Spindle birefringence of isolated mitotic apparatus analysed by pressure treatment

1976 ◽  
Vol 20 (2) ◽  
pp. 309-327
Author(s):  
A. Forer ◽  
A.M. Zimmerman
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Dimethyl Sulphoxide ◽  
Pressure Treatment ◽  
Mitotic Apparatus ◽  
Isolation Medium ◽  
Hexylene Glycol ◽  
X 24 ◽  
Induced Changes

Sea-urchin zygote mitotic apparatus (MA) isolated in a glycerol/dimethylsulphoxide medium were treated with pressure. Pressure treatment had no effect on spindle birefringence when MA were in full-strength isolation medium. After placing MA in quarter-strength isolation medium, pressures of 4-0 X 10(3)-1-8 X 10(4) lbf in.-2 (2 X 76 X 10(4)-I X 24 X 10(5) k N m-2) for 15 min caused reduction of birefringence which occurred in 2 steps: firstly 20–30% of the birefringence was lost, and then, at higher pressures, the rest of the birefringence was lost. Electron microscopy suggested that pressure-induced changes were in non-microtubule material. Pressure treatment had no effect on MA isolated with hexylene glycol when the MA were pressurized in hexylene glycol; but pressure treatment did cause loss of birefringence when MA isolated in hexylene glycol were transferred immediately into glycerol/dimethylsulphoxide medium and were subsequently treated with pressure (after dilution into quarter-strength glycerol/dimethyl-sulphoxide). We discuss the differences in response between isolated MA and in vivo MA, and we discuss the possibility that 2 components contribute to MA birefringence.

Spindle birefringence of isolated mitotic apparatus analysed by treatments with cold, pressure, and diluted isolation medium

1976 ◽  
Vol 20 (2) ◽  
pp. 329-339
Author(s):  
A. Forer ◽  
A.M. Zimmerman
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Half Life ◽  
Cold Treatment ◽  
Pressure Treatment ◽  
Mitotic Apparatus ◽  
Isolation Medium ◽  
Spindle Structure ◽  
Cold Pressure

Mitotic apparatus (MA) were isolated from sea-urchin zygotes using glycerol-dimethyl-sulphoxide. Cold treatment had no effect on MA birefringence when MA were in isolation medium, but caused a 10–15% reduction of MA birefringence when MA were in quarter-strength isolation medium. Pressure treatment also caused a reduction in MA birefringence, but the cold and pressure treatments were not additive, suggesting that both treatments affected the same MA component. MA were not stable in quarter-strength isolation medium, and birefringence gradually decayed, with a half-life of about 40 h. Electron microscopy after cold treatment, or after decay of 55% of the MA birefringence showed abundant, normal-looking microtubules, suggesting that alterations in non-microtubule components cause the reductions in birefringence. Addition of EGTA eliminates the effect of cold treatment, suggesting that Ca2+ has a role in maintenance of spindle structure. We discuss possible reasons why isolated MA do not respond to cold treatment like MA in vivo.

Characteristics of Sea-Urchin Mitotic Apparatus Isolated Using a Dimethyl Sulphoxide/Glycerol Medium

1974 ◽  
Vol 16 (3) ◽  
pp. 481-497
Author(s):  
A. FORER ◽  
A. M. ZIMMERMAN
Keyword(s):  
Sea Urchin ◽  
Phosphate Buffer ◽  
Dry Matter ◽  
Room Temperature ◽  
Dimethyl Sulphoxide ◽  
Mitotic Apparatus ◽  
Isolation Medium ◽  
Hexylene Glycol ◽  
Brain Microtubules ◽  
Glycerol Medium

A method for isolating sea-urchin zygote mitotic apparatus (MA) is described which is based on the Filner-Behnke method of isolating brain microtubules. MA were isolated in 50% (v/v) glycerol, 10%(v/v) dimethyl sulphoxide, 5 mM MgCl2, 0.1 mM EGTA, and 5 mM Sørensen's phosphate buffer at a final pH of 6.8. MA stored at room temperature in isolation medium had stable birefringence, stable microtubules and stable solubility properties (in 0.5 M KCl) over a period of 10 days to 2 weeks. These MA also seem to have more dry matter per volume than do MA isolated using hexylene glycol. The biggest disadvantages of the method are that zygotes often are difficult to lyse, and that cytoplasmic debris the same size as the MA sometimes contaminates the MA pellet.

