The effects of mitotic inhibitors on the structure of vinblastine-induced tubulin paracrystals from sea-urchin eggs

1976 ◽  
Vol 20 (1) ◽  
pp. 91-100
Author(s):  
D. Starling
Keyword(s):  
Sea Urchin ◽  
Ultrastructural Level ◽  
Tubulin Polymerization ◽  
Sea Urchin Eggs ◽  
Mitotic Inhibitors ◽  
Vinblastine Sulphate ◽  
Tubulin Paracrystals ◽  
Careful Standardization

Vinblastine sulphate (VLB) is known to induce in vivo formation of tubulin paracrystals in sea-urchin eggs. Corresponding paracrystals have been prepared in the presence of both vinblastine sulphate and other mitoclasic agents. Careful standardization of conditions was required to restrict the formation of alternative forms of the paracrystals induced by vinblastine alone. Comparisons were made between preparations in terms of paracrystal shape, size, proportion of eggs containing paracrystals, number per egg and their relative times of first appearance. A correlation between such properties were established. Comparison of paracrystals at the ultrastructural level showed them all to be similar regardless of the drugs present during their formation. The implications of tubulin polymerization in the presence of mitoclasic agents are discussed and mechanisms for paracrystal enhancement by combinations of such drugs are suggested. Some similarities of paracrystal and microtubule seeding are discussed together with the activation of tubulin in the pool.

Two ultrastructurally distinct tubulin paracrystals induced in sea-urchin eggs by vinblastine sulphate

1976 ◽  
Vol 20 (1) ◽  
pp. 79-89 ◽  
Author(s):  
D. Starling
Keyword(s):  
Sea Urchin ◽  
Binding Sites ◽  
Mammalian Cells ◽  
The Other ◽  
Sea Urchin Eggs ◽  
Vincristine Sulphate ◽  
Vinblastine Sulphate ◽  
Available Information ◽  
Tubulin Paracrystals

Two types of ultrastructurally distinct tubulin paracrystals have been induced in sea-urchin eggs with vinblastine sulphate (VLB) under different sets of conditions. One type of paracrystal appears to consist of hexagonally-close packed microtubules and closely resembles paracrystals present in mammalian cells treated with vinblastine or vincristine sulphate, but not previously reported in sea-urchin eggs. The other type is also made up of tubulin subunits, but these do not seem to have polymerized into microtubules. Both types of paracrystal are induced in sea-urchin eggs in the presence of VLB at a time when tubulin subunits would not normally polymerize. Possible mechanisms for tubulin activation and the induction of paracrystal formation are discussed in respect to the available information on the binding sites of the tubulin subunits.

scholarly journals Effects of caffeine and other methylxanthines on the development and metabolism of sea urchin eggs. Involvement of NADP and glutathione.

1976 ◽  
Vol 68 (3) ◽  
pp. 440-450 ◽  
Author(s):  
J Nath ◽  
J I Rebhun
Keyword(s):  
Cell Division ◽  
Sea Urchin ◽  
Reductase Activity ◽  
Inhibitory Effect ◽  
Pentose Phosphate ◽  
Nad Kinase ◽  
Mitotic Apparatus ◽  
Sea Urchin Eggs ◽  
Glutathione Reductase Activity

Methylxanthines (MX) inhibit cell division in sea urchin and clam eggs. This inhibitory effect is not mediated via cAMP. MX also inhibit respiration in marine eggs, at concentrations which inhibit cleavage. Studies showed that no changes occurred in ATP and ADP levels in the presence of inhibitory concentrations of MX, indicating an extra-mitochondrial site of action for the drug. Subsequent studies revealed decreased levels of NADP+ and NADPH, when eggs were incubated with inhibitory concentrations of MX, but no change in levels of NAD+ and NADH. MX did not affect the pentose phosphate shunt pathway and did not have any effect on the enzyme NAD+ -kinase. Further studies showed a marked inhibitory effect on the glutathione reductase activity of MX-treated eggs. Reduced glutathione (GSH) could reverse the cleavage inhibitory effect of MX. Moreover, diamide, a thiol-oxidizing agent specific for GSH in living cells, caused inhibition of cell division in sea urchin eggs. Diamide added to eggs containing mitotic apparatus (MA) could prevent cleavage by causing a dissolution of the formed MA. Both MX and diamide inhibit a Ca2+-activated ATPase in whole eggs. The enzyme can be reactivated by sulfhydryl reducing agents added in the assay mixture. In addition, diamide causes an inhibition of microtubule polymerization, reversible with dithioerythritol. All experimental evidence so far suggests that inhibition of mitosis in sea urchin eggs by MX is mediated by perturbations of the in vivo thiol-disulfide status of target systems, with a primary effect on glutathione levels.

