The cytoskeleton of isolated murine primitive erythrocytes

Summary Cytoskeletons of primitive erythrocytes have been isolated from the embryos of day 12 pregnant C57/Bl mice and examined by transmission electron microscopy, immunofluorescence microscopy, and SDS-polyacrylamide gel electrophoresis. Microtubules are the most prominent cytoskeletal component. They are found either singly or organized into loose bundles just under the plasma membrane, but do not form classical marginal bands in most cells. Immunofluorescence with a polyclonal tubulin antiserum confirms this distribution and further reveals numerous mitotic figures among the cells. Rhodamine-conjugated phalloidin and heavy meromyosin labeling reveal that actin is localized in the cortex of the primitive erythrocyte in the form of 6 nm filaments. Antibody directed against avian erythrocyte alpha spectrin demonstrates that spectrin is also found in the cortex. Occasional 10-nm intermediate filaments, observed in the primitve erythrocytes by electron microscopy, are believed to be of the vimentin class based on positive reaction of the cells with vimentin-specific antiserum. In addition, a band in erythrocyte cytoskeletons comigrates in SDS-polyacrylamide gels with vimentin isolated from mouse kidney. Spectrin and actin were also found to be associated with the membrane of primitive erythrocytes when membrane ghost preparations were analyzed by SDS-polyacrylamide gel electrophoresis.     

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.     

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.     

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.     

Density-gradient enrichment of newly-formed mouse erythrocytes: Application to the micronucleus test

To facilitate scoring micronuclei in peripheral blood erythrocytes, we have developed a centrifugation method to concentrate polychromatic and newly-formed normochromatic erythrocytes from microliter quantities of blood in a Percoll density gradient. Erythrocytes were separated into two discrete bands in a continuous gradient generated in situ in a microhematocrit capillary tube. The upper band contained white blood cells and a mixture of polychromatic and young normochromatic erythrocytes with a density of 1.080–1.082 g/ml. More than 75% of the polychromatic erythrocytes in samples of normal blood were recovered in the upper band. Older normochromatic erythrocytes migrated to the lower band. The frequency of polychromatic erythrocytes was increased from approximately 2% in whole blood to 60–80% in the upper band. After clastogen treatments, the elevated frequencies of micronuclei in the upper band polychromatic erythrocytes were similar to those in unfractionated blood. The frequencies of micronucleated normochromatic erythrocytes in the upper band were higher than those in whole blood at 48, 72 and 96 h after clastogen treatment, consistent with the expectation that the low-density normochromatic cells are newly derived from polychromatic erythrocytes. This density-gradient centrifugation technique enhances the efficiency of scoring micronuclei in the acute peripheral blood micronucleus test.     

Density-gradient enrichment of newly-formed mouse erythrocytes. Application to the micronucleus test

To facilitate scoring micronuclei in peripheral blood erythrocytes, we have developed a centrifugation method to concentrate polychromatic and newly-formed normochromatic erythrocytes from microliter quantities of blood in a Percoll density gradient. Erythrocytes were separated into two discrete bands in a continuous gradient generated in situ in a microhematocrit capillary tube. The upper band contained white blood cells and a mixture of polychromatic and young normochromatic erythrocytes with a density of 1.080-1.082 g/ml. More than 75% of the polychromatic erythrocytes in samples of normal blood were recovered in the upper band. Older normochromatic erythrocytes migrated to the lower band. The frequency of polychromatic erythrocytes was increased from approximately 2% in whole blood to 60-80% in the upper band. After clastogen treatments, the elevated frequencies of micronuclei in the upper band polychromatic erythrocytes were similar to those in unfractionated blood. The frequencies of micronucleated normochromatic erythrocytes in the upper band were higher than those in whole blood at 48, 72 and 96 h after clastogen treatment, consistent with the expectation that the low-density normochromatic cells are newly derived from polychromatic erythrocytes. This density-gradient centrifugation technique enhances the efficiency of scoring micronuclei in the acute peripheral blood micronucleus test.     

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 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.     

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.     

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.     

Acute ischemia causes 'dark cell' change of strial marginal cells in gerbil cochlea

