1,699 research outputs found
Role of the kinesin neck region in processive microtubule-based motility.
Kinesin is a dimeric motor protein that can move along a microtubule for several microns without releasing (termed processive movement). The two motor domains of the dimer are thought to move in a coordinated, hand-over-hand manner. A region adjacent to kinesin's motor catalytic domain (the neck) contains a coiled coil that is sufficient for motor dimerization and has been proposed to play an essential role in processive movement. Recent models have suggested that the neck enables head-to-head communication by creating a stiff connection between the two motor domains, but also may unwind during the mechanochemical cycle to allow movement to new tubulin binding sites. To test these ideas, we mutated the neck coiled coil in a 560-amino acid (aa) dimeric kinesin construct fused to green fluorescent protein (GFP), and then assayed processivity using a fluorescence microscope that can visualize single kinesin-GFP molecules moving along a microtubule. Our results show that replacing the kinesin neck coiled coil with a 28-aa residue peptide sequence that forms a highly stable coiled coil does not greatly reduce the processivity of the motor. This result argues against models in which extensive unwinding of the coiled coil is essential for movement. Furthermore, we show that deleting the neck coiled coil decreases processivity 10-fold, but surprisingly does not abolish it. We also demonstrate that processivity is increased by threefold when the neck helix is elongated by seven residues. These results indicate that structural features of the neck coiled coil, although not essential for processivity, can tune the efficiency of single molecule motility
Directional instability of microtubule transport in the presence of kinesin and dynein, two opposite polarity motor proteins.
Kinesin and dynein are motor proteins that move in opposite directions along microtubules. In this study, we examine the consequences of having kinesin and dynein (ciliary outer arm or cytoplasmic) bound to glass surfaces interacting with the same microtubule in vitro. Although one might expect a balance of opposing forces to produce little or no net movement, we find instead that microtubules move unidirectionally for several microns (corresponding to hundreds of ATPase cycles by a motor) but continually switch between kinesin-directed and dynein-directed transport. The velocities in the plus-end (0.2-0.3 microns/s) and minus-end (3.5-4 microns/s) directions were approximately half those produced by kinesin (0.5 microns/s) and ciliary dynein (6.7 microns/s) alone, indicating that the motors not contributing to movement can interact with and impose a drag upon the microtubule. By comparing two dyneins with different duty ratios (percentage of time spent in a strongly bound state during the ATPase cycle) and varying the nucleotide conditions, we show that the microtubule attachment times of the two opposing motors as well as their relative numbers determine which motor predominates in this assay. Together, these findings are consistent with a model in which kinesin-induced movement of a microtubule induces a negative strain in attached dyneins which causes them to dissociate before entering a force-generating state (and vice versa); reversals in the direction of transport may require the temporary dissociation of the transporting motor from the microtubule. The bidirectional movements described here are also remarkably similar to the back-and-forth movements of chromosomes during mitosis and membrane vesicles in fibroblasts. These results suggest that the underlying mechanical properties of motor proteins, at least in part, may be responsible for reversals in microtubule-based transport observed in cells
Purified plasma membranes inhibit polypeptide growth factor-induced DNA synthesis in subconfluent 3T3 cells.
Plasma membranes derived from NR-6 cells, a variant line of Swiss mouse 3T3 cells that does not have cell surface receptors for epidermal growth factor (EGF), inhibited EGF-induced stimulation of DNA synthesis by 50% in serum-starved, subconfluent 3T3 cells. Membranes derived from SV3T3 cells were much less effective in inhibiting EGF-induced DNA synthesis. This inhibition on DNA synthesis by NR-6 membranes was not a direct effect of membranes on EGF, nor could it be overcome by high concentrations of EGF. NR-6 membranes were most effective when added 3 h before EGF addition and had little effect when added 2 h or more after EGF. NR-6 membranes also reduced the stimulation of DNA synthesis induced by platelet-derived growth factor or fibroblast growth factor in serum-starved 3T3 cells. These findings indicate that membrane-membrane interactions between nontransformed cells may diminish their ability to proliferate in response to serum polypeptide growth factors
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Nucleotide specificity of the enzymatic and motile activities of dynein, kinesin, and heavy meromyosin.
