Marginal Band Of Microtubules Research Articles

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.

In vivo coassembly of a divergent beta-tubulin subunit (c beta 6) into microtubules of different function.

alpha- and beta-Tubulin are encoded in vertebrate genomes by a family of approximately 6-7 functional genes whose polypeptide products differ in amino acid sequence. In the chicken, one beta-tubulin isotype (c beta 6) has previously been found to be expressed only in thrombocytes and erythroid cells, where it is assembled into a circumferential ring of marginal band microtubules. In light of its unique in vivo utilization and its divergent assembly properties in vitro, we used DNA transfection to test whether this isotype could be assembled in vivo into microtubules of divergent functions. Using an antibody specific to c beta 6, we have found that upon transfection this polypeptide is freely coassembled into an extensive array of interphase cytoplasmic microtubules and into astral and pole-to-chromosome or pole-to-pole microtubules during mitosis. Further, examination of developing chicken erythrocytes reveals that both beta-tubulins that are expressed in these cells (c beta 6 and c beta 3) are found as co-polymers of the two isoforms. These results, in conjunction with efforts that have localized various other beta-tubulin isotypes, demonstrate that to the resolution limit afforded by light microscopy in vivo microtubules in vertebrates are random copolymers of available isotypes. Although these findings are consistent with functional interchangeability of beta-tubulin isotypes, we have also found that in vivo microtubules enriched in c beta 3 polypeptides are more sensitive to cold depolymerization than those enriched in c beta 6. This differential quantitative utilization of the two endogenous isotypes documents that some in vivo functional differences between isotypes do exist.

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.

Xenopus marginal band disassembly by calcium-activated cytoplasmic factors.

The marginal band microtubules of isolated Xenopus erythrocyte cytoskeletons possess the stability properties of non-steady-state microtubules. They are unperturbed by low temperatures, a variety of microtubule inhibitors, hypotonic treatment and the direct action of calcium. These microtubules can be rapidly depolymerized by erythrocyte lysis in the presence of calcium or by exposure of cytoskeletons obtained and washed in calcium-free media to calcium-containing supernatants of other cell lysates. Thus, marginal band microtubules are calcium-sensitive only in the presence of cytoplasm. The calcium-activated disassembly of the marginal band does not appear to be the result of general or tubulin-specific proteolysis and is prevented by the calmodulin inhibitor, trifluoperazine. On sodium dodecyl sulphate/polyacrylamide gels, samples of calcium-induced, marginal band disassembled cytoskeletons are always tubulin-depleted and also possess a new high molecular weight polypeptide doublet that is believed to constitute stable partial degradation products of spectrin. In the presence of calcium, addition of calmodulin and ATP to cytoskeletons washed free of cytoplasm does not initiate marginal band disassembly. Therefore, if calmodulin mediates marginal band disassembly, it requires cytoplasmic binding proteins or cytoplasmic cofactors.

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.

Studies on the cytoskeletal and nuclear architecture of Xenopus erythrocytes.

A proteinaceous cytoskeletal network is present in nucleated erythrocytes, which is obscured ultrastructurally in whole cells due to the presence of haemoglobin. Lysis of Xenopus erythrocytes in solutions containing Triton X-100 reveals a cytoskeleton that contains a centrally positioned nucleus, which is linked to the cell surface-associated cytoskeleton by intermediate filaments. The marginal band microtubules are also preserved in these structures. In addition, a single or a pair of perinuclear centrioles is frequently observed in thin sections. These structures are surrounded by a mass of intermediate filaments and fibrogranular material. In contrast to the centrioles in invertebrate erythrocytes those in Xenopus erythrocytes are not associated with the marginal band. Cytonuclear skeletons were obtained by DNase I digestion and subsequent high-salt extraction of cytoskeletons. The resulting structures were chromatin-depleted and consisted of a nuclear lamina that was maintained in the same overall shape and position as that of intact nuclei. With the exception of the marginal band, the remaining cytoskeletal elements persisted after these treatments. Although marginal bands were not detectable by electron microscopy, the cytonuclear skeletons contained roughly the same amount of tubulin as cytoskeletons, as indicated by immunoblotting with affinity-purified anti-tubulin antibodies. When intact erythrocytes were exposed to the ionophore A23187 in the presence of calcium, the cell shape and centric nuclear position were altered. Nuclear dislodgement may be attributable to the disruption of intermediate filament associations with the subsurface cytoskeletal shell. Indirect immunofluorescent staining of cytoskeletons lysed in buffers containing either EGTA or calcium indicates that in the absence of calcium, the intermediate filament network extends to the cell periphery. In structures lysed in calcium, however, the filaments are restricted to the vicinity of the nucleus.

