-
Cell Motility and the Cytoskeleton 1992Injection of CREST antikinetochore antiserum (AKA) containing antibodies to the kinetochore into living prometaphase PtK2 cells decreased chromosome velocity to near...
Injection of CREST antikinetochore antiserum (AKA) containing antibodies to the kinetochore into living prometaphase PtK2 cells decreased chromosome velocity to near zero. Injection of either phosphate-buffered saline or CREST antiserum without antikinetochore antibodies (antikinetochore negative: AKN) had no effect on prometaphase oscillations. AKA antiserum injected into anaphase cells at the beginning of chromatid separation had no effect on anaphase chromosome velocity, spindle elongation, or cytokinesis. Visible binding of antikinetochore antibodies in prometaphase cells at room temperature occurred between 5 and 15 minutes after injection. Anaphase cells injected at the beginning of chromatid separation had bound antibody at the end of anaphase. AKA antiserum recognizes in Western blots proteins associated with the primary constriction: CENP-B, -C, and -D, as reported by other workers. The control antiserum, AKN, does not recognize these proteins. These results imply that the antigens recognized by CREST antibodies are important for chromosome movement. Whether or not these antigens are themselves motor molecules cannot be addressed by the present data. In addition, the results suggest that these antigens are not involved in an important way in anaphase movement.
Topics: Anaphase; Animals; Autoantibodies; Autoimmune Diseases; Cell Division; Cell Line, Transformed; Centromere; Chromosomes; Fluorescent Antibody Technique; Macropodidae; Metaphase; Scleroderma, Systemic; Spindle Apparatus
PubMed: 1451187
DOI: 10.1002/cm.970230208 -
Molecular Biology of the Cell Jan 2004Mitosis requires the concerted activities of multiple microtubule (MT)-based motor proteins. Here we examined the contribution of the chromokinesin, KLP3A, to mitotic...
Mitosis requires the concerted activities of multiple microtubule (MT)-based motor proteins. Here we examined the contribution of the chromokinesin, KLP3A, to mitotic spindle morphogenesis and chromosome movements in Drosophila embryos and cultured S2 cells. By immunofluorescence, KLP3A associates with nonfibrous punctae that concentrate in nuclei and display MT-dependent associations with spindles. These punctae concentrate in indistinct domains associated with chromosomes and central spindles and form distinct bands associated with telophase midbodies. The functional disruption of KLP3A by antibodies or dominant negative proteins in embryos, or by RNA interference (RNAi) in S2 cells, does not block mitosis but produces defects in mitotic spindles. Time-lapse confocal observations of mitosis in living embryos reveal that KLP3A inhibition disrupts the organization of interpolar (ip) MTs and produces short spindles. Kinetic analysis suggests that KLP3A contributes to spindle pole separation during the prometaphase-to-metaphase transition (when it antagonizes Ncd) and anaphase B, to normal rates of chromatid motility during anaphase A, and to the proper spacing of daughter nuclei during telophase. We propose that KLP3A acts on MTs associated with chromosome arms and the central spindle to organize ipMT bundles, to drive spindle pole separation and to facilitate chromatid motility.
Topics: Anaphase; Animals; Cell Nucleus; Cells, Cultured; Chromatids; Chromosome Positioning; Cloning, Molecular; Drosophila Proteins; Drosophila melanogaster; Embryo, Nonmammalian; Kinesins; Metaphase; Microinjections; Microscopy, Fluorescence; Microtubules; Models, Molecular; Mutation; RNA, Small Interfering; Spindle Apparatus; Telophase
PubMed: 14528012
DOI: 10.1091/mbc.e03-07-0489 -
Physical Review. E, Statistical,... Jul 2002Primitive cells had to divide using very few biological mechanisms. This work proposes physicochemical mechanisms, based upon nanoscale electrostatics, which explain and...
Primitive cells had to divide using very few biological mechanisms. This work proposes physicochemical mechanisms, based upon nanoscale electrostatics, which explain and unify the motions of chromosomes during prometaphase, metaphase, and anaphase A. In the cytoplasmic medium that exists in biological cells, electrostatic fields are subject to strong attenuation by ionic screening, and therefore decrease rapidly over a distance equal to several Debye lengths. However, the presence of microtubules within cells completely changes the situation. Microtubule dimer subunits are electric dipolar structures, and can act as intermediaries that extend the reach of the electrostatic interaction over cellular distances. Experimental studies have shown that intracellular pH rises to a peak at mitosis, then decreases through cytokinesis. This result, in conjunction with the electric dipole nature of microtubule subunits, is sufficient to explain the dynamics of the above mitotic motions, including their timing and sequencing. The physicochemical mechanisms utilized by primitive eukaryotic cells could provide important clues regarding our understanding of cell division in modern eukaryotic cells.
