scholarly journals Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate.

1991 ◽  
Vol 114 (1) ◽  
pp. 73-81 ◽  
Author(s):  
R A Walker ◽  
N K Pryer ◽  
E D Salmon
Keyword(s):  
Dynamic Instability ◽  
Elongation Rate ◽  
Gtp Hydrolysis ◽  
Microtubule Stability ◽  
Assembly Mechanism ◽  
High Elongation ◽  
Microtubule Growth ◽  
Hydrolysis Of ◽  
Brain Tubulin

Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 microM) was assembled at 37 degrees C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution). Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length. Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated lifetime of the stable cap after infinite dilution was less than 3-4 s for plus and minus ends, much shorter than the approximately 200 s observed at steady state (Walker, R. A., E. T. O'Brien, N. K. Pryer, M. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. 1988. J. Cell Biol. 107:1437-1448.). We conclude that during elongation, both plus and minus ends are stabilized by a short region (approximately 200 dimers or less) and that the size of the stable cap is independent of 10-fold variation in elongation rate. These results eliminate models of dynamic instability which predict extensive "build-up" stabilizing caps and support models which constrain the cap to the elongating tip. We propose that the cell may take advantage of such an assembly mechanism by using "catastrophe factors" that can promote frequent catastrophe even at high elongation rates by transiently binding to microtubule ends and briefly inhibiting GTP-tubulin association.

scholarly journals EB3-informed dynamics of the microtubule stabilizing cap during stalled growth

2021 ◽  
Author(s):  
Maurits Kok ◽  
Florian Huber ◽  
Svenja-Marei Kalisch ◽  
Marileen Dogterom
Keyword(s):  
Growth Velocity ◽  
Hydrolysis Rate ◽  
Exact Relation ◽  
Gtp Hydrolysis ◽  
Lifetime Distributions ◽  
Microtubule Stability ◽  
In Vitro Reconstitution ◽  
1D Model ◽  
Microtubule Growth

Microtubule stability is known to be governed by a stabilizing GTP/GDP-Pi cap, but the exact relation between growth velocity, GTP hydrolysis and catastrophes remains unclear. We investigate the dynamics of the stabilizing cap through in vitro reconstitution of microtubule dynamics in contact with micro-fabricated barriers, using the plus-end binding protein GFP-EB3 as a marker for the nucleotide state of the tip. The interaction of growing microtubules with steric objects is known to slow down microtubule growth and accelerate catastrophes. We show that the lifetime distributions of stalled microtubules, as well as the corresponding lifetime distributions of freely growing microtubules, can be fully described with a simple phenomenological 1D model based on noisy microtubule growth and a single EB3-dependent hydrolysis rate. This same model is furthermore capable of explaining both the previously reported mild catastrophe dependence on microtubule growth rates and the catastrophe statistics during tubulin washout experiments.

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 Lysine and Polyamines Are Substrates for Transglutamination of Rho by the BordetellaDermonecrotic Toxin

2001 ◽  
Vol 69 (12) ◽  
pp. 7663-7670 ◽  
Author(s):  
Gudula Schmidt ◽  
Udo-Michael Goehring ◽  
Joerg Schirmer ◽  
Sandrine Uttenweiler-Joseph ◽  
Matthias Wilm ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Affinity Purification ◽  
Dependent Manner ◽  
Rho Proteins ◽  
Concentration Dependent Manner ◽  
Gtp Hydrolysis ◽  
Dermonecrotic Toxin ◽  
Hydrolysis Of ◽  
Active Fragment

ABSTRACT Bordetella dermonecrotic toxin (DNT) catalyzes the transglutamination of glutamine-63/61 of Rho GTPases, thereby constitutively activating Rho proteins. Here we identified second substrates for transglutamination of RhoA by DNT. The enzymatically active fragment of DNT (residues 1136 to 1451, ΔDNT) induced the incorporation of l-[14C]lysine in RhoA in a concentration-dependent manner. Also, Rac and Cdc42, but not Ras, were transglutaminated with lysine by ΔDNT. Transglutamination of the GTPase with l-lysine inhibited intrinsic and Rho-GAP-stimulated GTP hydrolysis of RhoA. In contrast to lysine, treatment of RhoA with alanine, arginine, and glutamine were not able to substitute for lysine in the transglutamination reaction. DNT increased the incorporation of l-[14C]lysine into embryonic bovine lung cells. Microinjection of GST-RhoA together with the enzymatically active DNT fragment intoXenopus oocytes, subsequent affinity purification of modified GST-RhoA, and mass spectrometry identified attachment of putrescine or spermidine at glutamine-63 of RhoA. A comparison of putrescine, spermidine, and lysine as substrates for DNT-induced transglutamination of RhoA revealed that lysine is a preferred second substrate at least in vitro.

scholarly journals Bacterial dynamin-like protein DynA mediates lipid and content mixing and shows phospholipid specificity

2018 ◽  
Author(s):  
Lijun Guo ◽  
Marc Bramkamp
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Stress ◽  
Gtp Hydrolysis ◽  
Cellular Processes ◽  
Organelle Division ◽  
Membrane Tethering ◽  
Hydrolysis Of

