scholarly journals Chromosome Fragments Possessing Only One Kinetochore Can Congress to the Spindle Equator

1997 ◽  
Vol 136 (2) ◽  
pp. 229-240 ◽  
Author(s):  
Alexey Khodjakov ◽  
Richard W. Cole ◽  
Bruce F. McEwen ◽  
Karolyn F. Buttle ◽  
Conly L. Rieder
Keyword(s):  
Three Dimensional ◽  
Spindle Pole ◽  
The Other ◽  
Dimensional Electron ◽  
Laser Microsurgery ◽  
Electron Microscopic ◽  
Spindle Poles ◽  
Chromosome Congression ◽  
Chromosome Fragments ◽  
Multiple Domains

We used laser microsurgery to cut between the two sister kinetochores on bioriented prometaphase chromosomes to produce two chromosome fragments containing one kinetochore (CF1K). Each of these CF1Ks then always moved toward the spindle pole to which their kinetochores were attached before initiating the poleward and away-from-the-pole oscillatory motions characteristic of monooriented chromosomes. CF1Ks then either: (a) remained closely associated with this pole until anaphase (50%), (b) moved (i.e., congressed) to the spindle equator (38%), where they usually (13/19 cells) remained stably positioned throughout the ensuing anaphase, or (c) reoriented and moved to the other pole (12%). Behavior of congressing CF1Ks was indistinguishable from that of congressing chromosomes containing two sister kinetochores. Three-dimensional electron microscopic tomographic reconstructions of CF1Ks stably positioned on the spindle equator during anaphase revealed that the single kinetochore was highly stretched and/or fragmented and that numerous microtubules derived from the opposing spindle poles terminated in its structure. These observations reveal that a single kinetochore is capable of simultaneously supporting the function of two sister kinetochores during chromosome congression and imply that vertebrate kinetochores consist of multiple domains whose motility states can be regulated independently.

scholarly journals Sld5 Ensures Centrosomal Resistance to Congression Forces by Preserving Centriolar Satellites

2017 ◽  
Vol 38 (2) ◽  
Author(s):  
Manpreet Kaur ◽  
Raksha Devi ◽  
Tanushree Ghosh ◽  
Md Muntaz Khan ◽  
Praveen Kumar ◽  
...  
Keyword(s):  
Dna Replication ◽  
Unknown Function ◽  
Motor Protein ◽  
Spindle Pole ◽  
Replication Protein ◽  
Traction Forces ◽  
Centrosomal Protein ◽  
Spindle Poles ◽  
Chromosome Congression ◽  
Pulling Forces

ABSTRACT The migration of chromosomes during mitosis is mediated primarily by kinesins that bind to the chromosomes and move along the microtubules, exerting pulling and pushing forces on the centrosomes. We report that a DNA replication protein, Sld5, localizes to the centrosomes, resisting the microtubular pulling forces experienced during chromosome congression. In the absence of Sld5, centriolar satellites, which normally cluster around the centrosomes, are dissipated throughout the cytoplasm, resulting in the loss of their known function of recruiting the centrosomal protein, pericentrin. We observed that Sld5-deficient centrosomes lacking pericentrin were unable to endure the CENP-E- and Kid-mediated microtubular forces that converge on the centrosomes during chromosome congression, resulting in monocentriolar and acentriolar spindle poles. The minus-end-directed kinesin-14 motor protein, HSET, sustains the traction forces that mediate centrosomal fragmentation in Sld5-depleted cells. Thus, we report that a DNA replication protein has an as yet unknown function of ensuring spindle pole resistance to traction forces exerted during chromosome congression.

scholarly journals The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles

2001 ◽  
Vol 154 (6) ◽  
pp. 1135-1146 ◽  
Author(s):  
Aime A. Levesque ◽  
Duane A. Compton
Keyword(s):  
Cultured Cells ◽  
Time Lapse ◽  
Spindle Pole ◽  
Mitotic Spindles ◽  
Differential Interference Contrast Microscopy ◽  
Spindle Poles ◽  
Ejection Force ◽  
Chromosome Congression ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy

Chromokinesins have been postulated to provide the polar ejection force needed for chromosome congression during mitosis. We have evaluated that possibility by monitoring chromosome movement in vertebrate-cultured cells using time-lapse differential interference contrast microscopy after microinjection with antibodies specific for the chromokinesin Kid. 17.5% of cells injected with Kid-specific antibodies have one or more chromosomes that remain closely opposed to a spindle pole and fail to enter anaphase. In contrast, 82.5% of injected cells align chromosomes in metaphase, progress to anaphase, and display chromosome velocities not significantly different from control cells. However, injected cells lack chromosome oscillations, and chromosome orientation is atypical because chromosome arms extend toward spindle poles during both congression and metaphase. Furthermore, chromosomes cluster into a mass and fail to oscillate when Kid is perturbed in cells containing monopolar spindles. These data indicate that Kid generates the polar ejection force that pushes chromosome arms away from spindle poles in vertebrate-cultured cells. This force increases the efficiency with which chromosomes make bipolar spindle attachments and regulates kinetochore activities necessary for chromosome oscillation, but is not essential for chromosome congression.

