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T H MacRae

Publications and source records attributed to T H MacRae.

At least 37 records · Page 2Linked to original sources
PubMed1992

Microtubule organization by cross-linking and bundling proteins.

To understand microtubule function the factors regulating their spatial organization and their interaction with cellular organelles, including other microtubules, must be elucidated. Many proteins are implicated in these organizational events and the known consequences of their actions within the cell are increasing. For example, the function of microtubule bundles at the surfaces of polarized cells has recently received attention, as has the action in cortical rotation of a transient arrangement of microtubules found beneath the vegetal surface of fertilized frog eggs. The in vivo association of microtubules during early Xenopus oogenesis has added interest as microtubules bundled in cell-free extracts are protected against the action of a severing protein found in this animal. A 52 kDa F-actin bundling protein purified from Physarum polycephalum organizes microtubules and causes the cobundling of microtubules and microfilaments. These observations, in concert with others that are presented, emphasize the diversity within the family of microtubule cross-linking proteins. The challenge is to determine which proteins are relevant from a physiological perspective, to ascertain their molecular mechanisms of action and to describe how they affect cytoplasmic organization and cell function. To realize this objective, the proteins which cross-link and bundle microtubules must be investigated by techniques which reveal different but related aspects of their properties. Cloning and sequencing of genes for cross-linking proteins, their subcellular localization especially as microtubule-related changes in cell morphology are occurring and the application of genetic studies are necessary. Study of the neural MAP provides the best example of just how powerful current experimental approaches are and at the same time shows their limits. The neural MAP have long been noted for their enhancement of tubulin assembly and microtubule stability. Their spatial distribution has been studied during the morphogenesis of neural cells. Sequencing of cloned genes has revealed the functional domains of neural MAP including carboxy-terminal microtubule-binding sites. Similarities to microtubule binding proteins from other cell types stimulate interest in the neural MAP and further suggest their importance in microtubule organization. For example, MAP4 enjoys a wide cellular distribution and has microtubule-binding sequences very similar to those in the neural MAP. Moreover, the nontubulin proteins of marginal bands are immunologically related to neural MAP, indicating shared structural/functional domains. Even with these findings the mechanism by which neural MAP cross-link microtubules remains uncertain. Indeed, some researchers express doubt that microtubule cross-linking is actually a function of neural MAP in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Epithelial morphogenesis in developing Artemia: the role of cell replication, cell shape change, and the cytoskeleton.

The roles of cell replication and shape change as morphogenetic forces in epithelial invagination were examined in instar II Artemia. The epidermal cells underwent a fixed pattern of cell division during the first 5 hr of instar II. Greater cell replication in the thoracopod bud (ThB) than in the arthrodial membrane (AM) region resulted in a higher density of epidermal cells in the ThB region (differential cell density). The ratio of cell density (AM/ThB) declined from 1.0 to less than 0.80 by Hour 2 of instar II. Invagination of the AM occurred during Hour 4 when the AM/ThB reached 0.75. A 2-hr pulse with 5'-fluorodeoxyuridine (FudR) during instar I delayed completion of the cell replication pattern and development of transverse cell files in the ThB region for a period equal to the length of the exposure. The delay in the cell division program resulted in a cell density ratio of 0.93 at Hour 4, a value normally observed in Hour 2 larvae, and evagination of the epidermis did not occur at apolysis (Hour 4). The FudR treatment did not perturb the cytoskeleton or the initial steps in cell shape change and the larvae formed small segments during instar III. Cell shape change within the AM began during Hour 4 as this region became significantly thinner than the neighboring ThB region (thickness ratio, AM/ThB = 0.77). Before apolysis the AM cells became wedge shaped, a change which occurred when the basal region of the cell enlarged. The microtubules and microfilaments were reorganized from the apical cytoplasm to the lateral border of apposing AM cells. Following apolysis (Late Hour 4) shape change was completed as the cells attained a thin spindle form, with microtubule- and microfilament-rich filopodial extensions which overlapped adjacent AM cells. As contact with ThB cells shifted from lateral to apicolateral, the AM cells formed the innermost edge of the invagination. Microtubules in the differentiating AM cells contained tyrosinated, detyrosinated, and acetylated alpha-tubulin isoforms. Treatment with nocodazole, colchicine, taxol, or cytochalasin B blocked AM cell shape change and inhibited segmentation, but did not affect the mitotic pattern or differential cell density. We conclude that the specific pattern of cell division led to differential cell density which, along with AM cell shape change, established the conditions necessary to achieve epidermal evagination.

