Centrosomal components immunologically related to tektins from ciliary and flagellar microtubules

1994 ◽  
Vol 107 (8) ◽  
pp. 2095-2105 ◽  
Author(s):  
W. Steffen ◽  
E.A. Fajer ◽  
R.W. Linck
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Immunoblot Analysis ◽  
Surf Clam ◽  
Alpha Tubulin ◽  
Sea Urchin Sperm

Centrosomes are critical for the nucleation and organization of the microtubule cytoskeleton during both interphase and cell division. Using antibodies raised against sea urchin sperm flagellar microtubule proteins, we characterize here the presence and behavior of certain components associated with centrosomes of the surf clam Spisula solidissima and cultured mammalian cells. A Sarkosyl detergent-resistant fraction of axonemal microtubules was isolated from sea urchin sperm flagella and used to produce monoclonal antibodies, 16 of which were specific- or cross-specific for the major polypeptides associated with this microtubule fraction: tektins A, B and C, acetylated alpha-tubulin, and 77 and 83 kDa polypeptides. By 2-D isoelectric focussing/SDS polyacrylamide gel electrophoresis the tektins separate into several polypeptide spots. Identical spots were recognized by monoclonal and polyclonal antibodies against a given tektin, indicating that the different polypeptide spots are isoforms or modified versions of the same protein. Four independently derived monoclonal anti-tektins were found to stain centrosomes of S. solidissima oocytes and CHO and HeLa cells, by immunofluorescence microscopy. In particular, the centrosome staining of one monoclonal antibody specific for tektin B (tekB3) was cell-cycle-dependent for CHO cells, i.e. staining was observed only from early prometaphase until late anaphase. By immuno-electron microscopy tekB3 specifically labeled material surrounding the centrosome, whereas a polyclonal anti-tektin B recognized centrioles as well as the centrosomal material throughout the cell cycle. Finally, by immunoblot analysis tekB3 stained polypeptides of 48–50 kDa in isolated spindles and centrosomes from CHO cells.

scholarly journals Two proteins isolated from sea urchin sperm flagella: structural components common to the stable microtubules of axonemes and centrioles

1998 ◽  
Vol 111 (5) ◽  
pp. 585-595 ◽  
Author(s):  
E.H. Hinchcliffe ◽  
R.W. Linck
Keyword(s):  
Sea Urchin ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Sea Urchins ◽  
Polyclonal Antibodies ◽  
Chinese Hamster ◽  
Basal Bodies ◽  
Spindle Poles ◽  
Biochemical Fractionation ◽  
Sea Urchin Sperm

Biochemical fractionation of axonemal microtubules yields the protofilament ribbon (pf-ribbon), an insoluble structure of 3–4 longitudinal protofilaments composed primarily of alpha/beta tubulin, tektins A, B and C, and two previously uncharacterized polypeptides of 77 kDa and 83 kDa. We have isolated the 77/83 kDa polypeptides (termed Sp77 and Sp83) from sperm flagella of the sea urchin Stronglyocentrotus purpuratus and raised polyclonal antibodies against them. Sp77 and Sp83 copurify exclusively with the pf-ribbon. Both the anti-Sp77 and anti-Sp83 antibodies detected the nine outer doublets and the basal bodies of sea urchin sperm by immunofluorescence microscopy. In addition, the anti-Sp83 antibody, but not the anti-Sp77 antibody, detected a single 83 kDa polypeptide on immunoblots of unfertilized sea urchin egg cytoplasm, and a single polypeptide of 80 kDa on blots of isolated mitotic spindles from Chinese hamster ovary (CHO) cells. Previous studies have shown that tektins are present in the basal bodies and centrosomes/centrioles of cells ranging from clam to human. We found that anti-Sp83 decorates the spindle poles in sea urchin zygotes, and the interphase centrosome and spindle poles in CHO cells. In CHO cells arrested in S phase with aphidicolin, anti-Sp83 detects multiple centrosomes. The staining of the centrosome was not disrupted by prolonged nocodazole treatment, suggesting that the 80 kDa polypeptide is associated with the centrioles themselves. Our observations demonstrate that, like tektins, Sp77 and Sp83 are structural proteins associated with stable doublet microtubules, and may be components of basal bodies and centrioles of sea urchins and mammalian cells.

