In vitro morphogenesis of amphibian erythroblasts.

Non-mammalian vertebrate erythrocytes are flattened nucleated ellipsoids containing marginal bands (MBs) of microtubules that assemble during cellular morphogenesis. Earlier work suggested that pointed erythroid cells containing pointed MBs were intermediate stages in terminal differentiation, rather than aberrant forms, but direct evidence was lacking. Here we report on morphogenesis in individual post-cytokinetic amphibian erythroblasts in culture. Daughter cells remained adjacent in pairs, and developed pointed morphology over 1-2 h in the following sequence: (a) ends opposite the cytokinetic furrow became pointed, producing a spheroidal singly-pointed stage; (b) furrow ends usually became pointed, yielding doubly-pointed cells; (c) furrow-end points disappeared, producing a second singly-pointed stage that was flattening. Over a longer term, the single points sometimes disappeared, yielding a flattened discoid. These observations support the hypothesis that pointed cells are normal intermediates in a biogenetic program in which post-mitotic centrosomes organize MBs while occupying the singly-pointed ends of differentiating erythroblasts.

Elliptical versus circular erythrocyte marginal bands: isolation, shape conversion, and mechanical properties.

Differentiation of nucleated erythrocytes involves transformation from spheroids to flattened discoids to mature flattened ellipsoids. The marginal band (MB) of microtubules is required for this process and continues to play a role in maintaining mature ellipsoidal cell shape. One hypothesis for MB function is that cell ellipticity is generated and maintained by asymmetric application of force across a flexible, circular MB frame by the membrane skeleton or other transverse elements. This is based on an earlier finding that isolated erythrocyte MBs are much more circular than MBs in situ. However, our present studies of salamander erythrocyte MBs isolated by a detergent-based method challenge this hypothesis. Most of these isolated MBs are initially elliptical, even though they lack transverse material (= E-MBs). They can be stabilized in that form for long periods and can be converted experimentally into the circular form (= C-MBs) by extended incubation in isolation medium or by treatment with elastase or subtilisin. We have tested an alternative hypothesis for generation and maintenance of ellipsoidal MBs, one based on intrinsic differential bending resistance and supported by construction of models. Using laser microsurgical transection to compare mechanical responses of isolated E-MBs and C-MBs, we have found their behavior to be quite different. Whereas C-MBs linearize, most E-MBs do not, instead retaining considerable curvature. These results are incompatible with the differential bending resistance hypothesis, which predicts both C-MB and E-MB linearization. However, they are consistent with a third model, in which material bound to the MB stabilizes it in the mature ellipsoidal form, and indicate that the mechanism for maintenance of MB ellipticity differs from that involved in its generation.

Distinctive Cytoskeletal Organization in Erythrocytes of the Cold-Seep Vesicomyid Clam, Calyptogena kilmeri.

Erythrocytes have long served as model cells, useful for analyzing cytoskeletal structure and function. In non-mammalian vertebrates, erythrocytes are typically highly flattened, nucleated ellipsoids in which a marginal band (MB) of microtubules interacts with the membrane skeleton (MS) to generate and maintain cell shape. Though relatively rare, erythrocytes also occur in representatives of many invertebrate phyla, including the arcid and vesicomyid molluscs, but the structure and function of these cells are not well understood. Previous work has shown arcid erythrocytes to be highly flattened ellipsoids containing the MB-MS cytoskeletal system, similar to vertebrates but with an additional interesting feature: a functional centriole-containing centrosome associated with each MB. In the present study we have examined, for the first time, erythrocyte morphology and cytoskeletal structure in a vesicomyid. Using Calyptogena kilmeri, the dominant invertebrate at many Pacific cold seeps, we have found that the erythrocytes are only slightly flattened and do not contain MBs. Rather, their cytoskeletons display a peripheral centriole-containing centrosome with radiating fibers, a distinctive type of organization not observed previously in mature erythrocytes from any species.

Assembly and bundling of marginal band microtubule protein: role of tau.

