Effect of plant tubulin kinetic diversification on microtubule lengths

Microtubules (MTs) are dynamic polymers vital for cellular physiology. Bulk tubulin polymerization is nucleation dependent, while individual filaments exhibit 'dynamic instability' driven by GTP hydrolysis rates. Although MTs assembled from well-studied animal brain tubulins have very comparable nucleation and GTP-hydrolysis rates, the kinetic rates of evolutionarily more distant species could diverge. Here we focus on a plant tubulin, the legume Vigna sp. (mung bean) to test the effect of kinetic diversification on MT polymerization. We activity purify tubulin from seedlings and find MT filaments are fewer and shorter than animal brain tubulin. We find mung tubulin polymerization kinetics is nucleation dependent with a high rate of GTP hydrolysis and a critical concentration lower than previously reported for tubulins. A computational model of the kinetics based on the relative influence of rates of nucleation and hydrolysis demonstrates increased rates of hydrolysis can affect MT filament numbers and their lengths, as compared to increasing nucleation rates. Our approach provides a framework to compare the effect of evolutionary diversification of MT nucleation and elongation.

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Polymerization of FtsZ, a Bacterial Homolog of Tubulin

Journal of Biological Chemistry ◽

10.1074/jbc.m009033200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 11743-11753 ◽

Cited By ~ 160

Author(s):

Laura Romberg ◽

Martha Simon ◽

Harold P. Erickson

Keyword(s):

Steady State ◽

Critical Concentration ◽

Gtp Hydrolysis ◽

Nucleotide Hydrolysis ◽

High Affinity ◽

Hydrolysis Rates ◽

Bacterial Homolog ◽

Nucleation Phase ◽

Maximum Steady State

FtsZ is a bacterial homolog of tubulin that is essential for prokaryotic cytokinesis.In vitro, GTP induces FtsZ to assemble into straight, 5-nm-wide polymers. Here we show that the polymerization of these FtsZ filaments most closely resembles noncooperative (or “isodesmic”) assembly; the polymers are single-stranded and assemble with no evidence of a nucleation phase and without a critical concentration. We have developed a model for the isodesmic polymerization that includes GTP hydrolysis in the scheme. The model can account for the lengths of the FtsZ polymers and their maximum steady state nucleotide hydrolysis rates. It predicts that unlike microtubules, FtsZ protofilaments consist of GTP-bound FtsZ subunits that hydrolyze their nucleotide only slowly and are connected by high affinity longitudinal bonds with a nanomolarKD.

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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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Bacterial Microtubules Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis

10.1101/112987 ◽

2017 ◽

Author(s):

César Díaz-Celis ◽

Viviana I. Risca ◽

Felipe Hurtado ◽

Jessica K. Polka ◽

Scott D. Hansen ◽

...

Keyword(s):

Molecular Motors ◽

Intracellular Transport ◽

Complex Dynamics ◽

Critical Concentration ◽

Polarized Growth ◽

Gtp Hydrolysis ◽

Filament Dynamics ◽

Mixed Polarity ◽

Self Association

AbstractBacteria of the genusProsthecobacterexpress homologs of eukaryotic α-and β-tubulin, called BtubA and BtubB, that have been observed to assemble into bacterial microtubules (bMTs). ThebtubABgenes likely entered theProsthecobacterlineage via horizontal gene transfer and may derive from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is GTP-dependent and reversible and that BtubA/B folding does not require chaperones. To better understand bMT behavior and gain insight into the evolution of microtubule dynamics, we characterizedin vitrobMT assembly using a combination of polymerization kinetics assays, and microscopy. Like eukaryotic microtubules, bMTs exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by bMT polymerization drives a stochastic mechanism of bMT disassembly that occurs via polymer breakage. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of bMT fragments. Unlike MTs, polymerization of bMTs requires KCl, which reduces the critical concentration for BtubA/B assembly and induces bMTs to form stable mixed-orientation bundles in the absence of any additional bMT-binding proteins. Our results suggest that at potassium concentrations resembling that inside the cytoplasm ofProsthecobacter, bMT stabilization through self-association may be a default behavior. The complex dynamics we observe in both stabilized and unstabilized bMTs may reflect common properties of an ancestral eukaryotic tubulin polymer.ImportanceMicrotubules are polymers within all eukaryotic cells that perform critical functions: they segregate chromosomes in cell division, organize intracellular transport by serving as tracks for molecular motors, and support the flagella that allow sperm to swim. These functions rely on microtubules remarkable range of tunable dynamic behaviors. Recently discovered bacterial microtubules composed of an evolutionarily related protein are evolved from a missing link in microtubule evolution, the ancestral eukaryotic tubulin polymer. Using microscopy and biochemical approaches to characterize bacterial microtubules, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules, but differ in how they self-associate into bundles and become destabilized. Our results demonstrate the diversity of mechanisms that microtubule-like filaments employ to promote filament dynamics and monomer turnover.

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Multi-functional regulator MapZ controls both positioning and timing of FtsZ polymerization

Biochemical Journal ◽

10.1042/bcj20190138 ◽

2019 ◽

Vol 476 (10) ◽

pp. 1433-1444 ◽

Cited By ~ 4

Author(s):

Zhang Feng ◽

Jiahai Zhang ◽

Da Xu ◽

Yong-Liang Jiang ◽

Cong-Zhao Zhou ◽

...

