Mutations of oncoprotein 18/stathmin identify tubulin-directed regulatory activities distinct from tubulin association.
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Oncoprotein 18/stathmin (Op18) is a recently identified phosphorylation-responsive regulator of the microtubule (MT) system. It was originally proposed that Op18 specifically regulates dynamic properties of MTs by associating with tubulin, but it has subsequently been proposed that Op18 acts simply by sequestering of tubulin heterodimers. We have dissected the mechanistic action of Op18 by generation of two distinct classes of mutants. One class has interruptions of the heptad repeats of a potential coiled-coil region of Op18, and the other involves substitution at all four phosphorylation sites with negatively charged Glu residues. Both types of mutation result in Op18 proteins with a limited decrease in tubulin complex formation. However, the MT-destabilizing activities of the coiled-coil mutants are more severely reduced in transfected leukemia cells than those of the Glu-substituted Op18 derivative, providing evidence for tubulin-directed regulatory activities distinct from tubulin complex formation. Analysis of Op18-mediated regulation of tubulin GTPase activity and taxol-promoted tubulin polymerization showed that while wild-type and Glu-substituted Op18 derivatives are active, the coiled-coil mutants are essentially inactive. This suggests that Op18-tubulin contact involves structural motifs that deliver a signal of regulatory importance to the MT system. (+info)
A processive single-headed motor: kinesin superfamily protein KIF1A.
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A single kinesin molecule can move "processively" along a microtubule for more than 1 micrometer before detaching from it. The prevailing explanation for this processive movement is the "walking model," which envisions that each of two motor domains (heads) of the kinesin molecule binds coordinately to the microtubule. This implies that each kinesin molecule must have two heads to "walk" and that a single-headed kinesin could not move processively. Here, a motor-domain construct of KIF1A, a single-headed kinesin superfamily protein, was shown to move processively along the microtubule for more than 1 micrometer. The movement along the microtubules was stochastic and fitted a biased Brownian-movement model. (+info)
Vibrio parahaemolyticus thermostable direct hemolysin modulates cytoskeletal organization and calcium homeostasis in intestinal cultured cells.
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Vibrio parahaemolyticus is a marine bacterium known to be the leading cause of seafood gastroenteritis worldwide. A 46-kDa homodimer protein secreted by this microorganism, the thermostable direct hemolysin (TDH), is considered a major virulence factor involved in bacterial pathogenesis since a high percentage of strains of clinical origin are positive for TDH production. TDH is a pore-forming toxin, and its most extensively studied effect is the ability to cause hemolysis of erythrocytes from different mammalian species. Moreover, TDH induces in a variety of cells cytotoxic effects consisting mainly of cell degeneration which often leads to loss of viability. In this work, we examined the cellular changes induced by TDH in monolayers of IEC-6 cells (derived from the rat crypt small intestine), which represent a useful cell model for studying toxins from enteric bacteria. In experimental conditions allowing cell survival, TDH induces a rapid transient increase in intracellular calcium as well as a significant though reversible decreased rate of progression through the cell cycle. The morphological changes seem to be dependent on the organization of the microtubular network, which appears to be the preferential cytoskeletal element involved in the cellular response to the toxin. (+info)
Gibberellic acid stabilises microtubules in maize suspension cells to cold and stimulates acetylation of alpha-tubulin.
(4/10496)
Gibberellic acid is known to stabilise microtubules in plant organs against depolymerisation. We have now devised a simplified cell system for studying this. Pretreatment of a maize cell suspension with gibberellic acid for just 3 h stabilised protoplast microtubules against depolymerisation on ice. In other eukaryotes, acetylation of alpha-tubulin is known to correlate with microtubule stabilisation but this is not established in plants. By isolating the polymeric tubulin fraction from maize cytoskeletons and immunoblotting with the antibody 6-11B-1, we have demonstrated that gibberellic acid stimulates the acetylation of alpha-tubulin. This is the first demonstrated link between microtubule stabilisation and tubulin acetylation in higher plants. (+info)
Evidence for a correlation between the number of marginal band microtubules and the size of vertebrate erthrocytes.