ABSTRACTThe dynamins family of GTPases is involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. The GTP hydrolysis cycle of dynamin translates to a conformational change in the protein structure, which forces the underlying lipid layer into an energetically unstable conformation that promotes membrane rearrangements. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. In recent years, our group has reported that the dynamin-like protein DynA from Bacillus subtilis mediates nucleotide-independent membrane tethering in vitro and contributes to the innate immunity of bacteria against membrane stress and phage infection. However, so far the mechanism of membrane stress response and the role of GTP hydrolysis remain unclear. Here, we employed content mixing and lipid mixing assays in reconstituted systems to study if the dynamin-like protein DynA from B. subtilis induces membrane full fusion, and further test the possibility that GTP hydrolysis of DynA may act on the fusion-through-hemifusion pathway. Our results based on fluorescence resonance energy transfer (FRET) indicated that DynA could induce aqueous content mixing even in absence of GTP. Moreover, DynA-induced membrane fusion in vitro is a thermo-promoted slow response. Surprisingly, digestion of protein mediated an instantl rise of content exchange, supporting the assumption that disassembly of DynA is the fundamental power for fusion-through-hemifusion.

scholarly journals XMAP215 promotes microtubule catastrophe by disrupting the growing microtubule end

2020 ◽  
Author(s):  
Veronica Farmer ◽  
Göker Arpağ ◽  
Sarah Hall ◽  
Marija Zanic
Keyword(s):  
Growth Rate ◽  
Growth Rates ◽  
Fast Growth ◽  
Fast Growing ◽  
Microtubule Stability ◽  
Large Fluctuations ◽  
Microtubule Growth ◽  
Enhanced Stability

ABSTRACTThe GTP-tubulin cap is widely accepted to protect microtubules against catastrophe. The GTP-cap size is thought to increase with the microtubule growth rate, presumably endowing fast-growing microtubules with enhanced stability. It is unknown what GTP-cap properties permit frequent microtubule catastrophe despite fast growth. Here, we investigate microtubules grown in vitro in the presence and absence of the microtubule polymerase XMAP215. Using EB1 as a GTP-cap marker, we find that GTP-cap size increases regardless of whether growth acceleration is achieved by increasing tubulin concentration or by XMAP215. In spite of the increased mean GTP-cap size, microtubules grown with XMAP215 display increased catastrophe frequency, in contrast to microtubules grown with more tubulin, for which catastrophe is abolished. However, microtubules polymerized with XMAP215 have large fluctuations in growth rate and EB1 intensity; display tapered and curled ends; and undergo catastrophe at faster growth rates and with higher EB1 end-localization. Our results underscore the role of growth irregularities in overall microtubule stability.

scholarly journals Dynamic instability 30 years later: complexities in microtubule growth and catastrophe

2015 ◽  
Vol 26 (7) ◽  
pp. 1207-1210 ◽  
Author(s):  
Gary J. Brouhard
Keyword(s):  
Dynamic Instability ◽  
Gtp Hydrolysis ◽  
Microtubule Growth

Microtubules are not like other polymers. Whereas polymers such as F-actin will grow continuously as long as the subunit concentration is high enough, a steadily growing microtubule can suddenly shrink even when there is ample αβ-tubulin around. This remarkable behavior was discovered in 1984 when Tim Mitchison and Marc Kirschner deduced that microtubules switch from growth to shrinkage when they lose their GTP caps. Here, I review the canonical explanation of dynamic instability that was fleshed out in the years after its discovery. Many aspects of this explanation have been recently subverted, particularly those related to how GTP-tubulin forms polymers and why GTP hydrolysis disrupts them. I describe these developments and speculate on how our explanation of dynamic instability can be changed to accommodate them.

scholarly journals Modulation of microtubule stability by kinetochores in vitro.

1990 ◽  
Vol 110 (5) ◽  
pp. 1607-1616 ◽  
Author(s):  
A A Hyman ◽  
T J Mitchison
Keyword(s):  
Dynamic Behavior ◽  
Mitotic Spindle ◽  
Dynamic Instability ◽  
Full Range ◽  
Chromosome Movement ◽  
Vitro Assay ◽  
Microtubule Stability ◽  
Rigor State

The interface between kinetochores and microtubules in the mitotic spindle is known to be dynamic. Kinetochore microtubules can both polymerize and depolymerize, and their dynamic behavior is intimately related to chromosome movement. In this paper we investigate the influence of kinetochores on the inherent dynamic behavior of microtubules using an in vitro assay. The dynamics of microtubule plus ends attached to kinetochores are compared to those of free plus ends in the same solution. We show that microtubules attached to kinetochores exhibit the full range of dynamic instability behavior, but at altered transition rates. Surprisingly, we find that kinetochores increase the rate at which microtubule ends transit from growing to shrinking. This result contradicts our previous findings (Mitchison, T. J., and M. W. Kirschner, 1985b) for technical reasons which are discussed. We suggest that catalysis of the growing to shrinking transition by kinetochores may account for selective depolymerization of kinetochore microtubules during anaphase in vivo. We also investigate the effects of a nonhydrolyzable ATP analogue on kinetochore microtubule dynamics. We find that 5' adenylylimido diphosphate induces a rigor state at the kinetochore-microtubule interface, which prevents depolymerization of the microtubule.

