The effect of solution composition on microtubule dynamic instability

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.

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Mathematical modeling of microtubule dynamic instability: new insight into the link between gtp-hydrolysis and microtubule aging

ESAIM Mathematical Modelling and Numerical Analysis ◽

10.1051/m2an/2017025 ◽

2018 ◽

Vol 52 (6) ◽

pp. 2433-2456 ◽

Cited By ~ 2

Author(s):

Ayuna Barlukova ◽

Diana White ◽

Gérard Henry ◽

Stéphane Honoré ◽

Florence Hubert

Keyword(s):

Dynamic Instability ◽

Dynamic Properties ◽

Model Parameters ◽

Microtubule Dynamic ◽

Gtp Hydrolysis ◽

Unique Type ◽

Microtubule Targeting Agents ◽

Age Dependent ◽

Type Of Behavior ◽

Insight Into

Microtubules (MTs) are protein polymers that exhibit a unique type of behavior referred to as dynamic instability. That is, they undergo periods of growth (through the addition of GTP-tubulin) and shortening (through the subtraction of GDP-tubulin). Shortening events are very fast, where this transition is referred to as a catastrophe. There are many processes that regulate MT dynamic instability, however, recent experiments show that MT dynamics may be highly regulated by a MTs age, where young MTs are less likely to undergo shortening events than older ones. In this paper, we develop a novel modeling approach to describe how the age of a MT affects its dynamic properties. In particular, we extend on a previously developed model that describes MT dynamics, by proposing a new concept for GTP-tubulin hydrolysis (the process by which newly incorporated GTP-tubulin is hydrolyzed to lower energy GDP-tubulin). In particular, we assume that hydrolysis is mainly vectorial, age-dependent and delayed according to the GTP-tubulin incorporation into the MT. Through numerical simulation, we are able to show how MT age affects certain properties that define MT dynamics. For example, simulations illustrate how the aging process leads to an increase in the rate of GTP-tubulin hydrolysis for older MTs, as well as increases in catastrophe frequency. Also, since it has been found that MT dynamic instability is affected by chemotherapy microtubule-targeting agents (MTAs), we highlight the fact that our model can be used to investigate the action of MTAs on MT dynamics by varying certain model parameters.

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

The Journal of Cell Biology ◽

10.1083/jcb.138.1.105 ◽

1997 ◽

Vol 138 (1) ◽

pp. 105-117 ◽

Cited By ~ 77

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.

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A Lateral Cap model of microtubule dynamic instability

FEBS Letters ◽

10.1016/0014-5793(89)81523-6 ◽

1989 ◽

Vol 259 (1) ◽

pp. 181-184 ◽

Cited By ~ 27

Author(s):

Peter Bayley ◽

Maria Schilstra ◽

Stephen Martin

Keyword(s):

Dynamic Instability ◽

Microtubule Dynamic ◽

Cap Model

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Enhanced mechanosensing of cells in synthetic 3D matrix with controlled biophysical dynamics

Nature Communications ◽

10.1038/s41467-021-23120-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Boguang Yang ◽

Kongchang Wei ◽

Claudia Loebel ◽

Kunyu Zhang ◽

Qian Feng ◽

...

Keyword(s):

Stem Cells ◽

Rate Constant ◽

Dynamic Properties ◽

3D Culture ◽

Binding Constants ◽

Dissociation Rate ◽

Dissociation Rate Constant ◽

Cellular Functions ◽

The Matrix ◽

3D Matrix

Abstract3D culture of cells in designer biomaterial matrices provides a biomimetic cellular microenvironment and can yield critical insights into cellular behaviours not available from conventional 2D cultures. Hydrogels with dynamic properties, achieved by incorporating either degradable structural components or reversible dynamic crosslinks, enable efficient cell adaptation of the matrix and support associated cellular functions. Herein we demonstrate that given similar equilibrium binding constants, hydrogels containing dynamic crosslinks with a large dissociation rate constant enable cell force-induced network reorganization, which results in rapid stellate spreading, assembly, mechanosensing, and differentiation of encapsulated stem cells when compared to similar hydrogels containing dynamic crosslinks with a low dissociation rate constant. Furthermore, the static and precise conjugation of cell adhesive ligands to the hydrogel subnetwork connected by such fast-dissociating crosslinks is also required for ultra-rapid stellate spreading (within 18 h post-encapsulation) and enhanced mechanosensing of stem cells in 3D. This work reveals the correlation between microscopic cell behaviours and the molecular level binding kinetics in hydrogel networks. Our findings provide valuable guidance to the design and evaluation of supramolecular biomaterials with cell-adaptable properties for studying cells in 3D cultures.

