| 1. |
- Amin, M., et al.
(författare)
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Treponema denticola Msp-deduced peptide conjugate, P34BSA, promotes RhoA-dependent actin stress fiber formation independent of its internalization by fibroblasts
- 2008
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Ingår i: Cell Motility and the Cytoskeleton. - : Wiley. - 0886-1544 .- 1097-0169. ; 65:5, s. 406-421
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Tidskriftsartikel (refereegranskat)abstract
- P34BSA, a BSA conjugate of a synthetic 10-mer peptide deduced from Treponema denticola major outer sheath protein (Msp), stabilizes actin filaments in fibroblasts and retards cell motility. We reported previously that it is internalized by cells, binds and bundles actin filaments in vitro, and activates RhoA, yet, its site and mechanism of action were not defined. We have assessed P34BSA's modes of interaction with and signaling to fibroblasts. At 4°C, P34BSA was not internalized, but it bound to the plasma membrane and promoted actin stress fiber formation at ~80% capacity compared with 37°C controls, casting doubt that cellular uptake is a critical step for its cytoskeleton-stabilizing property. In Rho G-LISA™ and co-immunoprecipitation assays, P34BSA was found to activate RhoA, even at 4°C, to promote its interaction with guanosine nucleotide exchange factor p114RhoGEF. It also caused phosphorylation of cofilin. Upon RhoA inhibition, either by C3 transferase RhoA inhibitor or by transfection with a dominant negative RhoA construct, P34BSA did not achieve the stress fiber formation seen with P34BSA alone. By inhibiting phosphatidylinositol-3 kinase (PI 3-K) with LY294002, the P34BSA effects were completely blocked. Depletion of cholesterol with methyl-ß-cyclodextrin (MßCD) partially inhibited P34BSA signaling via the plasma membrane to the cytoskeleton. This suggests that multivalent P34BSA activation of lipid raft components requires active PI 3-K, and initiates the pathway through a RhoGEF and RhoA, which mediates stress fiber formation in fibroblasts. Hence, P34BSA may represent a novel tool to investigate RhoA-dependent processes, such as remodeling filamentous actin in eukaryotic cells. © 2008 Wiley-Liss, Inc.
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| 2. |
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| 3. |
- Aspengren, Sara, 1977, et al.
(författare)
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Effects of acrylamide, latrunculin, and nocodazole on intracellular transport and cytoskeletal organization in melanophores
- 2006
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Ingår i: Cell Motility and the Cytoskeleton. - : Wiley. - 0886-1544 .- 1097-0169. ; 63:7, s. 423-436
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Tidskriftsartikel (refereegranskat)abstract
- The effects of acrylamide (ACR), nocodazole, and latrunculin were studied on intracellular transport and cytoskeletel morphology in cultured Xenopus laevis melanophores, cells that are specialized for regulated and bidirectional melanosome transport. We used three different methods; light microscopy, fluorescence microscopy, and spectrophotometry. ACR affected the morphology of both microtubules and actin filaments in addition to inhibiting retrograde transport of melanosomes but leaving dispersion unaffected. Using the microtubule-inhibitor nocodazole and the actin filament-inhibitor latrunculin we found that microtubules and actin filaments are highly dependent on each other, and removing either component dramatically changed the organization of the other. Both ACR and latrunculin induced bundling of microtubules, while nocodazole promoted formation of filaments resembling stress fibers organized from the cell center to the periphery. Removal of actin filaments inhibited dispersion of melanosomes, further concentrated the central pigment mass in aggregated cells, and induced aggregation even in the absence of melatonin. Nocodazole, on the other hand, prevented aggregation and caused melanosomes to cluster and slowly disperse. Dispersion of nocodazole-treated cells was induced upon addition of alpha-melanocyte-stimulating hormone (MSH), showing that dispersion can proceed in the absence of microtubules, but the distribution pattern was altered. It is well established that ACR has neurotoxic effects, and based on the results in the present study we suggest that ACR has several cellular targets of which the minus-end microtubule motor dynein and the melatonin receptor might be involved. When combining morphological observations with qualitative and quantitative measurements of intracellular transport, melanophores provide a valuable model system for toxicological studies.
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| 4. |
- Billger, Martin, et al.
(författare)
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Dynamic instability of microtubules from cold-living fishes.
