Microtubule-associated protein 4 (MAP4) regulates assembly, protomer-polymer partitioning and synthesis of tubulin in cultured cells.
(41/1933)
We depleted MAP4, a ubiquitously expressed microtubule (MT)-associated protein previously shown to be capable of stabilizing MTs, from HeLa cells by stably expressing antisense RNA. These HeLa-AS cells, in which the MAP4 level was decreased to 33% of the wild-type level, displayed decreased content of total tubulin (65% of the wild-type level). The partitioning of cellular tubulin into protomer and polymer was altered in HeLa-AS cells: polymeric tubulin was decreased to 46% of the level in control cells, while protomeric tubulin was increased to 226% of the level in control cells. Tubulin protein synthesis was decreased, consistent with the tubulin autoregulation model, which proposes that tubulin protomer inhibits its own synthesis. Following release from drug-induced depolymerization, MTs in HeLa-AS cells reformed more slowly, and showed an increased focus on the centrosome, as compared to control cells. HeLa-AS cells also appeared to be less bipolar in shape and flatter than control cells. Our data suggest that MAP4 regulates assembly level of MTs and, perhaps through this mechanism, is involved in controlling spreading and shape of cells. (+info)
(-)-Phenylahistin arrests cells in mitosis by inhibiting tubulin polymerization.
(42/1933)
(-)-Phenylahistin, a fungal diketopiperazine metabolite composed of phenylalanine and isoprenylated dehydrohistidine, arrested cells in mitosis and inhibited the proliferation of A549 cells. The microtubule network in A549 cells was disrupted by (-)-phenylahistin, which also inhibited the polymerization of both microtubule protein from bovine brain and phosphocellulose-purified tubulin in vitro. Competitive binding studies indicated that (-)-phenylahistin interacted with the colchicine binding site on tubulin but not with the vinblastine binding site. (+info)
Polymer-in-a-box mechanism for the thermal stabilization of collagen molecules in fibers.
(43/1933)
Collagen molecules in solution unfold close to the maximum body temperature of the species of animal from which the molecules are extracted. It is therefore vital that collagen is stabilized during fiber formation. In this paper, our concept that the collagen molecule is thermally stabilized by loss of configurational entropy of the molecule in the fiber lattice, is refined by examining the process theoretically. Combining an equation for the entropy of a polymer-in-a-box with our previously published rate theory analysis of collagen denaturation, we have derived a hyperbolic relationship between the denaturation temperature, Tm, and the volume fraction, epsilon, of water in the fiber. DSC data were consistent with the model for water volume fractions greater than 0.2. At a water volume fraction of about 0.2, there was an abrupt change in the slope of the linear relationship between 1/Tm and epsilon. This may have been caused by a collapse of the gap-overlap fiber structure at low hydrations. At more than 6 moles water per tripeptide, the enthalpy of denaturation on a dry tendon basis was independent of hydration at 58.55 +/- 0.59 J g-1. Between about 6 and 1 moles water per tripeptide, dehydration caused a substantial loss of enthalpy of denaturation, caused by a loss of water bridges from the hydration network surrounding the triple helix. At very low hydrations (less than 1 mole of water per tripeptide), where there was not enough water to form bridges and only sufficient to hydrogen bond to primary binding sites on the peptide chains, the enthalpy was approximately constant at 11.6 +/- 0.69 J g-1. This was assigned mainly to the breaking of the direct hydrogen bonds between the alpha chains. (+info)
Water in actin polymerization.
(44/1933)
We have addressed the question whether water is part of the G- to F-actin polymerization reaction. Under osmotic stress, the critical concentration for G-Ca-ATP actin was reduced for six different osmolytes. These results are interpreted as showing that reducing water activity favored the polymerized state. The magnitude of the effect correlated, then saturated, with increasing MW of the osmolyte and suggested that up to 10-12 fewer water molecules were associated with actin when it polymerized. By contrast, osmotic effects were insignificant for Mg-ATP actin. The nucleotide binding site of the Mg conformation is more closed than the Ca and more closely resembles the closed actin conformation in the polymerized state. These results suggest that the water may come from the cleft of the nucleotide binding site. (+info)
The Drosophila forked protein induces the formation of actin fiber bundles in vertebrate cells.