The substrate specificities of dynein, kinesin, and myosin substrate turnover activity and cytoskeletal filament-driven translocation were examined using 15 ATP analogues. The dyneins were more selective in their substrate utilization than bovine brain kinesin or muscle heavy meromyosin, and even different types of dyneins, such as 14S and 22S dynein from Tetrahymena cilia and the beta-heavy chain-containing particle from the outer-arm dynein of sea urchin flagella, could be distinguished by their substrate specificities. Although bovine brain kinesin and muscle heavy meromyosin both exhibited broad substrate specificities, kinesin-induced microtubule translocation varied over a 50-fold range in speed among the various substrates, whereas heavy meromyosin-induced actin translocation varied only by fourfold. With both kinesin and heavy meromyosin, the relative velocities of filament translocation did not correlate well with the relative filament-activated substrate turnover rates. Furthermore, some ATP analogues that did not support the filament translocation exhibited filament-activated substrate turnover rates. Filament-activated substrate turnover and power production, therefore, appear to become uncoupled with certain substrates. In conclusion, the substrate specificities and coupling to motility are distinct for different types of molecular motor proteins. Such nucleotide "fingerprints" of enzymatic activities of motor proteins may prove useful as a tool for identifying what type of motor is involved in powering a motility-related event that can be reconstituted in vitro
Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells.
To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOH-terminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesin-like protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with translational regulation in certain mRNAs. After transient expression in CV-1 cells, the kinesin heavy chain showed both a diffuse distribution and a filamentous staining pattern that coaligned with microtubules but not vimentin intermediate filaments. Altering the number and distribution of microtubules with taxol or nocodazole produced corresponding changes in the localization of the expressed kinesin heavy chain. The expressed NH2-terminal motor and the COOH-terminal tail domains, but not the alpha-helical coiled coil rod domain, also colocalized with microtubules. The finding that both the kinesin motor and tail domains can interact with cytoplasmic microtubules raises the possibility that kinesin could crossbridge and induce sliding between microtubules under certain circumstances
Is the even distribution of insecticide-treated cattle essential for tsetse control? Modelling the impact of baits in heterogeneous environments
Background:
Eliminating Rhodesian sleeping sickness, the zoonotic form of Human African Trypanosomiasis, can be achieved only through interventions against the vectors, species of tsetse (Glossina). The use of insecticide-treated cattle is the most cost-effective method of controlling tsetse but its impact might be compromised by the patchy distribution of livestock. A deterministic simulation model was used to analyse the effects of spatial heterogeneities in habitat and baits (insecticide-treated cattle and targets) on the distribution and abundance of tsetse.
Methodology/Principal Findings:
The simulated area comprised an operational block extending 32 km from an area of good habitat from which tsetse might invade. Within the operational block, habitat comprised good areas mixed with poor ones where survival probabilities and population densities were lower. In good habitat, the natural daily mortalities of adults averaged 6.14% for males and 3.07% for females; the population grew 8.46in a year following a 90% reduction in densities of adults and pupae, but expired when the population density of males was reduced to <0.1/km2; daily movement of adults averaged 249 m for males and 367 m for females. Baits were placed throughout the operational area, or patchily to simulate uneven distributions of cattle and targets. Gaps of 2–3 km between baits were inconsequential provided the average imposed mortality per km2 across the entire operational area was maintained. Leaving gaps 5–7 km wide inside an area where baits killed 10% per day delayed effective control by 4–11 years. Corrective measures that put a few baits within the gaps were more effective than deploying extra baits on the edges.
