scholarly journals A comparison of the distribution of actin and tubulin in the mammalian mitotic spindle as seen by indirect immunofluorescence.

1977 ◽  
Vol 72 (3) ◽  
pp. 552-567 ◽  
Author(s):  
W Z Cande ◽  
E Lazarides ◽  
J R McIntosh
Keyword(s):  
Indirect Immunofluorescence ◽  
Mitotic Spindle ◽  
Metaphase Plate ◽  
Spindle Pole ◽  
Early Prophase ◽  
Chromosome Movement ◽  
Late Prophase ◽  
Small Ball ◽  
Nuclear Envelope Breakdown ◽  
Daughter Cells

Rabbit antibodies against actin and tubulin were used in an indirect immunofluorescence study of the structure of the mitotic spindle of PtK1 cells after lysis under conditions that preserve anaphase chromosome movement. During early prophase there is no antiactin staining associated with the mitotic centers, but by late prophase, as the spindle is beginning to form, a small ball of actin antigenicity is found beside the nucleus; After nuclear envelope breakdown, the actiactin stains the region around each mitotic center, and becomes organized into fibers that run between the chromosomes and the poles. Colchicine blocks this organization, but does not disrupt the staining at the poles. At metaphase the antiactin reveals a halo of ill-defined radius around each spindle pole and fibers that run from the poles to the metaphase plate. Antitubulin shows astral rays, fibers running from chromosomes to poles, and some fibers that run across the metaphase plate. At anaphase, there is a shortening of the antiactin-stained fibers, leaving a zone which is essentially free of actin-staining fluorescence between the separating chromosomes. Antitubulin stains the region between chromosomes and poles, but also reveals substantial fibers running through the zone between separating chromosomes. Cells fixed during cytokinesis show actin in the region of the cleavage furrow, while antitubulin reveals the fibrous spindle remnant that runs between daughter cells. These results suggest that actin is a component of the mammalian mitotic spindle, that the distribution of actin differs from that of tubulin and that the distributions of these two fibrous proteins change in different ways during anaphase.

scholarly journals GTSE1 regulates spindle microtubule dynamics to control Aurora B kinase and Kif4A chromokinesin on chromosome arms

2017 ◽  
Vol 216 (10) ◽  
pp. 3117-3132 ◽  
Author(s):  
Aaron R. Tipton ◽  
Jonathan D. Wren ◽  
John R. Daum ◽  
Joseph C. Siefert ◽  
Gary J. Gorbsky
Keyword(s):  
Mitotic Spindle ◽  
Meta Analysis ◽  
Metaphase Plate ◽  
Spindle Pole ◽  
Aurora B ◽  
Chromosome Movement ◽  
Aurora B Kinase ◽  
Nuclear Envelope Breakdown ◽  
M Phase ◽  
Spindle Microtubules

In mitosis, the dynamic assembly and disassembly of microtubules are critical for normal chromosome movement and segregation. Microtubule turnover varies among different mitotic spindle microtubules, dictated by their spatial distribution within the spindle. How turnover among the various classes of spindle microtubules is differentially regulated and the resulting significance of differential turnover for chromosome movement remains a mystery. As a new tactic, we used global microarray meta-analysis (GAMMA), a bioinformatic method, to identify novel regulators of mitosis, and in this study, we describe G2- and S phase–expressed protein 1 (GTSE1). GTSE1 is expressed exclusively in late G2 and M phase. From nuclear envelope breakdown until anaphase onset, GTSE1 binds preferentially to the most stable mitotic spindle microtubules and promotes their turnover. Cells depleted of GTSE1 show defects in chromosome alignment at the metaphase plate and in spindle pole integrity. These defects are coupled with an increase in the proportion of stable mitotic spindle microtubules. A consequence of this reduced microtubule turnover is diminished recruitment and activity of Aurora B kinase on chromosome arms. This decrease in Aurora B results in diminished binding of the chromokinesin Kif4A to chromosome arms.

