An Estimate of the Amount of Microtubule Protein in the Isolated Mitotic Apparatus

1970 ◽  
Vol 6 (1) ◽  
pp. 159-176
Author(s):  
W. D. COHEN ◽  
L. I. REBHUN
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Cross Sections ◽  
Mitotic Apparatus ◽  
Arbacia Punctulata ◽  
Molecular Weights ◽  
Cellular Factors ◽  
Microtubule Protein ◽  
Specific Orientation ◽  
Existing Data

The microtubule content of the isolated mitotic apparatus of sea-urchin eggs (Arbacia punctulata has been investigated by electron microscopy. Cross-sections were made through asters or spindles of flat-embedded mitotic apparatuses of known mitotic stage and specific orientation in the block. Cross-sections between chromosomes and poles of five metaphase half-spindles revealed approximately 2000-2300 sectioned microtubules. The number was somewhat higher in three anaphase half-spindles examined, approximately 2400-2600. A method was devised for calculating the total number of microtubules in an aster, based upon the number of microtubules appearing in cross-sections. Application of this method to selected mitotic apparatuses enabled calculation of the total number of microtubules in metaphase mitotic apparatuses of average dimensions. Using a 13-protofilament model of the microtubule and existing data on possible monomer sizes and molecular weights, the total amount of microtubule protein in the isolated mitotic apparatus was calculated. The values obtained are in the range of about 1-2 x 10-8 mg microtubule protein per isolated mitotic apparatus. These values are close to those reported for the 4-5s protein of the isolated mitotic apparatus, but are considerably lower than the amount of 22s protein. The results are discussed with respect to cellular factors which determine microtubule number, and the possible sources and origin of mitotic microtubule protein.

C-Microtubules in Isolated Mitotic Spindles

1971 ◽  
Vol 9 (3) ◽  
pp. 603-619
Author(s):  
W. D. COHEN ◽  
T. GOTTLIEB
Keyword(s):  
Cross Section ◽  
Sea Urchin ◽  
Cross Sections ◽  
Metaphase Chromosome ◽  
Inverse Relationship ◽  
Mitotic Spindles ◽  
Cylindrical Structure ◽  
Arbacia Punctulata ◽  
Alternate Possibility

Microtubules with incomplete cylindrical structure are present in isolated mitotic spindles of the sea urchin, Arbacia punctulata. In cross-section they appear C-shaped, and are thus similar to the ‘C-microtubules’ or ‘C-filaments’ observed previously in other systems. The C-microtubules are not uniformly distributed within isolated spindles, but are typically numerous in the interzonal region of anaphase spindles and in the metaphase chromosome ‘plate’. In chromosome-to-pole regions they are seen much less frequently, and microtubules with the usual O-configuration predominate. Counts of C- and O-microtubules in anaphase spindle cross-sections of known location show an inverse relationship between the number of C-microtubules present and the total number of microtubules present. The observations suggest that the C-microtubules are not simple artifacts of fixation or isolation, but rather may represent a stage of microtubule disassembly which occurs in the interzone during isolation or during anaphase in vivo. The alternate possibility of assembly is not excluded, however. The significance of C-microtubules is further discussed with respect to their occurrence in other systems, and to potential differences between mitotic microtubules.

scholarly journals IMMUNOCHEMICAL STUDIES OF 22S PROTEIN FROM ISOLATED MITOTIC APPARATUS

1969 ◽  
Vol 41 (2) ◽  
pp. 577-590 ◽  
Author(s):  
Thomas Bibring ◽  
Jane Baxandall
Keyword(s):  
Sea Urchin ◽  
Morphological Features ◽  
Mitotic Apparatus ◽  
Antibody Preparation ◽  
Sea Urchin Eggs ◽  
Microtubule Protein ◽  
Mild Acid

Evidence is presented that the "22S protein" of mitotic apparatus isolated from sea urchin eggs is not microtubule protein. An antibody preparation active against 22S protein is described, and immunochemical studies of the distribution of 22S protein in various cellular fractions and among morphological features of mitotic apparatus are reported. The protein is ubiquitous in the metaphase egg fractions that were tested but is not found in sperm flagella. It is immunologically distinct from proposed microtubule protein isolated from mitotic apparatus by the method of Sakai, and from proposed microtubule protein obtained after extraction with mild acid. It exists in nontubule material of isolated mitotic apparatus but is not detectable in microtubules.

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.

