scholarly journals Purification and characterization of oocyte cytoplasmic tubulin and meiotic spindle tubulin of the surf clam Spisula solidissima.

1984 ◽  
Vol 98 (1) ◽  
pp. 253-266 ◽  
Author(s):  
K A Suprenant ◽  
L I Rebhun
Keyword(s):  
Peptide Mapping ◽  
Critical Concentration ◽  
Meiotic Spindle ◽  
Spisula Solidissima ◽  
Surf Clam ◽  
Alpha Tubulin ◽  
One Dimensional ◽  
Unfertilized Oocyte ◽  
Meiotic Spindles

Assembly-competent tubulin was purified from the cytoplasm of unfertilized and parthogenetically activated oocytes, and from isolated meiotic spindles of the surf clam, Spisula solidissima. At 22 degrees C or 37 degrees C, Spisula tubulin assembled into 48-51-nm macrotubules during the first cycle of polymerization and 25-nm microtubules during the third and subsequent cycles of assembly. Macrotubules were formed from sheets of 26-27 protofilaments helically arranged at a 36 degree angle relative to the long axis of the polymer and were composed of alpha and beta tubulins and several other proteins ranging in molecular weight from 30,000 to 270,000. Third cycle microtubules contained 14-15 protofilaments in cross-section and were composed of greater than 95% alpha and beta tubulins. After three cycles of polymerization at 37 degrees C, unfertilized and activated oocyte tubulin self-assembled into microtubules at a critical concentration (Ccr) of 0.09 mg/ml. At the physiological temperature of 22 degrees C, unfertilized oocyte tubulin assembled into microtubules at a Ccr of 0.36 mg/ml, activated oocyte tubulin assembled at a Ccr of 0.42 mg/ml, and isolated meiotic spindle tubulin assembled at a Ccr of 0.33 mg/ml. The isoelectric points of tubulin from both unfertilized oocytes and isolated meiotic spindles were 5.8 for alpha tubulin and 5.6 for beta tubulin. In addition, one dimensional peptide maps of oocyte and spindle alpha and beta tubulins were very similar, if not identical. These results indicate that unfertilized oocyte tubulin and tubulin isolated from the first meiotic spindle are indistinguishable on the basis of assembly properties, isoelectric focusing, and one dimensional peptide mapping. These results suggest that the transition of tubulin from the quiescent oocyte state to that competent to form spindle microtubules in vivo does not require special modification of tubulin but may involve changes in the availability of microtubule organizing centers or assembly-promoting microtubule-associated proteins.

Parthenogenesis and cytoskeletal organization in ageing mouse eggs

Development ◽  
1986 ◽  
Vol 95 (1) ◽  
pp. 131-145
Author(s):  
Michelle Webb ◽  
Sarah K. Howlett ◽  
Bernard Maro
Keyword(s):  
Critical Concentration ◽  
Metaphase Plate ◽  
Meiotic Spindle ◽  
Cytoskeletal Organization ◽  
Spindle Poles ◽  
Different Types ◽  
Mouse Egg ◽  
Parthenogenetic Embryos

The cytoskeletal organization of the mouse egg changes during ageing in vivo and in vitro. The earliest change observed is the disappearance of the microfilament-rich area overlying the meiotic spindle. This is followed by the migration of the spindle towards the centre of the egg. Finally the spindle breaks down and the chromosomes are no longer organized on a metaphase plate. This spindle disruption may result from changes in the microtubule nucleating material found at the spindle poles and from an increase in the critical concentration for tubulin polymerization. It is possible to correlate the changes in the cytoskeletal organization of the egg occurring during ageing with the different types of parthenogenetic embryos obtained after ethanol activation. These observations strengthen the hypothesis that the actin-rich cortical area that overlies the meiotic spindle forms a domain to which the meiotic cleavage furrow is restricted and provides some insights into the mechanisms by which different types of parthenogenetic embryos are generated.

