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”).

Kinetic stabilization of microtubule dynamic instability in vitro by vinblastine

Biochemistry ◽  
1993 ◽  
Vol 32 (5) ◽  
pp. 1285-1293 ◽  
Author(s):  
Robert J. Toso ◽  
Mary Ann Jordan ◽  
Kevin W. Farrell ◽  
Brian Matsumoto ◽  
Leslie Wilson
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic

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

Ultraviolet microbeam irradiation of chromosomal spindle fibres shears microtubules and permits study of the new free ends in vivo

1988 ◽  
Vol 91 (4) ◽  
pp. 455-468 ◽  
Author(s):  
P.J. Wilson ◽  
A. Forer
Keyword(s):  
Ultraviolet Light ◽  
Relative Stability ◽  
Dynamic Instability ◽  
Spindle Fibre ◽  
Microtubule Depolymerization ◽  
Immunofluorescence Analysis ◽  
Ultraviolet Microbeam ◽  
Spindle Fibres

Irradiation of birefringent chromosomal spindle fibres in crane-fly spermatocytes in metaphase I or anaphase I produces an area of reduced birefringence (ARB) on the fibre. This ARB moves poleward and is lost at the pole. Ultrastructural and immunofluorescence analysis of ARBs obtained by irradiation with monochromatic ultraviolet light of wavelength 260 nm shows that the microtubules in the irradiated area are depolymerized, though the rest of the spindle appears unaffected. The area of microtubule depolymerization moves poleward with the ARB, and once the ARB reaches the pole the irradiated half-spindle appears normal. The motion of the ARB, therefore, appears to be due to the behaviour of the sheared microtubules in the chromosomal spindle fibre. The relative stability of the sheared microtubules shows that chromosomal fibre microtubules are not dynamically unstable, as are microtubules under certain conditions in vitro. However, ARB motion may be due to a moderated version of dynamic instability. Possible models for ARB motion are discussed.

scholarly journals Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A

2013 ◽  
Vol 202 (4) ◽  
pp. 605-621 ◽  
Author(s):  
Ricardo Nunes Bastos ◽  
Sapan R. Gandhi ◽  
Ryan D. Baron ◽  
Ulrike Gruneberg ◽  
Erich A. Nigg ◽  
...  
Keyword(s):  
Dynamic Instability ◽  
Phosphorylation Site ◽  
Size Control ◽  
Aurora B ◽  
Microtubule Dynamic ◽  
Central Spindle ◽  
Specific Subset ◽  
Mutant Forms

Anaphase central spindle formation is controlled by the microtubule-stabilizing factor PRC1 and the kinesin KIF4A. We show that an MKlp2-dependent pool of Aurora B at the central spindle, rather than global Aurora B activity, regulates KIF4A accumulation at the central spindle. KIF4A phosphorylation by Aurora B stimulates the maximal microtubule-dependent ATPase activity of KIF4A and promotes its interaction with PRC1. In the presence of phosphorylated KIF4A, microtubules grew more slowly and showed long pauses in growth, resulting in the generation of shorter PRC1-stabilized microtubule overlaps in vitro. Cells expressing only mutant forms of KIF4A lacking the Aurora B phosphorylation site overextended the anaphase central spindle, demonstrating that this regulation is crucial for microtubule length control in vivo. Aurora B therefore ensures that suppression of microtubule dynamic instability by KIF4A is restricted to a specific subset of microtubules and thereby contributes to central spindle size control in anaphase.

scholarly journals Okadaic acid induces interphase to mitotic-like microtubule dynamic instability by inactivating rescue.

1992 ◽  
Vol 119 (5) ◽  
pp. 1271-1276 ◽  
Author(s):  
N R Gliksman ◽  
S F Parsons ◽  
E D Salmon
Keyword(s):  
High Resolution ◽  
Sea Urchin ◽  
Okadaic Acid ◽  
Dynamic Instability ◽  
Major Change ◽  
Microtubule Dynamics ◽  
Regulatory Pathway ◽  
Microtubule Dynamic ◽  
Interphase Cells ◽  
Frequent Switching

We used high-resolution video microscopy to visualize microtubule dynamic instability in extracts of interphase sea urchin eggs and to analyze the changes that occur upon addition of 0.8-2.5 microM okadaic acid, an inhibitor of phosphatase 1 and 2A (PP1, PP2a) (Bialojan, D., and A. Takai. 1988. Biochem. J. 256:283-290). Microtubule plus-ends in these extracts oscillated between the elongation and shortening phases of dynamic instability at frequencies typical for interphase cells. Switching from elongation to shortening (catastrophe) was frequent, but microtubules persisted and grew long because of frequent switching back to elongation (rescue). Addition of okadaic acid to the extract induced rapid (< 5 min) conversion to short, dynamic microtubules typical of mitosis. The frequency of catastrophe doubled and the velocities of elongation and shortening increased slightly; however, the major change was an elimination of rescue. Thus, modulation of the rescue frequency by phosphorylation-dependent mechanisms may be a major regulatory pathway for selectively controlling microtubule dynamics without dramatically changing velocities of microtubule elongation and shortening.

scholarly journals Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.

1997 ◽  
Vol 8 (6) ◽  
pp. 973-985 ◽  
Author(s):  
R J Vasquez ◽  
B Howell ◽  
A M Yvon ◽  
P Wadsworth ◽  
L Cassimeris
Keyword(s):  
Dynamic Instability ◽  
Light Level ◽  
Pause Duration ◽  
Microtubule Assembly ◽  
Differential Interference Contrast Microscopy ◽  
Microtubule Dynamic ◽  
State Behavior ◽  
Dose Dependent Decrease

Previous studies demonstrated that nanomolar concentrations of nocodazole can block cells in mitosis without net microtubule disassembly and resulted in the hypothesis that this block was due to a nocodazole-induced stabilization of microtubules. We tested this hypothesis by examining the effects of nanomolar concentrations of nocodazole on microtubule dynamic instability in interphase cells and in vitro with purified brain tubulin. Newt lung epithelial cell microtubules were visualized by video-enhanced differential interference contrast microscopy and cells were perfused with solutions of nocodazole ranging in concentration from 4 to 400 nM. Microtubules showed a loss of the two-state behavior typical of dynamic instability as evidenced by the addition of a third state where they exhibited little net change in length (a paused state). Nocodazole perfusion also resulted in slower elongation and shortening velocities, increased catastrophe, and an overall decrease in microtubule turnover. Experiments performed on BSC-1 cells that were microinjected with rhodamine-labeled tubulin, incubated in nocodazole for 1 h, and visualized by using low-light-level fluorescence microscopy showed similar results except that nocodazole-treated BSC-1 cells showed a decrease in catastrophe. To gain insight into possible mechanisms responsible for changes in dynamic instability, we examined the effects of 4 nM to 12 microM nocodazole on the assembly of purified tubulin from axoneme seeds. At both microtubule plus and minus ends, perfusion with nocodazole resulted in a dose-dependent decrease in elongation and shortening velocities, increase in pause duration and catastrophe frequency, and decrease in rescue frequency. These effects, which result in an overall decrease in microtubule turnover after nocodazole treatment, suggest that the mitotic block observed is due to a reduction in microtubule dynamic turnover. In addition, the in vitro results are similar to the effects of increasing concentrations of GDP-tubulin (TuD) subunits on microtubule assembly. Given that nocodazole increases tubulin GTPase activity, we propose that nocodazole acts by generating TuD subunits that then alter dynamic instability.

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