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.

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.

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.

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.

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.

scholarly journals Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein.

1985 ◽  
Vol 101 (5) ◽  
pp. 1858-1870 ◽  
Author(s):  
N Hirokawa ◽  
R Takemura ◽  
S Hisanaga
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Mitotic Spindle ◽  
Cytoplasmic Dynein ◽  
High Salt ◽  
Mitotic Spindles ◽  
Mitotic Apparatus ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Spindle Microtubules

We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three-dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button-like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule-associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75-kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic immunocytochemistry with a monoclonal antibody (D57) against sea urchin sperm flagellar 21S dynein and colloidal gold-labeled second antibody. Immunogold particles were closely associated with spindle microtubules. 76% of these were within 50 nm and 55% were within 20 nm from the surface of the microtubules. These gold particles were sporadically found on both polar and kinetochore microtubules of half-spindles at both metaphase and anaphase. They localized also on the microtubules between sister chromatids in late anaphase. These data indicate that cytoplasmic dynein is attached to the microtubules in sea urchin mitotic spindles.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

scholarly journals BIREFRINGENCE OF SPERMATOZOA

1972 ◽  
Vol 55 (2) ◽  
pp. 501-510 ◽  
Author(s):  
Irwin J. Bendet ◽  
James Bearden
Keyword(s):  
Refractive Index ◽  
Thermal Denaturation ◽  
The Other ◽  
Refractive Indices ◽  
Sperm Cells ◽  
Form Birefringence ◽  
Before And After ◽  
Bull Sperm ◽  
Influence Of Solvents ◽  
Intrinsic Birefringence

In thermal denaturation experiments on sperm cells, described in the accompanying paper, it was found that squid sperm, when melted, lose both birefringence and morphological shape. Bull sperm, on the other hand, show no change of morphology, but their initial negative birefringence becomes positive. Since this suggested the existence of form birefringence, the influence of solvents of different refractive indices on the observed birefringence was investigated, using a new derivation of the Wiener form birefringence equations which allows direct comparison of Wiener's theory with experimental results. Bull sperm showed form birefringence both before and after melting, while squid sperm showed none. Quantitative application of the general form of the Wiener equations to these results gave values for the refractive index and intrinsic birefringence of bull sperm cells. Application of the specific forms of the Wiener equations showed that neither of these descriptions of idealized systems was adequate to describe completely the form birefringence of bull sperm, but that the equation for platelike submicroscopic structures was more nearly an accurate fit to the data than that for rodlike structures.

scholarly journals REFRACTIVE INDEX OF THE PROTOPLASM IN SEA URCHIN EGGS*

1979 ◽  
Vol 21 (2) ◽  
pp. 141-153 ◽  
Author(s):  
Y. HIRAMOTO ◽  
SHUMEI SHIMODA ◽  
YOKO SHOJI
Keyword(s):  
Refractive Index ◽  
Sea Urchin ◽  
Sea Urchin Eggs

scholarly journals Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy.

1975 ◽  
Vol 66 (1) ◽  
pp. 198-200 ◽  
Author(s):  
D Mazia ◽  
G Schatten ◽  
W Sale
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Mammalian Cells ◽  
Experimental Treatment ◽  
The Body ◽  
Transmission Electron ◽  
Coated Plate ◽  
Living Material ◽  
Coated Surfaces ◽  
Triton X

Cells of many kinds adhere firmly to glass or plastic surfaces which have been pretreated with polylysine. The attachment takes place as soon as the cells make contact with the surfaces, and the flattening of the cells against the surfaces is quite rapid. Cells which do not normally adhere to solid surfaces, such as sea urchin eggs, attach as well as cells which normally do so, such as amebas or mammalian cells in culture. The adhesion is interpreted simply as the interaction between the polyanionic cell surfaces and the polycationic layer of adsorbed polylysine. The attachment of cells to the polylysine-treated surfaces can be exploited for a variety of experimental manipulations. In the preparation of samples for scanning or transmission electron microscopy, the living material may first be attached to a polylysine-coated plate or grid, subjected to some experimental treatment (fertilization of an egg, for example), then transferred rapidly to fixative and further passed through processing for observation; each step involves only the transfer of the plate or grid from one container to the next. The cells are not detached. The adhesion of the cell may be so firm that the body of the cell may be sheared away, leaving attached a patch of cell surface, face up, for observation of its inner aspect. For example, one may observe secretory vesicles on the inner face of the surface (3) or may study the association of filaments with the inner surface (Fig. 1). Subcellular structures may attach to the polylysine-coated surfaces. So far, we have found this to be the case for nuclei isolated from sea urchin embryos and for the microtubules of flagella, which are well displayed after the membrane has been disrupted by Triton X-100 (Fig. 2).

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