scholarly journals Temperature and pH govern the self-assembly of microtubules from unfertilized sea-urchin egg extracts

1987 ◽  
Vol 87 (1) ◽  
pp. 71-84 ◽  
Author(s):  
K.A. Suprenant ◽  
J.C. Marsh
Keyword(s):  
Sea Urchin ◽  
Self Assembly ◽  
Ionic Composition ◽  
New Method ◽  
Temperature Dependent ◽  
Microtubule Associated Proteins ◽  
Sea Urchin Egg ◽  
The Pacific ◽  
Egg Extracts ◽  
Associated Proteins

A new method for microtubule purification from unfertilized sea-urchin eggs was developed in order to obtain large quantities of calcium- and cold-labile microtubules that contained microtubule-associated components important for mitosis. By taking into consideration the pH, ionic composition of egg cytoplasm, and the physiological temperature for growth of the Pacific coast sea-urchin Strongylocentrotus purpuratus, methods were developed for the assembly of intact microtubules directly from unfertilized egg extracts. The microtubules obtained by cycles of temperature-dependent assembly and disassembly are composed of tubulin and abundant microtubule-associated proteins. These microtubules are cold- and calcium-labile and assemble at a critical protein concentration of 0.11 mg ml-1 at 24 degrees C. The yield of microtubule protein obtained by this new method is equivalent to that obtained with taxol (6–8 mg/20 ml packed eggs). Microtubules that have been fixed and prepared for electron microscopy are decorated with large, globular projections that are attached to the microtubule by thin stalks.

scholarly journals Temperature-dependent reversible assembly of taxol-treated microtubules.

1987 ◽  
Vol 105 (6) ◽  
pp. 2847-2854 ◽  
Author(s):  
C A Collins ◽  
R B Vallee
Keyword(s):  
Electron Microscopy ◽  
Self Assembly ◽  
Temperature Dependent ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Microtubule Associated Proteins ◽  
Negative Stain ◽  
Microtubule Protein ◽  
Associated Proteins ◽  
Negative Stain Electron Microscopy

Taxol is a plant alkaloid that binds to and strongly stabilizes microtubules. Taxol-treated microtubules resist depolymerization under a variety of conditions that readily disassemble untreated microtubules. We report here that taxol-treated microtubules can be induced to disassemble by a combination of depolymerizating conditions. Reversible cycles of disassembly and reassembly were carried out using taxol-containing microtubules from calf brain and sea urchin eggs by shifting temperature in the presence of millimolar levels of Ca2+. Microtubules depolymerized completely, yielding dimers and ring-shaped oligomers as revealed by negative stain electron microscopy and Bio-Gel A-15m chromatography, and reassembled into well-formed microtubule polymer structures. Microtubule-associated proteins (MAPs), including species previously identified only by taxol-based purification such as MAP 1B and kinesin, were found to copurify with tubulin through reversible assembly cycles. To determine whether taxol remained bound to tubulin subunits, we subjected depolymerized taxol-treated microtubule protein to Sephadex G-25 chromatography, and the fractions were assayed for taxol content by reverse-phase HPLC. Taxol was found to be dissociated from the depolymerized microtubules. Protein treated in this way was found to be competent to reassemble, but now required conditions comparable with those for protein that had never been exposed to taxol. Thus, the binding of taxol to tubulin can be reversed. This has implications for the mechanism of taxol action and for the purification of microtubules from a wide variety of sources for use in self-assembly experiments.

Chapter 9 Actin Gelation in Sea Urchin Egg Extracts

1982 ◽  
pp. 175-199 ◽  
Author(s):  
Joseph Bryan ◽  
Robert E. Kane
Keyword(s):  
Sea Urchin ◽  
Sea Urchin Egg ◽  
Egg Extracts

Traces of brain microtubule-associated proteins affect dynamic properties of microtubules

1990 ◽  
Vol 68 (10) ◽  
pp. 1202-1209 ◽  
Author(s):  
Robert A. B. Keates
Keyword(s):  
Self Assembly ◽  
Dynamic Properties ◽  
Microtubule Assembly ◽  
Microtubule Associated Proteins ◽  
End Point ◽  
Associated Proteins ◽  
Polymerization Mixture ◽  
Low Levels

