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.

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.

Mutants of the microtubule motor protein, nonclaret disjunctional, affect spindle structure and chromosome movement in meiosis and mitosis

1992 ◽  
Vol 101 (3) ◽  
pp. 547-559 ◽  
Author(s):  
M. Hatsumi ◽  
S.A. Endow
Keyword(s):  
Motor Protein ◽  
Spindle Motor ◽  
Meiotic Spindle ◽  
Chromosome Movement ◽  
Chromosome Distribution ◽  
Early Embryos ◽  
Microtubule Motor ◽  
Microtubule Motor Protein ◽  
Spindle Structure ◽  
Meiosis I

The Drosophila microtubule motor protein, nonclaret disjunctional (ncd), is required for proper chromosome distribution in meiosis and mitosis. We have examined the meiotic and mitotic divisions in wild-type Drosophila oocytes and early embryos, and the effects of three ncd mutants (cand, ncd and ncdD) on spindle structure and chromosome movement. The ncd mutants cause abnormalities in spindle structure early in meiosis I, and abnormal chromosome configurations throughout meiosis I and II. Defective divisions continue in early embryos of the motor null mutant, cand, with abnormal early mitotic spindles. The effects of mutants on spindle structure suggest that ncd is required for proper meiotic spindle assembly, and may play a role in forming or maintaining spindle poles in meiosis. The disruption of normal meiotic and mitotic chromosome distribution by ncd mutants can be attributed to its role as a spindle motor, although a role for ncd as a chromosome-associated motor protein is not excluded. The ncd motor protein functions not only in meiosis, but also performs an active role in the early mitotic divisions of the embryo.

scholarly journals Mutants of the Drosophila ncd microtubule motor protein cause centrosomal and spindle pole defects in mitosis

1994 ◽  
Vol 107 (4) ◽  
pp. 859-867 ◽  
Author(s):  
S.A. Endow ◽  
R. Chandra ◽  
D.J. Komma ◽  
A.H. Yamamoto ◽  
E.D. Salmon
Keyword(s):  
Cell Cycle ◽  
Coiled Coil ◽  
Motor Protein ◽  
Spindle Pole ◽  
Opposite Polarity ◽  
Microtubule Assembly ◽  
Loss Of Function ◽  
Spindle Poles ◽  
Microtubule Motor ◽  
Microtubule Motor Protein

Nonclaret disjunctional (ncd) is a kinesin-related microtubule motor protein required for meiotic and early mitotic chromosome distribution in Drosophila. ncd translocates on microtubules with the opposite polarity to kinesin, toward microtubule minus ends, and is associated with spindles in chromosome/spindle preparations. Here we report a new mutant of ncd caused by partial deletion of the predicted coiled-coil central stalk. The mutant protein exhibits a velocity of translocation and ability to generate torque in motility assays comparable to near full-length ncd, but only partially rescues a null mutant for chromosome mis-segregation. Antibody staining experiments show that the partial loss-of-function and null mutants cause centrosomal and spindle pole defects, including centrosome splitting and loss of centrosomes from spindle poles, and localize ncd to centrosomes as well as spindles of wild-type embryos. Association of ncd with spindles and centrosomes is microtubule- and cell cycle-dependent: inhibition of microtubule assembly with colchicine abolishes ncd staining and centrosomal staining is observed in prometaphase, metaphase and anaphase, but diminishes in late anaphase/telophase. The cell cycle dependence of centrosomal staining and the defects of mutants provide clear evidence for activity of the ncd motor protein near or at the spindle poles in mitosis. The ncd motor may interact with centrosomal microtubules and spindle fibers to attach centrosomes to spindle poles, and mediate poleward translocation (flux) of kinetochore fibers, a process that may underlie poleward movement of chromosomes in mitosis. Together with previous work, our findings indicate that ncd is important in maintaining spindle poles in mitosis as well as in meiosis.

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 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.

scholarly journals Failure of pronuclear migration and repeated divisions of polar body nuclei associated with MTOC defects in polo eggs of Drosophila

2000 ◽  
Vol 113 (18) ◽  
pp. 3341-3350 ◽  
Author(s):  
M.G. Riparbelli ◽  
G. Callaini ◽  
D.M. Glover
Keyword(s):  
Polar Body ◽  
Motor Protein ◽  
Meiotic Spindle ◽  
Wild Type ◽  
Female Meiosis ◽  
Male Pronucleus ◽  
Female Pronucleus ◽  
Sperm Aster ◽  
Meiosis I

The meiotic spindle of Drosophila oocytes is acentriolar but develops an unusual central microtubule organising centre (MTOC) at the end of meiosis I. In polo oocytes, this common central pole for the two tandem spindles of meiosis II was poorly organised and in contrast to wild-type failed to maintain its associated Pav-KLP motor protein. Furthermore, the polar body nuclei failed to arrest at metaphase, and the four products of female meiosis all underwent repeated haploid division cycles on anastral spindles. This was linked to a failure to form the astral array of microtubules with which the polar body chromosomes are normally associated. The MTOC associated with the male pronucleus was also defective in polo eggs, and the sperm aster did not grow. Migration of the female pronucleus did not take place and so a gonomeric spindle could not form. We discuss these findings in relation to the known roles of polo like kinases in regulating the behaviour of MTOCs.

Differential regulation of maternal and paternal chromosome condensation in mitotic zygotes

2002 ◽  
Vol 115 (14) ◽  
pp. 2931-2940 ◽  
Author(s):  
Jacqueline Bomar ◽  
Pedro Moreira ◽  
John J. Balise ◽  
Philippe Collas
Keyword(s):  
Differential Regulation ◽  
Chromosome Condensation ◽  
Meiotic Spindle ◽  
Oocyte Activation ◽  
Anchoring Protein ◽  
Female Pronucleus ◽  
Mouse Zygotes ◽  
Mitotic Chromosome Condensation ◽  
Metaphase Ii Oocytes

A-kinase anchoring protein AKAP95 is implicated in somatic mitotic chromosome condensation by recruiting the condensin complex. Here, we report a differential regulation of condensation of maternal and paternal chromosomes mediated by AKAP95 in mitotic mouse zygotes. AKAP95 is synthesized upon oocyte activation, targeted to the female pronucleus and specifically associates with maternal chromosomes at mitosis. AKAP95 mRNA is highly restricted to the vicinity of the meiotic spindle in metaphase II oocytes. In vivo displacement of endogenous AKAP95 in female pronuclei by microinjection of competitor peptides and rescue experiments show that AKPA95 is required for recruitment of the mCAP-D2 condensin subunit to, and condensation of, maternal chromosomes. In contrast, AKAP95 is dispensable for mCAP-D2 recruitment to,and condensation of, paternal chromosomes. Our results indicate that at first embryonic mitosis, paternal chromosomes target condensins and condense independently of AKAP95, whereas maternal chromosomes require AKAP95 for condensin recruitment and condensation. We propose a concept whereby condensation of chromosomes in gametes, zygotes and somatic cells involves related but distinct mechanisms.

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.

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).

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