DNA precursor pools and ribonucleotide reductase activity: distribution between the nucleus and cytoplasm of mammalian cells

1985 ◽  
Vol 5 (12) ◽  
pp. 3443-3450
Author(s):  
J M Leeds ◽  
M B Slabaugh ◽  
C K Mathews
Keyword(s):  
Cell Cycle ◽  
Ribonucleotide Reductase ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Reductase Activity ◽  
Cell Activity ◽  
S Phase ◽  
Whole Cell ◽  
Ribonucleotide Reductase Activity

Nuclear and whole-cell deoxynucleoside triphosphate (dNTP) pools were measured in HeLa cells at different densities and throughout the cell cycle of synchronized CHO cells. Nuclei were prepared by brief detergent (Nonidet P-40) treatment of subconfluent monolayers, a procedure that solubilizes plasma membranes but leaves nuclei intact and attached to the plastic substratum. Electron microscopic examination of monolayers treated with Nonidet P-40 revealed protruding nuclei surrounded by cytoskeletal remnants. Control experiments showed that nuclear dNTP pool sizes were stable during the time required for isolation, suggesting that redistribution of nucleotides during the isolation procedure was minimal. Examination of HeLa whole-cell and nuclear dNTP levels revealed that the nuclear proportion of each dNTP was distinct and remained constant as cell density increased. In synchronized CHO cells, all four dNTP whole-cell pools increased during S phase, with the dCTP pool size increasing most dramatically. The nuclear dCTP pool did not increase as much as the whole-cell dCTP pool during S phase, lowering the relative nuclear dCTP pool. Although the whole-cell dNTP pools decreased after 30 h of isoleucine deprivation, nuclear pools did not decrease proportionately. In summary, nuclear dNTP pools in synchronized CHO cells maintained a relatively constant concentration throughout the cell cycle in the face of larger fluctuations in whole-cell dNTP pools. Ribonucleotide reductase activity was measured in CHO cells throughout the cell cycle, and although there was a 10-fold increase in whole-cell activity during S phase, we detected no reductase in nuclear preparations at any point in the cell cycle.

Calcium and cell cycle control

Development ◽  
1990 ◽  
Vol 108 (4) ◽  
pp. 525-542 ◽  
Author(s):  
M. Whitaker ◽  
R. Patel
Keyword(s):  
Cell Cycle ◽  
Sea Urchin ◽  
Mammalian Cells ◽  
Cell Cycle Regulation ◽  
Sea Urchins ◽  
Cell Cycle Control ◽  
Control Point ◽  
Control Cell ◽  
Free Calcium ◽  
Cycle Regulation

The cell division cycle of the early sea urchin embryo is basic. Nonetheless, it has control points in common with the yeast and mammalian cell cycles, at START, mitosis ENTRY and mitosis EXIT. Progression through each control point in sea urchins is triggered by transient increases in intracellular free calcium. The Cai transients control cell cycle progression by translational and post-translational regulation of the cell cycle control proteins pp34 and cyclin. The START Cai transient leads to phosphorylation of pp34 and cyclin synthesis. The mitosis ENTRY Cai transient triggers cyclin phosphorylation. The motosis EXIT transient causes destruction of phosphorylated cyclin. We compare cell cycle regulation by calcium in sea urchin embryos to cell cycle regulation in other eggs and oocytes and in mammalian cells.

