scholarly journals Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs.

1987 ◽  
Vol 105 (5) ◽  
pp. 2191-2201 ◽  
Author(s):  
D L Gard ◽  
M W Kirschner
Keyword(s):  
Xenopus Oocytes ◽  
Critical Concentration ◽  
Specific Inhibitor ◽  
Bovine Brain ◽  
High Rate ◽  
Microtubule Assembly ◽  
Microtubule Networks ◽  
Brain Tubulin

We have investigated the differences in microtubule assembly in cytoplasm from Xenopus oocytes and eggs in vitro. Extracts of activated eggs could be prepared that assembled extensive microtubule networks in vitro using Tetrahymena axonemes or mammalian centrosomes as nucleation centers. Assembly occurred predominantly from the plus-end of the microtubule with a rate constant of 2 microns.min-1.microM-1 (57 s-1.microM-1). At the in vivo tubulin concentration, this corresponds to the extraordinarily high rate of 40-50 microns.min-1. Microtubule disassembly rates in these extracts were -4.5 microns.min-1 (128 s-1) at the plus-end and -6.9 microns.min-1 (196 s-1) at the minus-end. The critical concentration for plus-end microtubule assembly was 0.4 microM. These extracts also promoted the plus-end assembly of microtubules from bovine brain tubulin, suggesting the presence of an assembly promoting factor in the egg. In contrast to activated eggs, assembly was never observed in extracts prepared from oocytes, even at tubulin concentrations as high as 20 microM. Addition of oocyte extract to egg extracts or to purified brain tubulin inhibited microtubule assembly. These results suggest that there is a plus-end-specific inhibitor of microtubule assembly in the oocyte and a plus-end-specific promoter of assembly in the eggs. These factors may serve to regulate microtubule assembly during early development in Xenopus.

scholarly journals Microtubule assembly and disassembly at alkaline pH

1981 ◽  
Vol 89 (1) ◽  
pp. 45-53 ◽  
Author(s):  
CS Regula ◽  
Pfeiffer JR ◽  
RD Berlin
Keyword(s):  
Steady State ◽  
Critical Concentration ◽  
Ph Dependence ◽  
Intrinsic Rate ◽  
Bovine Brain ◽  
Microtubule Assembly ◽  
Alkaline Ph ◽  
Direct Assembly

Although it is now apparent that the intracellular pH may rise considerably above neutrality under physiological conditions, information on the effect of alkaline pH on microtubule assembly and disassembly is still quite fragmentay. We have studied the assembly/disassembly of bovine brain microtubule protein at alkaline pH in vitro. When microtubules are assembled to a new steady state at pH less than 7 and pH is then made more alkaline, they undergo a rapid disassembly to a new steady state. This disassembly is reversed by acidification. The degree of disassembly is determined largely by the pH- dependence of the critical concentration, which increases five to eight times, from pH 7 to 8. A fraction of assembly-incompetent tubulin is identified that increases with pH, but its incompetency is largely reversed with acidification. Measurements of microtubule lengths are used to indicate that disassembly occurs by uniform shortening of microtubules. A comparison of shortening by alkalinization with dilution suggests that the intrinsic rate of disassembly is accelerated by increasing pH. The capacity for initiating assembly is progressively lost with incubation at alkaline pH (although some protection is afforded by sulfhydryl-reducing agents). However, direct assembly from depolymerized mixtures is possible at least up to pH 8.3, and the steady state achieved at these alkaline pH values is stable. Such preparations are readily disassembled by cold and podophyllotoxin (PLN). Disassembly induced by PLN is also markedly enhanced at alkaline pH, suggesting a corresponding enhancement of "treadmilling." The implications of physiological events leading to alkaline shifts of pH for microtubule assembly/disassembly are discussed, particularly in the light of recent hypotheses regarding treadmilling and its role in controlling the distribution of microtubules in vivo.

Inability to detect Chlamyodomonas microtubule assembly in vitro: possible implications to the in vivo regulation of microtubule assembly

1975 ◽  
Vol 17 (3) ◽  
pp. 669-681
Author(s):  
K.W. Farrell ◽  
R.G. Burns
Keyword(s):  
Column Chromatography ◽  
Divalent Cation ◽  
Microtubule Assembly ◽  
Mammalian Brain ◽  
Wide Range ◽  
In Vitro Assembly ◽  
Brain Extracts ◽  
Brain Tubulin

It has been demonstrated that the in vitro assembly of microtubules from Chlamydomonas preparations does not occur under a wide range of conditions, including those efficacious for mammalian brain tubulin. This incompetence of Chlamydomonas extracts to form microtubules is independent of the tubulin concentration, the presence of added nucleotides or an added seed, temperature, or the concentration of divalent cation. However, an amorphous aggregate was observed under certain conditions, who composition was mainly tubulin. The in vitro reassembly of microtubules in gerbil brain extracts is inhibited by Chlamydomonas preparations. Fractionation of the Chlamydomonas extracts by column chromatography suggests that the inhibitory component is Chlamydomonas tubulin itself. The mechanism of this inhibition is unknown, but reassembly experiments indicate that the 2 types of tubulins cannot copolymerize. We suggest that the Chlamydomonas tubulin, derived from a cytoplasmic pool, requires to be activated prior to its in vivo polymerization into microtubules.

