| 1. |
- de Pereda, J M, et al.
(author)
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Comparative study of the colchicine binding site and the assembly of fish and mammalian microtubule proteins.
- 1995
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In: Cell motility and the cytoskeleton. - : Wiley. - 0886-1544 .- 1097-0169. ; 30:2, s. 153-63
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Journal article (peer-reviewed)abstract
- Isolated microtubules from cod (Gadus morhua) are apparently more stable to colchicine than bovine microtubules. In order to further characterize this difference, the effect of the colchicine analogue 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cyclo heptatrien-1-one (MTC) was studied on assembly, as measured by turbidity and sedimentation analysis, and on polymer morphology. MTC has the advantage to bind fast and reversible to the colchicine binding site of tubulin even at low temperatures. It was found to bind to one site in cod brain tubulin, with affinity (6.5 +/- 1.5) x 10(5)M-1 at both low or high temperature, similarly to bovine brain tubulin. However, the effect of the binding differed. At substoichiometric concentrations of MTC bovine brain microtubule assembly was almost completely inhibited, while less effect was seen on the mass of polymerized cod microtubule proteins. A preformed bovine tubulin-colchicine complex inhibited the assembly of both cod and bovine microtubules at substoichiometric concentrations, but the effect on the assembly of cod microtubules was less. At higher concentrations (5 x 10(-5) to 1 x 10(-3) M), MTC induced a large amount of cold-stable spirals of cod proteins, whereas abnormal polymers without any defined structure were formed from bovine proteins. Spirals of cod microtubule proteins were only formed in the presence of microtubule associated proteins (MAPs), indicating that the morphological effect of MTC can be modulated by MAPs. The effects of colchicine and MTC differed. At 10(-5) M colchicine no spirals were formed, while at 10(-4) M and 10(-3) M, a mixture of spirals and aggregates was found. The morphology of the spirals differed both from vinblastine spirals and from the spirals previously found when cod microtubule proteins polymerize in the presence of high Ca2+ concentrations. The present data show that even if the colchicine binding site is conserved between many different species, the bindings have different effects which seem to depend on intrinsic properties of the different tubulins.
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| 2. |
- Klotz, A, et al.
(author)
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Polyglutamylation of atlantic cod tubulin: immunochemical localization and possible role in pigment granule transport.
- 1999
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In: Cell motility and the cytoskeleton. - 0886-1544. ; 44:4, s. 263-73
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Journal article (peer-reviewed)abstract
- In higher organisms, there is a large variety of tubulin isoforms, due to multiple tubulin genes and extensive post-translational modification. The properties of microtubules may be modulated by their tubulin isoform composition. Polyglutamylation is a post-translational modification that is thought to influence binding of both structural microtubule associated proteins (MAPs) and mechano-chemical motors to tubulin. The present study investigates the role of tubulin polyglutamylation in a vesicle transporting system, cod (Gadus morhua) melanophores. We did this by microinjecting an antibody against polyglutamylated tubulin into these cells. To put our results into perspective, and to be able to judge their universal application, we characterized cod tubulin polyglutamylation by Western blotting technique, and compared it to what is known from mammals. We found high levels of polyglutamylation in tissues and cell types whose functions are highly dependent on interactions between microtubules and motor proteins. Microinjection of the anti-polyglutamylation antibody GT335 into cultured melanophores interfered with pigment granule dispersion, while dynein-dependent aggregation was unaffected. Additional experiments showed that GT335-injected cells were able to aggregate pigment even when actin filaments were depolymerized, indicating that the maintained ability of pigment aggregation in these cells was indeed microtubule-based and did not depend upon actin filaments. The results indicate that dynein and the kinesin-like dispersing motor protein in cod melanophores bind to tubulin on slightly different sites, and perhaps depend differentially on polyglutamylation for their interaction with microtubules. The binding site of the dispersing motor may bind directly to the polyglutamate chain, or more closely than dynein.
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| 3. |
- Modig, C, et al.
(author)
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Identification of betaIII- and betaIV-tubulin isotypes in cold-adapted microtubules from Atlantic cod (Gadus morhua): antibody mapping and cDNA sequencing.