scholarly journals Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs.

1976 ◽  
Vol 70 (1) ◽  
pp. 75-85 ◽  
Author(s):  
G Sluder
Keyword(s):  
Nuclear Envelope ◽  
Sea Urchin ◽  
Maximum Rate ◽  
Spindle Assembly ◽  
Experimental Manipulation ◽  
Lytechinus Variegatus ◽  
Tubulin Polymerization ◽  
Treated Cell ◽  
Sea Urchin Eggs ◽  
Nuclear Envelope Breakdown

Spindle assembly is studied in the eggs of the sea urchin Lytechinus variegatus by experimentally varying the amount of polymerizable tubulin within the egg. Aliquots of fertilized eggs from the same female are individually pulsed for 1-6 min with 1 X 10(-6) M Colcemid at least 20 min before first nuclear envelope breakdown. This treatment inactivates a portion of the cellular tubulin before the spindle is formed. Upon entering mitosis, treated eggs form functional spindles that are reduced in length and birefringent retardation but not width. With increased exposure to Colcemid, the length and retardation of the metaphase spindles are progressively reduced. Similar results are obtained by pulsing the eggs with Colcemid before fertilization, which demonstrates that the tubulin found in unfertilized sea urchin eggs is later used in spindle formation. Spindles, once assembled, are responsive to increases in the amount of polymerizable tubulin within the cell. Rapid increases in the amount of polymerizable tubulin within a Colcemid-treated cell can be experimentally effected by irradiating the cells with 366-nm light. This treatment photochemically inactivates the Colcemid, thereby freeing the tubulin to polymerize. Upon irradiation, the small prometaphase spindles of Colcemid-treated cells immediately increase in length and retardation. In these irradiated cells, spindle length and retardation increase as much as four times faster than they do during prometaphase for normal spindles. This suggests that the rate of the normal prometaphase increase in retardation and spindle size may be determined by factors other than the maximum rate of tubulin polymerization in the cell.

scholarly journals Cell cycle-dependent phosphorylation of the 77 kDa echinoderm microtubule-associated protein (EMAP) in vivo and association with the p34cdc2 kinase

1996 ◽  
Vol 109 (12) ◽  
pp. 2885-2893 ◽  
Author(s):  
E. Brisch ◽  
M.A. Daggett ◽  
K.A. Suprenant
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
Time Course ◽  
Mitotic Apparatus ◽  
Sea Urchin Eggs ◽  
Spindle Poles ◽  
A Cell ◽  
Cycle Dependent ◽  
Phosphopeptide Mapping

The most abundant microtubule-associated protein in sea urchin eggs and embryos is the 77 kDa echinoderm microtubule-associated protein (EMAP). EMAP localizes to the mitotic spindle as well as the interphase microtubule array and is a likely target for a cell cycle-activated kinase. To determine if EMAP is phosphorylated in vivo, sea urchin eggs and embryos were metabolically labeled with 32PO4 and a monospecific antiserum was used to immunoprecipitate EMAP from 32P-labeled eggs and embryos. In this study, we demonstrate that the 77 kDa EMAP is phosphorylated in vivo by two distinct mechanisms. In the unfertilized egg, EMAP is constitutively phosphorylated on at least five serine residues. During the first cleavage division following fertilization, EMAP is phosphorylated with a cell cycle-dependent time course. As the embryo enters mitosis, EMAP phosphorylation increases, and as the embryo exits mitosis, phosphorylation decreases. During mitosis, EMAP is phosphorylated on 10 serine residues and two-dimensional phosphopeptide mapping reveals a mitosis-specific site of phosphorylation. At all stages of the cell cycle, a 33 kDa polypeptide copurifies with the 77 kDa EMAP, regardless of phosphorylation state. Antibodies against the cdc2 kinase were used to demonstrate that the 33 kDa polypeptide is the p34cdc2 kinase. The p34cdc2 kinase copurifies with the mitotic apparatus and immunostaining indicates that the p34cdc2 kinase is concentrated at the spindle poles. Models for the interaction of the p34cdc2 kinase and the 77 kDa EMAP are presented.