A Relatively Inexpensive Microprocessor-Linked Digital Plamimeter for Electron Microscopic Morphometry

1981 ◽  
Vol 39 ◽  
pp. 266-269
Author(s):  
Lee D. Peachey
Keyword(s):  
Theoretical Basis ◽  
Structural Parameters ◽  
Three Dimensional ◽  
Structural Data ◽  
Large Set ◽  
Dimensional Electron ◽  
Electron Microscopic ◽  
Morphometric Methods ◽  
Electron Microscopic Morphometry ◽  
Separate Step

Stereology provides a theoretical basis for powerful morphometric methods for the estimation of three-dimensional structural parameters from two-dimensional electron micrographs of cells and tissues. These methods assume at the start that one has a sufficiently large set of micrographs containing valid structural data. The task of obtaining from these micrographs the large quantity of data needed to get statistically valid results has been eased in two general ways. Sampling of data in the micrograph can be done rapidly by point and intersection counting methods. An alternate method, planimetry, obtains all the data in the micrograph, but in general is more time-consuming than point and intersection counting. Some of the relative inefficiency of planimetry is compensated when a digital planimeter is coupled with a computer. Areas and lengths can be computed simultaneously as fast as profiles are traced. Furthermore, rapid and numerically accurate compilation and statistical analysis of the data can be done automatically as the planimetry is done, not as a separate step after the data have been obtained.

Somatic nuclear division in the sporidia of Ustilago violacea. III. Ultrastructural observations

1976 ◽  
Vol 22 (4) ◽  
pp. 495-506 ◽  
Author(s):  
N. H. Poon ◽  
A. W. Day
Keyword(s):  
Daughter Cell ◽  
Daughter Nucleus ◽  
Nuclear Division ◽  
Spindle Pole ◽  
The Other ◽  
Parent Cell ◽  
Electron Microscopic ◽  
Ustilago Violacea ◽  
Left Behind ◽  
Spindle Pole Bodies

The paper provides detailed ultrastructural observations on nuclear division in the smut fungus Ustilago violacea and is based on previous light-microscopic work outlining the division in living and stained cells. The division as in many other Basidiomycetes is not intranuclear, but occurs within a partially disrupted membrane. The division takes place after migration of most of the nucleus into the bud cell, after limited breakdown of the nuclear membrane, and after the formation of a spindle between two spindle-pole bodies (SPB). The remaining part of the nucleus containing the nucleolus is left behind in the parent cell and degenerates there. The SPB, as in other Basidiomycetes, is a dome-shaped relatively structureless body, quite distinct from the flat plaques of many Ascomycetes and the elaborate centrioles of Phycomycetes. The SPB divides shortly before migration into the daughter cell and invariably is located at the apex of the migrating nucleus. Nuclear division is completed when the two masses of chromatin clustered about each of the SPB's are separated as the spindle elongates. One daughter nucleus reforms in the bud and the other is reformed in the mother cell.Cells fixed and stained by conventional light-microscopic methods were examined in the light of the electron-microscopic observations to determine whether these procedures induce artefacts. Aceto-orcein and Giemsa when used cold were found to produce relatively artefact-free preparations. However, previous results in which the cells were warmed gently in these stains are now seen to contain artefacts in the form of contracted chromatinic granules often arranged in chains. These artefacts may provide useful information but clearly they must be interpreted cautiously until the nature of the changes induced by heating are known.

scholarly journals A Mutation in γ-Tubulin Alters Microtubule Dynamics and Organization and Is Synthetically Lethal with the Kinesin-like Protein Pkl1p

2000 ◽  
Vol 11 (4) ◽  
pp. 1225-1239 ◽  
Author(s):  
Janet L. Paluh ◽  
Eva Nogales ◽  
Berl R. Oakley ◽  
Kent McDonald ◽  
Alison L. Pidoux ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Three Dimensional ◽  
Time Lapse ◽  
Spindle Pole ◽  
Microtubule Dynamics ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Cytoplasmic Microtubule ◽  
Spindle Poles ◽  
Tubulin Protein

Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. γ-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in γ-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30°C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant γ-tubulin is like the wild-type protein. Prediction of γ-tubulin structure indicates that non-α/β-tubulin protein–protein interactions could be affected. The kinesin-like protein (klp)Pkl1p localizes to the spindle poles and spindle and is essential for viability of the γ-tubulin mutant and in multicopy for normal cell morphology at 30°C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for γ-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of γ-tubulin that involves non-tubulin protein–protein interactions, presumably with a second motor, MAP, or MTOC component.