Topics: Anaphase; Biophysical Phenomena; Biophysics; Chromosomes; Hydrogen-Ion Concentration; Kinetochores; Metaphase; Microtubules; Models, Biological; Movement; Static Electricity
PubMed: 12241378
DOI: 10.1103/PhysRevE.66.011901 -
The Journal of Cell Biology Mar 1989Microtubule (MT) dynamics in PtK2 cells have been investigated using in vivo injection of unmodified Paramecium ciliary tubulin and time-lapse fixation. The sites of...
Microtubule dynamics investigated by microinjection of Paramecium axonemal tubulin: lack of nucleation but proximal assembly of microtubules at the kinetochore during prometaphase.
Microtubule (MT) dynamics in PtK2 cells have been investigated using in vivo injection of unmodified Paramecium ciliary tubulin and time-lapse fixation. The sites of incorporation of the axonemal tubulin were localized using a specific antibody which does not react with vertebrate cytoplasmic tubulin (Adoutte, A., M. Claisse, R. Maunoury, and J. Beisson. 1985. J. Mol. Evol. 22:220-229), followed by immunogold labeling, Nanovid microscopy, and ultrastructural observation of the same cells. We confirm data from microinjection of labeled tubulins in other cell types (Soltys, B. J., and G. G. Borisy. 1985. J. Cell Biol. 100:1682-1689; Mitchison, T., L. Evans, E. Schulze, and M. Kirschner. 1986. Cell. 45:515-527; Schulze, E., and M. Kirschner. 1986. J. Cell Biol. 102:1020-1031). In agreement with the dynamic instability model (Mitchison, T., and M. Kirschner. 1984. Nature (Lond.). 312:237-242), during interphase, fast (2.6 microns/min) distal growth of MTs occurs, together with new centrosomal nucleation. Most of the cytoplasmic MT complex is replaced within 15-30 min. During mitosis, astral MTs display the same pattern of renewal, but the turnover of the MT system is much faster (approximately 6 min). We have concentrated on the construction of the kinetochore fibers during prometaphase and observe that (a) incorporation of tubulin in the vicinity of the kinetochores is not seen during prophase and early prometaphase as long as the kinetochores are not yet connected to a pole by MTs; (b) proximal time-dependent incorporation occurs only into preexisting kinetochore MTs emanating from centrosomes. Consequently, in undisturbed prometaphase cells, the kinetochores probably do not act as independent nucleation sites. This confirms a model in which, at prometaphase, fast probing centrosomal MTs are grabbed by the kinetochores, where tubulin incorporation then takes place.
Topics: Animals; Cell Line; Centromere; Chromosomes; Fluorescent Antibody Technique; Immunohistochemistry; Interphase; Metaphase; Microinjections; Microscopy, Electron; Microtubules; Paramecium; Prophase; Spindle Apparatus; Tubulin
PubMed: 2646309
DOI: 10.1083/jcb.108.3.939 -
The Journal of Biological Chemistry May 2007Plk1, an evolutionarily conserved M phase kinase, associates with not only spindle poles but also kinetochores during prometaphase. However, the role of Plk1 at...
Plk1, an evolutionarily conserved M phase kinase, associates with not only spindle poles but also kinetochores during prometaphase. However, the role of Plk1 at kinetochores has been poorly understood. Here we show that BubR1 mediates the action of Plk1 at kinetochores for proper chromosome alignment. Our results show that BubR1 colocalizes with Plk1 at kinetochores of unaligned chromosomes and physically interacts with Plk1 in prometaphase cells. Down-regulation of Plk1 by small interfering RNA abolished the mobility-shifted, hyperphosphorylated form of BubR1 in the prometaphase-arrested cells. In addition, BubR1 was phosphorylated by Plk1 in vitro at two Plk1 consensus sites in the kinase domain of BubR1. The add-back of either wild-type BubR1 or BubR1 2E, in which the two Plk1 phosphorylation sites were replaced by glutamic acids, but not that of BubR1 2A, an unphosphorylatable mutant, rescued the chromosome alignment defects in BubR1-deficient cells. Moreover, when both Plk1 and BubR1 were down-regulated, the add-back of BubR1 2E, but not that of wild-type BubR1, rescued the chromosome alignment defects. These results taken together suggest that Plk1 facilitates chromosome alignment during prometaphase through BubR1.