Unorthodox male meiosis in Trichosia pubescens (Sciaridae). Chromosome elimination involves polar organelle degeneration and monocentric spindles in first and second division

1994 ◽  
Vol 107 (1) ◽  
pp. 299-312 ◽  
Author(s):  
H. Fuge
Keyword(s):  
Meiotic Division ◽  
Metaphase Plate ◽  
Chromosome Elimination ◽  
Spindle Pole ◽  
Male Meiosis ◽  
Early Prophase ◽  
Late Prophase ◽  
Section Electron ◽  
The One ◽  
Polar Organelle

Male meiosis in Trichosia pubescens (Sciaridae) was investigated by means of serial section electron microscopy and immunofluorescence light microscopy. From earlier studies of another sciarid fly, Sciara coprophila (Phillips (1967) J. Cell. Biol. 33, 73–92), it is known that the spindle poles in sciarid spermatogonia are characterized by pairs of ‘giant centrioles’, ring-shaped organelles composed of large numbers of singlet microtubules. In the present study spermatocytes in early prophase of Trichosia were found to possess single giant centrioles at opposite sides of the nucleus. The obvious reduction in centriole number from the spermatogonial to the spermatocyte stage is suggested to be the result of a suppression of daughter centriole formation. In late prophase, a large aster is developed around the centriole at one pole. At the opposite pole no comparable aster is formed. Instead, a number of irregular centriolar components appear in this region, a process that is understood to be a degeneration of the polar organelle. The components of the degenerate pole migrate into a cytoplasmic protrusion (‘bud’), which later is also utilized for the elimination of paternal chromosomes. The existence of only one functional polar centre is the reason for the formation of a monopolar monocentric spindle in first meiotic division, which in turn is one of the prerequisites for the elimination of paternal chromosomes. While the set of maternal and L chromosomes orientates and probably moves towards the pole, paternal chromosomes seem to be unable to contact the pole, possibly due to an inactivation of their kinetochores. Retrograde (‘away from the pole’) chromosome motion not involving kinetochores is assumed. Eventually, paternal chromosomes move into the pole-distal bud and are eliminated by casting off, together with the components of the degenerate polar organelle. Chromosome elimination can be delayed until the second meiotic division. The spindle of the second meiotic division is bipolar and monocentric. One spindle pole is marked by the polar centre of first division. The opposite spindle apex is devoid of a polar centre. It is assumed that spindle bipolarity in the second division is induced by the amphi-orientated chromosomes themselves. The maternal and L chromosome set (except the non-disjunctional X chromosome, which is found near the polar centre) congress in a metaphase plate, divide and segregate. Of the two daughter nuclei resulting from the second meiotic division, the one containing the X chromatids is retained as the nucleus of the future spermatozoon. The other nucleus becomes again eliminated within a second cytoplasmic bud.

Probing The Mechanisms Underlying Kinetochore Behavior In Vertebate Cells Using Combinations of Advanced Light and 3-D Electron Microscopy

1997 ◽  
Vol 3 (S2) ◽  
pp. 217-218
Author(s):  
B. F. McEwen ◽  
A.B. Heagle ◽  
C.L. Rieder
Keyword(s):  
Electron Microscopy ◽  
Mitotic Spindle ◽  
Attachment Site ◽  
Spindle Pole ◽  
Nuclear Envelope Breakdown ◽  
Daughter Cells ◽  
Primary Constriction ◽  
Sister Kinetochore ◽  
To Receive ◽  
Chromosome Motion

For daughter cells to receive equal copies of the genome during mitosis, the replicated chromosomes must attach to and move bi-directionally on the mitotic spindle. A chromosome becomes attached to the spindle via a pair specialized structures, known as kinetochores, that are positioned on opposite sides of its primary constriction (one on each of the two chromatids). In addition to being the spindle attachment site, kinetochores are also involved in producing and/or transmitting the forces for chromosome motion. In vertebrates the kinetochore closest to a spindle pole at the time of nuclear envelope breakdown usually is the first to attach to the spindle. As a result of this attachment the now “monooriented” chromosome moves toward the closest pole where its only attached kinetochore initiates oscillatory motions toward and away from that pole until the unattached sister kinetochore acquires microtubules (Mts) from the opposite spindle pole.

Nuclear envelope breakdown in mammalian cells involves stepwise lamina disassembly and microtubule-drive deformation of the nuclear membrane

1997 ◽  
Vol 110 (17) ◽  
pp. 2129-2140 ◽  
Author(s):  
S.D. Georgatos ◽  
A. Pyrpasopoulou ◽  
P.A. Theodoropoulos
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Nuclear Lamina ◽  
Early Prophase ◽  
Nuclear Surface ◽  
Late Prophase ◽  
Lamin B ◽  
Nuclear Envelope Breakdown