The Structure and Some Properties of the Isolated Mitotic Apparatus

1969 ◽  
Vol 4 (1) ◽  
pp. 179-209
Author(s):  
R. D. GOLDMAN ◽  
L. I. REBHUN
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Structural Changes ◽  
Close Association ◽  
Light And Electron Microscopy ◽  
Mitotic Apparatus ◽  
Isolation Medium ◽  
Oriented Parallel ◽  
Differential Interference Microscope

The morphology of the isolated sea-urchin mitotic apparatus (MA) was examined by light and electron microscopy. With the polarization microscope and the Nomarski differential interference microscope, the isolated MAs appeared to be similar to in vivo MAs. Electron microscopy of the isolated MAs revealed the presence of microtubules, ribosome-like particles and vesicles. A close association between the ribosome-like particles and the MA microtubules resulted in the appearance of chains of particles running along the length of the microtubules. Isolated MAs washed two to three times in isolation medium showed fine-structural changes in the electron microscope, which were reflected by lower retardation values obtained with the polarization microscope. The addition of magnesium and calcium or sucrose to the washing medium prevented these structural changes. Varying the pH of the isolation medium also resulted in changes in birefringence and ultrastructure of unwashed MAs. Isolated MAs stored in the original isolation medium gradually became less birefringent and lost their microtubules. At pH 6.1 and pH 6.2 a residual birefringence was retained, even after several weeks of storage. Electron microscopy of these MAs revealed the presence of linear aggregates of ribosome-like particles oriented parallel to the long axis of the spindle. On the other hand, at pH 6.3and pH 6.4, MAs lost their birefringence completely, and the ribosome-like particles became more randomly dispersed. 2M sucrose or 0.003 M Mg2+ greatly retarded the loss of birefringence in stored MAs. Glutaraldehyde-fixed MAs stained intensely with azure B bromide, demonstrating the presence of RNA. Treatment with RNase resulted in a loss of this staining. RNase-treated MAs examined with the electron microscope, revealed changes in the ribosome-like particles. The results are discussed in the light of recent biochemical analyses of the isolated MA, structural similarities to in situ MAs and the interpretation of the birefringence of the MA.

Experimental determinations of tubulin in the in vivo mitotic apparatus of sea urchin zygotes

1980 ◽  
Vol 58 (11) ◽  
pp. 1277-1285 ◽  
Author(s):  
Arthur Forer ◽  
D. E. Larson ◽  
A. M. Zimmerman
Keyword(s):  
Sea Urchin ◽  
Gel Electrophoresis ◽  
Dry Matter ◽  
Strongylocentrotus Purpuratus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mitotic Apparatus ◽  
Cell Lysate ◽  
Hexylene Glycol

Mitotic apparatus (MA) were isolated from zygotes of a sea urchin (Strongylocentrotus purpuratus), using hexylene glycol (pH 6.4) as lysing–stabilizing agent. Protein was measured in the MA pellet and in the remainder of the cell lysate (using the Lowry procedure). Tubulin was measured in the MA pellet and in the remainder of the cell lysate (using microdensitometry of stained gels after sodium dodecyl sulphate – polyacrylamide gel electrophoresis). From these data we calculated the maximum possible amounts of tubulin in the isolated MA and in the MA in vivo; in these calculations we assumed that all the tubulin in the cell is associated with the MA, and we assumed that, as reported in the literature, the MA lose 90% of their dry matter during the isolation. We conclude that tubulin probably comprises less than 7% of the protein in the in vivo MA, and, even if there are very large errors, tubulin is considerably less than haf the protein in the MA.

Induction of cleavage in nucleated and enucleated frog eggs by injection of isolated sea-urchin mitotic apparatus

1978 ◽  
Vol 31 (1) ◽  
pp. 117-135
Author(s):  
Y. Masui ◽  
A. Forer ◽  
A.M. Zimmerman
Keyword(s):  
Sea Urchin ◽  
Room Temperature ◽  
Rana Pipiens ◽  
Sea Urchins ◽  
Cooperative Interaction ◽  
Nuclear Materials ◽  
Cytological Examination ◽  
Mitotic Apparatus ◽  
Frog Brain ◽  
Hexylene Glycol

Mitotic apparatus (MA) were isolated in glycerol-dimethylsulphoxide solution (MTME) from zygotes of sea urchins (Stronglyocentrotus purpuratus). Freshly isolated MA were stored in 1/10 strength MTME for varying periods of time and were then injected into unfertilized frog (Rana pipiens) eggs. These injections induced 40–60% of the recipient frog eggs to initiate cleavage, resulting in the formation of blastula cell clusters. The cleavage-inducing activity of MA stored in 1/10 MTME at room temperature decreased with time of storage in 1/10 strength MTME, and disappeared by about 6 h. There was no change in the ultrastructure of MA during storage. MA isolated and stored in MTME at room temperature had a constant level of cleavage-inducing activity during the first 48 h of storage, but this activity slowly declined upon further storage; almost no activity was left after 3 weeks. MA isolated in hexylene glycol (HG) and immediately transferred into MTME were compared with MA isolated in MTME; both MA had the same cleavage-inducing activity on the day of isolation, after which the MA isolated in HG quickly lost activity. On the other hand, MA isolated and stored in HG had little cleavage-inducing activity when tested 3 h following isolation. Cleavage-inducing agent (CIA) isolated from frog brains induced cleavage and blastula formation when injected into nucleated frog eggs, but had no such activity when injected into enucleated frog eggs. MA isolated in MTME induced cleavage and blastula formation in enucleated frog eggs as well as in nucleated frog eggs. Cytological examination revealed that blastula cells which developed from MA-injected enucleated eggs contained Feulgennegative nuclei, whereas cells which developed from CIA-injected nucleated eggs contained Feulgen-positive nuclei. These results suggest that sea-urchin nuclear materials participate in mitosis in frog eggs. Isolated MA which had been stored in MTME for 3 weeks and which exhibited little cleavage-inducing activity were injected together with frog brain CIA into either normal or enucleated eggs; normal recipient eggs cleaved with significantly higher frequencies (70%) than those injected with CIA alone (40%). Furthermore, enucleated eggs injected with CIA alone failed to cleave, while those injected with MA and CIA together cleaved with significant frequencies (overall 29%). This result suggests a cooperative interaction between CIA and the inactivated MA to restore the cleavage-inducing activity of MA.