Microtubule protein extracted from dogfish erythrocyte cytoskeletons by disassembly of marginal bands at low temperature formed linear microtubule (MT) bundles upon reassembly at 22 degrees C. The bundles, which were readily visible by video-enhanced phase contrast or DIC microscopy, increased in length and thickness with time. At steady state after 1 hour, most bundles were 6-11 microns in length and 2-5 MTs in thickness. No inter-MT cross-bridges were visible by negative staining. The bundles exhibited mechanical stability in flow as well as flexibility, in this respect resembling native marginal bands. As analyzed by SDS-PAGE and immunoblotting, our standard extraction conditions yielded MT protein preparations and bundles containing tau protein but not high molecular weight MAPs such as MAP-2 or syncolin. In addition, late fractions of MT protein obtained by gel filtration were devoid of high molecular weight proteins but still produced MT bundles. The marginal band tau was salt-extractable and heat-stable, bound antibodies to mammalian brain tau, and formed aggregates upon desalting. Antibodies to tau blocked MT assembly, but both assembly and bundling occurred in the presence of antibodies to actin or syncolin. The MTs were "unbundled" by subtilisin or by high salt (0.5-1 M KCl or NaCl), consistent with tau involvement in bundling. High salt extracts retained bundling activity, and salt-induced unbundling was reversible with desalting. However, reversibility was observed only after salt-induced MT disassembly had occurred. Reconstitution experiments showed that addition of marginal band tau to preassembled MTs did not produce bundles, whereas tau presence during MT reassembly did yield bundles. Thus, in this system, tau appears to play a role in both MT assembly and bundling, serving in the latter function as a coassembly factor.

Localization of tau and other proteins of isolated marginal bands.

To determine which proteins were associated with and intrinsic to the marginal band (MB) of microtubules (MTs), we studied protein components of MBs isolated from nucleated erythrocytes by differential detergent solubilization of the membrane skeleton (MS). MBs isolated from dogfish erythrocytes contained major proteins in the tubulin M(r) range. A high molecular weight protein of approximately 290 kD that bound antibody to syncolin and to heat-stable brain MAPs was present in the whole cytoskeleton. However, most of it was solubilized by the MB isolation medium, together with the MS. Dogfish erythrocyte cytoskeletons and isolated MBs were examined with polyclonal and monoclonal antibodies against mammalian brain tau and chicken erythrocyte tau. As shown by immunofluorescence and immunoblotting, these antibodies bound to proteins in the 50 to 67 kD range, located along the length of isolated MBs. Two-dimensional SDS-PAGE revealed isolated MB proteins of pI approximately 6.8 in the same molecular weight range, as well as alpha- and beta-tubulin with pI approximately 5.4. Subtilisin or high-salt treatment of isolated MBs resulted in unbundling of MTs, indicating involvement of MAPs. MBs isolated from chicken erythrocyte cytoskeletons also contained tau as shown by anti-mammalian brain tau immunofluorescence. Both chicken and dogfish isolated MBs also bound phalloidin, but the binding was usually discontinuous and, for any given MB, matched the pattern of anti-syncolin binding. Both syncolin and F-actin were part of the MS remnant remaining after MT disassembly, supporting their assignment to a specialized MS region at the MB/MS interface. In contrast, tau protein appears to be intrinsic to the MB, where it may have an MT stabilizing and bundling function.

Detergent-based isolation of marginal bands of microtubules from nucleated erythrocytes.