Anti-tubulin antibodies locate the blepharoplast during spermatogenesis in the fern Platyzoma microphyllum R.Br.: a correlated immunofluorescence and electron-microscopic study

1986 ◽  
Vol 81 (1) ◽  
pp. 243-265
Author(s):  
J.H. Doonan ◽  
C.W. Lloyd ◽  
J.G. Duckett
Keyword(s):  
Basal Body ◽  
De Novo ◽  
Spindle Pole ◽  
Electron Microscopic ◽  
Mitotic Apparatus ◽  
Strong Argument ◽  
Body Formation ◽  
Spindle Poles ◽  
Tubulin Antibodies ◽  
Cytoplasmic Microtubules

The discovery that the monoclonal anti-tubulin antibody YOL 1/34 recognizes a microtubule organizing centre, the blepharoplast (which arises de novo during the latter stages of spermatogenesis in the fern, Platyzoma microphyllum), has enabled us to follow it and associated microtubules throughout most of its ontogeny. By correlating electron-microscopic and immunofluorescence observations, YOL 1/34 is seen to stain the blepharoplast uniformly at a time when no microtubules are present within the organelle. Later, staining becomes intense at the surface, concomitant with the re-location of cylindrical channels to the periphery of the blepharoplast. During anaphase of the ultimate division of the spermatid mother cell the blepharoplast moves to the spindle poles and sharpens the otherwise barrel-shaped mitotic apparatus. Prior to this stage the blepharoplast is, however, off-centre and at variable positions around the poles. Later still, in the differentiating spermatids, the blepharoplast is the focus for radiating cytoplasmic microtubules that abut directly onto the electron-dense organelle, penetrating the ribosome-free halo. The three main conclusions are: that tubulin in a pre-microtubular form is associated with the cylindrical channels that arise de novo within the previously amorphous blepharoplast and act as a template in basal body formation; that the late appearance of the blepharoplast as a focus for the spindle poles during the final mitosis provides strong argument against its functioning during spindle pole initiation (despite its ability to sharpen the poles at anaphase); that the blepharoplast does seem to act as a microtubule organizing centre in the mitotically quiescent spermatid.

scholarly journals Kinetochore Fiber Maturation in PtK1 Cells and Its Implications for the Mechanisms of Chromosome Congression and Anaphase Onset

1997 ◽  
Vol 137 (7) ◽  
pp. 1567-1580 ◽  
Author(s):  
Bruce F. McEwen ◽  
Amy B. Heagle ◽  
Grisel O. Cassels ◽  
Karolyn F. Buttle ◽  
Conly L. Rieder
Keyword(s):  
Time Course ◽  
Spindle Pole ◽  
Metaphase Chromosomes ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Chromosome Congression ◽  
Early Anaphase ◽  
Full Complement ◽  
Spindle Microtubules ◽  
Chromosome Motion

Kinetochore microtubules (kMts) are a subset of spindle microtubules that bind directly to the kinetochore to form the kinetochore fiber (K-fiber). The K-fiber in turn interacts with the kinetochore to produce chromosome motion toward the attached spindle pole. We have examined K-fiber maturation in PtK1 cells using same-cell video light microscopy/serial section EM. During congression, the kinetochore moving away from its spindle pole (i.e., the trailing kinetochore) and its leading, poleward moving sister both have variable numbers of kMts, but the trailing kinetochore always has at least twice as many kMts as the leading kinetochore. A comparison of Mt numbers on sister kinetochores of congressing chromosomes with their direction of motion, as well as distance from their associated spindle poles, reveals that the direction of motion is not determined by kMt number or total kMt length. The same result was observed for oscillating metaphase chromosomes. These data demonstrate that the tendency of a kinetochore to move poleward is not positively correlated with the kMt number. At late prometaphase, the average number of Mts on fully congressed kinetochores is 19.7 ± 6.7 (n = 94), at late metaphase 24.3 ± 4.9 (n = 62), and at early anaphase 27.8 ± 6.3 (n = 65). Differences between these distributions are statistically significant. The increased kMt number during early anaphase, relative to late metaphase, reflects the increased kMt stability at anaphase onset. Treatment of late metaphase cells with 1 μM taxol inhibits anaphase onset, but produces the same kMt distribution as in early anaphase: 28.7 ± 7.4 (n = 54). Thus, a full complement of kMts is not sufficient to induce anaphase onset. We also measured the time course for kMt acquisition and determined an initial rate of 1.9 kMts/min. This rate accelerates up to 10-fold during the course of K-fiber maturation, suggesting an increased concentration of Mt plus ends in the vicinity of the kinetochore at late metaphase and/or cooperativity for kMt acquisition.

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