Actin Cytoskeleton↗
PubMed1991

Spatial distribution of posttranslationally modified tubulins in polarized cells of developing Artemia.

In many differentiated cells, posttranslationally modified tubulins exhibit restricted subcellular distribution, leading to the proposal that they are required for the production and maintenance of polarity. To study this possibility, we used immunological approaches to examine tubulin isoforms in developing Artemia larvae and to determine their location in several types of cells within the organism. The amount of tubulin in relation to total protein remained relatively constant during early larval development while detyrosinated tubulin increased, an event correlated with the differentiation of larval gut muscle cells. Except for epidermal cells of the developing thorax, each type of cell within the Artemia larvae exhibited characteristic staining patterns which were very similar for each antitubulin antibody. Within epidermal cells, microtubules containing acetylated tubulin appeared patchy or punctate in their distribution, an image not seen with the other antibodies. In most polarized cells, staining for tubulin and actin colocalized in discrete areas, demonstrating enrichment of both proteins within the same cellular compartment and suggesting functional interactions. Mitotic figures were stained with qualitatively equal intensity by all of the antitubulin antibodies, but asters were not observed. Midbodies were intensely stained with phalloidin as well as the antibodies to tubulin. It was clear that microtubules exhibited a preferential localization in cells of Artemia but in no case was a tubulin isoform found exclusively in one area of a cell. The results support the contention that microtubules influence the organization of polarized cell structure and function but they do not permit the conclusion that this capability is dependent on the localization of posttranslationally modified tubulins to restricted subcellular positions.

Actins↗
PubMed1991

Effects of metals on early life stages of the brine shrimp, Artemia: a developmental toxicity assay.

The need for simple, fast and inexpensive tests to study metal pollution in the marine environment has become more pressing as utilization of coastal waters increases. To address this concern, the influence of four metals, cupric sulfate, lead nitrate, zinc sulfate and nickel sulfate, on emergence and hatching of the brine shrimp, Artemia, has been assessed. Occurrence of these easily recognized developmental milestones provides a convenient method to assay metal effects on development. Copper and lead were about equally toxic, reducing the rate and extent of Artemia development at or below concentrations of 0.1 microM. Zinc was somewhat less toxic than copper and lead, while nickel was the least toxic. Emerging Artemia are much more sensitive to metals than are larvae and adults. Furthermore, in contrast to results obtained with larvae and adults, the toxicity of lead is high when prelarval stages are considered. It is apparent from the findings that use of early stages of Artemia is an alternative to the examination of slower growing animals for the study of metal pollution in coastal marine waters.

Animals↗
PubMed1991

Synthesis of tubulin during early postgastrula development of Artemia: isotubulin generation and translational regulation.

Isotubulin diversity and the synthesis of tubulin were examined during development of the brine shrimp, Artemia. It was found, by Northern and dot-blot analyses, that Artemia possess constant amounts of one size class of mRNA each for alpha- and beta-tubulin during the first 24 hr of postgastrula development. Two-dimensional gel electrophoresis and fluorography, following the in vitro translation of developmentally staged poly(A)+ mRNA, yielded one alpha- and one beta-tubulin. Clearly, the isotubulin diversity seen on Coomassie blue-stained two-dimensional gels of Artemia tubulin is not generated by differential gene transcription during postgastrula growth, nor is development accompanied by synthesis of novel isotubulins resolvable by the methods employed. Characterization of polysomal poly(A)+ mRNA, and of proteins synthesized in vivo, indicated very little tubulin was synthesized in Artemia as they developed from gastrula to first instar larvae. The results suggest control of tubulin synthesis in Artemia by a mechanism that restricts binding of the message to ribosomes. Of general significance, it appears that a complex metazoan animal is able to undergo extensive growth with limited tubulin synthesis and in the absence of differential expression of tubulin genes. Moreover, the capacity of microtubules to assume changing and/or increased functions associated with cellular development is seemingly not dependent on the synthesis of new tubulin isoforms.