- 1999
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In: Cell motility and the cytoskeleton. - 0886-1544. ; 42:4, s. 315-30
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Journal article (peer-reviewed)abstract
- Isolated microtubule proteins from the Atlantic cod (Gadus morhua) assemble at temperatures between 8 and 30 degrees C. The cold-adaptation is an intrinsic property of the tubulin molecules, but the reason for it is unknown. To increase our knowledge of tubulin diversity and its role in cold-adaptation we have further characterized cod tubulins using alpha- and beta-tubulin site-directed antibodies and antibodies towards posttranslationally modified tubulin. In addition, one cod brain beta-tubulin isotype has been sequenced. In mammals there are five beta-tubulins (betaI, betaII, betaIII, betaIVa and betaIVb) expressed in brain. A cod betaIII-tubulin was identified by its electrophoretic mobility after reduction and carboxymethylation. The betaIII-like tubulin accounted for more than 30% of total brain beta-tubulins, the highest yield yet observed in any animal. This tubulin corresponds most probably with an additional band, designated beta(x), which was found between alpha- and beta-tubulins on SDS-polyacrylamide gels. It was found to be phosphorylated and neurospecific, and constituted about 30% of total cod beta-tubulin isoforms. The sequenced cod tubulin was identified as a betaIV-tubulin, and a betaIV-isotype was stained by a C-terminal specific antibody. The amount of staining indicates that this isotype, as in mammals, only accounts for a minor part of the total brain beta-tubulin. Based on the estimated amounts of betaIII- and betaIV-tubulins in cod brain, our results indicate that cod has at least one additional beta-tubulin isotype and that beta-tubulin diversity evolved early during fish evolution. The sequenced cod betaIV-tubulin had four unique amino acid substitutions when compared to beta-tubulin sequences from other animals, while one substitution was in common with Antarctic rockcod beta-tubulin. Residues 221, Thr to Ser, and 283, Ala to Ser, correspond in the bovine tubulin dimer structure to loops that most probably interact with other tubulin molecules within the microtubule, and might contribute to cold-adaptation of microtubules.
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| 4. |
- Modig, C, et al.
(author)
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MAP 0, a 400-kDa microtubule-associated protein unique to teleost fish.
- 1997
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In: Cell motility and the cytoskeleton. - 0886-1544. ; 38:3, s. 258-69
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Journal article (peer-reviewed)abstract
- Microtubules from neural tissues of the Atlantic cod, Gadus morhua, and of several species of Antarctic teleosts are composed of tubulin and several microtubule-associated proteins (MAPs), one of which has an apparent molecular weight of approximately 400-430 kDa. Because its apparent molecular weight exceeds those of the MAP 1 proteins, we designate this high molecular weight teleost protein MAP 0. Cod MAP 0 failed to cross-react with antibodies specific for MAPs 1A, 1B and 2 of mammalian brain, for MAP H1 of squid optic lobe, and for chicken erythrocyte syncolin, which suggests that it has a novel structure. Similarly, MAP 0 from the Antarctic fish was not recognized by an antibody specific for bovine MAP 2. Together, these observations suggest that MAP 0 is a novel MAP that may be unique to fish. To determine the tissue specificity and phylogenetic distribution of this protein, we generated a rabbit polyclonal antibody against cod MAP 0. Using this antibody, we found that MAP 0 was present in microtubule proteins isolated from cod brain tissues and spinal cord but was absent in microtubules from heart, liver, and spleen. At the subcellular level, MAP 0 was distributed in cod brain cells in a punctate pattern coincident with microtubules but was absent in skin cells. MAP 0 was also detected in cells of the peripheral nervous system. A survey of microtubule proteins from chordates and invertebrates showed that anti-MAP 0-reactive homologs were present in five teleost species but not in more primitive fish and invertebrates or in higher vertebrates. MAP 0 bound to cod microtubules by ionic interaction at a site recognized competitively by bovine MAP 2. Although its function is unknown, MAP 0 does not share the microtubule-binding properties of the motor proteins kinesin and dynein. We propose that MAP 0 is a unique, teleost-specific MAP.
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| 5. |
- Nilsson, Helen, 1970, et al.
(author)
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Evidence for several roles of dynein in pigment transport in melanophores
- 1997
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In: Cell Motility and the Cytoskeleton. - 0886-1544. ; 38:4, s. 397-409
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Journal article (peer-reviewed)abstract
- Melanophores are specialized cells that transport pigment granules to and from the cell center, giving animals the ability to change skin color. A kinesin-related plus-end motor has previously been shown to be responsible for pigment granule dispersion [V.I. Rodionov, F.K. Gyoeva, and V.I. Gelfand. Proc. Natl. Acad. Sci. USA. 1991, 88:4956-4960]. Here, we have microinjected a dynein antibody (70.1) into cultured cod (Gadus morhua) melanophores and used the dynein inhibitor vanadate on permeabilized melanophores in skin pieces, to examine the role of the microtubule minus-end motor dynein in these cells. Both pigment granule aggregation and maintenance of the spherical central pigment mass (CPM) were inhibited by the antibody and by vanadate. Vanadate or antibody treatment of cells with aggregated pigment did not induce pigment dispersion. However, when the antibody-injected cells were induced to disperse pigment, the pigment moved farther to the cell periphery, which resulted in a depletion of pigment in the cell center. Similar superdispersion of previously uniformly distributed pigment was also seen when the antibody was injected in melanophores with dispersed pigment. Our results demonstrate that both pigment aggregation and maintenance of the CPM are dynein-dependent processes. Our data further show that dynein is involved in the homogeneous distribution of dispersed pigment. These results suggest that both dynein and kinesin are active in keeping pigment granules dispersed throughout the cytoplasm, transporting pigment granules in opposite directions. The possibility that dynein is continuously active during both aggregation and dispersion, while kinesin might be the target for regulation, is discussed. (C) 1997 Wiley-Liss, Inc.