scholarly journals Regulation of microtubule dynamic instability by the carboxy-terminal tail of β-tubulin

2018 ◽  
Vol 1 (2) ◽  
pp. e201800054 ◽  
Author(s):  
Colby P Fees ◽  
Jeffrey K Moore
Keyword(s):  
Dynamic Instability ◽  
Intrinsic Property ◽  
In Vivo Studies ◽  
Microtubule Dynamic ◽  
Microtubule Stability ◽  
Microtubule Networks ◽  
Carboxy Terminal ◽  
Β Tubulin

Dynamic instability is an intrinsic property of microtubules; however, we do not understand what domains of αβ-tubulins regulate this activity or how these regulate microtubule networks in cells. Here, we define a role for the negatively charged carboxy-terminal tail (CTT) domain of β-tubulin in regulating dynamic instability. By combining in vitro studies with purified mammalian tubulin and in vivo studies with tubulin mutants in budding yeast, we demonstrate that β-tubulin CTT inhibits microtubule stability and regulates the structure and stability of microtubule plus ends. Tubulin that lacks β-tubulin CTT polymerizes faster and depolymerizes slower in vitro and forms microtubules that are more prone to catastrophe. The ends of these microtubules exhibit a more blunted morphology and rapidly switch to disassembly after tubulin depletion. In addition, we show that β-tubulin CTT is required for magnesium cations to promote depolymerization. We propose that β-tubulin CTT regulates the assembly of stable microtubule ends and provides a tunable mechanism to coordinate dynamic instability with ionic strength in the cell.

scholarly journals In vitro assembly and GTP hydrolysis by bacterial tubulins BtubA and BtubB

2005 ◽  
Vol 169 (2) ◽  
pp. 233-238 ◽  
Author(s):  
Christopher A. Sontag ◽  
James T. Staley ◽  
Harold P. Erickson
Keyword(s):  
Critical Concentration ◽  
Three Dimensional ◽  
Outer Diameter ◽  
Gtpase Activity ◽  
Gtp Hydrolysis ◽  
Assembly Mechanism ◽  
In Vitro Assembly ◽  
Cooperative Assembly ◽  
Versus Concentration

Arecent study identified genuine tubulin proteins, BtubA and BtubB, in the bacterial genus Prosthecobacter. We have expressed BtubA and BtubB in Escherichia coli and studied their in vitro assembly. BtubB by itself formed rings with an outer diameter of 35–36 nm in the presence of GTP or GDP. Mixtures of BtubB and BtubA formed long protofilament bundles, 4–7 protofilaments wide (20–30 protofilaments in the three-dimensional bundle). Regardless of the starting stoichiometry, the polymers always contained equal concentrations of BtubA and BtubB, suggesting that BtubA and B alternate along the protofilament. BtubA showed negligible GTP hydrolysis, whereas BtubB hydrolyzed 0.40 mol GTP per min per mol BtubB. This GTPase activity increased to 1.37 per min when mixed 1:1 with BtubA. A critical concentration of 0.4–1.0 μM was indicated by light scattering experiments and extrapolation of GTPase versus concentration, thus suggesting a cooperative assembly mechanism.

A simple formulation of microtubule dynamics: quantitative implications of the dynamic instability of microtubule populations in vivo and in vitro

1989 ◽  
Vol 93 (2) ◽  
pp. 241-254
Author(s):  
P.M. Bayley ◽  
M.J. Schilstra ◽  
S.R. Martin
Keyword(s):  
Steady State ◽  
Dynamic Instability ◽  
Microtubule Assembly ◽  
Simple Extension ◽  
Simple Formulation ◽  
Exchange Processes ◽  
Microtubule Growth ◽  
Kinetics Of

A simple formulation of microtubule dynamic instability is presented, which is based on the experimental observations by T. Horio and H. Hotani of coexisting, interconverting growing and shrinking microtubules. Employing only three independent, experimentally determined parameters for a given microtubule end, this treatment accounts quantitatively for the principal features of the observed dynamic behaviour of steady-state tubulin microtubules in vitro. Experimental data are readily reproduced for microtubule length redistribution, and for the kinetics of tubulin exchange processes, including pulse-chase properties. The relative importance of dynamic incorporation and that due to treadmilling are assessed. Dynamic incorporation is found to dominate the overall exchange properties; polarized incorporation due to treadmilling generally becomes significant only when the dynamics are largely suppressed. This treatment also permits simulation of certain cellular phenomena, showing how microtubule renucleation can control microtubule growth, by means of changes in microtubule number concentration in a system at constant microtubule mass. A relatively simple extension of the formulation accounts quantitatively for non-steady-state microtubule properties, e.g. dilution-induced rapid disassembly and the oscillatory mode of microtubule assembly. The principles relating dynamic instability and oscillatory behaviour are clearly indicated. Possible mechanisms of the switching of microtubules are briefly discussed.

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