Animals↗
PubMed1991

Post-translationally modified tubulins in Artemia: prelarval development in the absence of detyrosinated tubulin.

The synthesis of post-translationally modified tubulins was examined during Artemia development. Tubulin, either purified to homogeneity or in cell-free extracts, was blotted to nitrocellulose and probed with a panel of antibodies. When purified tubulin was examined, tyrosinated tubulin underwent a large decrease as development progressed and this was accompanied by the appearance of detyrosinated tubulin in samples from organisms developed 24 hr. The inclusion of carboxypeptidase inhibitors had a small effect on the relative amounts of tyrosinated and detyrosinated tubulins in 24-hr preparations. The amount of alpha- and beta-tubulin in cell-free extracts of Artemia either remained relatively constant during development or increased slightly. The same result was obtained for acetylated and tyrosinated tubulin. Detyrosinated tubulin first appeared in 24-hr cell-free extracts and was only post-translationally modified tubulin to increase, relative to the total amount of tubulin, as the brine shrimp developed. As revealed by immunofluorescence staining, detyrosinated tubulin occurred in many cell types of developing nauplii and was prominently displayed in mitotic figures. Artemia, a complex metazoan animal, is thus able to grow for an extended period of time in the absence of detyrosinated tubulin. This isoform is however, synthesized in early larvae and may be required for the development of elongated cells including those which encircle the gut. Detyrosination remains as the only developmentally related change observed for brine shrimp tubulin.

Animals↗
PubMed1991

Toxicity of organic mercury compounds to the developing brine shrimp, Artemia.

Mercury can be coupled to a wide variety of organic compounds but there is limited information concerning the influence of such substitutions on the toxicity of mercury within the marine environment. We therefore determined the effects of six organomercuries on the emergence and hatching of the brine shrimp, Artemia. The relative toxicities of the organic mercuries were unaffected by the ability of the compounds to ionize, whereas the sizes of the compounds appeared to be important. Thus, brine shrimp were equally sensitive to five of the organic mercuries while diphenylmercury, the largest of the organic mercuries tested, was the least toxic. In the presence of 0.1 microM diphenylmercury the final amount of hatching was similar to that in the absence of metal but in this situation there was an easily measured reduction in the rate of development. By determining the rates of emergence and hatching it is apparent that Artemia are adversely affected by organic mercuries at concentrations less than 0.1 microM, the lowest level examined in this study. The work extends our earlier findings with cadmium and zinc, supporting the proposal that Artemia is an excellent alternative to more complex, slow-growing animals for the study of biochemical/physiological aspects of marine pollution.

Animals↗
PubMed1990

Purification and assembly in vitro of tubulin from Trypanosoma brucei brucei.

Trypanosome tubulin was purified to near homogeneity by chromatography on DEAE-Sephadex, Amicon filtration and assembly-disassembly in vitro. Polymerization of the tubulin in vitro yielded long, structurally normal, microtubules and some sheet structures on addition of GTP and incubation at 37 degrees C, in either the presence or the absence of Mg2+. Tubulin assembly was disrupted by glycerol and a selection of microtubule-reactive drugs. Immunological analysis of the purified tubulin revealed tyrosinated and acetylated alpha-tubulin, in addition to defining the migration characteristics of the alpha- and beta-tubulin on one-dimensional SDS/polyacrylamide gels. This is the first isolation of trypanosome tubulin with the ability to form structurally normal microtubules independent of the addition of taxol or nucleating microtubule fragments. The development of the purification procedure thus provides an important step for subsequent study of microtubule-associated protein-tubulin and plasma-membrane-microtubule cytoskeleton interactions of trypanosomes, and increases the potential for development of tubulin-based anti-trypanosome drugs.