In vitro lens transdifferentiation of Xenopus laevis outer cornea induced by Fibroblast Growth Factor (FGF)

Development ◽  
1997 ◽  
Vol 124 (2) ◽  
pp. 421-428
Author(s):  
L. Bosco ◽  
G. Venturini ◽  
D. Willems
Keyword(s):  
Growth Factor ◽  
Xenopus Laevis ◽  
Fibroblast Growth Factor ◽  
Specific Inhibitor ◽  
Bovine Brain ◽  
Fibroblast Growth ◽  
Acidic Fibroblast Growth Factor ◽  
Lens Fibers

It has been shown that lens regeneration from outer cornea of larval Xenopus laevis is dependent on neural retina both in vivo and in tissue culture. The isolated outer cornea cultured in the presence of bovine brain-derived acidic Fibroblast Growth Factor (aFGF) is able to reprogram the differentiation into lens fibers, although this transdifferentiative process is not coupled with the formation of a normally organized lens. The capacity of aFGF to promote lens differentiation from cornea is not linked to its mitogenic activity. The cultured corneal cells can transdifferentiate into lens fibers in the presence of aFGF when DNA replication and cell proliferation are prevented by addition of aphidicolin, a specific inhibitor of DNA polymerase in eukaryotes, to the culture medium.

scholarly journals Human chromosomes and centrioles as nucleating sites for the in vitro assembly of microtubules from bovine brain tubulin.

1975 ◽  
Vol 67 (1) ◽  
pp. 189-199 ◽  
Author(s):  
M McGill ◽  
B R Brinkley
Keyword(s):  
Mammalian Cells ◽  
Experimental Studies ◽  
Bovine Brain ◽  
Microtubule Assembly ◽  
Direct Result ◽  
Metaphase Chromosomes ◽  
Microtubule Formation ◽  
Triton X ◽  
Brain Tubulin

Treatment of HeLa cells with Colcemid at concentrations of 0.06-0.10 mug/ml leads to irreversible arrest in mitosis. Colcemid-arrested cells contained few microtubules, and many kinetochores and centrioles were free of microtubule association. When these cells were exposed to microtubule reassembly buffer containing Triton X-100 and bovine brain tubulin at 37 degrees C, numerous microtubules were reassembled at all kinetochores of metaphase chromosomes and in association with centriole pairs. When bovine brain tubulin was eliminated from the reassembly system, microtubules failed to assemble at these sites. Similarly, when EGTA was eliminated from the reassembly system, microtubules failed to polymerize. These results are consistent with other investigations of in vitro microtubule assembly and indicate that HeLa chromosomes and centrioles can serve as nucleating sites for the assembly of microtubules from brain tubulin. Both chromosomes and centrioles became displaced from their C-metaphase configurations during tubulin reassembly, indicating that their movements were a direct result of microtubule formation. Although both kinetochore- and centriole-associated microtubules were assembled and movement occurred, we did not observe direct extension of microtubules from kinetochores to centrioles. This system should prove useful for experimental studies of spindle microtubule formation and chromosome movement in mammalian cells.

scholarly journals The periodic association of MAP2 with brain microtubules in vitro.

1979 ◽  
Vol 80 (2) ◽  
pp. 266-276 ◽  
Author(s):  
H Kim ◽  
L I Binder ◽  
J L Rosenbaum
Keyword(s):  
Critical Concentration ◽  
Microtubule Assembly ◽  
Molar Ratio ◽  
Thin Sections ◽  
Microtubule Associated Proteins ◽  
Heat Stable ◽  
Associated Proteins ◽  
Brain Microtubules ◽  
Brain Tubulin

Several high molecular weight polypeptides have been shown to quantitatively copurify with brain tubulin during cycles of in vitro assembly-disassembly. These microtubule-associated proteins (MAPs) have been shown to influence the rate and extent of microtubule assembly in vitro. We report here that a heat-stable fraction highly enriched for one of the MAPs, MAP2 (mol wt approximately 300,000 daltons), devoid of MAP1 (mol wt approximately 350,000 daltons), has been purified from calf neurotubules. This MAP2 fraction stoichiometrically promotes microtubule assembly, lowering the critical concentration for tubulin assembly to 0.05 mg/ml. Microtubules saturated with MAP2 contain MAP2 and tubulin in a molar ratio of approximately 1 mole of MAP2 to 9 moles of tubulin dimer. Electron microscopy of thin sections of the MAP2-saturated microtubules fixed in the presence of tannic acid demonstrates a striking axial periodicity of 32 +/- 8 nm.