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In 23 species of vertebrates the dimensions of erythrocytes and the number of their marginal band microtubules were examined. A positive correlation was found between the size of erythrocytes and the number of microtubules. The absence of microtubules in diskoid erythrocytes of mammals-Camelidae-is discussed. (+info)
The preprophase band: possible involvement in the formation of the cell wall.
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Numerous vesicles were observed among the microtubules of the "preprophase" band in prophase cells from root tips of Allium cepa. The content of these vesicles looks similar to the matrix of adjacent cell walls, and these vesicles often appear to be involved in exocytosis. In addition, the cell walls perpendicular to the plane of (beneath) the preprophase band are often differentially thickened compared to the walls lying parallel to the plane of the band. Our interpretation of these observations is that the preprophase band may direct or channel vesicles containing precursors of the cell wall to localized regions of wall synthesis. The incorporation of constituents of the cell wall into a narrow region defined by the position of the preprophase band may be a mechanism that ensures unidirecitonal growth of meristematic cells. (+info)
EB1, a protein which interacts with the APC tumour suppressor, is associated with the microtubule cytoskeleton throughout the cell cycle.
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The characteristics of the adenomatous polyposis coli (APC) associated protein EB1 were examined in mammalian cells. By immunocytochemistry EB1 was shown to be closely associated with the microtubule cytoskeleton throughout the cell cycle. In interphase cells EB1 was associated with microtubules along their full length but was often particularly concentrated at their tips. During early mitosis, EB1 was localized to separating centrosomes and associated microtubules, while at metaphase it was associated with the spindle poles and associated microtubules. During cytokinesis EB1 was strongly associated with the midbody microtubules. Treatment with nocodazole caused a diffuse redistribution of EB1 immunoreactivity, whereas treatment with cytochalasin D had no effect. Interestingly, treatment with taxol abolished the EB1 association with microtubules. In nocodazole washout experiments EB1 rapidly became associated with the centrosome and repolymerizing microtubules. In taxol wash-out experiments EB1 rapidly re-associated with the microtubule cytoskeleton, resembling untreated control cells within 10 min. Immunostaining of SW480 cells, which contain truncated APC incapable of interaction with EB1, showed that the association of EB1 with microtubules throughout the cell cycle was not dependent upon an interaction with APC. These results suggest a role for EB1 in the control of microtubule dynamics in mammalian cells. (+info)
Opposing motor activities of dynein and kinesin determine retention and transport of MHC class II-containing compartments.
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MHC class II molecules exert their function at the cell surface by presenting to T cells antigenic fragments that are generated in the endosomal pathway. The class II molecules are targetted to early lysosomal structures, termed MIIC, where they interact with antigenic fragments and are subsequently transported to the cell surface. We previously visualised vesicular transport of MHC class II-containing early lysosomes from the microtubule organising centre (MTOC) region towards the cell surface in living cells. Here we show that the MIIC move bidirectionally in a 'stop-and-go' fashion. Overexpression of a motor head-deleted kinesin inhibited MIIC motility, showing that kinesin is the motor that drives its plus end transport towards the cell periphery. Cytoplasmic dynein mediates the return of vesicles to the MTOC area and effectively retains the vesicles at this location, as assessed by inactivation of dynein by overexpression of dynamitin. Our data suggest a retention mechanism that determines the perinuclear accumulation of MIIC, which is the result of dynein activity being superior over kinesin activity. The bidirectional nature of MIIC movement is the result of both kinesin and dynein acting reciprocally on the MIIC during its transport. The motors may be the ultimate targets of regulatory kinases since the protein kinase inhibitor staurosporine induces a massive release of lysosomal vesicles from the MTOC region that is morphologically similar to that observed after inactivation of the dynein motor. (+info)