Insertions of up to 17 amino acids into a region of alpha-tubulin do not disrupt function in vivo

1987 ◽  
Vol 7 (10) ◽  
pp. 3799-3805
Author(s):  
P J Schatz ◽  
G E Georges ◽  
F Solomon ◽  
D Botstein
Keyword(s):  
Amino Acids ◽  
Variable Region ◽  
Yeast Cells ◽  
Meiotic Spindle ◽  
Nuclear Movement ◽  
Yeast Saccharomyces Cerevisiae ◽  
Alpha Tubulin ◽  
Essential Components ◽  
Altered Proteins

Microtubules in yeasts are essential components of the mitotic and meiotic spindle and are necessary for nuclear movement during cell division and mating. The yeast Saccharomyces cerevisiae has two alpha-tubulin genes, TUB1 and TUB3, either of which alone is sufficient for these processes when present in a high enough copy number. Comparisons of sequences from several species reveals the presence of a variable region near the amino terminus of alpha-tubulin proteins. We perturbed the structure of this region in TUB3 by inserting into it 3, 9, or 17 amino acids and tested the ability of these altered proteins to function as the only alpha-tubulin protein in yeast cells. We found that each of these altered proteins was sufficient on its own for mitotic growth, mating, and methods of yeast. We conclude that this region can tolerate considerable variation without losing any of the highly conserved functions of alpha-tubulin. Our results suggest that variability in this region occurs because it can be tolerated, not because it specifies an important function for the protein.

scholarly journals Effect of taxol on first and second meiotic spindle formation in oocytes of the surf clam, Spisula solidissima

1986 ◽  
Vol 84 (1) ◽  
pp. 153-164
Author(s):  
R. Kuriyama
Keyword(s):  
Untreated Control ◽  
Polar Body ◽  
Immunofluorescence Staining ◽  
Meiotic Spindle ◽  
Spisula Solidissima ◽  
Microtubule Depolymerization ◽  
Spindle Formation ◽  
Surf Clam ◽  
First Polar Body ◽  
Spindle Microtubules

The effect of taxol on first and second meiotic spindle formation was examined in oocytes of the surf clam, Spisula solidissima, by immunofluorescence staining with anti-tubulin antibody. The first meiotic spindle appeared to form as in untreated control cells. However, the spindle did not migrate toward the periphery of taxol-activated oocytes, resulting in blockage of the formation of the first polar body. In spite of inhibited microtubule depolymerization and failure of spindle disappearance, the pole separation in telophase that is typical of this material began at the same time as in untreated cells. Polymerization of the second spindle microtubules onto the spindle persisting from the first meiosis led to the formation of a triple form of spindle connected at the poles of each other. The subsequent emergence of ring-shaped microtubule-containing structures in mature activated eggs was not affected by taxol. The mechanism of meiotic spindle formation thus seemsto be different from that in mitosis, where taxol has been shown to block spindle formation completely.

scholarly journals Katanin maintains meiotic metaphase chromosome alignment and spindle structure in vivo and has multiple effects on microtubules in vitro

2014 ◽  
Vol 25 (7) ◽  
pp. 1037-1049 ◽  
Author(s):  
Karen McNally ◽  
Evan Berg ◽  
Daniel B. Cortes ◽  
Veronica Hernandez ◽  
Paul E. Mains ◽  
...  
Keyword(s):  
Point Mutations ◽  
Metaphase Plate ◽  
Meiotic Spindle ◽  
Loss Of Function ◽  
Microtubule Organization ◽  
Bipolar Structure ◽  
Microtubule Severing ◽  
Meiotic Spindles

Assembly of Caenorhabditis elegans female meiotic spindles requires both MEI-1 and MEI-2 subunits of the microtubule-severing ATPase katanin. Strong loss-of-function mutants assemble apolar intersecting microtubule arrays, whereas weaker mutants assemble bipolar meiotic spindles that are longer than wild type. To determine whether katanin is also required for spindle maintenance, we monitored metaphase I spindles after a fast-acting mei-1(ts) mutant was shifted to a nonpermissive temperature. Within 4 min of temperature shift, bivalents moved off the metaphase plate, and microtubule bundles within the spindle lengthened and developed a high degree of curvature. Spindles eventually lost bipolar structure. Immunofluorescence of embryos fixed at increasing temperature indicated that MEI-1 was lost from spindle microtubules before loss of ASPM-1, indicating that MEI-1 and ASPM-1 act independently at spindle poles. We quantified the microtubule-severing activity of purified MEI-1/MEI-2 complexes corresponding to six different point mutations and found a linear relationship between microtubule disassembly rate and meiotic spindle length. Previous work showed that katanin is required for severing at points where two microtubules intersect in vivo. We show that purified MEI-1/MEI-2 complexes preferentially sever at intersections between two microtubules and directly bundle microtubules in vitro. These activities could promote parallel/antiparallel microtubule organization in meiotic spindles.

scholarly journals Insertions of up to 17 amino acids into a region of alpha-tubulin do not disrupt function in vivo.