(45/1933)
The forked protein is an actin binding protein involved in the formation of large actin fiber bundles in developing Drosophila bristles. These are the largest example of a type of actin bundle characterized by parallel, hexagonally packed actin fibers, also found in intestinal microvilli, kidney proximal tubule microvilli, and stereocilia in the ear. Understanding how these structures are constructed and how that construction is regulated is an important question in cell and developmental biology. Because the timing of forked gene expression coincides with the formation of the actin fiber bundles, and since the forked protein is localized at the site of initiation of these bundles before they form, it has been proposed that the forked protein is an initiator of actin bundle formation. In this paper we show that the forked protein can induce the formation of bundles and increase actin polymerization in vertebrate cells. We use this system to identify regions of the forked protein which are essential for bundle formation and actin co-localization. (+info)
Hyaluronan stimulates tumor cell migration by modulating the fibrin fiber architecture.
(46/1933)
The glycosaminoglycan hyaluronan, which supports tumor cell migration and metastasis, interferes with fibrin polymerization and leads to increased fiber size and porosity of fibrin clots. Here we have studied the proportionate effect of fibrin polymerization on hyaluronan-mediated migration of glioblastoma cells. The structural and physical properties of hyaluronan-containing fibrin gels were analyzed by turbidity measurement, laser scanning microscopy, compaction assay, and calculation of pore size by liquid permeation. When fibrin polymerized in the presence of hyaluronan or dextran, the resulting gels strongly stimulated cell migration, and migration significantly correlated with fiber mass-to-length ratios and pore diameters. In contrast, cell migration was not induced by addition of hyaluronan to supernatants of already polymerized gels. Hyaluronan-mediated migration was inhibited in fibrin gels by antibodies to alphav- and beta1integrins and the disintegrin echistatin, but not by antibodies to the hyaluronan receptor CD44 (up to 50 microg/ml). As a control, we show that anti-CD44 (10 microg/ml) inhibited cell migration on a pure hyaluronan matrix using a two-dimensional Boyden chamber system. In contrast to three-dimensional migration, the migration of cells on the surfaces of variably structured fibrin gels was not significantly different, indicating that increased gel permeability (porosity) may account for hyaluronan-mediated migration. We conclude that, in complex three-dimensional substrates, the predominant effect of hyaluronan on cell migration might be indirect and requires modulation of fibrin polymerization. (+info)
Vinblastine induces an interaction between FtsZ and tubulin in mammalian cells.
(47/1933)
The Escherichia coli cell division protein FtsZ was expressed in Chinese hamster ovary cells, where it formed a striking array of dots that were independent of the mammalian cytoskeleton. Although FtsZ appears to be a bacterial homolog of tubulin, its expression had no detectable effects on the microtubule network or cell growth. However, treatment of the cells with vinblastine at concentrations that caused microtubule disassembly rapidly induced a network of FtsZ filaments that grew from and connected the dots, suggesting that the dots are an active storage form of FtsZ. Cells producing FtsZ also exhibited vinblastine- and calcium-resistant tubulin polymers that colocalized with the FtsZ network. The FtsZ polymers could be selectively disassembled, indicating that the two proteins were not copolymerized. The vinblastine effects were readily reversible by washing out the drug or by treating the cells with the vinblastine competitor, maytansine. These results demonstrate that FtsZ assembly can occur in the absence of bacterial chaperones or cofactors, that FtsZ and tubulin do not copolymerize, and that tubulin-vinblastine complexes have an enhanced ability to interact with FtsZ. (+info)
Cutting edge: trimolecular interaction of TCR with MHC class II and bacterial superantigen shows a similar affinity to MHC:peptide ligands.
(48/1933)
Bacterial superantigens such as Staphylococcus aureus enterotoxin A (SEA) are very potent stimulators of T cells. They bind to the Vbeta region of the TCR and to MHC class II, stimulating T cells at nanomolar concentrations. Using surface plasmon resonance measurements, we find that binding between the individual components of the complex (TCR-class II, TCR-SEA, SEA-class II) is very weak, but that the stability of the trimolecular complex is considerably enhanced, reaching an affinity similar to that found for TCR interactions with MHC:peptide ligand. Thus, the potency of SEA in stimulation of T cells is not due to particularly strong affinities between the proteins, but to a cooperative effect of interactions in the TCR-SEA-MHC class II trimolecular complex that brings the kinetics into a similar range to binding of conventional Ags. This range may be the optimum for T cell activation. (+info)