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 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.

Isolation and characterization of a novel dynein that contains C and A heavy chains from sea urchin sperm flagellar axonemes

1994 ◽  
Vol 107 (2) ◽  
pp. 345-351 ◽  
Author(s):  
E. Yokota ◽  
I. Mabuchi
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Heavy Chain ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Heavy Chains ◽  
Isolation And Characterization ◽  
Sea Urchin Sperm

A novel dynein (C/A dynein), which is composed of C and A heavy chains, two intermediate chains and several light chains, was isolated from sea urchin sperm flagella. The C/A dynein was released by the treatment with 0.7 M NaCl plus 5 mM ATP from the axonemes depleted of outer arm 21 S dynein. Sedimentation coefficient of this dynein was estimated by sucrose density gradient centrifugation to be 22–23 S. The C/A dynein particle appeared to be composed of three distinct domains; two globular head domains and one rod domain as seen by negative staining electron microscopy. The mobility of ‘A’ heavy chain of C/A dynein on SDS-gel electrophoresis was similar to that of A heavy chains (A alpha and A beta) of 21 S dynein. However, UV-cleavage patterns of C and A heavy chains of C/A dynein were different from those of A heavy chains of 21 S dynein. Furthermore, an antiserum raised against A heavy chain of C/A dynein did not crossreact with A heavy chains of 21 S dynein. Under the conditions in which the C/A dynein was released, some of inner arms were removed concomitantly from axonemes as observed by electron microscopy. These results suggested that C/A dynein is a component of the inner arms.

X-ray and optical diffraction studies on the outer fibres of sea-urchin sperm tail flagella

1972 ◽  
Vol 257 (1) ◽  
pp. 30-36 ◽  
Author(s):  
Takashi Yamaguchi ◽  
Masao Hayashi ◽  
Katsuzo Wakabayashi ◽  
Sugie Higashi-Fujime
Keyword(s):  
Sea Urchin ◽  
Optical Diffraction ◽  
Sperm Tail ◽  
X Ray ◽  
Sea Urchin Sperm

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.

scholarly journals The axonemal axis and Ca2+-induced asymmetry of active microtubule sliding in sea urchin sperm tails.

1986 ◽  
Vol 102 (6) ◽  
pp. 2042-2052 ◽  
Author(s):  
W S Sale
Keyword(s):  
Sea Urchin ◽  
Pair Interaction ◽  
Central Pair ◽  
Sperm Tail ◽  
Lytechinus Pictus ◽  
Motile Cells ◽  
Definition Of ◽  
Triton X ◽  
Explicit Discussion ◽  
Sea Urchin Sperm

Structural studies of stationary principal bends and of definitive patterns of spontaneous microtubule sliding disruption permitted description of the bending axis in sea urchin sperm tail axonemes. Lytechinus pictus sperm were demembranated in a buffer containing Triton X-100 and EGTA. Subsequent resuspension in a reactivation buffer containing 0.4 mM CaCl2 and 1.0 mM MgATP2- resulted in quiescent, rather than motile, cells and each sperm tail axoneme took on an extreme, basal principal bend of 5.2 rad. Thereafter, such flagellar axonemes began to disrupt spontaneously into two subsets of microtubules by active sliding requiring ATP. Darkfield light microscopy demonstrated that subset "1" is composed of microtubules from the inside edge of the principal bend. Subset "2" is composed of microtubules from the outside edge of the principal bend and always scatters less light in darkfield than subset 1. Subset 2, which always slides in the proximal direction, relative to subset 1, results in a basal loop of microtubules, and the subset 2 loop is restricted to the bend plane during sliding disruption. Electron microscopy revealed that doublets 8, 9, 1, 2, 3 and the central pair comprise subset 1, and doublets 4, 5, the bridge, 6, and 7 comprise subset 2. The microtubules of isolated subset 2 are maintained in a circular arc in the absence of spoke-central pair interaction. Longitudinal sections show that the bending plane bisects the central pair. We therefore conclude that the bend plane passes through doublet 1 and the 5-6 bridge and that doublet 1 is at the inside edge of the principal bend. Experimental definition of the axis permits explicit discussion of the location of active axonemal components which result in Ca2+-induced stationary basal bends and explicit description of components responsible for alternating basal principal and reverse bends.

scholarly journals THE MECHANISM OF ACTION OF COLCHICINE

1967 ◽  
Vol 34 (2) ◽  
pp. 535-548 ◽  
Author(s):  
G. G. Borisy ◽  
E. W. Taylor
Keyword(s):  
Sea Urchin ◽  
Mammalian Cells ◽  
Major Protein ◽  
Mitotic Apparatus ◽  
Sedimentation Constant ◽  
A Subunit ◽  
Low Ionic Strength ◽  
Whole Egg

Colchicine forms a complex in vivo with a protein present in fertilized or unfertilized sea urchin eggs; similar binding was obtained in vitro with the soluble fraction from egg homogenates. Kinetic parameters and binding equilibrium constant were essentially the same in vivo and in vitro. The binding site protein was shown to have a sedimentation constant of 6S by zone centrifugation. The protein was present in extracts of the isolated mitotic apparatus at a concentration which was several times higher than in whole-egg homogenates. It was extracted from the mitotic apparatus at low ionic strength under conditions which lead to the disappearance of microtubules. No binding could be detected to the 27S protein, previously described by Kane, which is a major protein component of the isolated mitotic apparatus. The properties of the colchicine-bindinG protein, (binding constant, sedimentation constant, Sephadex elution volume) are similar to those obtained with the protein from mammalian cells, sea-urchin sperm tails, and brain tissue, and thus support the conclusion that the protein is a subunit of microtubules.

Localization of sea urchin egg cytoplasmic dynein in mitotic apparatus studied by using a monoclonal antibody against sea urchin sperm flagellar 21S dynein

1987 ◽  
Vol 7 (2) ◽  
pp. 97-109 ◽  
Author(s):  
Shin-Ichi Hisanaga ◽  
Takeshi Tanaka ◽  
Tomoh Masaki ◽  
Hikoichi Sakai ◽  
Issei Mabuchi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Sea Urchin ◽  
Cytoplasmic Dynein ◽  
Mitotic Apparatus ◽  
Sea Urchin Egg ◽  
Sea Urchin Sperm

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.

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