Animals↗
PubMed1990

Production and characterization of monoclonal antibodies to the mammalian sperm cytoskeleton.

The cytoskeleton exerts a direct effect on the function of sperm by influencing the distribution of subcellular organelles and plasma membrane molecules. We have prepared six monoclonal antibodies to Triton X-100-insoluble components of the bull sperm cytoskeleton. One of the antibodies reacts with a detachable portion of the bull sperm acrosome. The remainder include an antibody that recognizes the principal and end piece of the tail and another that is specific to the middle piece. Two of the antibodies yield dissimilar staining patterns of the neck region and the tail, and the final monoclonal antibody stains the subacrosomal region and a detachable acrosomal domain of bull sperm. The cross reactivities of the antibodies with hamster sperm and PtK2 cells are described, as is the recognition of bull sperm polypeptides on western blots. The results suggest that these antibodies will provide interesting insights concerning the role of the cytoskeleton in sperm development and function.

Animals↗
PubMed1990

Tubulin isoforms in the brine shrimp, Artemia: primary gene products and their posttranslational modification.

The brine shrimp, Artemia, contains 3 alpha- and 2 beta-tubulins as shown by Coomassie Blue staining of two-dimensional gels. In order to study the biosynthetic origins of the isotubulins, we hybridized cloned Drosophila tubulin genes, under stringent conditions, to blots of Artemia DNA and RNA. Southern blot analyses indicate a tubulin gene family of limited complexity. One size class of alpha- and beta-tubulin mRNA at 1800 bases was observed on Northern blots. Fluorograms of Artemia tubulin synthesized in vitro, revealed one alpha- and one beta-tubulin on two-dimensional gels, indicating that each mRNA is translated into one polypeptide and that additional tubulin spots observed on Coomassie-stained two-dimensional gels may arise posttranslationally. Artemia tubulin, which was either purified to homogeneity, or in crude cell-free extracts, was analyzed with a panel of tubulin-specific antibodies. The presence of acetylated tubulin, restricted to one of the three major alpha-tubulin spots on two-dimensional gels, demonstrated that Artemia tubulin diversity is partially generated by posttranslational mechanisms. Artemia tubulin reacted very well with an antibody to tyrosinated tubulin, but there was no, or very little, detectable detyrosinated tubulin unless the purified Artemia tubulin was exposed to carboxypeptidase. The results suggest that all microtubule-dependent events in Artemia, a complex metazoan animal, are accomplished with microtubules composed from a limited repertoire of tubulins and that none of these events require appreciable amounts of detyrosinated tubulin.

Animals↗
PubMed1989

Cross-linking of microtubules by microtubule-associated proteins (MAPs) from the brine shrimp, Artemia.

Microtubules induced with taxol to assemble in cell-free extracts of the brine shrimp, Artemia, are cross-linked by microtubule-associated proteins (MAPs). When the MAPs, extracted from taxol-stabilized microtubules with 1 M-NaCl are co-assembled with purified Artemia or mammalian neural tubulin, reconstitution of cross-linking between microtubules occurs. The most prominent non-tubulin protein associated with reconstituted cross-linked microtubules has a molecular weight of 49,000 but we cannot yet exclude the possibility that other proteins may be responsible for the cross-linking. Cross-linkers are separated by varying distances while cross-linked microtubules, prepared under different conditions, are 6.9-7.7 nm apart. Cross-linking of microtubules by MAPs occurs whether MAPs are added to assembling tubulin or to microtubules, and it is not disrupted by ATP. The MAPs are heat-sensitive and do not stabilize microtubules to cold. Immunological characterization of Artemia MAPs on Western blots indicates that Artemia lack MAP 1, MAP 2 and tau. Our results clearly demonstrate that Artemia contain novel MAPs with the ability to cross-link microtubules from phylogenetically disparate organisms in an ATP-independent manner.

