Correlation between myofilament response to Ca2+ and altered dynamics of contraction and relaxation in transgenic cardiac cells that express beta-tropomyosin.
(57/12201)
We compared the dynamics of the contraction and relaxation of single myocytes isolated from nontransgenic (NTG) mouse hearts and from transgenic (TG-beta-Tm) mouse hearts that overexpress the skeletal isoform of tropomyosin (Tm). Compared with NTG controls, TG-beta-Tm myocytes showed significantly reduced maximal rates of contraction and relaxation with no change in the extent of shortening. This result indicated that the depression in contraction dynamics determined in TG-beta-Tm isolated hearts is intrinsic to the cells. To further investigate the effect of Tm isoform switching on myofilament activity and regulation, we measured myofilament force and ATPase rate as functions of pCa (-log of [Ca2+]). Compared with controls, force generated by myofilaments from TG-beta-Tm hearts and myofibrillar ATPase activity were both more sensitive to Ca2+. However, the shift in pCa50 (half-maximally activating pCa) caused by changing sarcomere length from 1.8 to 2.4 microm was not significantly different between NTG and TG-beta-Tm fiber preparations. To test directly whether isoform switching affected the economy of contraction, force versus ATPase rate relationships were measured in detergent-extracted fiber bundles. In both NTG and TG-beta-Tm preparations, force and ATPase rate were linear and identically correlated, which indicated that crossbridge turnover was unaffected by Tm isoform switching. However, detergent extracted fibers from TG-beta-Tm demonstrated significantly less maximum tension and ATPase activity than NTG controls. Our results provide the first evidence that the Tm isoform population modulates the dynamics of contraction and relaxation of single myocytes by a mechanism that does not alter the rate-limiting step of crossbridge detachment. Our results also indicate that differences in sarcomere-length dependence of activation between cardiac and skeletal muscle are not likely due to differences in the isoform population of Tm. (+info)
Cytosolic ATPases, p97 and NSF, are sufficient to mediate rapid membrane fusion.
(58/12201)
Much recent work has focussed on the role of membrane-bound components in fusion. We show here that p97 and NSF are sufficient to mediate rapid membrane fusion. Fractionation of cytosol revealed that p97 and its co-factor, p47, constitutes the major fusion activity. This was confirmed by depleting p97 from the cytosol, which resulted in an 80% decrease in fusion. Using purified protein, p97 or NSF was found to be sufficient to mediate rapid fusion in an ATP-dependent manner. A regulatory role was observed for their corresponding co-factors, p47 and alpha-SNAP. When present at a molar ratio half of that of the ATPase, both co-factors increased fusion activity significantly. Intriguingly, at this ratio the ATPase activity of the complex measured in solution was at its lowest, suggesting that the co-factor stabilizes the ATP state. The fusion event involved mixing of both leaflets of the opposing membranes and contents of liposomes. We conclude from these data that p97, NSF and perhaps other related ATPases catalyse rapid and complete fusion between lipid bilayers on opposing membranes. This highlights a new role for p97 and NSF and prompts a re-evaluation of current fusion models. (+info)
Structures of P-type transporting ATPases and chromosomal locations of their genes.
(59/12201)
P-type ATPases (E1E2-ATPases) are primary active transporters which form phospho-intermediates during their catalytic cycle. They are classified into P1 to P4 based on the primary structure and potential transmembrane segments. Although the classic P-type ATPases are cation transporters, two new members have recently been found; one is a flippase catalyzing the flip-flop movement of aminophospholipids, but the substrate and function of the other one remain unknown. It would be interesting to determine whether the cations and aminophospholipids are transported by similar or different mechanisms. P-type ATPases are believed to have been derived from a common ancestor, and their genes are found to be distributed in various chromosomal loci. However, gene duplication events can be traced from the tandem arrangement of genes and their linkage map. Na+/K+- and H+/K+-ATPases have not only closely related a subunits but also similar beta subunits. Renal Na+/K+-ATPase has an additional subunit gamma. Similar small polypeptides (phospholemman, Mat-8 and CHIF), which induce Cl- and K+ currents, have been found. The idea of their functional and structural coupling with P-type ATPases, especially with H+/K+-ATPase, is intriguing. Each P-type ATPase must have specific domains or sequences for its intracellular trafficking (sorting, retention and recycling). Identification of such regions and studies on the molecules playing role in their recognition may facilitate the unveiling of various cellular processes regulated by P-type ATPases. (+info)
Transmembrane regulation of intracellular calcium by a plasma membrane sodium/calcium exchanger in mouse ova.