Cell cycle progression and cell division are sensitive to hypoxia in Drosophila melanogaster embryos

2001 ◽  
Vol 280 (5) ◽  
pp. R1555-R1563 ◽  
Author(s):  
Robert M. Douglas ◽  
Tian Xu ◽  
Gabriel G. Haddad
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Immunoblot Analysis ◽  
Embryonic Cell ◽  
Cell Cycle Checkpoints ◽  
Cycle Phase ◽  
Embryonic Cells

We and others recently demonstrated that Drosophila melanogaster embryos arrest development and embryonic cells cease dividing when they are deprived of O2. To further characterize the behavior of these embryos in response to O2 deprivation and to define the O2-sensitive checkpoints in the cell cycle, embryos undergoing nuclear cycles 3–13 were subjected to O2deprivation and examined by confocal microscopy under control, hypoxic, and reoxygenation conditions. In vivo, real-time analysis of embryos carrying green fluorescent protein-kinesin demonstrated that cells arrest at two major points of the cell cycle, either at the interphase (before DNA duplication) or at metaphase, depending on the cell cycle phase at which O2 deprivation was induced. Immunoblot analysis of embryos whose cell divisions are synchronized by inducible String (cdc25 homolog) demonstrated that cyclin B was degraded during low O2 conditions in interphase-arrested embryos but not in those arrested in metaphase. Embryos resumed cell cycle activity within ∼20 min of reoxygenation, with very little apparent change in cell cycle kinetics. We conclude that there are specific points during the embryonic cell cycle that are sensitive to the O2 level in D. melanogaster. Given the fact that O2deprivation also influences the growth and development of other species, we suggest that similar hypoxia-sensitive cell cycle checkpoints may also exist in mammalian cells.

The polyglutamylated lateral chain of alpha-tubulin plays a key role in flagellar motility

1996 ◽  
Vol 109 (6) ◽  
pp. 1545-1553 ◽  
Author(s):  
C. Gagnon ◽  
D. White ◽  
J. Cosson ◽  
P. Huitorel ◽  
B. Edde ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Cortical Microtubule ◽  
Flagellar Motility ◽  
Microtubule Network ◽  
Alpha Tubulin ◽  
Oxyrrhis Marina ◽  
Terminal Domain ◽  
Flagellar Beat ◽  
Sea Urchin Sperm

To investigate whether a specific isotype of tubulin is involved in flagellar motility, we have developed and screened a panel of monoclonal antibodies (mAb) generated against sea urchin sperm axonemal proteins. Antibodies were selected for their ability to block the motility of permeabilized sperm models. The antitubulin mAb B3 completely inhibited, at low concentrations, the flagellar motility of permeabilized sperm models from four sea urchin species. On immunoblots, B3 recognized predominantly alpha-tubulin in sea urchin sperm axonemes and equally well brain alpha- and beta-tubulins. Subtilisin cleavage of tubulin removed the B3 epitope, indicating that it was restricted to the last 13 amino acid residues of the C-terminal domain of alpha-tubulin. In enzyme-linked immunosorbant assays, B3 reacted with glutamylated alpha-tubulin peptides from sea urchin or mouse brain but did not bind to the unmodified corresponding peptide, indicating that it recognized polyglutamylated motifs in the C-terminal domain of alpha-tubulin. On the other hand, other tubulin antibodies directed against various epitopes of the C-terminal domain, with the exception of the antipolyglutamylated mAb GT335, had no effect on motility while having binding properties similar to that of B3. B3 and GT335 acted by decreasing the beating amplitude without affecting the flagellar beat frequency. B3 and GT335 were also capable of inhibiting the motility of flagella of Oxyrrhis marina, a 400,000,000 year old species of dinoflagellate, and those of human sperm models. Localization of the antigens recognized by B3 and GT335 by immunofluorescence techniques revealed their presence along the whole axoneme of sea urchin spermatozoa and flagella of O. marina, except for the distal tip and the cortical microtubule network of the dinoflagellate. Taken together, the data reported here indicate that the polyglutamylated lateral chain of alpha-tubulin plays a dynamic role in a dynein-based motility process.

scholarly journals The NUF1 gene encodes an essential coiled-coil related protein that is a potential component of the yeast nucleoskeleton.

