scholarly journals Behaviors of individual microtubules and microtubule populations relative to critical concentrations: dynamic instability occurs when critical concentrations are driven apart by nucleotide hydrolysis

2020 ◽  
Vol 31 (7) ◽  
pp. 589-618 ◽  
Author(s):  
Erin M. Jonasson ◽  
Ava J. Mauro ◽  
Chunlei Li ◽  
Ellen C. Labuz ◽  
Shant M. Mahserejian ◽  
...  
Keyword(s):  
Dynamic Instability ◽  
Hydrolysis Rate ◽  
Nucleotide Hydrolysis ◽  
Critical Concentrations

We show that polymers displaying dynamic instability (DI) have at least two experimentally distinguishable critical concentrations (CCs), typical DI occurs between these two CCs, and the separation between the CCs depends on the NTP hydrolysis rate. We demonstrate how these CCs relate to various existing experimental and theoretical definitions of CC.

scholarly journals Behaviors of individual microtubules and microtubule populations relative to critical concentrations: Dynamic instability occurs when critical concentrations are driven apart by nucleotide hydrolysis

2018 ◽  
Author(s):  
Erin M. Jonasson ◽  
Ava J. Mauro ◽  
Chunlei Li ◽  
Ellen C. Norby ◽  
Shant M. Mahserejian ◽  
...  
Keyword(s):  
Steady State ◽  
Dynamic Instability ◽  
Critical Concentration ◽  
Growth Phases ◽  
Nucleotide Hydrolysis ◽  
Critical Concentrations ◽  
Degree Of Separation ◽  
Polymer Assembly

ABSTRACTThe concept of critical concentration (CC) is central to understanding behaviors of microtubules and other cytoskeletal polymers. Traditionally, these polymers are understood to have one CC, measured multiple ways and assumed to be the subunit concentration necessary for polymer assembly. However, this framework does not incorporate dynamic instability (DI), and there is work indicating that microtubules have two CCs. We use our previously established simulations to confirm that microtubules have (at least) two experimentally relevant CCs and to clarify the behaviors of individuals and populations relative to the CCs. At free subunit concentrations above the lower CC (CCIndGrow), growth phases of individual filaments can occur transiently; above the higher CC (CCPopGrow), the population’s polymer mass will increase persistently. Our results demonstrate that most experimental CC measurements correspond to CCPopGrow, meaning “typical” DI occurs below the concentration traditionally considered necessary for polymer assembly. We report that [free tubulin] at steady state does not equal CCPopGrow, but instead approaches CCPopGrow asymptotically as [total tubulin] increases and depends on the number of stable microtubule seeds. We show that the degree of separation between CCIndGrow and CCPopGrow depends on the rate of nucleotide hydrolysis. This clarified framework helps explain and unify many experimental observations.

Nucleotide hydrolysis regulates the dynamics of actin filaments and microtubules

1992 ◽  
Vol 336 (1276) ◽  
pp. 93-97 ◽  
Keyword(s):  
Protein Interactions ◽  
Actin Filaments ◽  
Metal Ion ◽  
Dynamic Instability ◽  
Dynamic Properties ◽  
Monomer Concentration ◽  
Nonlinear Dependence ◽  
Nucleotide Hydrolysis ◽  
Conformational Switch

Actin filaments and microtubules are major dynamic components of the cytoskeleton of eukaryotic cells. Assembly of these polymers from monomeric actin or tubulin occurs with expenditure of energy, because ATP (or GTP) tightly bound to actin (or tubulin) is irreversibly hydrolysed during polymerization. Therefore, actin filaments an microtubules are dissipative structures. Our purpose has been to understand how the dissipation of chemical energy perturbs the laws of reversible helical polymerization defined by Oosawa, and affects the dynamics of these polymers. A kinetic study has shown that nucleotide is hydrolysed on the polymer within at least two steps consecutive to the incorporation of the monomer: cleavage of the γ-phosphoester bond followed by the slower release of P i ; only the second reaction appears reversible. P i release, and not cleavage of the γ-phosphate, is linked to the destabilization of protein-protein interactions in the polymer, and therefore plays the role of a conformational switch. The dynamic properties of the polymer in the NTP- and NDP-P i intermediate states of the assembly process have been investigated using non-hydrolysable analogues of nucleotides and structural analogues of P i , AIF 4 - and (BeF 3 - , H 2 O). Because nucleotide hydrolysis is uncoupled from polymerization, actin filaments and microtubules grow with a ‘cap’ of terminal NTP- and NDP-P i - subunits that interact strongly, and prevent the rapid depolymerization of the unstable core of the polymer formed of NDP-subunits. The fact that the dynamic properties of the polymer are affected by bound nucleotide results in a nonlinear dependence of the rate of elongation on monomer concentration. This nonlinearity accounts for the dynamic instability behaviour of microtubules, which is an important feature of their function, and explains the oscillatory polymerization kinetics in a population of synchronized microtubules. The above analysis provides the basis for anticipating possible modes of regulation of cytoskeletal assembly via modulation of the rate of nucleotide hydrolysis. The role of the metal ion (Ca 2+ , Mg 2+ ) chelated to the β- and γ-phosphates of ATP (or GTP) and the stereochemistry of nucleotide binding to actin and tubulin have been studied using the CrATP and CrGTP β γ-bidentate analogues of MgATP and MgGTP. The changes in the environment of the triphosphate moiety of the nucleotide following the release of P i ; on Factin, and of Mg 2+ and P i ; on microtubules, is proposed as being part of the conformational switch leading to the destabilization of the polymers.