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| 6. |
- Nilsson, H, et al.
(author)
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Localization of kinesin and cytoplasmic dynein in cultured melanophores from Atlantic cod, Gadus morhua.
- 1996
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In: Cell motility and the cytoskeleton. - 0886-1544. ; 33:3, s. 183-96
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Journal article (peer-reviewed)abstract
- In this study we have analyzed pigment translocation in cultured melanophores from the cold-tempered Atlantic cod, Gadus morhua. The transport process was found to be cold-adapted, as it proceeded at low temperatures. Both the typical morphology of the melanophores with long cytoplasmic processes, and the ability to translocate pigment granules, were found to be highly dependent on microtubules. Microtubules in melanophores were relatively stable to vinblastine treatment compared to microtubules in other skin cells. Extensive posttranslational modifications of tubulin were found. Detyrosinated and polyglutamylated microtubules were frequent, while acetylated microtubules only comprised a subpopulation or domains of microtubules. Both cod kinesin and dynein were distributed in a punctate pattern throughout the melanophores in close proximity to microtubules. The motors accumulated together with pigment granules during aggregation and were dispersed during translocation of pigment granules to the periphery. Individual melanosomes were occasionally found to rapidly change direction during translocation. Our data raise the interesting possibility that both kinesin and dynein are bound to pigment granules. This is of functional significance, since pigment granules are transported back and forth in the melanophores, and may be activated differently during aggregation and dispersion to generate translocation.
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| 7. |
- Nilsson, Helen, 1970, et al.
(author)
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Microtubule aster formation by dynein-dependent organelle transport.
- 1998
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In: Cell motility and the cytoskeleton. - 0886-1544. ; 41:3, s. 254-63
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Journal article (peer-reviewed)abstract
- The interplay between microtubules and the motor enzyme, cytoplasmic dynein, is essential for organisation of the cytoplasm, organelle transport, and cell division in eukaryotic cells. During mitosis, cytoplasmic dynein organises microtubules into two spindle pole asters, as well as the comparable multiple cytoplasmic asters induced by the microtubule-stabilising agent taxol. The mechanisms behind this cell cycle-regulated organisation are, however, not fully understood. We report here that the unidirectional dynein-dependent pigment organelle aggregation in taxol-treated melanophores from Atlantic cod, induces multiple microtubule asters. Usually, the pigment aggregates to a central pigment mass in the cell center, but pigment aggregation in taxol-treated cells induces formation of several peripheral pigment clusters that each localise to the center of a microtubule aster formation. When a cell with previously formed peripheral pigment clusters redisperse pigment, the asters disappear. Upon a subsequent reaggregation of the pigment, the aster formations reappear. The results indicate that the pigment aggregation process organises the microtubules into these formations. Immuno-electron microscopy of isolated pigment organelles indicates the presence of several dynein molecules on each pigment organelle, making it possible for each organelle to interact with several microtubules and thereby focusing microtubule minus ends. The possibility of unidirectional dynein-dependent organelle movement for organising microtubules into asters during cell division, and similarities in signal transduction between mitosis and pigment movement, are discussed.
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| 8. |
- Wallin, Margareta, 1952, et al.
(author)
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Coassembly of bovine and cod microtubule proteins: the ratio of the different tubulins within hybrid microtubules determines the ability to assemble at low temperatures, MAPs dependency and effects of Ca2+.
- 1997
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In: Cell motility and the cytoskeleton. - 0886-1544. ; 38:3, s. 297-307
-
Journal article (peer-reviewed)abstract
- Cod and bovine microtubule proteins (MTP) differ from each other in many respects, e.g., tubulin isoforms and microtubule-associated proteins (MAPs) but only cod MTP are cold-adapted. We used these differences to determine how tubulin isoform composition affects microtubule properties. Mixtures of cod and bovine MTP coassembled at 30 degrees C as shown by light scattering and immunoelectron microscopy, with no apparent preference for one set of MAPs over the other. Bovine tubulin was, in contrast to cod tubulin, unable to assemble in the absence of MAPs, while 50%/50% mixtures of bovine and cod tubulin, respectively, coassembled readily without exclusion of cod or bovine tubulin isoforms in the hybrids, as shown by two-dimensional gel electrophoresis. Alteration in MAPs dependency was also confirmed by the use of the MAPs-binding microtubule inhibitor estramustine phosphate. Addition of 10 mM Ca2+ to microtubules induced formation of spirals or rings depending on the ratio of the cod and bovine MTP, respectively. Bovine MTP were unable to assemble at low temperatures, while cod MTP are cold-adapted and assembled efficiently at 14 degrees C in the presence of MAPs. Amounts of cod MTP as low as 33% were enough to induce assembly of bovine/cod MTP hybrids. The critical concentration for assembly of a 50%/50% mixture was similar to that of 100% cod MTP. Taken together, the results show that the divergent cod and bovine MTP can coassemble, and that alterations in tubulin isotype/isoform composition above certain thresholds significantly modulate microtubule properties such as MAPs dependency, effects of Ca2+, and ability to assemble at low temperatures.
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