Keyword(s):

Critical Concentration ◽

Dynamic State ◽

Protein Z ◽

Gtp Hydrolysis ◽

Intracellular Domain ◽

Precise Positioning ◽

Daughter Cells ◽

Division Site ◽

Z Ring ◽

Key Residues

AbstractThe tubulin-like GTPase protein FtsZ, which forms a discontinuous cytokinetic ring at mid-cell, is a central player to recruit the division machinery to orchestrate cell division. To guarantee the production of two identical daughter cells, the assembly of FtsZ, namely Z-ring, and its precise positioning should be finely regulated. In Streptococcus pneumoniae, the positioning of Z-ring at the division site is mediated by a bitopic membrane protein MapZ (mid-cell-anchored protein Z) through direct interactions between the intracellular domain (termed MapZ-N (the intracellular domain of MapZ)) and FtsZ. Using nuclear magnetic resonance titration experiments, we clearly assigned the key residues involved in the interactions. In the presence of MapZ-N, FtsZ gains a shortened activation delay, a lower critical concentration for polymerization and a higher cooperativity towards GTP hydrolysis. On the other hand, MapZ-N antagonizes the lateral interactions of single-stranded filaments of FtsZ, thus slows down the formation of highly bundled FtsZ polymers and eventually maintains FtsZ at a dynamic state. Altogether, we conclude that MapZ is not only an accelerator to trigger the polymerization of FtsZ, but also a brake to tune the velocity to form the end-product, FtsZ bundles. These findings suggest that MapZ is a multi-functional regulator towards FtsZ that controls both the precise positioning and proper timing of FtsZ polymerization.

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Effects of organic acids on tubulin polymerization and associated GTP hydrolysis

Biochemistry ◽

10.1021/bi00532a014 ◽

1982 ◽

Vol 21 (3) ◽

pp. 503-509 ◽

Cited By ~ 34

Author(s):

Ernest Hamel ◽

Anthony A. Del Campo ◽

Michael C. Lowe ◽

Phyllis G. Waxman ◽

Chii M. Lin

Keyword(s):

Organic Acids ◽

Tubulin Polymerization ◽

Gtp Hydrolysis

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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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Slow Polymerization of Mycobacterium tuberculosis FtsZ

Journal of Bacteriology ◽

10.1128/jb.182.14.4028-4034.2000 ◽

2000 ◽

Vol 182 (14) ◽

pp. 4028-4034 ◽

Cited By ~ 92

Author(s):

E. Lucile White ◽

Larry J. Ross ◽

Robert C. Reynolds ◽

Lainne E. Seitz ◽

Georgia D. Moore ◽

...

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Tubulin Polymerization ◽

Gtpase Activity ◽

Gtp Hydrolysis ◽

Minimum Concentration ◽

E Coli ◽

Cell Division Protein ◽

Mycobacterial Protein

ABSTRACT The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coliand purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90° light scattering. The mycobacterial protein reaches maximum polymerization much more slowly (∼10 min) than E. coliFtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg2+and GTP. The minimum concentration of FtsZ needed for polymerization is 3 μM. Electron microscopy shows that polymerized M. tuberculosis FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments. Ethyl 6-amino-2,3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits M. tuberculosis FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.

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Dynamics of microtubules from erythrocyte marginal bands.

Molecular Biology of the Cell ◽

10.1091/mbc.4.3.323 ◽

1993 ◽

Vol 4 (3) ◽

pp. 323-335 ◽

Cited By ~ 35

Author(s):

B Trinczek ◽

A Marx ◽

E M Mandelkow ◽

D B Murphy ◽

E Mandelkow

Keyword(s):

Phosphate Buffer ◽

Dynamic Instability ◽

Critical Concentration ◽

Mammalian Brain ◽

Tubulin Isoforms ◽

Marginal Band ◽

Associated Proteins ◽

Free Microtubules ◽

The Difference ◽

Brain Microtubules

Microtubules can adjust their length by the mechanism of dynamic instability, that is by switching between phases of growth and shrinkage. Thus far this phenomenon has been studied with microtubules that contain several components, that is, a mixture of tubulin isoforms, with or without a mixture of microtubule-associated proteins (MAPs), which can act as regulators of dynamic instability. Here we concentrate on the influence of the tubulin component. We have studied MAP-free microtubules from the marginal band of avian erythrocytes and compared them with mammalian brain microtubules. The erythrocyte system was selected because it represents a naturally stable aggregate of microtubules; second, the tubulin is largely homogeneous, in contrast to brain tubulin. Qualitatively, erythrocyte microtubules show similar features as brain microtubules, but they were found to be much less dynamic. The critical concentration of elongation, and the rates of association and dissociation of tubulin are all lower than with brain microtubules. Catastrophes are rare, rescues frequent, and shrinkage slow. This means that dynamic instability can be controlled by the tubulin isotype, independently of MAPs. Moreover, the extent of dynamic behavior is highly dependent on buffer conditions. In particular, dynamic instability is strongly enhanced in phosphate buffer, both for erythrocyte marginal band and brain microtubules. The lower stability in phosphate buffer argues against the hypothesis that a cap of tubulin.GDP.Pi subunits stabilizes microtubules. The difference in dynamics between tubulin isotypes and between the two ends of microtubules is preserved in the different buffer systems.

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