scholarly journals The size of helical pitch is important for microtubule plus end dynamic instability

2020 ◽  
Author(s):  
Chenshu Liu ◽  
Louis Prahl ◽  
Yu He ◽  
Yan Wang ◽  
Ruijun Zhu ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Dynamic Instability ◽  
Chemotherapeutic Agents ◽  
Stochastic Simulations ◽  
Microtubule Assembly ◽  
Hydrolysis Rate ◽  
Cellular Functions ◽  
Helical Pitch ◽  
Division Cell ◽  
The Impact

ABSTRACTMicrotubule (MT) dynamic instability is a conserved phenomenon underlying essential cellular functions such as cell division, cell migration and intracellular transport, and is a key target of many chemotherapeutic agents. However, it remains unclear how the organization of tubulin dimers at the nanometer scale translates into dynamic instability as an emergent property at the micrometer scale. Tubulin dimers are organized into left-handed helical MT lattice, and most present-day MTs converge at a 1.5 dimer helical pitch that causes a seam in an otherwise symmetric helix. Because presently there are no experimental methods that can precisely manipulate tubulin subunit with sub-dimer resolution, the impact of helical pitch on dynamic instability remains unknown. Here by using stochastic simulations of microtubule assembly dynamics we demonstrate that helical pitch plays essential roles in MT plus end dynamic instability. By systematically altering helical pitch size, one half-dimer at a time, we found that a helical pitch as small as one half-dimer is sufficient to inhibit short-term MT length plateaus associated with diminishing GTP-tubulin cap. Notably, MT plus end dynamics quantitatively scale with the size of helical pitch, rather than being clustered by the presence or absence of helical symmetry. Microtubules with a 1.5 dimer helical pitch exhibit growth and shrinkage phases and undergo catastrophe and rescue similar to experimentally observed microtubules. Reducing helical pitch to 0 promotes rapid disassembly, while increasing it causes microtubules to undergo persistent growth, and it is the 1.5 dimer helical pitch that yields the highest percentage of MTs that undergo alternating growth and shrinkage without being totally disassembled. Finally, although the 1.5 dimer helical pitch is conserved among most present-day MTs, we find that other parameters, such as GTP hydrolysis rate, can partially compensate for changes in helical pitch. Together our results indicate that helical pitch is a determinant of MT plus end dynamic instability and that the evolutionarily conserved 1.5 dimer helical pitch promotes dynamic instability required for microtubule-dependent cellular functions.

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”).

Time-Resolved Cryo-Electron Microscopy of Oscillating Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 502-503
Author(s):  
Eva-Maria Mandelkow ◽  
Ron Milligan
Keyword(s):  
Electron Microscopy ◽  
Dynamic Instability ◽  
Entire Solution ◽  
Reaction Scheme ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
A Chain ◽  
Ray Scattering

Microtubules form part of the cytoskeleton of eukaryotic cells. They are hollow libers of about 25 nm diameter made up of 13 protofilaments, each of which consists of a chain of heterodimers of α-and β-tubulin. Microtubules can be assembled in vitro at 37°C in the presence of GTP which is hydrolyzed during the reaction, and they are disassembled at 4°C. In contrast to most other polymers microtubules show the behavior of “dynamic instability”, i.e. they can switch between phases of growth and phases of shrinkage, even at an overall steady state [1]. In certain conditions an entire solution can be synchronized, leading to autonomous oscillations in the degree of assembly which can be observed by X-ray scattering (Fig. 1), light scattering, or electron microscopy [2-5]. In addition such solutions are capable of generating spontaneous spatial patterns [6].In an earlier study we have analyzed the structure of microtubules and their cold-induced disassembly by cryo-EM [7]. One result was that disassembly takes place by loss of protofilament fragments (tubulin oligomers) which fray apart at the microtubule ends. We also looked at microtubule oscillations by time-resolved X-ray scattering and proposed a reaction scheme [4] which involves a cyclic interconversion of tubulin, microtubules, and oligomers (Fig. 2). The present study was undertaken to answer two questions: (a) What is the nature of the oscillations as seen by time-resolved cryo-EM? (b) Do microtubules disassemble by fraying protofilament fragments during oscillations at 37°C?