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

The Journal of Cell Biology ◽

10.1083/jcb.108.3.931 ◽

1989 ◽

Vol 108 (3) ◽

pp. 931-937 ◽

Cited By ~ 87

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.

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Dynamic properties of nucleated microtubules: GTP utilisation in the subcritical concentration regime

Journal of Cell Science ◽

10.1242/jcs.109.11.2755 ◽

1996 ◽

Vol 109 (11) ◽

pp. 2755-2766

Author(s):

M.F. Symmons ◽

S.R. Martin ◽

P.M. Bayley

Keyword(s):

Dynamic Instability ◽

Dynamic Properties ◽

Critical Concentration ◽

Specific Inhibitor ◽

Microtubule Assembly ◽

Microtubule Dynamic ◽

Assembly Kinetics ◽

Microtubule Growth ◽

Growth Features

Microtubule assembly kinetics have been studied quantitatively under solution conditions supporting microtubule dynamic instability. Purified GTP-tubulin (Tu-GTP) and covalently cross-linked short microtubule seeds (EGS-seeds; Koshland et al. (1988) Nature 331, 499) were used with and without biotinylation. Under sub-critical concentration conditions ([Tu-GTP] < 5.3 microM), significant microtubule growth of limited length was observed on a proportion of the EGS-seeds by immuno-electron microscopy. A sensitive fluorescence assay for microtubule GDP production was developed for parallel assessment of GTP utilisation. This revealed a correlation between the detected microtubule growth and the production of tubulin-GDP, deriving from the shortening phase of the dynamic microtubules. This correlation was confirmed by the action of nocodazole, a specific inhibitor of microtubule assembly, that was found to abolish the GDP release. The variation of the GDP release with tubulin concentration (Jh(c) plot) was determined below the critical concentration (Cc). The GDP production observed was consistent with the elongation of the observed seeded microtubules with an apparent rate constant of 1.5 × 10(6) M-1 second-1 above a threshold of approximately 1 microM tubulin. The form of this Jh(c) plot for elongation below Cc is reproduced by the Lateral Cap model for microtubule dynamic instability adapted for seeded assembly. The behaviour of the system is contrasted with that previously studied in the absence of detectable microtubule elongation (Caplow and Shanks (1990) J. Biol. Chem. 265, 8935–8941). The approach provides a means of monitoring microtubule dynamics at concentrations inaccessible to optical microscopy, and shows that essentially the same dynamic mechanisms apply at all concentrations. Numerical simulation of the subcritical concentration regime shows dynamic growth features applicable to the initiation of microtubule growth in vivo.

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A mutation uncouples the tubulin conformational and GTPase cycles, revealing allosteric control of microtubule dynamics

eLife ◽

10.7554/elife.10113 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 59

Author(s):

Elisabeth A Geyer ◽

Alexander Burns ◽

Beth A Lalonde ◽

Xuecheng Ye ◽

Felipe-Andres Piedra ◽

...

Keyword(s):

Conformational Change ◽

Dynamic Instability ◽

Dynamic Properties ◽

Microtubule Dynamics ◽

Gtpase Activity ◽

Microtubule Dynamic ◽

Gtp Hydrolysis ◽

Allosteric Control

Microtubule dynamic instability depends on the GTPase activity of the polymerizing αβ-tubulin subunits, which cycle through at least three distinct conformations as they move into and out of microtubules. How this conformational cycle contributes to microtubule growing, shrinking, and switching remains unknown. Here, we report that a buried mutation in αβ-tubulin yields microtubules with dramatically reduced shrinking rate and catastrophe frequency. The mutation causes these effects by suppressing a conformational change that normally occurs in response to GTP hydrolysis in the lattice, without detectably changing the conformation of unpolymerized αβ-tubulin. Thus, the mutation weakens the coupling between the conformational and GTPase cycles of αβ-tubulin. By showing that the mutation predominantly affects post-GTPase conformational and dynamic properties of microtubules, our data reveal that the strength of the allosteric response to GDP in the lattice dictates the frequency of catastrophe and the severity of rapid shrinking.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102699 ◽

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”).

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