- 1994
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Ingår i: Cell motility and the cytoskeleton. - : Wiley. - 0886-1544 .- 1097-0169. ; 28:4, s. 327-32
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Tidskriftsartikel (refereegranskat)abstract
- The dynamic instability of microtubules free of microtubule-associated proteins from two genera of cold-living fishes was measured, by means of video-enhanced differential-interference-contrast microscopy, at temperatures near those of their habitats. Brain microtubules were isolated from the boreal Atlantic cod (Gadus morhua; habitat temperature approximately 2-15 degrees C) and from two austral Antarctic rockcods (Notothenia gibberifrons and N. coriiceps neglecta; habitat temperature approximately -1.8 to + 2 degrees C). Critical concentrations for polymerization of the fish tubulins were in the neighborhood of 1 mg/ml, consistent with high interdimer affinities. Rates of elongation and frequencies of growth-to-shortening transitions ("catastrophes") for fish microtubules were significantly smaller than those for mammalian microtubules. Slow dynamics is therefore an intrinsic property of these fish tubulins, presumably reflecting their adaptation to low temperatures. Two-dimensional electrophoresis showed striking differences between the isoform compositions of the cod and the rockcod tubulins, which suggests that the cold-adapted microtubule phenotypes of northern and southern fishes may have arisen independently.
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| 5. |
- de Pereda, J M, et al.
(författare)
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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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Ingår i: Cell motility and the cytoskeleton. - : Wiley. - 0886-1544 .- 1097-0169. ; 30:2, s. 153-63
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Tidskriftsartikel (refereegranskat)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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| 6. |
- Fridén, B, et al.
(författare)
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Different assembly properties of cod, bovine, and rat brain microtubules.
- 1992
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Ingår i: Cell motility and the cytoskeleton. - : Wiley. - 0886-1544. ; 21:4, s. 305-12
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Tidskriftsartikel (refereegranskat)abstract
- Assembly properties of cod, bovine, and rat brain microtubules were compared. Estramustine phosphate, heparin, poly-L-aspartic acid, as well as NaCl, inhibited the assembly and disassembled both bovine and rat microtubules by inhibition of the binding between tubulin and MAPs. The assembly of cod brain microtubules was in contrast only marginally affected by these agents, in spite of a release of the MAPs. The results suggest that cod tubulin has a high intrinsic ability to assemble. This was confirmed by studies on phosphocellulose-purified cod tubulin, since the critical concentration for assembly was independent of the presence or absence of MAPs. The results show therefore that cod brain tubulin has, in contrast to bovine and rat brain tubulins, a high propensity to assembly under conditions which normally require the presence of MAPs. Even if cod MAPs, which have an unusual protein composition, were not needed for the assembly of cod microtubules, they were able to induce assembly of bovine brain tubulin. Both cod and bovine MAPs bound to cod microtubules, and bovine MAP1 and MAP2 bound to, and substituted at least the 400 kDa cod protein. This suggests that the tubulin-binding sites and the assembly-stimulatory ability of MAPs are common properties of MAPs from different species, independent of the tubulin assembly propensity.
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| 7. |
- Klotz, A, et al.
(författare)
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Polyglutamylation of atlantic cod tubulin: immunochemical localization and possible role in pigment granule transport.
- 1999
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Ingår i: Cell motility and the cytoskeleton. - 0886-1544. ; 44:4, s. 263-73
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Tidskriftsartikel (refereegranskat)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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| 8. |
- Loitto, Vesa, et al.
(författare)
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Water Flux in Cell Motility : Expanding the Mechanisms of Membrane Protrusion
- 2009
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Ingår i: CELL MOTILITY AND THE CYTOSKELETON. - : Wiley. - 0886-1544 .- 1097-0169. ; 66:5, s. 237-247
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Forskningsöversikt (refereegranskat)abstract
- Transmembrane water fluxes through aquaporins (AQPs) are suggested to play, pivotal roles in cell polarization and directional cell motility. Local dilution by W water influences the dynamics of the subcortical actin polymerization and directs the formation of nascent membrane protrusions. In this paper. recent evidence is discussed in support of such a central role of AQP in membrane protrusion formation, and cell migration as a basis for our Understanding AQP9 Underlying molecular mechanisms of directional motility. Specifically. AQP9 in a physiological context controls transmembrane water fluxes driving, membrane protrusion formation, as an initial cellular response to a chemoattractant or other migratory signals. The importance of AQP-facilitated water fluxes in directional cell motility is underscored the observation that blocking or modifying specific sites in AQP9 also interferes with the molecular machinery that govern actin-mediated cellular shape changes. Cell Motil.
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| 9. |
- Modig, C, et al.
(författare)
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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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Ingår i: Cell motility and the cytoskeleton. - 0886-1544. ; 42:4, s. 315-30
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Tidskriftsartikel (refereegranskat)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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| 10. |
- Modig, C, et al.
(författare)
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MAP 0, a 400-kDa microtubule-associated protein unique to teleost fish.
- 1997
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Ingår i: Cell motility and the cytoskeleton. - 0886-1544. ; 38:3, s. 258-69
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Tidskriftsartikel (refereegranskat)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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