scholarly journals A Metastable Intermediate State of Microtubule Dynamic Instability That Differs Significantly between Plus and Minus Ends

1997 ◽  
Vol 138 (1) ◽  
pp. 105-117 ◽  
Author(s):  
P.T. Tran ◽  
R.A. Walker ◽  
E.D. Salmon
Keyword(s):  
Intermediate State ◽  
Transition Rate ◽  
Original Work ◽  
Dynamic Instability ◽  
Kinetic Properties ◽  
Uv Damage ◽  
Microtubule Dynamic ◽  
Cap Model ◽  
The Stability ◽  
Mechanical Cutting

The current two-state GTP cap model of microtubule dynamic instability proposes that a terminal crown of GTP-tubulin stabilizes the microtubule lattice and promotes elongation while loss of this GTP-tubulin cap converts the microtubule end to shortening. However, when this model was directly tested by using a UV microbeam to sever axoneme-nucleated microtubules and thereby remove the microtubule's GTP cap, severed plus ends rapidly shortened, but severed minus ends immediately resumed elongation (Walker, R.A., S. Inoué, and E.D. Salmon. 1989. J. Cell Biol. 108: 931–937). To determine if these previous results were dependent on the use of axonemes as seeds or were due to UV damage, or if they instead indicate an intermediate state in cap dynamics, we performed UV cutting of self-assembled microtubules and mechanical cutting of axoneme-nucleated microtubules. These independent methods yielded results consistent with the original work: a significant percentage of severed minus ends are stable after cutting. In additional experiments, we found that the stability of both severed plus and minus ends could be increased by increasing the free tubulin concentration, the solution GTP concentration, or by assembling microtubules with guanylyl-(α,β)-methylene-diphosphonate (GMPCPP). Our results show that stability of severed ends, particularly minus ends, is not an artifact, but instead reveals the existence of a metastable kinetic intermediate state between the elongation and shortening states of dynamic instability. The kinetic properties of this intermediate state differ between plus and minus ends. We propose a three-state conformational cap model of dynamic instability, which has three structural states and four transition rate constants, and which uses the asymmetry of the tubulin heterodimer to explain many of the differences in dynamic instability at plus and minus ends.

scholarly journals A Lateral Cap model of microtubule dynamic instability

FEBS Letters ◽  
1989 ◽  
Vol 259 (1) ◽  
pp. 181-184 ◽  
Author(s):  
Peter Bayley ◽  
Maria Schilstra ◽  
Stephen Martin
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Cap Model

scholarly journals The effect of solution composition on microtubule dynamic instability

1991 ◽  
Vol 277 (3) ◽  
pp. 839-847 ◽  
Author(s):  
M J Schilstra ◽  
P M Bayley ◽  
S R Martin
Keyword(s):  
Rate Constant ◽  
Dynamic Instability ◽  
Dynamic Properties ◽  
Intrinsic Rate ◽  
Dissociation Rate ◽  
Microtubule Dynamic ◽  
Exchange Method ◽  
Dynamic Activity ◽  
Cap Model ◽  
Good Agreement

The exchange of tubulin dimer into steady-state microtubules was studied over a range of solution conditions, in order to assess the effects of various common buffer components on the dynamic instability of microtubules. In comparison with standard buffer conditions (100 mM-Pipes buffer, pH 6.5, containing 0.1 mM-EGTA, 1.8 mM-MgC12 and 1 M-glycerol), the rate and extent of exchange, and thus of dynamic instability, are suppressed by increasing the concentration of glycerol above 2 M. Exchange is enhanced by the addition of further Mg2+ (up to 17 mM) or by the addition of Ca2+ (up to 0.4 mM). Phosphate ion (150 mM) has relatively little effect on the dynamic behaviour of microtubules, as judged by the exchange method. The findings are interpreted within the framework of the Lateral Cap model for microtubule dynamic instability, in terms of the effects of these changes on the intrinsic rate constants of the system. By contrast, the extent of tubulin exchange depends selectively on the value of the dissociation rate constant for tubulin-GDP. A decrease in the extent of exchange, and hence in dynamic activity, is associated with a decreased value for this rate constant, and vice versa. The results also show good agreement of predictions of the model in treating the observed variations in the dynamic properties of individual microtubules, induced by different solution conditions.

scholarly journals Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro.