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.

scholarly journals Water ordering during the cell cycle: nuclear magnetic resonance studies of the sea-urchin egg

1985 ◽  
Vol 79 (1) ◽  
pp. 247-257
Author(s):  
S. Zimmerman ◽  
A.M. Zimmerman ◽  
G.D. Fullerton ◽  
R.F. Luduena ◽  
I.L. Cameron
Keyword(s):  
Cell Cycle ◽  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Low Temperature ◽  
Sea Urchin ◽  
Water Proton ◽  
Proton Relaxation ◽  
Mitotic Apparatus ◽  
Microtubule Protein ◽  
Nuclear Magnetic

Nuclear magnetic resonance was used to measure spin-lattice water proton relaxation times (T1) during the first cell cycle in sea-urchin zygotes of packed Strongylocentrotus purpuratus. Following insemination there was a 90% increase in the T1 value. The increase in T1 at fertilization could be accounted for by the accumulation of extracellular fluid between the egg surface and the fertilization envelope. The T1 value then remained without change during the first cell cycle, except at metaphase when there was a significant 13% decrease. The lowered T1 values measured at metaphase were not related to a change in the water content of the packed cells, which remained fairly constant throughout the cell cycle. High hydrostatic pressure, low temperature and colchicine (agents that depolymerize mitotic apparatus microtubules) did not affect the T1 values in fertilized eggs. Treatment in vitro of a microtubule protein preparation with low temperature and colchicine resulted in an increased T1, which accompanied the depolymerization of microtubule protein. Since depolymerization of the microtubules associated with the mitotic apparatus by high pressure, colchicine or low temperature does not alter the T1 of water protons in the cell, it is proposed that the increased state of ordered water molecules at metaphase is maintained by nonmicrotubular factor(s) of the metaphase egg.

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.

scholarly journals SELECTIVE EXTRACTION OF ISOLATED MITOTIC APPARATUS

1971 ◽  
Vol 48 (2) ◽  
pp. 324-339 ◽  
Author(s):  
Thomas Bibring ◽  
Jane Baxandall
Keyword(s):  
Sea Urchin ◽  
Protein Extraction ◽  
Sedimentation Coefficient ◽  
Selective Extraction ◽  
Major Protein ◽  
Mitotic Apparatus ◽  
Sperm Tail ◽  
Microtubule Protein ◽  
Related Proteins ◽  
Sea Urchin Sperm

Mitotic apparatus isolated from sea urchin eggs has been treated with meralluride sodium under conditions otherwise resembling those of its isolation. The treatment causes a selective morphological disappearance of microtubules while extracting a major protein fraction, probably consisting of two closely related proteins, which constitutes about 10% of mitotic apparatus protein. Extraction of other cell particulates under similar conditions yields much less of this protein. The extracted protein closely resembles outer doublet microtubule protein from sea urchin sperm tail in properties considered typical of microtubule proteins: precipitation by calcium ion and vinblastine, electrophoretic mobility in both acid and basic polyacrylamide gels, sedimentation coefficient, molecular weight, and, according to a preliminary determination, amino acid composition. An antiserum against a preparation of sperm tail outer doublet microtubules cross-reacts with the extract from mitotic apparatus. On the basis of these findings it appears that microtubule protein is selectively extracted from isolated mitotic apparatus by treatment with meralluride, and is a typical microtubule protein.

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.

Centrosomes are phosphorylated in sea urchin eggs throughout the cell cycle, during artificial activation, and when microtubule growth is inhibited

1994 ◽  
Vol 52 ◽  
pp. 296-297
Author(s):  
Heide Schatten ◽  
Amitabha Chakrabarti
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Transmission Electron Microscopy ◽  
Sea Urchin ◽  
Immunofluorescence Microscopy ◽  
Mitotic Apparatus ◽  
Zygote Nucleus ◽  
Transmission Electron ◽  
Microtubule Growth ◽  
Artificial Activation

In most animal systems, microtubules are nucleated and organized by centrosomes which undergo considerable modifications during the cell cycle. Typically, centrosomes are phoshorylated at the transition from interphase to mitosis as shown with MPM2, an antibody directed against phosphoproteins. Using the MPM2 antibody, we show in this paper with transmission electron microscopy (TEM) and immunofluorescence microscopy (IFM) that in the sea urchin system centrosomes are phosphorylated in the sperm before fertilization and at every stage of the first cell cycle. MPM2 exhibits identical staining patterns as Ah-6 and 5051 previously shown to reliably identify centrosomal material in sea urchin cells. After centrosomal material is brought into the egg by the sperm, it spreads around the zygote nucleus where it gets distributed and becomes bipolar and compacted to form the mitotic apparatus. Typically, at these mitotic stages, centrosomal material exhibits the brightest staining with MPM2, Ah-6 and 5051 Since in this system phoshorylated centrosomal material is contributed by the sperm, the egg's competence for centrosome phosphorylation was analyzed by activating centrosomal material in the unfertilized egg by treatment with either A23187, ammonia, D2O, or taxol.

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