1992 ◽  
Vol 116 (6) ◽  
pp. 1319-1332 ◽  
Author(s):  
C Mirzayan ◽  
C S Copeland ◽  
M Snyder
Keyword(s):  
Mammalian Cells ◽  
Polyclonal Antibodies ◽  
Coiled Coil ◽  
Immunoblot Analysis ◽  
Expression Library ◽  
Amino Terminal ◽  
Eukaryotic Nucleus ◽  
Nonionic Detergent ◽  
Potential Component ◽  
Carboxy Terminal

In an attempt to identify structural components of the yeast nucleus, subcellular fractions of yeast nuclei were prepared and used as immunogens to generate complex polyclonal antibodies. One such serum was used to screen a yeast genomic lambda gt11 expression library. A clone encoding a gene called NUF1 (for nuclear filament-related) was identified and extensively characterized. Antibodies to NUF1 fusion proteins were generated, and affinity-purified antibodies were used for immunoblot analysis and indirect immunofluorescence localization. The NUF1 protein is 110 kD in molecular mass and localizes to the yeast nucleus in small granular patches. Intranuclear staining is present in cells at all stages of the cell cycle. The NUF1 protein of yeast is tightly associated with the nucleus; it was not removed by extraction of nuclei with nonionic detergent or salt, or treatment with RNAse and DNAse. Sequence analysis of the NUF1 gene predicts a protein 945 amino acids in length that contains three domains: a large 627 residue central domain predicted to form a coiled-coil structure flanked by nonhelical amino-terminal and carboxy-terminal regions. Disruption of the NUF1 gene indicates that it is necessary for yeast cell growth. These results indicate that NUF1 encodes an essential coiled-coil protein within the yeast nucleus; we speculate that NUF1 is a component of the yeast nucleoskeleton. In addition, immunofluorescence results indicate that mammalian cells contain a NUF1-related nuclear protein. These data in conjunction with those in the accompanying manuscript (Yang et al., 1992) lead to the hypothesis that an internal coiled-coil filamentous system may be a general structural component of the eukaryotic nucleus.

scholarly journals Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms.

1985 ◽  
Vol 101 (6) ◽  
pp. 2085-2094 ◽  
Author(s):  
G Piperno ◽  
M T Fuller
Keyword(s):  
Monoclonal Antibodies ◽  
Sea Urchin ◽  
Human Sperm ◽  
Preliminary Evidence ◽  
Alpha Tubulin ◽  
Tubulin Isoforms ◽  
Cilia And Flagella ◽  
Sea Urchin Sperm

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.

scholarly journals THE SYNTHESIS OF ACIDIC CHROMOSOMAL PROTEINS DURING THE CELL CYCLE OF HELA S-3 CELLS

1972 ◽  
Vol 52 (2) ◽  
pp. 292-307 ◽  
Author(s):  
G. S. Stein ◽  
T. W. Borun
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Nuclear Proteins ◽  
Regulatory Function ◽  
Nuclear Fraction ◽  
Tightly Coupled ◽  
Acidic Proteins

The synthesis and accumulation of acidic proteins in the tightly bound residual nuclear fraction goes on throughout the cell cycle of continuously dividing populations of HeLa S-3 cells; however, during late G1 there is an increased rate of synthesis and accumulation of these proteins which precedes the onset of DNA synthesis. Unlike that of the histones, whose synthesis is tightly coupled to DNA replication, the synthesis of acidic residual nuclear proteins is insensitive to inhibitors of DNA synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of acidic residual nuclear proteins shows different profiles during the G1, S, and G2 phases of the cell cycle. These results suggest that, in contrast to histones whose synthesis appears to be highly regulated, the acidic residual proteins may have a regulatory function in the control of cell proliferation in continuously dividing mammalian cells.