(60/12201)
Regulation of cytoplasmic free calcium concentration ([Ca2+)]i) is a key factor for maintenance of viability of cells, including oocytes. Indeed, during fertilization of an ovum, [Ca2+]i is known to undergo oscillations, but it is unknown how basal [Ca2+]i or calcium oscillations are regulated. In the present study we investigated the role of the plasma membrane in regulating [Ca2+]i of metaphase II-arrested mouse oocytes (ova). Ova were collected from B6C3F1 mice treated with eCG (10 IU) and hCG (5 IU), and intracellular calcium was determined by means of fura-2. Extracellular calcium flux across the zona pellucida was detected noninvasively by a calcium ion-selective, self-referencing microelectrode that was positioned by a computer-controlled micromanipulator. Under basal conditions ova exhibited a calcium net efflux of 20.6 +/- 5.2 fmol/cm2 per sec (n = 69). Treatment of ova with ethanol (7%) or thapsigargin (25 nM-2.5 microM) transiently increased intracellular calcium and stimulated calcium efflux that paralleled levels of [Ca2+]i. The presence of a Na+/Ca2+ exchanger was indicated by experiments employing both bepridil, an inhibitor of Na+/Ca2+ exchange, and sodium-depleted media. In the presence of bepridil, a net influx of calcium was revealed across the zona pellucida, which was reflected by an increase in the [Ca2+]i. In addition, replenishment of extracellular sodium to ova that had been incubated in sodium-depleted media induced a large calcium efflux, consistent with the actions of Na+/Ca2+ exchange. Sodium/calcium exchange in mouse ova may be an important mechanism that regulates [Ca2+]i. (+info)
An essential role for katanin in severing microtubules in the neuron.
(61/12201)
Several lines of evidence suggest that microtubules are nucleated at the neuronal centrosome, and then released for transport into axons and dendrites. Here we sought to determine whether the microtubule-severing protein known as katanin mediates microtubule release from the neuronal centrosome. Immunomicroscopic analyses on cultured sympathetic neurons show that katanin is present at the centrosome, but is also widely distributed throughout the neuron. Microinjection of an antibody that inactivates katanin results in a dramatic accumulation of microtubules at the centrosome, indicating that katanin is indeed required for microtubule release from the centrosome. However, the antibody also causes an inhibition of axon outgrowth that is more immediate than expected on this basis alone. It may be that katanin severs microtubules throughout the cell body to keep them sufficiently short to be efficiently transported into developing processes. Consistent with this idea, there were significantly fewer free ends of microtubules in the cell bodies of neurons that had been injected with the katanin antibody compared with controls. These results indicate that microtubule-severing by katanin is essential for releasing microtubules from the neuronal centrosome, and also for regulating the length of the microtubules after their release. (+info)
Role of the copper-binding domain in the copper transport function of ATP7B, the P-type ATPase defective in Wilson disease.
(62/12201)
We have analyzed the functional effect of site-directed mutations and deletions in the copper-binding domain of ATP7B (the copper transporting P-type ATPase defective in Wilson disease) using a yeast complementation assay. We have shown that the sixth copper-binding motif alone is sufficient, but not essential, for normal ATP7B function. The N-terminal two or three copper-binding motifs alone are not sufficient for ATP7B function. The first two or three N-terminal motifs of the copper-binding domain are not equivalent to, and cannot replace, the C-terminal motifs when placed in the same sequence position with respect to the transmembrane channel. From our data, we propose that the copper-binding motifs closest to the channel are required for the copper-transport function of ATP7B. We propose that cooperative copper binding to the copper-binding domain of ATP7B is not critical for copper transport function, but that cooperative copper binding involving the N-terminal two or three copper-binding motifs may be involved in initiating copper-dependent intracellular trafficking. Our data also suggest a functional difference between the copper-binding domains of ATP7A and ATP7B. (+info)
Escherichia coli DNA helicase II is active as a monomer.
(63/12201)
Helicases are thought to function as oligomers (generally dimers or hexamers). Here we demonstrate that although Escherichia coli DNA helicase II (UvrD) is capable of dimerization as evidenced by a positive interaction in the yeast two-hybrid system, gel filtration chromatography, and equilibrium sedimentation ultracentrifugation (Kd = 3.4 microM), the protein is active in vivo and in vitro as a monomer. A mutant lacking the C-terminal 40 amino acids (UvrDDelta40C) failed to dimerize and yet was as active as the wild-type protein in ATP hydrolysis and helicase assays. In addition, the uvrDDelta40C allele fully complemented the loss of helicase II in both methyl-directed mismatch repair and excision repair of pyrimidine dimers. Biochemical inhibition experiments using wild-type UvrD and inactive UvrD point mutants provided further evidence for a functional monomer. This investigation provides the first direct demonstration of an active monomeric helicase, and a model for DNA unwinding by a monomer is presented. (+info)
Vesicular ATPase-overexpressing cells determine the distribution of malaria parasite oocysts on the midguts of mosquitoes.
(64/12201)
In Plasmodium-infected mosquitoes, oocysts are preferentially located at the posterior half of the posterior midgut. Because mosquitoes rest vertically after feeding, the effect of gravity on the ingested blood has been proposed as the cause of such a biased distribution. In this paper, we examined the oocyst distribution on the midguts of mosquitoes that were continuously rotated to nullify the effect of gravity and found that the typical pattern of oocyst distribution did not change. Invasion of the midgut epithelium by ookinetes was similarly found to be biased toward the posterior part of the posterior midgut. We examined whether the distribution of oocysts depends on the distribution of vesicular ATPase (V-ATPase)-overexpressing cells that Plasmodium ookinetes preferentially use to cross the midgut epithelium. An antiserum raised against recombinant Aedes aegypti V-ATPase B subunit indicated that the majority of V-ATPase-overexpressing cells in Ae. aegypti and Anopheles gambiae are localized at the posterior part of the posterior midgut. We propose that the typical distribution of oocysts on the mosquito midgut is attributable to the presence and the spatial distribution of the V-ATPase-overexpressing cells in the midgut epithelium. (+info)