Mesoscopic gelation of chitosan and genipin at below critical concentrations

2017 ◽  
Author(s):  
Christoph Engwer ◽  
Ronja Loy ◽  
Ioannis S. Chronakis ◽  
Ana C. Mendes ◽  
Francisco M. Goycoolea
Keyword(s):  
Sol Gel ◽  
Crosslinking Agent ◽  
Small Deformation ◽  
Macroscopic Appearance ◽  
Critical Concentrations ◽  
Gelation Kinetics ◽  
Gardenia Jasminoides ◽  
Sol Gel Transition ◽  
Low Cytotoxicity ◽  
High Concentrations

Genipin is increasingly used as a crosslinking agent for chitosans due to its low cytotoxicity as a naturally occurring extract of the plant <i>Gardenia jasminoides</i>. Genipin reacts with the primary amino groups of chitosan to form blue hydrogels. We studied the gelation kinetics of different chitosans varying in their properties (molar mass 34 000-213 000 g mol<sup>-1</sup>, degree of acetylation 9-20%) and genipin in detail. We found that critical sol-gel transition times obtained from dynamic light scattering were in good agreement with the results obtained by small deformation oscillatory rheometry and microviscosimetry at high concentrations of chitosan. However, at below critical concentrations, we found a second regime of gelation that followed the same Ross-Murphy's gelation kinetics. The macroscopic appearance of these samples was a suspension of weak gel-like particles that were sensitive to mechanical forces. We believe that the material is a mesoscopic gel, as described for other polymers. To the best of our knowledge, this is the first time that this phenomenon has been described for the gelling system of chitosan and genipin.

Evaluation of treatability for two textile mill effluents

1999 ◽  
Vol 40 (1) ◽  
pp. 145-152 ◽  
Author(s):  
F. Germirli Babuna ◽  
B. Soyhan ◽  
G. Eremektar ◽  
D. Orhon
Keyword(s):  
Experimental Investigation ◽  
Biological Treatment ◽  
Chemical Oxidation ◽  
Domestic Sewage ◽  
Hydrolysis Rate ◽  
Textile Mill ◽  
Acrylic Fiber ◽  
Partial Chemical

The study emphasizes wastewater characteristics of two different textile plants as they apply to biological treatment. Although conventional characterization reveals no major differences, the effluents from the acrylic fiber and yarn dyeing plant exhibit all the properties of a non-biodegradable wastewater. Appropriate pretreatment consisting of partial chemical oxidation with H2O2 reduces its COD content to 700 mg l−1, almost entirely biodegradable whereas COD fractionation indicates that the effluents of the cotton knit dyeing plant contain 9% residual fractions. Experimental investigation shows that most kinetic and stoichiometric properties of both wastewaters are compatible with that of domestic sewage with the exception of a much slower hydrolysis rate.

In-vitro Kinetic Hydrolysis Study of Metronidazole Derivatives with Carvacrol and Eugenol Using Validated RP-HPLC Method

2020 ◽  
Vol 16 ◽  
Author(s):  
M. Alarjah
Keyword(s):  
Half Life ◽  
Hplc Method ◽  
Hydrolysis Rate ◽  
First Order ◽  
First Order Kinetics ◽  
Rp Hplc ◽  
Pharmacokinetic Properties ◽  
Pseudo First Order ◽  
Kinetic Hydrolysis

Background: Prodrugs principle is widely used to improve the pharmacological and pharmacokinetic properties of some active drugs. Much effort was made to develop metronidazole prodrugs to enhance antibacterial activity and or to improve pharmacokinetic properties of the molecule or to lower the adverse effects of metronidazole. Objective: In this work, the pharmacokinetic properties of some of monoterpenes and eugenol pro metronidazole molecules that were developed earlier were evaluated in-vitro. The kinetic hydrolysis rate constants and half-life time estimation of the new metronidazole derivatives were calculated using the validated RP-HPLC method. Method: Chromatographic analysis was done using Zorbbax Eclipse eXtra Dense Bonding (XDB)-C18 column of dimensions (250 mm, 4.6 mm, 5 μm), at ambient column temperature. The mobile phase was a mixture of sodium dihydrogen phosphate buffer of pH 4.5 and methanol in gradient elution, at 1ml/min flow rate. The method was fully validated according to the International Council for Harmonization (ICH) guidelines. The hydrolysis process carried out in an acidic buffer pH 1.2 and in an alkaline buffer pH 7.4 in a thermostatic bath at 37ºC. Results: The results followed pseudo-first-order kinetics. All metronidazole prodrugs were stable in the acidic pH, while they were hydrolysed in the alkaline buffer within a few hours (6-8 hr). The rate constant and half-life values were calculated, and their values were found to be 0.082- 0.117 hr-1 and 5.9- 8.5 hr., respectively. Conclusion: The developed method was accurate, sensitive, and selective for the prodrugs. For most of the prodrugs, the hydrolysis followed pseudo-first-order kinetics; the method might be utilised to conduct an in-vivo study for the metronidazole derivatives with monoterpenes and eugenol.

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