1989 ◽  
Vol 108 (3) ◽  
pp. 931-937 ◽  
Author(s):  
R A Walker ◽  
S Inoué ◽  
E D Salmon
Keyword(s):  
Sea Urchin ◽  
Molecular Basis ◽  
Dynamic Instability ◽  
Second Step ◽  
Microtubule Dynamic ◽  
Asymmetric Behavior ◽  
Ultraviolet Microbeam ◽  
Cap Model ◽  
Flagellar Axoneme

The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a "GTP cap." A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules. Phosphocellulose-purified tubulin was assembled onto the plus and minus ends of sea urchin flagellar axoneme fragments at 21-22 degrees C. The assembly dynamics of individual microtubules were recorded in real time using video microscopy. When the tip of an elongating plus end microtubule was cut off, the severed plus end microtubule always rapidly shortened back to the axoneme at the normal plus end rate. However, when the distal tip of an elongating minus end microtubule was cut off, no rapid shortening occurred. Instead, the severed minus end resumed elongation at the normal minus end rate. Our results show that some form of "stabilizing cap," possibly a GTP cap, governs the transition (catastrophe) from elongation to rapid shortening at the plus end. At the minus end, a simple GTP cap is not sufficient to explain the observed behavior unless UV induces immediate recapping of minus, but not plus, ends. Another possibility is that a second step, perhaps a structural transformation, is required in addition to GTP cap loss for rapid shortening to occur. This transformation would be favored at plus, but not minus ends, to account for the asymmetric behavior of the ends.

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

scholarly journals Temperature-jump studies of microtubule dynamic instability.

1988 ◽  
Vol 263 (21) ◽  
pp. 10344-10352
Author(s):  
M Caplow ◽  
J Shanks ◽  
R L Ruhlen
Keyword(s):  
Temperature Jump ◽  
Dynamic Instability ◽  
Microtubule Dynamic

scholarly journals Influence of rapid changes in cytosolic pH on oxidative phosphorylation in skeletal muscle: theoretical studies

2002 ◽  
Vol 365 (1) ◽  
pp. 249-258 ◽  
Author(s):  
Bernard KORZENIEWSKI ◽  
Jerzy A. ZOLADZ
Keyword(s):  
Skeletal Muscle ◽  
Creatine Kinase ◽  
Oxidative Phosphorylation ◽  
Kinetic Properties ◽  
Main Role ◽  
Anaerobic Glycolysis ◽  
Cytosolic Ph ◽  
Intensive Exercise ◽  
Proton Production ◽  
The Stability

Cytosolic pH in skeletal muscle may vary significantly because of proton production/consumption by creatine kinase and/or proton production by anaerobic glycolysis. A computer model of oxidative phosphorylation in intact skeletal muscle developed previously was used to study the kinetic effect of these variations on the oxidative phosphorylation system. Two kinds of influence were analysed: (i) via the change in pH across the inner mitochondrial membrane and (ii) via the shift in the equilibrium of the creatine kinase-catalysed reaction. Our simulations suggest that cytosolic pH has essentially no impact on the steady-state fluxes and most metabolite concentrations. On the other hand, rapid acidification/alkalization of cytosol causes a transient decrease/increase in the respiration rate. Furthermore, changes in pH seem to affect significantly the kinetic properties of transition between resting state and active state. An increase in pH brought about by proton consumption by creatine kinase at the onset of exercise lengthens the transition time. At intensive exercise levels this pH increase could lead to loss of the stability of the system, if not compensated by glycolytic H+ production. Thus our theoretical results stress the importance of processes/mechanisms that buffer/compensate for changes in cytosolic proton concentration. In particular, we suggest that the second main role of anaerobic glycolysis, apart from additional ATP supply, may be maintaining the stability of the system at intensive exercise.

scholarly journals Motile kinetochores and polar ejection forces dictate chromosome position on the vertebrate mitotic spindle

1994 ◽  
Vol 124 (3) ◽  
pp. 223-233 ◽  
Author(s):  
CL Rieder ◽  
ED Salmon
Keyword(s):  
Mitotic Spindle ◽  
Constant Velocity ◽  
Molecular Mechanisms ◽  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Pole Motion ◽  
Microtubule Motors ◽  
Chromosome Position ◽  
Pulling Forces