Cytostellin: a novel, highly conserved protein that undergoes continuous redistribution during the cell cycle

1992 ◽  
Vol 103 (2) ◽  
pp. 381-388 ◽  
Author(s):  
S.L. Warren ◽  
A.S. Landolfi ◽  
C. Curtis ◽  
J.S. Morrow
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Immunoblot Analysis ◽  
Cytoplasmic Proteins ◽  
Spindle Apparatus ◽  
Nuclease Digestion ◽  
Cell Processes ◽  
A Cell ◽  
Cycle Dependent

Cytostellin, a 240 kDa protein, has been purified from mammalian cells by immunoaffinity chromatography using monoclonal antibody H5. Immunofluorescence microscopy shows diffuse and punctate cytostellin immunoreactivity in interphase nuclei. Nuclease digestion and salt extraction are not required to expose the epitope. The onset of prophase is marked by the appearance of multiple intensely immunofluorescent cytostellin-containing ‘bodies’ within the nucleus. Nuclear disassembly is heralded by the movement of cytostellin bodies from the nucleus to multiple positions throughout the cell. Cytostellin bodies in metaphase, anaphase and telophase cells are widely dispersed, including some in cell processes far removed from the mitotic spindle apparatus. However, a distinct subset of larger, more intensely staining bodies surrounds the mitotic spindle apparatus. Cytostellin bodies remain in the cytoplasm of the daughter cells and disappear after the appearance of nascent nuclei. Cytostellin is immunologically distinct from other nuclear and cytoplasmic proteins, and it has been detected by immunoblot analysis in all species tested from yeast to humans. Based upon these findings, we postulate that cytostellin has a cell cycle-dependent function which is conserved in higher and lower eukaryotic cells.

A monoclonal antibody, raised against mammalian centrosomes and screened by recognition of plant microtubule organizing centers, identifies a pericentriolar component in different cell types

1992 ◽  
Vol 101 (4) ◽  
pp. 823-835 ◽  
Author(s):  
V. Chevrier ◽  
S. Komesli ◽  
A.C. Schmit ◽  
M. Vantard ◽  
A.M. Lambert ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Monoclonal Antibody ◽  
Mammalian Cells ◽  
Cell Types ◽  
Immunoblot Analysis ◽  
Dependent Manner ◽  
Specific Staining ◽  
Microtubule Organizing Centers ◽  
Pericentriolar Material ◽  
Cycle Dependent

We have used monoclonal antibodies raised against isolated native calf thymus centrosomes to probe the structure and composition of the pericentriolar material. To distinguish prospective antibodies as specific to conserved elements of this material, we screened clones by their identification of microtubule organizing centers (MTOCs) in different animal and plant cells. Among the clonal antibodies that reacted with MTOCs in both plant and mammalian cells, we describe one (mAb 6C6) that was found to immunostain centrosomes in a variety of bovine and human cells. In cycling cells this signal persisted through the entire cell cycle. Microscopy showed that the mAb 6C6 antigen was a component of the pericentriolar material and this was confirmed by biochemical analysis of centrosomes. Using immunoblot analysis of protein fractions derived from purified components of centrosomes, we have characterized the mAb 6C6 antigen as a 180 kDa polypeptide. We conclude that we have identified a protein component permanently associated with the pericentriolar material. Surprisingly, monoclonal antibody 6C6 also stained other mitotic organelles in mammalian cells, in a cell-cycle-dependent manner. During prometaphase and metaphase the antibody stained both centrosomes and kinetochores. At the onset of anaphase the kinetochore-specific staining dissociated from chromosomes and was subsequently redistributed onto a newly characterized organelle, the telophase disc while the centrosomal stain remained intact. It is not known if the 180 kDa centrosomal protein itself redistributes during mitosis, or if the pattern observed represents other antigens with shared epitopes. The pericentriolar material is thought to be composed of conserved elements, which appeared very early during the evolution of eukaryotes. Our results strongly suggest that mAb 6C6 identifies one of these elements.

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