Microvesiculation and sphingomyelinase activation in chicken erythrocytes treated with ionophore A23187 and Ca 2+

Treatment of chicken erythrocytes with ionophore A23187 and Ca 2+ leads to the disappearance of the marginal band of microtubules and to a release of the constraints which normally maintain the nucleus in a central position in the cells. The consequent close apposition of the nucleus to the plasma membrane may allow nuclear-plasma membrane fusion to occur and subsequently results in the release of microvesicles from the hybrid surface membrane. The remnant cells are spherical, and have nuclei which appear to be partly exocytosed. Concomitant with these morphological changes, there is a breakdown of 20–30% of the total cell sphingomyelin by an endogenous sphingomyelinase which does not require Ca 2+ and which releases phosphorylcholine only into the cell interior. It is suggested that the pool of sphingomyelin which is broken down as a consequence of Ca 2+ entry into the cells is present in the nuclear membrane and that it becomes available to the plasma membrane sphingomyelinase as a result of the close apposition of nucleus and plasma membrane induced by Ca 2+.

The cytoskeletal system of nucleated erythrocytes. II. presence of a high molecular weight calmodulin-binding protein.

Calmodulin was detected in dogfish erythrocyte lysates by means of phosphodiesterase activation. Anucleate dogfish erythrocyte cytoskeletons bound calmodulin. Binding of calmodulin was calcium-dependent, concentration-dependent, and saturable. Cytoskeletons consisted of a marginal band of microtubules containing primarily tubulin, and trans-marginal band material containing actin and spectrinlike proteins. Dogfish erythrocyte ghosts and cytoskeletons were found to contain a calcium-dependent calmodulin-binding protein, CBP, by two independent techniques: (a) 125I-calmodulin binding to cytoskeletal proteins separated by SDS PAGE, and (b) in situ azidocalmodulin binding in whole anucleate ghosts and cytoskeletons. CBP, with an apparent molecular weight of 245,000, co-migrated with the upper band of human and dogfish erythrocyte spectrin. CBP was present in anucleate ghosts devoid of marginal bands and absent from isolated marginal bands. CBP therefore appears to be localized in the trans-marginal band material and not in the marginal band. Similarities between CBP and high molecular weight calmodulin-binding proteins from mammalian species are discussed.

Structural associations of synemin and vimentin filaments in avian erythrocytes revealed by immunoelectron microscopy

Intermediate filament structure and distribution were studied by antibody decoration and low-angle shadowing of sonicated chicken erythrocytes and embryonic erythroid cells. Intermediate filaments containing vimentin and synemin form a three-dimensional network in these cells, interlinking the nucleus and plasma membrane. This filament network is spatially segregated from the marginal band of microtubules, indicating that these two systems do not interact directly in the development or maintenance of cell shape. Incubation of sonicated cells with an antiserum specific for vimentin results in uniform decoration of the intermediate filaments; incubation with antisynemin results in decoration of periodically spaced foci. Measurement of the synemin periodicity under a specified set of sample preparation conditions gives average values of 180 nm for adult erythrocytes and 230 nm for 10 day old embryonic erythroid cells, suggesting some fundamental change in the structure of the filaments during erythropoiesis. Registration of these foci in laterally associated filaments, and decoration of bridges between slightly separated filaments, suggest that synemin mediates crosslinking of intermediate filaments through self-interaction.

The cytoskeletal system of nucleated erythrocytes. I. Composition and function of major elements.