Topics: Amino Acid Substitution; Animals; COS Cells; Cell Cycle Proteins; Chlorocebus aethiops; Chromosomes, Human; Down-Regulation; HeLa Cells; Humans; Kinetochores; Metaphase; Mutation, Missense; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Proto-Oncogene Proteins; Polo-Like Kinase 1
PubMed: 17376779
DOI: 10.1074/jbc.M611053200 -
Oral Oncology Apr 2010One apparent feature of cancerous cells is genomic instability, which may include various types of chromosomal aberrations, such as translocation, aneuploidy, and the...
One apparent feature of cancerous cells is genomic instability, which may include various types of chromosomal aberrations, such as translocation, aneuploidy, and the presence of micronuclei inside the cells. Mutagenic factors that promote the emergence of genomic instability are recognized as risk factors for the development of human malignancies. In Asia, betel quid (BQ) chewing is one of such risk factors for oral cancer. Areca nut is an essential constitute of BQ and is declared as a group I carcinogen by the International Agency for Research on Cancer. However, the molecular and cellular mechanisms regarding the carcinogenicity of areca nut are not fully explored. Here we reported that arecoline, a major alkaloid of areca nut, could arrest cells at prometaphase with large amounts of misaligned chromosomes. This prometaphase arrest was evidenced by condensed chromosome pattern, increased histone H3 phosphorylation, and accumulation of mitotic proteins, including aurora A and cyclin B(1). To investigate the molecular mechanisms accounting for arecoline-induced prometaphase arrest, we found that arecoline could stabilize mitotic spindle assembly, which led to distorted organization of mitotic spindles, misalignment of chromosomes, and up-regulation of spindle assembly checkpoint (SAC) genes. The SAC proteins BubR1 and Mps1 were differentially modified between the cells treated with arecoline and nocodazole. This together with aurora A overexpression suggested that SAC might be partly suppressed by arecoline. As a result, the arecoline-exposed cells might produce progeny that contained various chromosomal aberrations and exhibited genomic instability.
Topics: Areca; Arecoline; Carcinogens; Cell Line, Tumor; Cell Transformation, Neoplastic; Cells, Cultured; Genomic Instability; Histones; Humans; Mitosis; Mouth Neoplasms; Prometaphase; Spindle Apparatus; Tumor Suppressor Protein p53
PubMed: 20138568
DOI: 10.1016/j.oraloncology.2010.01.003 -
Cell Structure and Function Feb 2002We previously reported that exogenous histone H1, when injected into mitotic cells, disrupts the synchronous progression of mitotic events by delaying chromosome...
We previously reported that exogenous histone H1, when injected into mitotic cells, disrupts the synchronous progression of mitotic events by delaying chromosome decondensation. This strategy was utilized to determine whether any other interphase proteins are also able to disrupt normal mitotic processes, when introduced into the mitotic phase. We found that a chromatin subfraction from bovine liver nuclei induced postmitotic micronuclei formation in a dose-dependent manner when injected into the prometaphase of rat kangaroo kidney epithelial (PtK(2)) cells. Close observation showed that, in the case of injected mitotic cells, the mitotic spindles were disrupted, chromosomes became scattered throughout the cytoplasm, and actin filaments were organized ectopically. In addition, when the fraction was injected into interphase cells, extra actin filaments were formed and microtubule organization was affected. In order to determine whether the micronuclei formation resulted from the ectopic formation of actin filaments, we examined the effect of the actin polymerization inhibitor, cytochalasin D. The results showed that the drug inhibited micronuclei formation. From these findings, we concluded that this chromatin subfraction contains actin polymerization activity, thus causing the disruption of mitotic spindles.
Topics: Actin Cytoskeleton; Animals; Cattle; Chromatin; Chromosomes; Cytochalasin D; Epithelial Cells; Histones; Interphase; Liver; Macropodidae; Metaphase; Microinjections; Micronuclei, Chromosome-Defective; Spindle Apparatus
PubMed: 11937717
DOI: 10.1247/csf.27.39 -
BioRxiv : the Preprint Server For... Dec 2023To ensure genomic fidelity a series of spatially and temporally coordinated events are executed during prometaphase of mitosis, including bipolar spindle formation,...