1987 ◽  
Vol 7 (10) ◽  
pp. 3799-3805 ◽  
Author(s):  
P J Schatz ◽  
G E Georges ◽  
F Solomon ◽  
D Botstein
Keyword(s):  
Amino Acids ◽  
Variable Region ◽  
Yeast Cells ◽  
Meiotic Spindle ◽  
Nuclear Movement ◽  
Yeast Saccharomyces Cerevisiae ◽  
Alpha Tubulin ◽  
Essential Components ◽  
Altered Proteins

Microtubules in yeasts are essential components of the mitotic and meiotic spindle and are necessary for nuclear movement during cell division and mating. The yeast Saccharomyces cerevisiae has two alpha-tubulin genes, TUB1 and TUB3, either of which alone is sufficient for these processes when present in a high enough copy number. Comparisons of sequences from several species reveals the presence of a variable region near the amino terminus of alpha-tubulin proteins. We perturbed the structure of this region in TUB3 by inserting into it 3, 9, or 17 amino acids and tested the ability of these altered proteins to function as the only alpha-tubulin protein in yeast cells. We found that each of these altered proteins was sufficient on its own for mitotic growth, mating, and methods of yeast. We conclude that this region can tolerate considerable variation without losing any of the highly conserved functions of alpha-tubulin. Our results suggest that variability in this region occurs because it can be tolerated, not because it specifies an important function for the protein.

scholarly journals Structurally similar Drosophila alpha-tubulins are functionally distinct in vivo.

1997 ◽  
Vol 8 (3) ◽  
pp. 481-500 ◽  
Author(s):  
J A Hutchens ◽  
H D Hoyle ◽  
F R Turner ◽  
E C Raff
Keyword(s):  
Germ Cells ◽  
Microtubule Assembly ◽  
Meiotic Spindle ◽  
Dominant Male ◽  
Developmentally Regulated ◽  
Male Germ Cells ◽  
Alpha Tubulin ◽  
Tubulin Isoforms ◽  
Tubulin Structure

We used transgenic analysis in Drosophila to compare the ability of two structurally similar alpha-tubulin isoforms to support microtubule assembly in vivo. Our data revealed that even closely related alpha-tubulin isoforms have different functional capacities. Thus, in multicellular organisms, even small changes in tubulin structure may have important consequences for regulation of the microtubule cytoskeleton. In spermatogenesis, all microtubule functions in the postmitotic male germ cells are carried out by a single tubulin heterodimer composed of the major Drosophila alpha-84B tubulin isoform and the testis-specific beta 2-tubulin isoform. We tested the ability of the developmentally regulated alpha 85E-tubulin isoform to replace alpha 84B in spermatogenesis. Even though it is 98% similar in sequence, alpha 85E is not functionally equivalent to alpha 84B. alpha 85E can support some functional microtubules in the male germ cells, but alpha 85E causes dominant male sterility if it makes up more than one-half of the total alpha-tubulin pool in the spermatids. alpha 85E does not disrupt meiotic spindle or cytoplasmic microtubules but causes defects in morphogenesis of the two classes of singlet microtubules in the sperm tail axoneme, the central pair and the accessory microtubules. Axonemal defects caused by alpha 85E are precisely reciprocal to dominant defects in doublet microtubules we observed in a previous study of ectopic germ-line expression of the developmentally regulated beta 3-tubulin isoform. These data demonstrate that the doublet and singlet axoneme microtubules have different requirements for alpha- and beta-tubulin structure. In their normal sites of expression, alpha 85E and beta 3 are coexpressed during differentiation of several somatic cell types, suggesting that alpha 85E and beta 3 might form a specialized heterodimer. Our tests of different alpha-beta pairs in spermatogenesis did not support this model. We conclude that if alpha 85E and beta 3 have specialized properties required for their normal functions, they act independently to modulate the properties of microtubules into which they are incorporated.

