, independently of MAPs. Moreover, the extent of dynamic behavior is highly dependent on buffer conditions. In particular, dynamic instability is strongly enhanced in phosphate buffer, both for erythrocyte marginal band and brain microtubules. The lower stability in phosphate buffer argues against the hypothesis that a cap of tubulin.GDP.Pi subunits stabilizes microtubules. The difference in dynamics between tubulin isotypes and between the two ends of microtubules is preserved in the different buffer systems.     

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.     

Bundling of microtubules by glyceraldehyde-3-phosphate dehydrogenase and its modulation by ATP

Glyceraldehyde-3-phosphate dehydrogenase from different origins (brain, muscle, erythrocytes) binds to microtubules polymerized from pure brain tubulin and causes bundle formation in vitro. ATP is shown to dissociate these bundles into individual microtubules, while the dehydrogenase is not displaced from the polymers by this nucleotide. ATP can be replaced by adenosine 5'-(beta, gamma-imido]triphosphate, a nonhydrolyzable analog of ATP. These data are interpreted in terms of dissociation of the glyceraldehyde-3-phosphate dehydrogenase tetramer into dimers by ATP. The enzyme is also efficiently purified by a tubulin-Sepharose affinity chromatography.     

Effects of brain microtubule-associated proteins on microtubule dynamics and the nucleating activity of centrosomes

In this paper, we report on the effect of brain microtubule-associated proteins (MAPs) on the dynamic instability of microtubules as well as on the nucleation activity of purified centrosomes. Under our experimental conditions, tau and MAP2 have similar effects on microtubule nucleation and dynamic instability. Tau increases the apparent elongation rate of microtubules in proportion to its molar ratio to tubulin, and we present evidence indicating that this is due to a reduction of microtubule instability rather than to an increase of the on rate of tubulin subunits at the end of growing microtubules. Increasing the molar ratio of tau over tubulin leads also to an increase in the average number of microtubules nucleated per centrosome. This number remains constant with time. This suggests that the number of centrosome-nucleated microtubules at steady state can be determined by factors that are not necessarily irreversibly bound to centrosomes but, rather, affect the dynamic properties of microtubules.     

Association of centrioles with the marginal band of a molluscan erythrocyte

Continuous circumferential bundles of microtubules, or marginal bands (MBs), are best known as a prominent structural feature of all nonmammalian vertebrate erythrocytes and mammalian blood platelets. Since their discovery in the late 19th century, MBs have been thought to play a cellular morphogenetic role, but no cytological clues to the mechanism of MB biogenesis have been reported. In previous work we have established the presence of MBs in serveral invertebrate blood cell types, including amebocytes and coelomocytes of certain Arthropod species and erythrocytes of a Sipunculan. We report here the occurrence of MBs in erythrocytes of the ark Anadara transversa (Mollusca) and four closely related species. The MBs of these arks have a striking structural feature; each is physically associated with a pair of centrioles. The centrioles are identified as such on the basis of morphological criteria: size, cylindrical shape, right-angle orientation, pairing, and 9-triplet ultrastructure. This intimate association between centrioles and MBs suggests that centrioles may be MB-organizing centers and invites comparative investigation of their possible role in vertebrate erythrocyte and platelet morphogenesis.