scholarly journals Stable, resealable pores formed in sea urchin eggs by electric discharge (electroporation) permit substrate loading for assay of enzymes in vivo.

1989 ◽  
Vol 1 (1) ◽  
pp. 65-74 ◽  
Author(s):  
R R Swezey ◽  
D Epel
Keyword(s):  
Small Molecules ◽  
Sea Urchin ◽  
Cell Types ◽  
Atp Content ◽  
Fluorescent Indicators ◽  
Sea Urchin Eggs ◽  
Minimal Loss ◽  
Endogenous Metabolites ◽  
Intracellular Milieu

We describe a simple electroporation procedure for loading suspensions of unfertilized sea urchin eggs with impermeant small molecules under conditions that allow close to 90% successful fertilization and development. Poration is carried out in a low-Ca2+ medium that mimicks the intracellular milieu. The induced pores remain open for several minutes in this medium, allowing loading of the cells; resealing is achieved by adding back millimolar calcium ions to the medium. While the pores are open, an influx of exogenous molecules and efflux of endogenous metabolites takes place, and the eggs can lose up to 40% of their ATP content and still survive. Introduced metabolites are utilized by the cells, e.g., introduced 3H-thymidine is incorporated into DNA. This procedure will be useful for loading impermeant substrates into eggs, permitting in vivo assessment of metabolism, and also for introducing other interesting impermeant molecules, such as inhibitors, fluorescent indicators, etc. Though the details may differ, the principle of electroporation in an intracellular-like medium may prove to be useful for loading other cell types with minimal loss of viability.

scholarly journals Fertilization triggers unmasking of maternal mRNAs in sea urchin eggs.

1987 ◽  
Vol 7 (11) ◽  
pp. 3947-3954 ◽  
Author(s):  
J L Grainger ◽  
M M Winkler
Keyword(s):  
Sea Urchin ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Salt ◽  
Translation System ◽  
Sea Urchin Eggs ◽  
Translational Activity

Fertilization of sea urchin eggs results in a large increase in the rate of protein synthesis which is mediated by the translation of stored maternal mRNA. The masked message hypothesis suggests that messenger ribonucleoprotein particles (mRNPs) from unfertilized eggs are translationally inactive and that fertilization results in alterations of the mRNPs such that they become translationally active. Previous workers have isolated egg mRNPs by sucrose gradient centrifugation and have assayed their translational activity in heterologous cell-free systems. The conflicting results they obtained are probably due to the sensitivity of mRNPs to artifactual activation and inactivation. Previously, we demonstrated that unfractionated mRNPs in a sea urchin cell-free translation system were translationally inactive. Now, using large-pore gel filtration chromatography, we partially purified egg mRNPs while retaining their translationally repressed state. Polysomal mRNPs from fertilized eggs isolated under the same conditions were translationally active. The changes in the pattern of proteins synthesized by fractionated unfertilized and fertilized mRNPs in vitro were similar to those changes observed in vivo. Treatment of egg mRNPs with buffers containing high salt and EDTA, followed by rechromatography, resulted in the activation of the mRNPs and the release of an inhibitor of translation from the mRNPs. Analysis of the inhibitory fraction on one-dimensional sodium dodecyl sulfate gels indicated that this fraction contains a complex set of proteins, several of which were released from high-salt-EDTA-activated mRNPs and not from inactive low-salt control mRNPs. One of the released proteins may be responsible for the repression of egg mRNPs in vitro and be involved in the unmasking of mRNPs at fertilization.

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