We have developed a new, detergent-based method for the isolation of marginal bands (MBs) of microtubules from non-mammalian vertebrate erythrocytes. The critical step in MB isolation is selective removal of the "membrane skeleton" network (MS), within which the MB is enclosed. To test potential MS solubilizing agents systematically, we prepared dogfish (Mustelus canis) erythrocyte cytoskeletons in the presence of protease inhibitors and stored them at -20 degrees C in medium containing 50% glycerol and 10 microM taxol to stabilize the MB. Using this as a standard starting material, we found that low concentrations of sodium dodecyl sulfate (SDS) (0.025-0.1%) in the presence of Triton X-100 (0.1-0.4%) released both MBs and nuclei intact from cytoskeletons. Either detergent alone was ineffective. MB release from cytoskeletons was blocked by excess Triton X-100 and slowed by glycerol, and this was useful for stopping the release reaction during quantitative time-course studies. Most MBs (greater than 90%) were liberated from cytoskeletons in 5 to 30 min, depending upon detergent concentrations and other conditions, and they were sufficiently stable for mass isolation by differential centrifugation. Added standard proteins were not proteolyzed during MB release, nor was release blocked by protease inhibitors, indicating that endogenous proteases were not involved. As observed in thin sections and negatively stained whole mounts (transmission electron microscopy) and in critical-point dried preparations (scanning electron microscopy), the isolated MBs consisted principally of bundled microtubules, with some additional adhering material. SDS-polyacrylamide gel electrophoresis showed the isolated MBs to be composed primarily of four tubulin region polypeptides, with the same stoichiometry as in the whole cytoskeleton. As determined by immunofluorescence microscopy, isolated MBs bound antibody to both chicken brain and erythrocyte tau, in addition to anti-tubulin. Thus, proteins of the tau family may be involved in bundling of MB microtubules. Unlike previous MB isolation methods, that employed here is applicable to erythrocytes of diverse species, including the marine toad (Bufo marinus) and the chicken (Gallus domestica), both of which should be of value for comparative studies.

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.

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.

Role of the marginal band in an invertebrate erythrocyte: evidence for a universal mechanical function.

Marginal bands of microtubules are present in erythrocytes of all nonmammalian vertebrates and some invertebrates, in which they are thought to play a role in erythrocyte morphogenesis. Recently, marginal bands have also been implicated in maintenance of shape in vertebrate erythrocytes and platelets subjected to external mechanical forces. Here we demonstrate that marginal bands in an invertebrate ("blood clam") erythrocyte act similarly. Cells with and without marginal bands at the same temperature were prepared by (a) nocodazole or colchicine inhibition of marginal band reassembly following 0 degree C disassembly, (b) taxol inhibition of marginal band disassembly at 0 degree C, and (c) taxol induction of marginal band reassembly at 0 degree C. As shown previously for temperature-induced marginal band reassembly in this species, taxol-induced reassembly at 0 degree C occurred in association with centrioles. When erythrocytes with and without marginal bands were compared for their response to the mechanical stress of fluxing through capillary tubes, many more of those without marginal bands tended to become folded or buckled regardless of the method used to prepare them. The results provide evidence that marginal bands have a universal mechanical function in mature erythrocytes.

The cytoskeletal system of nucleated erythrocytes. III. Marginal band function in mature cells.

Marginal bands (MBs) of microtubules are believed to function during morphogenesis of nonmammalian vertebrate erythrocytes, but there has been little evidence favoring a continuing role in mature cells. To test MB function, we prepared dogfish erythrocytes with and without MBs at the same temperature by (a) stabilization of the normally cold-labile MB at 0 degree C by taxol, and (b) inhibition of MB reassembly at room temperature by nocodazole or colchicine. We then compared the responses of these cells to mechanical stress by fluxing them through capillary tubes. Before fluxing , cells with or without MBs had normal flattened elliptical shape. After fluxing , deformation was consistently observed in a much greater percentage of cells lacking MBs. The difference in percent deformation between the two cell types was highly significant. That the MB is an effector of cell shape was further documented in studies of the formation of singly or doubly pointed dogfish erythrocytes that appear during long-term incubation of normal cells at room temperature. On-slide perfusion experiments revealed that the pointed cells contain MBs of corresponding pointed morphology. Incubation of cells with and without MBs showed that they become pointed only when they contain MBs, indicating that the MB acts as a flexible frame which can deform and support the cell surface from within. To test this idea further, cells with and without MBs were exposed to hyperosmotic conditions. Many of the cells without MBs collapsed and shriveled , whereas those with MBs did not. The results support the view that the MB has a continuing function in mature erythrocytes, resisting deformation and/or rapidly returning deformed cells to an efficient equilibrium shape in the circulation.