A method is described for measuring the quantities of stable and dynamic microtubules in a population in vitro. The method exploits the tendency of dynamic microtubules to depolymerize rapidly after being sheared. Stable microtubules, such as those protected by microtubule-associated proteins (MAPs), are broken to a smaller size by shearing, but do not depolymerize into subunits. The usual difficulty with this procedure is that the tubulin released from the dynamic microtubules rapidly repolymerizes before the end point of depolymerization can be measured. This has been overcome by including a small quantity of tubulin–colchicine complex in the mixture to block the repolymerization. For a total of 24 μM tubulin in a polymerization mixture, 10 μM of the sample polymerized originally under the conditions used. When 1.05 μM tubulin–colchicine complex was added at the time of shearing, the dynamic microtubules depolymerized, but the tubulin was released was unable to repolymerize and a small fraction of stable microtubules that resisted shear-induced depolymerization could then be detected. When traces of MAPs (0.23–2.8% by mass) were included in the tubulin mixture, the fraction of stable microtubules increased from 5% in the absence of added MAPs to 41% in the presence of 2.8% MAPs. All the MAPs in the mixture were found in the stable fraction and this stable fraction forms early during microtubule assembly. Calculations on the extent of enrichment of MAPs in the stable fraction indicated that as little as 4% MAPs in a microtubule protected it from shear-induced disassembly. The results suggest that low levels of MAPs may distribute nonrandomly in the microtubule population.Key words: dynamics, microtubules, tubulin, microtubule-associated proteins, self-assembly.

Isolation of Mitotic Microtubule-Associated Proteins from Sea Urchin Eggs

1986 ◽  
Vol 466 (1 Dynamic Aspec) ◽  
pp. 328-339 ◽  
Author(s):  
GEORGE S. BLOOM ◽  
FRANCIS C. LUCA ◽  
CHRISTINE A. COLLINS ◽  
RICHARD B. VALLEE
Keyword(s):  
Sea Urchin ◽  
Microtubule Associated Proteins ◽  
Sea Urchin Eggs ◽  
Associated Proteins

scholarly journals Yeast proteins associated with microtubules in vitro and in vivo.

1992 ◽  
Vol 3 (1) ◽  
pp. 29-47 ◽  
Author(s):  
G Barnes ◽  
K A Louie ◽  
D Botstein
Keyword(s):  
Self Assembly ◽  
Synthetic Peptides ◽  
Immunofluorescence Microscopy ◽  
The Self ◽  
Microtubule Associated Proteins ◽  
Amino Terminal ◽  
Novel Proteins ◽  
Associated Proteins

Conditions were established for the self-assembly of milligram amounts of purified Saccharomyces cerevisiae tubulin. Microtubules assembled with pure yeast tubulin were not stabilized by taxol; hybrid microtubules containing substoichiometric amounts of bovine tubulin were stabilized. Yeast microtubule-associated proteins (MAPs) were identified on affinity matrices made from hybrid and all-bovine microtubules. About 25 yeast MAPs were isolated. The amino-terminal sequences of several of these were determined: three were known metabolic enzymes, two were GTP-binding proteins (including the product of the SAR1 gene), and three were novel proteins not found in sequence databases. Affinity-purified antisera were generated against synthetic peptides corresponding to two of the apparently novel proteins (38 and 50 kDa). Immunofluorescence microscopy showed that both these proteins colocalize with intra- and extranuclear microtubules in vivo.

Evidence for an association between U1 RNA and interspersed repeat single-copy RNAs in the cytoplasm of sea urchin eggs

Development ◽  
1987 ◽  
Vol 101 (1) ◽  
pp. 107-116
Author(s):  
S. Ruzdijic ◽  
T. Pederson
Keyword(s):  
Sea Urchin ◽  
Messenger Rna ◽  
Single Copy ◽  
Mrna Splicing ◽  
Rna Molecules ◽  
Sea Urchin Egg ◽  
Selection Experiments ◽  
U1 Snrnp ◽  
Egg Extracts ◽  
Rna Complexes

Psoralen crosslinking of RNA-RNA intermolecular duplexes in sea urchin egg extracts reveals that some maternal poly(A)+ RNA molecules are complexed with U1 RNA, a cofactor in somatic nuclear pre-mRNA splicing. Reaction of egg extracts with a monoclonal antibody specific for U1 snRNP selects, in addition to U1, RNAs that contain repeated sequences interspersed with single-copy elements. Antibody-selection experiments with nucleate and anucleate egg halves demonstrate that most of the U1 RNA-interspersed RNA complexes are cytoplasmic, as is the egg's store of total U1 snRNP. These results raise the possibility that maternal interspersed RNAs include unprocessed pre-messenger RNA molecules in arrested complexes with splicing cofactors.

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