Alkaloids↗
PubMed1989

Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies.

The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.

Animals↗
PubMed1986

Temperature effects on solutions of vinblastine-induced polymers assembled from brine shrimp (Artemia) tubulin.

Purified Artemia tubulin in the presence of neural microtubule-associated proteins and vinblastine, or with vinblastine alone, forms extensive coils. Reduction in temperature of a coil-containing solution to 4 degrees C causes an increase in turbidity, which returns to previous levels once the solution is warmed. Examination of negatively stained samples indicates that the turbidity fluctuations are not accompanied by a pronounced change in coil structure nor by increased polymer formation. Bovine neural tubulin responds in the same way as Artemia tubulin to vinblastine and temperature. An interesting novel response to vinblastine, shared by tubulins from phylogenetically distinct organisms, is illustrated by our results.

Animals↗
PubMed1986

Vinblastine-induced aggregation of brine shrimp (Artemia) tubulin.

Tubulin from the brine shrimp Artemia readily assembles in vitro in the absence of microtubule-associated proteins under conditions which do not permit assembly of tubulin from brain. Heated microtubule-associated protein preparations from bovine brain do, however, interact with Artemia tubulin, resulting in stimulation of tubulin assembly and formation of morphologically normal cold-sensitive microtubules. Addition of vinblastine to mixtures containing microtubules assembled in the presence of neural microtubule-associated proteins caused a drop and then a rise in turbidity of the solution. The turbidity changes were accompanied by the appearance of coils, presumably derived from the microtubules which disappeared upon addition of vinblastine. Coils also resulted when microtubule-associated proteins and vinblastine were added to tubulin before polymerization was initiated. Vinblastine prevented normal assembly and caused disruption of Artemia microtubules polymerized in the absence of microtubule-associated proteins. Under these conditions clumped or compact coils, different in appearance from those formed in the presence of the microtubule-associated proteins, were observed. The data confirm that tubulin from Artemia, an organism that is phylogenetically far removed from mammals, has retained binding sites for vinblastine and microtubule-associated proteins and that the interrelationship of these sites has been at least partially preserved. The incomplete depolymerization of Artemia microtubules in response to vinblastine when microtubule-associated proteins are absent suggests that the longitudinal tubulin-tubulin interactions involved in microtubule formation are more stable for Artemia than for neural tubulin.

Animals↗
PubMed1985

N,N'-Ethylene-bis(iodoacetamide) as a probe for structural and functional characteristics of brine shrimp, squid, and bovine tubulins.

We have developed a simple probe for certain functionally significant features of the tubulin molecule. When bovine brain tubulin is treated with N,N'-ethylene-bis(iodoacetamide) (EBI), two intrachain cross-links, designated beta s and beta *, are formed in beta-tubulin, each one with a unique effect on the electrophoretic mobility of beta on gels containing sodium dodecyl sulfate. Formation of the beta * cross-link, which involves at least one assembly-critical sulfhydryl, is completely inhibited by colchicine and its congeners, while that of beta s is inhibited completely by maytansine and GTP and partly by vinblastine. To see how conserved this complex pattern is in evolution we examined tubulins from the brine shrimp Artemia and the squid Loligo. In both tubulins EBI forms the beta * cross-link in a reaction inhibitable by colchicine, podophyllotoxin, and nocodazole. In each tubulin, EBI appears to form a second intrachain cross-link in a reaction that can be inhibited completely by maytansine and GTP and partly by vinblastine. In Artemia, this cross-link alters the electrophoretic mobility to a slightly smaller extent than is the case for beta s in bovine brain, but in Loligo the alteration is much greater. It seems that the ligand-binding sites, the critical sulfhydryls, and their spatial interrelationships are strongly conserved and that the beta s sulfhydryls or the sequence between them are less strongly conserved in evolution.

Alkylation↗