We have studied the dogfish erythrocyte cytoskeletal system, which consists of a marginal band of microtubules (MB) and trans-marginal band material (TBM). The TBM appeared in whole mounts as a rough irregular network and in thin sections as a surface-delimiting layer completely enclosing nucleus and MB. In cells incubated at 0 degrees C for 30 min or more, the MB disappeared but the TBM remained. MB reassembly occurred with rewarming, and was inhibited by colchicine. Flattened elliptical erythrocyte morphology was retained even when MBs were absent. Total solubilization of MB and TBM at low pH, or dissolution of whole anucleate cytoskeletons, yielded components comigrating with actin, spectrin, and tubulin standards during gel electrophoresis. Mass-isolated MBs, exhibiting ribbonlike construction apparently maintained by cross-bridges, contained four polypeptides in the tubulin region of the gel. Only these four bands were noticeably increased in the soluble phase obtained from cells with 0 degrees C-disassembled MBs. The best isolated MB preparations contained tubulin but no components comigrating with high molecular weight microtubule-associated proteins, spectrin, or actin. Actin and spectrin therefore appear to be major TBM constituents, with tubulin localized in the MB. The results are interpreted in terms of an actin- and spectrin-containing subsurface cytoskeletal layer (TBM), related to that of mammalian erythrocytes, which maintains cell shape in the absence of MBs. Observations on abnormal pointed erythrocytes containing similarly pointed MBs indicate further that the MB can deform the TBM from within so as to alter cell shape. MBs may function in this manner during normal cellular morphogenesis and during blood flow in vivo.

Microtubules in mammalian erythroblasts. Are marginal bands present?

The purpose of this study was to determine if marginal bands, such as those present in mature nucleated red blood cells of other non-mammalian vertebrates and in primitive mammalian erythrocytes are present in definitive mammalian erythroblasts. In a small number of erythroblasts examined from mouse spleen, a bundle of 5-8 microtubules could be seen. These microtubules appeared similar to those previously identified by others as marginal band microtubules in liver and marrow erythroblasts. However, it was difficult to distinguish these bundles from remnants of mitotic spindle microtubules, or bundles of microtubules which extend to the midbody, a structure which is seen quite frequently in sections of erythroid cells. Triton extraction, a process which renders cytoskeletal elements such as microtubules more visible, also failed to confirm the presence of conventional marginal bands in these cells. It is suggested that use of the term "marginal band" be restricted to those cases in which it can be unequivocally demonstrated that a bundle of microtubules encircles the perimeter of the cell.

Structure and composition of the cytoskeleton of nucleated erythrocytes I. The presence of microtubule-associated protein 2 in the marginal band.

The marginal band (MB) of nucleated erythrocytes is composed of a bundle of microtubules that encircles the cell immediately beneath the plasma membrane. When cells are lysed in buffer containing Triton X-100 the MB microtubules remain intact, and the nucleus remains suspended at the cell center by a filamentous network called the trans-MB material that connects the nucleus to the peripheral MB. When these lysed cells are prepared for indirect immunofluorescence by use of an antibody to chick brain microtubule-associated protein 2 (MAP 2), intense staining of the MB results; no staining is evident in the areas occupied by the nucleus or the trans-MB material. Controls demonstrate that the staining is specific, because no staining occurs with fluorescent goat antirabbit serum alone or when nonimmune serum is used as the first antibody. Furthermore, the fluorescence of the MB is not affected by pretreatment of the immune serum with purified tubulin, but staining is prevented by pretreatment of the immune serum with purified MAP 2. To determine which protein component of the MB was responsible for the positive immunofluorescence results, 125I-protein A staining was used after the protein components of the isolated cytoskeleton had been resolved by SDS-polyacrylamide gels. Controls showed that the antiserum could react on SDS gels with MAP 2 from purified chick brain microtubules. The results with the cytoskeletal proteins demonstrated that the antiserum reacted only with a high molecular weight protein having a molecular weight similar, but not identical, to that of chick brain MAP 2. Thus, it is concluded that a protein with antigenic characteristics similar to those of chick brain MAP 2 is a component of the MB. The results are discussed in terms of the possible function of MAP 2 in the MB.

Marginal bands in camel erythrocytes

The elliptical, anucleate erythrocytes of camels have been examined for the presence of marginal bands and their constituent microtubules. Lysis of erythrocytes under microtubule-stabilizing conditions readily revealed marginal bands in at least 3 % of the cells, as observed by phase-contrast and darkfield light microscopy. Microtubules plus a marginal band-encompassing network of material are visible in lysed cell whole mounts with transmission electron microscopy. Marginal band microtubules are also evident in electron micrographs of thin-sectioned camel erythrocytes identifiable as reticuloyctes on the basis of submaximal electron density (reduced haemoglobin iron content) and presence of polysomes. The results suggest that marginal bands may be involved in morphogenesis of camel erythrocytes but are not required for maintenance of their ellipticity after cells are fully differentiated.