The Concentrations of Dry Matter in Mitotic Apparatuses in Vivo and After Isolation From Sea-Urchin Zygotes

1972 ◽  
Vol 10 (2) ◽  
pp. 387-418
Author(s):  
A. FORER ◽  
R. D. GOLDMAN
Keyword(s):  
Sea Urchin ◽  
Dry Matter ◽  
Mass Medium ◽  
Dry Mass ◽  
Interference Microscopy ◽  
High Mass ◽  
Isolation Medium ◽  
High Ph ◽  
Cytoplasmic Material

We have measured the concentrations of dry matter in mitotic apparatuses (MA) in vivo and after isolation from the same cell type. The isolation medium was hexylene glycol plus buffer. The MA were from sea-urchin zygotes (Echinus esculentus Linn. and Psammechinus miliaris Gmelin), and measurements were made using interference microscopy. MA as isolated have much lower concentrations of dry matter than do MA in vivo. The dry mass concentrations of isolated MA vary with the pH of the isolation medium, ranging from about 20 % of the in vivo concentration (at pH 7.3) to about 60 % of the in vivo concentration (at pH 5.3). The isolated MA were further characterized. Evidence is presented which suggests that non-specific cytoplasmic material adsorbs to MA, and thus that at least some of the material in isolated MA is not derived from in vivo MA. Some MA components are apparently changed during the isolation procedure: MA lysed in low pH (high mass) medium and quickly transferred to high pH (low mass) medium have higher concentrations of dry matter than do MA lysed in high pH medium. The isolation media as generally used do not have enough buffering capacity: the pH changes after the isolation. These data suggest that the isolation procedures need be improved before studies of isolated MA can give data relevant to the chemistry of in vivo MA. We discuss the problem of obtaining functional isolated MA, and also the relevance of our data to previous work on MA from other species.

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 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 ELECTRON MICROSCOPY OF MITOSIS IN AMEBAE

1967 ◽  
Vol 34 (1) ◽  
pp. 47-59 ◽  
Author(s):  
L. E. Roth
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Metaphase Plate ◽  
Test Sequence ◽  
Partial Loss ◽  
Mitotic Apparatus ◽  
In Vivo Test ◽  
Characteristic Sequences ◽  
Inhibitor Compounds

The mitotic apparatus (MA) of the giant ameba, Chaos carolinensis, has characteristic sequences of microtubule arrays and deployment of nuclear envelope fragments. If mitotic organisms are subjected to 2°C for 5 min, the MA microtubules are completely degraded, and the envelope fragments are released from the chromosomes which remain condensed but lose their metaphase-plate orientation. On warming, microtubules reform but show partial loss of their parallel alignment; displacement of the envelope fragments persists or is increased by microtubule reformation. This study demonstrates that cooling causes destruction of microtubules and intermicrotubular cross-bonds and further shows that such controlled dissolution and reformation can provide an in vivo test sequence for studies on the effects of inhibitor-compounds on microtubule subunit aggregation. Urea, at the comparatively low concentration of 0.8 M, inhibited reformation following cooling and rewarming but was ineffective in altering microtubules that had formed before treatment.

scholarly journals THE MITOTIC APPARATUS

1965 ◽  
Vol 25 (3) ◽  
pp. 31-39 ◽  
Author(s):  
R. E. Kane ◽  
Arthur Forer
Keyword(s):  
Electron Microscopy ◽  
Structural Change ◽  
Sea Urchin ◽  
Phase Contrast ◽  
Fibrous Structure ◽  
Polarization Microscopy ◽  
Mitotic Apparatus ◽  
Sea Urchin Eggs ◽  
Dense Granules

The fibrous structure of the mitotic apparatus (MA) isolated from dividing sea urchin eggs undergoes no changes visible in phase contrast during extended storage, but the solubility of the MA rapidly decreases after isolation. Polarization microscopy shows that a decrease in the birefringence of the MA also occurs after isolation and is correlated with the loss of solubility. This loss of birefringence indicates that some structural change takes place during this period, and such a change was demonstrated by means of electron microscopy. The tubular filaments which form the spindle of the intracellular MA and of the freshly isolated MA were found to break down during storage to rows of dense granules, this loss of continuity presumably accounting for the loss of birefringence. The interrelations of the observed changes and the significance of these observations for investigations on the isolated MA are discussed.

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