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.

scholarly journals Flagellar elongation and shortening in Chlamydomonas. III. structures attached to the tips of flagellar microtubules and their relationship to the directionality of flagellar microtubule assembly.

1977 ◽  
Vol 74 (3) ◽  
pp. 747-759 ◽  
Author(s):  
W L Dentler ◽  
J L Rosenbaum
Keyword(s):  
Microtubule Assembly ◽  
Central Pair ◽  
In Vitro Assembly ◽  
Flagellar Membrane ◽  
Brain Tubulin

Two structures on the distal ends of Chlamydomonas flagellar microtubules are described. One of these, the central microbutule cap, attaches the distal ends of the central pair microtubules to the tip of the flagellar membrane. In addition, filaments, called distal filaments, are observed attached to the ends of the A-tubules of the outer doublet microtubules. Inasmuch as earlier studies suggested that flagellar elongation in vivo occurs principally by the distal addition of sublnits and because it has been shown that brain tubulin assembles in vitro primarily onto the distal ends of both central and outer doublet microtubules, the presence of the cap and distal filaments was quantitated during flagellar resorption and elongation. The results showed that the cap remains attached to the central microtubules throughout flagellar resorption and elongation. The cap was also found to block the in vitro assembly of neurotubules onto the distal ends of the central microtubules. Conversely, the distal filaments apparently do not block the assembly of neurotubules onto the ends of the outer doublets. During flagellar elongation, the distal ends of the outer doublets are often found to form sheets of protofilaments similar to those observed on the elongating ends of neurotubules being assembled in vitro. These results suggest that the outer doublet microtubules elongate by the distal addition of subunits, whereas the two central microtubules assemble by the addition of subunits to the proximal ends.

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.

scholarly journals Importance of beta-lactamase inhibitor pharmacokinetics in the pharmacodynamics of inhibitor-drug combinations: studies with piperacillin-tazobactam and piperacillin-sulbactam.

1997 ◽  
Vol 41 (4) ◽  
pp. 721-727 ◽  
Author(s):  
P D Lister ◽  
A M Prevan ◽  
C C Sanders
Keyword(s):  
Antibacterial Activity ◽  
Critical Concentration ◽  
Specific Inhibitor ◽  
Logarithmic Phase ◽  
Critical Ratio ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Dosing Regimens ◽  
Lactamase Inhibitor

An in vitro pharmacokinetic model was used to study the pharmacodynamics of piperacillin-tazobactam and piperacillin-sulbactam against gram-negative bacilli producing plasmid-encoded beta-lactamases. Logarithmic-phase cultures were exposed to peak antibiotic concentrations observed in human serum after the administration of intravenous doses of 3 g of piperacillin and 0.375 g of tazobactam or 0.5 g of sulbactam. Piperacillin and inhibitor were either dosed simultaneously or piperacillin was dosed sequentially 0.5 h after dosing with the inhibitor. In studies with all four test strains, the pharmacodynamics observed after simultaneous dosing were similar to those observed with the sequential regimen. Since the ratio between piperacillin and tazobactam was in constant fluctuation after sequential dosing, these data suggest that the pharmacodynamics of the piperacillin-inhibitor combinations were not dependent upon maintenance of a critical ratio between the components. Furthermore, when regrowth was observed, the time at which bacterial counts began to increase was similar between the simultaneous and sequential dosing regimens. Since the pharmacokinetics of the inhibitors were the same for all regimens, these data suggest that the length of time that the antibacterial activity was maintained over the dosing interval with these combinations was dictated by the pharmacokinetics of the beta-lactamase inhibitor in the combination. The antibacterial activity of the combination appeared to be lost when the amount of inhibitor available fell below some critical concentration. This critical concentration varied depending upon the type and amount of enzyme produced, as well as the specific inhibitor used. These results indicate that the antibacterial activity of drug-inhibitor combinations, when dosed at their currently recommended ratios, is more dependent on the pharmacokinetics of the inhibitor than on those of the beta-lactam drug.

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