Conclusions/Significance:
The uneven distribution of cattle within settled areas is unlikely to compromise the impact of insecticide-treated cattle on tsetse. However, where areas of >3 km wide are cattle-free then insecticide-treated targets should be deployed to compensate for the lack of cattle
Dynein structure and power stroke
Dynein ATPases are microtubule motors that are critical to diverse processes such as vesicle transport and the beating of sperm tails; however, their mechanism of force generation is unknown. Each dynein comprises a head, from which a stalk and a stem emerge. Here we use electron microscopy and image processing to reveal new structural details of dynein c, an isoform from Chlamydomonas reinhardtii flagella, at the start and end of its power stroke. Both stem and stalk are flexible, and the stem connects to the head by means of a linker approximately 10 nm long that we propose lies across the head. With both ADP and vanadate bound, the stem and stalk emerge from the head 10 nm apart. However, without nucleotide they emerge much closer together owing to a change in linker orientation, and the coiled-coil stalk becomes stiffer. The net result is a shortening of the molecule coupled to an approximately 15-nm displacement of the tip of the stalk. These changes indicate a mechanism for the dynein power stroke
Improving the Cost-Effectiveness of Artificial Visual Baits for Controlling the Tsetse Fly Glossina fuscipes fuscipes
Tsetse flies, which transmit sleeping sickness to humans and nagana to cattle, are commonly controlled by stationary artificial baits consisting of traps or insecticide-treated screens known as targets. In Kenya the use of electrocuting sampling devices showed that the numbers of Glossina fuscipes fuscipes (Newstead) visiting a biconical trap were nearly double those visiting a black target of 100 cm×100 cm. However, only 40% of the males and 21% of the females entered the trap, whereas 71% and 34%, respectively, alighted on the target. The greater number visiting the trap appeared to be due to its being largely blue, rather than being three-dimensional or raised above the ground. Through a series of variations of target design we show that a blue-and-black panel of cloth (0.06 m2) flanked by a panel (0.06 m2) of fine black netting, placed at ground level, would be about ten times more cost-effective than traps or large targets in control campaigns. This finding has important implications for controlling all subspecies of G. fuscipes, which are currently responsible for more than 90% of sleeping sickness cases
Changes in standard of candidates taking the MRCP(UK) Part 1 examination, 1985 to 2002: Analysis of marker questions
The maintenance of standards is a problem for postgraduate medical examinations, particularly if they use norm-referencing as the sole method of standard setting. In each of its diets, the MRCP(UK) Part 1 Examination includes a number of marker questions, which are unchanged from their use in a previous diet. This paper describes two complementary studies of marker questions for 52 diets of the MRCP(UK) Part 1 Examination over the years 1985 to 2001 to assess whether standards have changed
Optimal strategies for controlling riverine tsetse flies using targets: a modelling study
Background: Tsetse flies occur in much of sub-Saharan Africa where they transmit the trypanosomes that cause the diseases of sleeping sickness in humans and nagana in livestock. One of the most economical and effective methods of tsetse control is the use of insecticide-treated screens, called targets, that simulate hosts. Targets have been ~1m2, but recently it was shown that those tsetse that occupy riverine situations, and which are the main vectors of sleeping sickness, respond well to targets only ~0.06m2. The cheapness of these tiny targets suggests the need to reconsider what intensity and duration of target deployments
comprise the most cost-effective strategy in various riverine habitats.
Methodology/Principal Findings: A deterministic model, written in Excel spreadsheets and managed by Visual Basic for Applications, simulated the births, deaths and movement of tsetse confined to a strip of riverine vegetation composed of segments of habitat in which the tsetse population was either selfsustaining, or not sustainable unless supplemented by immigrants. Results suggested that
in many situations the use of tiny targets at high density for just a few months per year would be the most cost-effective strategy for rapidly reducing tsetse densities by the ~90% expected to have a great impact on the incidence of sleeping sickness. Local elimination of tsetse becomes feasible when targets are deployed in isolated situations, or where the only invasion occurs from populations that are not self-sustaining.
Conclusion/Significance: Seasonal use of tiny targets deserves field trials. The ability to recognise habitat that contains tsetse populations which are not self-sustaining could improve the planning of all methods of tsetse control, against any species, in riverine, savannah or forest situations. Criteria to assist such recognition are suggested
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