Enhancement of the ncdD microtubule motor mutant by mutants of alpha Tub67C

1997 ◽  
Vol 110 (2) ◽  
pp. 229-237 ◽  
Author(s):  
D.J. Komma ◽  
S.A. Endow
Keyword(s):  
Motor Protein ◽  
Early Embryo ◽  
Meiotic Spindle ◽  
Molecular Defect ◽  
Loss Of Function ◽  
Alpha Tubulin ◽  
Microtubule Motor ◽  
Female Pronucleus ◽  
Allele Specific

Ncd is a kinesin-related microtubule motor protein required for chromosome segregation in Drosophila oocytes and early embryos. In tests for interactions with other proteins, we find that mutants of alpha Tub67C, which affect an oocyte- and early embryo-specific alpha-tubulin, enhance meiotic nondisjunction and zygotic loss of ncdD, a partial loss-of-function mutant of ncd. The enhancement is dominant and allele-specific with respect to alpha Tub67C, and depends on the recessive effects of ncdD. Cytologically, embryos of alpha Tub67C/+ show delayed meiotic divisions and defective female pronucleus formation, while meiotic spindle assembly is abnormal in embryos of ncdD/ncdD. Doubly mutant alpha Tub67C ncdD/ncdD embryos are rescued for female pronucleus formation, but show delayed meiotic progression and defective pronuclear conjugation or fusion. Delayed completion of meiosis, together with failure of pronuclear fusion, prevents normal interactions of maternal with paternal chromosomes, enhancing the ncdD mutant phenotype. The genetics and cytology of doubly mutant embryos and the molecular defect of NcdD provide evidence for interaction of Ncd with alpha Tub67C in vivo. These results imply that a specific alpha-tubulin isoform is required for normal cellular function of a kinesin motor protein.

scholarly journals Identification of microtubule-associated proteins in the meiotic spindle of surf clam oocytes.

1980 ◽  
Vol 84 (2) ◽  
pp. 235-245 ◽  
Author(s):  
D B Murphy
Keyword(s):  
Meiotic Spindle ◽  
Surf Clam ◽  
Microtubule Associated Proteins ◽  
Cytoplasmic Proteins ◽  
Associated Proteins ◽  
Meiotic Spindles

Meiotic spindles isolated from surf clam oocytes to morphological purity are biochemically complex, consisting of many polypeptides. These proteins fall into two classes: (a) polypeptides that are apparently cytoplasmic proteins and are not specifically associated with the spindle; and (b) polypeptides that are specifically associated with the spindle. A subset of the spindle-associated proteins, including a 250,000 mol wt component, remain with spindle tubulin through cycles of cold depolymerization and warm polymerization, showing that they are microtubule-associated proteins.

Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

1983 ◽  
Vol 41 ◽  
pp. 552-555
Author(s):  
Conly L. Rieder ◽  
S. Bowser ◽  
R. Nowogrodzki ◽  
K. Ross ◽  
G. Sluder
Keyword(s):  
Small Sample Size ◽  
Small Sample ◽  
Meiotic Spindle ◽  
Serial Sections ◽  
Mitotic Apparatus ◽  
Thin Sections ◽  
Cell Cycles ◽  
Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

scholarly journals Characterization of calcium-dependent membrane binding proteins of brain cortex

1985 ◽  
Vol 229 (3) ◽  
pp. 587-593 ◽  
Author(s):  
A R Rhoads ◽  
M Lulla ◽  
P B Moore ◽  
C E Jackson
Keyword(s):  
Brain Cortex ◽  
Peptide Mapping ◽  
Bovine Brain ◽  
Particulate Fraction ◽  
Structural Homology ◽  
One Dimensional ◽  
Calcium Dependent ◽  
Stokes Radius ◽  
The Brain

Proteins of Mr 68 000, 34 000 and 32 000 were selectively extracted by EGTA from brain cortex. The three proteins that were extracted along with calmodulin were acidic, monomeric, and did not exhibit structural homology, as demonstrated by one-dimensional peptide mapping. The Mr-68 000 protein was purified to homogeneity and had a Stokes radius of 3.54 nm and S20,W value of 5.1S. Purified calmodulin, Mr-68 000 protein and two proteins of Mr 34 000 and Mr 32 000, interacted with the brain particulate fraction, with half-maximal binding occurring at 3.5 microM, 8.3 microM and 150 microM-Ca2+ respectively. Proteins were bound independently of each other and calmodulin. Pretreatment of the particulate fraction with trypsin prevented the Ca2+-dependent binding of calmodulin; however, the binding of the Mr-68 000 protein or the Mr−32 000 and −34 000 proteins was unaffected. The Mr-68 000 protein of bovine brain did not cross-react immunologically with Mr-67 000 calcimedin from chicken gizzard.

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