Similarities between the Mr 245,000 calmodulin-binding protein of the dogfish erythrocyte cytoskeleton and alpha-fodrin.

The Mr 245,000 calmodulin-binding protein of the dogfish erythrocyte cytoskeleton (D245) has been compared with human erythrocyte spectrin and mammalian brain fodrin [J. Levine and M. Willard (1981) J. Cell Biol. 90, 631-643]. Mammalian erythrocyte alpha-spectrin, brain alpha-fodrin, and D245 are all localized in the cell surface-associated cytoskeleton, and have similar molecular weights. Like mammalian erythrocyte spectrin, D245 was extracted from erythrocyte ghosts under low-ionic-strength conditions. However, D245 failed to bind an antibody which reacted strongly with both subunits of human erythrocyte spectrin. Unlike mammalian erythrocyte alpha- and beta-spectrin, D245 bound calmodulin in the absence of urea both in a "gel-binding" assay and in situ using azidocalmodulin [D.C. Bartelt, R.K. Carlin, G.A. Scheele, and W.D. Cohen (1982) J. Cell Biol. 95, 278-284]. Striking similarities were noted between D245 and alpha-fodrin in that both exhibited (a) comparable calcium-dependent calmodulin binding properties, (b) strong reactivity with two different anti-fodrin antibody preparations, (c) similar reactivity with antibody to brain CBP-I, now believed to be fodrin, (d) proteolytic degradation yielding an Mr 150,000 calmodulin-binding fragment, and (e) lack of reactivity with an anti-spectrin antibody. A protein with calmodulin-binding and anti-fodrin-binding properties similar to D245 was detected in cytoskeletal preparations of chicken erythrocytes. Moderate and consistent cross-reactivity of anti-fodrin with human erythrocyte alpha-spectrin was also observed. The data indicate that D245 is functionally and immunologically more closely related to alpha-fodrin than to alpha-spectrin of the mammalian erythrocyte.

Centrioles as microtubule-organizing centers for marginal bands of molluscan erythrocytes.

The erythrocytes of blood clams (arcidae) are flattened, elliptical, and nucleated. They contain elliptical marginal bands (MBs) of microtubules, each physically associated with a pair of centrioles marginal bands (MBs) of microtubles, each physically associated with a pair of centrioles (Cohen, W., and I. Nemhauser, 1980, J. Cell Biol., 86:286-291). The MBs were found to be cold labile in living cells, disappearing within 1-2 h at 0 degrees C. After the cells had been rewarmed for 1-2 h, continuous MBs with associated centrioles were once again present. Time-course studies utilizing phase contrast, antitubulin immunofluorescence, and electron microscopy of cytoskeletons prepared during rewarming revealed structural evidence of centriole participation in MB reassembly. At the earliest stage of reassembly, a continuous MB was not present. Instead, relatively short and straight microtubules focused on a pointed centriolar "pole," and none were present elsewhere in the cytoskeleton. Thin continuous MBs then formed, still pointed in the centriolar region. Subsequently, the MBs regained ellipticity, with their thickness gradually increasing but not reaching that of controls even after several hours of rewarming. At these later time points, microtubules still radiated from the centrioles and joined the MBs some distance away. In the presence of 0.1 mM colchicines, MB reassembly was arrested at the pointed stage. Electron microscopic observations indicate that pericentriolar material is involved in microtubule nucleation in this system, rather than the centriolar triplets directly. The results suggest a model in which the centrioles and associated material nucleate assembly and growth of microtubules in diverging directions around the cell periphery. Microtubules of opposite polarity would then pass each other at the end of the cell distal to the centrioles, with continued elongation eventually closing the MB ellipse behind the centriole pair.