We argue that hypotheses for how chromosomes achieve a metaphase alignment, that are based solely on a tug-of-war between poleward pulling forces produced along the length of opposing kinetochore fibers, are no longer tenable for vertebrates. Instead, kinetochores move themselves and their attached chromosomes, poleward and away from the pole, on the ends of relatively stationary but shortening/elongating kinetochore fiber microtubules. Kinetochores are also "smart" in that they switch between persistent constant-velocity phases of poleward and away from the pole motion, both autonomously and in response to information within the spindle. Several molecular mechanisms may contribute to this directional instability including kinetochore-associated microtubule motors and kinetochore microtubule dynamic instability. The control of kinetochore directional instability, to allow for congression and anaphase, is likely mediated by a vectorial mechanism whose magnitude and orientation depend on the density and orientation or growth of polar microtubules. Polar microtubule arrays have been shown to resist chromosome poleward motion and to push chromosomes away from the pole. These "polar ejection forces" appear to play a key role in regulating kinetochore directional instability, and hence, positions achieved by chromosomes on the spindle.

Dynamic Instability in Quartering Seas—Part III: Nonlinear Effects on Periodic Motions

1997 ◽  
Vol 41 (03) ◽  
pp. 210-223 ◽  
Author(s):  
K. J. Spyrou
Keyword(s):  
Periodic Motion ◽  
Horizontal Plane ◽  
Dynamic Instability ◽  
Parametric Instability ◽  
Nonlinear Effects ◽  
Ship Motions ◽  
Nonlinear Phenomena ◽  
Specific Sequence ◽  
The Stability ◽  
Two Stages

The loss of stability of the horizontal-plane periodic motion of a steered ship in waves is investigated. In earlier reports we referred to the possibility of a broaching mechanism that will be intrinsic to the periodic mode, whereby there will exist no need for the ship to go through the surf-riding stage. However, about this point the discussion was essentially conjectural. In order to provide substance we present here a theoretical approach that is organized in two stages: Initially, we demonstrate the existence of a mechanism of parametric instability of yaw on the basis of a rudimentary, single-degree model of maneuvering motion in waves. Then, with a more elaborate model, we identify the underlying nonlinear phenomena that govern the large-amplitude horizontal ship motions, considering the ship as a multi-degree, nonlinear oscillator. Our analysis brings to light a very specific sequence of phenomena leading to cumulative broaching that involves a change in the stability of the ordinary periodic motion on the horizontal plane, a transition towards subharmonic response and, ultimately, a sudden jump to resonance. Possible means for controlling the onset of such undesirable behavior are also investigated.

Purification to homogeneity and characterization of nonproteolyzed potato (Solanum tuberosum) tuber hexokinase 1

Botany ◽  
2011 ◽  
Vol 89 (5) ◽  
pp. 289-299 ◽  
Author(s):  
Marie-Claude Moisan ◽  
Jean Rivoal
Keyword(s):  
Solanum Tuberosum ◽  
Specific Activity ◽  
Solanum Tuberosum L ◽  
Extraction Procedure ◽  
Hydrophobic Interaction Chromatography ◽  
Solanum Chacoense ◽  
Kinetic Properties ◽  
Chromatographic Separations ◽  
The Stability ◽  
Fast Flow

We have developed an extraction procedure that improves the stability of potato ( Solanum tuberosum L.) tuber hexokinase (HK) after extraction. Using this protocol, we showed that at least four HK isoforms are present in this tissue, and they can be separated by hydrophobic-interaction chromatography on a butyl-Sepharose™ 4 Fast Flow column. One of the main HK isoforms was purified to homogeneity using further chromatographic separations on red dye, DEAE Fractogel, hydroxyapatite, cibacron blue, and MonoQ matrices. HK-specific activity of this fraction (10.2 U·mg protein–1) corresponds to an enrichment of more than 5500-fold, with a yield of 0.9%. This is the highest reported HK-specific activity from a plant source. The purified enzyme consisted of a monomer with a subunit apparent Mr of 51 kDa when analyzed by SDS–PAGE. This polypeptide was recognized by affinity-purified anti- Solanum chacoense Bitt. recombinant HK IgGs. The protein was digested with trypsin and its digestion products were subjected to MS – MS sequencing after HPLC separation. The sequences of these tryptic peptides matched the predicted coding sequence of the S. tuberosum HK1 gene with a coverage of 57%. Examination of the kinetic properties of the purified protein HK1 indicates that it may be regulated by the internal O2 concentration of the tuber because of its sensitivity to acidic pHs and inhibition by ADP.

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