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.

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.

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.

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.

Correlative light and electron microscopy: Counting centrioles in individual sea urchin zygotes previously followed in vivo

1993 ◽  
Vol 51 ◽  
pp. 178-179
Author(s):  
Greenfield Sluder ◽  
Frederick J. Miller
Keyword(s):  
Sea Urchin ◽  
Large Population ◽  
Light And Electron Microscopy ◽  
Experimental System ◽  
Animal Cells ◽  
Mitotic Apparatus ◽  
Cell Cycles ◽  
Central Spindle ◽  
Pericentriolar Material

Centrosomes are the ensembles of structures that define the poles of the mitotic apparatus. In animal cells, a centrosome typically consists of a pair of orthogonally arranged centrioles associated with osmiophilic pericentriolar material which nucleates the microtubules of the aster and central spindle. The precise doubling, or reproduction, of the centrosome during interphase is an important event in the cell’s preparations for mitosis.To characterize the mechanisms that control centrosome reproduction we need to follow the behavior of centrosomes within individual cells over several cell cycles. We use sea urchin zygotes as an experimental system because they have a rapid cell cycle, are large enough (100 um diameter) to easily manipulate, and are optically clear. However, the experiments pose two challenges. The first is to follow the behavior of centrosomes within individual zygotes for several hours with the light microscope and then recover just those individuals from a large population for ultrastructural analysis.

scholarly journals Quantitative studies on the polarization optical properties of living cells II. The role of microtubules in birefringence of the spindle of the sea urchin egg.

1981 ◽  
Vol 89 (1) ◽  
pp. 121-130 ◽  
Author(s):  
Y Hiramoto ◽  
Y Hamaguchi ◽  
Y Shóji ◽  
T E Schroeder ◽  
S Shimoda ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Preceding Paper ◽  
Living Cells ◽  
Mitotic Apparatus ◽  
Form Birefringence ◽  
Quantitative Studies ◽  
Sea Urchin Egg ◽  
Interpolar Distance

Birefringence of the mitotic apparatus (MA) and its change during mitosis in sea urchin eggs were quantitatively determined using the birefringence detection apparatus reported in the preceding paper (Hiramoto el al., 1981, J. Cell Biol. 89:115-120). The birefringence and the form of the MA are represented by five parameters: peak retardation (delta p), through retardation (delta t), interpolar distance (D1), the distance (D2) between chromosome groups moving toward poles, and the distance (D3) between two retardation peaks. Distributions of birefringence retardation and the coefficient of birefringence in the spindle were quantitatively determined in MAs isolated during metaphase and anaphase. The distribution of microtubules (MTs) contained in the spindle is attributable to the form birefringence caused by regularly arranged MTs. The distribution coincided fairly well with the distribution of MTs in isolated MAs determined by electron microscopy. Under the same assumption, the distribution of MTS in the spindle in living cells during mitosis was determined. The results show that the distribution of MTs and the total amount of polymerized tubulin (MTs) in the spindle change during mitosis, suggesting the assembly and disassembly of MTs as well as the dislocation of MTs during mitosis.

scholarly journals ULTRASTRUCTURE AND BIREFRINGENCE OF THE ISOLATED MITOTIC APPARATUS OF MARINE EGGS

1967 ◽  
Vol 34 (3) ◽  
pp. 859-883 ◽  
Author(s):  
Lionel I. Rebhun ◽  
Greta Sander
Keyword(s):  
Electron Microscopy ◽  
Refractive Index ◽  
Sea Urchin ◽  
Major Part ◽  
Living Cells ◽  
Mitotic Apparatus ◽  
Positive Form ◽  
Form Birefringence ◽  
Microscopy Examination ◽  
Intrinsic Birefringence

Isolated mitotic apparatuses (MA) of clam and sea urchin eggs were investigated by polarizing and electron microscopy. Examination of fixed MA in oils of different refractive index revealed that at least 90% of the retardation of isolated MA is due to positive, form birefringence, the remaining retardation deriving from positive, intrinsic birefringence. Electron micrographs reveal the isolated MA to be composed of microtubules, ribosome-like particles, and a variety of vesicles. In the clam MA the number of vesicles and ribosome-like particles relative to the number of microtubules is much lower than in the sea urchin MA. In clam MA this allows form and intrinsic birefringence to be related directly to microtubules. The relation of birefringence to microtubules in isolated sea urchin MA is more complex since ribosome-like particles adhere to microtubules, are oriented by them, and are likely to contribute to the form birefringence of the isolated MA. However, comparison of values of retardation for clam and sea urchin MA, indicates that the major part of the birefringence in sea urchin MA is also due to microtubules. The interpretation of the structures giving rise to birefringence in the MA of the living cells is likely to be even more complex since masking substances, compression, or tension on the living MA may alter the magnitude or sign of the birefringence.

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