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.

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

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.

The microtubule marginal band of the newt erythrocyte: Observations on the isolated band

A marginal band of microtubules is present in nucleated erythrocytes of nonmammalian vertebrates. The role attributed to this system of microtubules is the development and maintenance of the flat ellipsoidal shape of the mature red blood cell. The present paper describes the structure of the marginal band in red cells, hemolyzed in order to better visualize the microtubules, and a method of isolation of the complete marginal band. The isolated marginal band can be considered a very convenient material for ultrastructural and biochemical studies of an integrated system of microtubules.

The fine structure of the haemocyte of the holothurian, Cucumaria miniata (Brandt)

The fine structure of the holothurian haemocyte is similar to that of the nucleated erythrocytes of lower vertebrates. Points of similarity include nuclear and nucleolar features, the distribution of haemoglobin, mitochondria, lysosomes, Golgi apparatus, and centrioles, and the presence of a marginal band of microtubules. Features apparently unique to this haemocyte are a canalicular system and an isolation membrane system. An interpretation of the functional relationships of the organelles is presented. Stages in the development and degradation of the haemocyte are also demonstrated. The morphological similarity of the holothurian haemocyte to lower vertebrate erythrocytes is attributed to functional analogy and parallel cellular adaptations, but not to homology.

Microfibrils of blood platelets: their relationship TO MICROTUBULES AND THE CONTRACTILE PROTEIN.

Human blood platelets were subjected to osmotic shock, brief sonication, pressure homogenization, or treatment with adenosine diphosphate (ADP). These procedures demonstrated an abundance of cytoplasmic microfibrils. The fibrils resembled those found on electron microscopy of partially purified thrombosthenin, the actomyosin-like protein isolated from platelets, and they also appeared to resemble the myofilaments of smooth muscle. Similar fibrils were not found in leukocytes studied under identical conditions. Treatment with colchicine (2 x 10(-5) mole/liter) resulted in the disappearance of microtubules but did not affect the morphology of the microfibrils or interfere with platelet-dependent clot retraction. Thus, microfibrils rather than microtubules may represent the morphologic counterpart of the contractile protein. Brief osmotic shock at low temperature or treatment with 10(-4) M ADP caused the marginal band of microtubules to be replaced by a bundle of intertwining microfibrils. The apparent inter-conversion of microtubules and microfibrils under a variety of conditions led to the hypothesis that fibrils and tubules consist of similar subunits whose degree of polymerization might be dependent on local cytoplasmic forces. Furthermore, on the basis of these observations, it is postulated that the contractile properties of the cells may be vested in the microfibrils, whereas the tubules may serve to maintain the highly asymmetric shape characteristic of circulating and irreversibly aggregated platelets.

Effects of ethylenediamine tetracetic acid (EDTA) on platelet structure.

The anticoagulant, EDTA, is known to produce changes in the external contour of platelets. In the present investigation the fine structural alterations which develop in EDTA platelets were examined in the electron microscope. Platelets exposed to EDTA at 37° C. lose their disc shape and become irregularly swollen with multiple pseudopods. This external transformation is identical to the change which occurs in platelets exposed to ADP, thrombin, or low temperature. Internal changes in EDTA platelets, however, are markedly different from those which occur under the influence of aggregating agents or cold. The differences indicate that the effects of EDTA on platelet shape are not due to dispersal of marginal band microtubules or to the trigging of an internal wave of contraction. Chelation of membrane calcium by EDTA appears to cause marked irregularities in the platelet wall and massive swelling of the canalicular system. Swelling of internal channals is the dominant alteration in EDTA platelets, and this change appears ultimately to be related to the breakdown of platelet granules.

Tubular Elements in Platelet Granules

Abstract Improved fixation of platelets has facilitated recognition of tubular elements in the hyaloplasm of the cells. Marginal band microtubules, hyaloplasmic microtubules, and a system of tubular channels have been dealt with in several publications. The purpose of this investigation is to describe a third type of tubular element in platelets which has not previously been reported. Observation of the substructure of hyaloplasmic particles in normal platelets has revealed small, tubelike elements occurring singly or in groups of two or three in many granules. Exposure of platelets to digitonin shock appears to increase the number of tubular elements inside the particles. The tubelike components in granules superficially resemble microtubules, but their origin, nature, and function remain obscure.