To ensure genomic fidelity a series of spatially and temporally coordinated events are executed during prometaphase of mitosis, including bipolar spindle formation, chromosome attachment to spindle microtubules at kinetochores, the correction of erroneous kinetochore-microtubule (k-MT) attachments, and chromosome congression to the spindle equator. Cyclin A/Cdk1 kinase plays a key role in destabilizing k-MT attachments during prometaphase to promote correction of erroneous k-MT attachments. However, it is unknown if Cyclin A/Cdk1 kinase regulates other events during prometaphase. Here, we investigate additional roles of Cyclin A/Cdk1 in prometaphase by using an siRNA knockdown strategy to deplete endogenous Cyclin A from human cells. We find that depleting Cyclin A significantly extends mitotic duration, specifically prometaphase, because chromosome alignment is delayed. Unaligned chromosomes display erroneous monotelic, syntelic, or lateral k-MT attachments suggesting that bioriented k-MT attachment formation is delayed in the absence of Cyclin A. Mechanistically, chromosome alignment is likely impaired because the localization of the kinetochore proteins BUB1 kinase, KNL1, and MPS1 kinase are reduced in Cyclin A-depleted cells. Moreover, we find that Cyclin A promotes BUB1 kinetochore localization independently of its role in destabilizing k-MT attachments. Thus, Cyclin A/Cdk1 facilitates chromosome alignment during prometaphase to support timely mitotic progression.
PubMed: 38187612
DOI: 10.1101/2023.12.21.572788 -
Chromosoma Nov 1978Rates of movement of univalents at prometaphase and of half-bivalents at anaphase in living cricket and grasshopper spermatocytes were determined as a function of the...
Rates of movement of univalents at prometaphase and of half-bivalents at anaphase in living cricket and grasshopper spermatocytes were determined as a function of the distance from the pole toward which the movement was directed. In the artificially produced univalents of cricket cells, correlation coefficients for rate versus distance form the pole were widely disparate from movement to movement and there was no consistent relationship between velocity and distance from the pole. However, in the naturally occurring univalents of grasshopper cells, there was a significant positive correlation between velocity and distance from the pole. In both cricket and grasshopper cells, there was no consistent correlation between rate of movement and distance from the pole for half-bivalents at anaphase. The prometaphase data from grasshopper cells support the simple hypothesis of Ostergren (1950) that congression results from the application to chromosomes of forces which increase with increasing distance from the pole. Furthermore, these data are consistent with models of force production which suppose that the relationship between force (reflected as velocity) and distance from the pole is a linear one.
Topics: Animals; Chromosomes; Grasshoppers; Male; Mitosis; Models, Biological; Motion Pictures; Spermatocytes
PubMed: 743900
DOI: 10.1007/BF00329921 -
IEEE Transactions on Information... Jul 2009Karyotype analysis is a widespread procedure in cytogenetics to assess the possible presence of genetics defects. The procedure is lengthy and repetitive, so that an...
Karyotype analysis is a widespread procedure in cytogenetics to assess the possible presence of genetics defects. The procedure is lengthy and repetitive, so that an automatic analysis would greatly help the cytogeneticist routine work. Still, automatic segmentation and full disentangling of chromosomes are open issues. We propose an automatic procedure to obtain the separated chromosomes, which are then ready for a subsequent classification step. The segmentation is carried out by means of a space-variant thresholding scheme, which proved to be successful even in presence of hyper- or hypofluorescent regions in the image. Then, the tree of choices to resolve touching and overlapping chromosomes is recursively explored, choosing the best combination of cuts and overlaps based on geometric evidence and image information. We show the effectiveness of the proposed method on routine data acquired with different microscope-camera setup at different laboratories: from 162 images of 117 cells totaling 6683 chromosomes, 94% of the chromosomes were correctly segmented, solving 90% of the overlaps and 90% of the touchings. In order to provide the scientific community with a public dataset, the data used in this paper are available for public download.
Topics: Algorithms; Chromosomes, Human; Cluster Analysis; Humans; Image Processing, Computer-Assisted; Karyotyping; Prometaphase
PubMed: 19193514
DOI: 10.1109/TITB.2009.2014464