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.

scholarly journals XMAP from Xenopus eggs promotes rapid plus end assembly of microtubules and rapid microtubule polymer turnover.

1994 ◽  
Vol 127 (4) ◽  
pp. 985-993 ◽  
Author(s):  
R J Vasquez ◽  
D L Gard ◽  
L Cassimeris
Keyword(s):  
Dynamic Instability ◽  
Fold Increase ◽  
Nucleation Site ◽  
Microtubule Assembly ◽  
Shortening Velocity ◽  
Threefold Increase ◽  
Cell Biol ◽  
Dic Microscopy

We have used video-enhanced DIC microscopy to examine the effects of XMAP, a Mr 215,000 microtubule-associated protein from Xenopus eggs (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), on the dynamic instability of microtubules nucleated from axoneme fragments in vitro. Our results indicate that XMAP substantially alters the parameters of microtubule assembly at plus ends. Specifically, addition of 0.2 microM XMAP resulted in (a) 7-10-fold increase in elongation velocity, (b) approximately threefold increase in shortening velocity, and (c) near elimination of rescue (the switch from rapid shortening to elongation). Thus, addition of XMAP resulted in the assembly of longer, but more dynamic, microtubules from the plus ends of axonemes which upon catastrophe disassembled back to the axoneme nucleation site. In agreement with previous observations (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), the effects of XMAP on the minus end were much less dramatic, with only a 1.5-3-fold increase in elongation velocity. These results indicate that XMAP, unlike brain MAPs, promotes both polymer assembly and turnover, and suggests that the interaction of XMAP with tubulin and the function of XMAP in vivo may differ from previously characterized MAPs.

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 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.

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

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.

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.

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”).

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

Anti-thrombotic Effect of a PAI-1 Inhibitor in Rats Given Endotoxin

1996 ◽  
Vol 75 (01) ◽  
pp. 118-126 ◽  
Author(s):  
T Abrahamsson ◽  
V Nerme ◽  
M Strömqvist ◽  
B Åkerblom ◽  
A Legnehed ◽  
...  
Keyword(s):  
Plasminogen Activator Inhibitor ◽  
Rat Plasma ◽  
Clot Lysis ◽  
Fibrin Deposition ◽  
Plasminogen Activator Inhibitor 1 ◽  
Fab Fragment ◽  
Dose Dependent Decrease ◽  
Pai 1

SummaryThe aim of this study was to investigate the anti-thrombotic effects of an inhibitor of the plasminogen activator inhibitor-1 (PAI-1) in rats given endotoxin. In studies in vitro, PRAP-1, a Fab-fragment of a polyclonal antibody against human PAI-1, was shown to inhibit PAI-1 activity in rat plasma as well as to stimulate clot-lysis of the euglobulin fraction derived from rat plasma. Endotoxin administered to anaesthetised rats produced a marked increase in plasma PAI-1 activity. To study fibrin formation and lysis in vivo after intravenous (i. v.) injection of the coagulant enzyme batroxobin, 125I-fibrinogen was administered to the animals. The thrombi formed by batroxobin were rapidly lysed in control animals, while the rate of lysis was markedly attenuated in rats given endotoxin. PRAP-1 was administered i.v. (bolus + infusion) to rats given endotoxin and batroxobin and the PAI-1 inhibitor caused a dose-dependent decrease in the 125I-fibrin deposition in the lungs. An immunohistochemical technique was used to confirm this decrease in density of fibrin clots in the tissue. Furthermore, PRAP-1 decreased plasma PAI-1 activity in the rats and this reduction was correlated to the decrease in lung 125I-fibrin deposition at the corresponding time point. It is concluded that in this experimental model the PAI-1 antibody PRAP-1 may indeed inhibit thrombosis in animals exposed to endotoxin.

scholarly journals A novel dephosphorylation targeting chimera selectively promoting tau removal in tauopathies

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Jie Zheng ◽  
Na Tian ◽  
Fei Liu ◽  
Yidian Zhang ◽  
Jingfen Su ◽  
...  
Keyword(s):  
Neurodegenerative Disorders ◽  
Protein Phosphatase ◽  
High Efficiency ◽  
Microtubule Assembly ◽  
Hyperphosphorylated Tau ◽  
Phosphatase 2A ◽  
Dependent Learning ◽  
The Brain

AbstractIntraneuronal accumulation of hyperphosphorylated tau is a hallmark pathology shown in over twenty neurodegenerative disorders, collectively termed as tauopathies, including the most common Alzheimer’s disease (AD). Therefore, selectively removing or reducing hyperphosphorylated tau is promising for therapies of AD and other tauopathies. Here, we designed and synthesized a novel DEPhosphorylation TArgeting Chimera (DEPTAC) to specifically facilitate the binding of tau to Bα-subunit-containing protein phosphatase 2A (PP2A-Bα), the most active tau phosphatase in the brain. The DEPTAC exhibited high efficiency in dephosphorylating tau at multiple AD-associated sites and preventing tau accumulation both in vitro and in vivo. Further studies revealed that DEPTAC significantly improved microtubule assembly, neurite plasticity, and hippocampus-dependent learning and memory in transgenic mice with inducible overexpression of truncated and neurotoxic human tau N368. Our data provide a strategy for selective removal of the hyperphosphorylated tau, which sheds new light for the targeted therapy of AD and related-tauopathies.

scholarly journals Drosophila Stathmin: A Microtubule-destabilizing Factor Involved in Nervous System Formation

2002 ◽  
Vol 13 (2) ◽  
pp. 698-710 ◽  
Author(s):  
Sylvie Ozon ◽  
Antoine Guichet ◽  
Olivier Gavet ◽  
Siegfried Roth ◽  
André Sobel
Keyword(s):  
Nervous System ◽  
System Development ◽  
Nervous System Development ◽  
Microtubule Assembly ◽  
Nervous Systems ◽  
Germ Cell Migration ◽  
Numerous Cell ◽  
First Time

Stathmin is a ubiquitous regulatory phosphoprotein, the generic element of a family of neural phosphoproteins in vertebrates that possess the capacity to bind tubulin and interfere with microtubule dynamics. Although stathmin and the other proteins of the family have been associated with numerous cell regulations, their biological roles remain elusive, as in particular inactivation of the stathmin gene in the mouse resulted in no clear deleterious phenotype. We identified stathmin phosphoproteins inDrosophila, encoded by a unique gene sharing the intron/exon structure of the vertebrate stathmin andstathmin family genes. They interfere with microtubule assembly in vitro, and in vivo when expressed in HeLa cells. Drosophila stathmin expression is regulated during embryogenesis: it is high in the migrating germ cells and in the central and peripheral nervous systems, a pattern resembling that of mammalian stathmin. Furthermore, RNA interference inactivation ofDrosophila stathmin expression resulted in germ cell migration arrest at stage 14. It also induced important anomalies in nervous system development, such as loss of commissures and longitudinal connectives in the ventral cord, or abnormal chordotonal neuron organization. In conclusion, a single Drosophilagene encodes phosphoproteins homologous to the entire vertebrate stathmin family. We demonstrate for the first time their direct involvement in major biological processes such as development of the reproductive and nervous systems.

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