The cochlear stria vascularis produces the endolymph and generates the endocochlear DC potential, two indispensable ingredients of an auditory transduction process. The marginal cell, one of the several cell types constituting the stria vascularis, is called 'the dark cell' on the basis of its appearance by transmission electron microscopy (TEM). To clarify whether this commonly observed 'dark appearance' is a normal characteristic of marginal cells, as conjectured in the literature, or an experimental artifact, we developed an in vivo fixation method for minimizing ischemic tissue damages. While under sustained systemic circulation with oxygenated blood, the stria vascularis of gerbils was chemically fixed by perilymphatic perfusion with a fixative, and the stria vascularis was observed by TEM. In contrast to a number of previous reports, the cytoplasm of marginal cells was not dark, and quantitative analysis showed that the difference between the cytoplasmic electron density of marginal cells and that of intermediate cells (another type of strial cells) was not statistically significant. For comparison, the gerbils were allowed to undergo 3 min of ischemia following decapitation. Under these conditions, marginal cells showed typical 'dark appearance', as reported previously, and their cytoplasmic electron density was 1.7 times higher than that of the intermediate cells. In addition, the volume of mitochondria in marginal cells undergoing 3 min of ischemia was higher than that fixed in vivo. We therefore conclude that the widely recognized 'dark cell' appearance of marginal cells following conventional fixation procedures reflects cell injury due to ischemia, which is inherent in the standard fixation procedures, but can be avoided by our fixation protocol here introduced.     

Osmotic and diffusive properties of intracellular water in camel erythrocytes: effect of hemoglobin crowdedness

Camel erythrocytes have exceptional osmotic resistance and is believed to be due to augmented water-binding associated with the high hydrophilicity of camel hemoglobin. In practical terms this means that the proportion of osmotically non-removable water in camel erythrocytes is nearly 3-fold greater than that in human erythrocytes (approximately 65 vs approximately 20%). The relationship between water diffusion and the osmotic characteristics of intracellular water is the subject of this report. The amount of osmotically inactive water is 2-fold greater in camel hemoglobin solution in vitro compared to that of human, but water diffusion does not differ in camel and human hemoglobin solutions. However, the evaluation of water diffusion by magnetic resonance measurements in camel erythrocytes revealed approximately 15% lower apparent diffusion coefficient (ADC) compared with human erythrocytes. When human erythrocytes were dehydrated to the level of camel erythrocytes, their osmotic and water diffusion properties were similar. These results show that a lower ADC is associated with a more pronounced increase in osmotically inactive water fraction. It is proposed that increased hemoglobin hydrophilicity allows not only augmented water-binding, but also a closer hemoglobin packaging in vivo, which in turn is associated with slower ADC and increased osmotic resistance.     

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.     

Cell separation in the buffy coat

One of the most rapid methods to determine cell counts in whole blood is by way of layer thickness measurements of the buffy coat. The purpose of this study was to determine the separation and purity of blood cells in the different layers of the buffy coat. Blood samples were centrifuged at 10,000 g in microhematocrit tubes with an inserted float to expand the buffy coat region. Whole blood from normal laboratory individuals separates by density into four regions: platelets, a layer of lymphocyte and monocytes, granulocytes and erythrocytes. A thin band of highly swollen red cells was discovered between the buffy coat layers of many normal volunteers and patients. Stereological analysis of electron micrographs showed that mixing of formed elements within the layers is less than 2% with the exception of some erythrocytes, which can make up a higher volume fraction in the lymphocyte/monocyte and granulocyte layers. The red cell column contains about 95.7% erythrocytes and is depleted of platelets and leukocytes. In approximately 5% of hospital blood samples, the granulocyte-erythrocyte interface was feathered and undetectable, and a significantly higher volume fraction of red cells was found among the granulocytes. Cell mass density determinations indicate that the erythrocytes in these abnormal granulocyte layers have a lowered mass density, overlapping with that of the granulocytes.     

Cellular morphogenesis and the formation of marginal bands in amphibian splenic erythroblasts

The spleen of Ambystoma mexicanum (axolotl) larvae develops as a closed sac containing differentiating nucleated erythrocytes, and is typically isolated from the general circulation for about 10 days post-hatching. Beginning 3-4 days posthatching, it can be removed intact for examination of the morphology and cytoskeletal structure of the erythropoietic cells. In the smallest (earliest) spleens, spheroidal cells predominate, while older ones contain a preponderance of cells exhibiting the flattened elliptical morphology typical of all non-mammalian vertebrate erythrocytes. Most striking in the splenic erythroid population are cells with singly or doubly pointed morphology. Though common in the developing spleen and circulation of young larvae, pointed cells are less frequently encountered in the circulation of older larvae, indicating that they are intermediate stages in the differentiation of spheroids to flattened ellipsoids. This is supported by structural observations on cytoskeletons prepared from the splenic cells. Incomplete singly and doubly pointed marginal bands of microtubules are observed, many of which contain a pair of centrioles within or close to a pointed end, suggestive of organizing center function. The observations are consistent with a sequence of changes in cell morphology from spherical to doubly pointed to singly pointed to flattened ellipse, causally linked to stages of marginal band biogenesis.     

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.     

Evidence for a correlation between the number of marginal band microtubules and the size of vertebrate erthrocytes.

In 23 species of vertebrates the dimensions of erythrocytes and the number of their marginal band microtubules were examined. A positive correlation was found between the size of erythrocytes and the number of microtubules. The absence of microtubules in diskoid erythrocytes of mammals-Camelidae-is discussed.