We have studied nuclear envelope disassembly in mammalian cells by morphological methods. The first signs of nuclear lamina depolymerization become evident in early prophase as A-type lamins start dissociating from the nuclear lamina and diffuse into the nucleoplasm. While B-type lamins are still associated with the inner nuclear membrane, two symmetrical indentations develop on antidiametric sites of the nuclear envelope. These indentations accommodate the sister centrosomes and associated astral microtubules. At mid- to late prophase, elongating microtubules apparently push on the nuclear surface and eventually penetrate the nucleus. At this point the nuclear envelope becomes freely permeable to large ligands, as indicated by experiments with digitonin-treated cells and by the massive release of solubilized A-type lamins into the cytoplasm. At the prophase/prometaphase transition, the B-type lamina is fragmented, but ‘islands’ of lamin B polymer can still be discerned on the tips of congressing chromosomes. Finally, at metaphase, the lamin B polymer breaks down into small pieces, which tend to concentrate in the area of the mitotic spindle. Nuclear envelope breakdown is not prevented when the microtubules are depolymerized by nocodazole; however, the mode of nuclear lamina fragmentation in the absence of microtubules is markedly different from the normal one and involves multiple raffles and gaps, which develop rapidly along the entire surface of the nuclear envelope. These data suggest that nuclear envelope disassembly is a stepwise process in which the microtubules play an important part.

Identification of a 102 kDa protein (cytocentrin) immunologically related to keratin 19, which is a cytoplasmically derived component of the mitotic spindle pole

1993 ◽  
Vol 106 (3) ◽  
pp. 967-981 ◽  
Author(s):  
E.C. Paul ◽  
A. Quaroni
Keyword(s):  
Cellular Localization ◽  
Spindle Pole ◽  
Early Prophase ◽  
Mitotic Apparatus ◽  
Nuclear Envelope Breakdown ◽  
Keratin 19 ◽  
Pericentriolar Material ◽  
Late Telophase ◽  
Cytoplasmic Microtubules ◽  
Cycle Dependent

The mAb RK7, previously shown to recognize keratin 19, was also found to cross-react with a biologically unrelated 102 kDa protein, which becomes associated with the poles of the mitotic apparatus. This newly identified protein, called cytocentrin, is a stable cellular component, may be at least in part phosphorylated, and displays a cell cycle-dependent cellular localization. In interphase cells, it is diffusely distributed in the cytosol and shows no affinity for cytoplasmic microtubules. It becomes localized to the centrosome in early prophase, prior to nuclear envelope breakdown, separation of replicated centrosomes, and nucleation of mitotic apparatus microtubules. During metaphase, cytocentrin is located predominately at the mitotic poles, often appearing as an aggregate of small globular sub-components; it also associates with some polar microtubules. In late anaphase/early telophase cytocentrin dissociates entirely from the mitotic apparatus and becomes temporarily localized with microtubules in the midbody, from which it disappears by late telophase. In taxol-treated cells cytocentrin was associated with the center of the miniasters but also showed affinity for some cytoplasmic microtubules. Studies employing G2-synchronized cells and nocodazole demonstrated that cytocentrin can become associated with mitotic centrosomes independently of tubulin polymerization and that microtubules regrow from antigen-containing foci. We interpret these results to suggest that cytocentrin is a cytoplasmic protein that becomes specifically activated or modified at the onset of mitosis so that it can affiliate with the mitotic poles where it may provide a link between the pericentriolar material and other components of the mitotic apparatus.

Nucleolus behaviour during the cell cycle of a primitive dinoflagellate eukaryote, Prorocentrum micans Ehr., seen by light microscopy and electron microscopy

1992 ◽  
Vol 102 (3) ◽  
pp. 475-485 ◽  
Author(s):  
MARIE-ODILE SOYER-GOBILLARD ◽  
MARIE-LINE GERAUD
Keyword(s):  
Cell Cycle ◽  
Light Microscopy ◽  
Early Prophase ◽  
Microscopy Observation ◽  
Prorocentrum Micans ◽  
Late Prophase ◽  
Daughter Cells ◽  
Nucleolar Material ◽  
Freeze Fixation ◽  
Higher Eukaryotes

Light-microscopy observation of the dinoflagellate Prorocentrum micans after silver-staining of the argyrophilic proteins of the nucleolar organizing region (Ag-NOR staining) showed the presence of nucleolar material throughout the vegetative cell cycle, and in particular during all the mitotic stages. This contrasts with the case in most higher eukaryotes, in which nucleoli disappear at the end of prophase and are reconstituted in daughter cells during telophase. Electron-microscope (EM) observations after conventional or fast-freeze fixation revealed that during interphase several functional nucleoli with three compartments (NORs, the fibrillogranular and the preribosomal granular compartments) are present in a nucleus in which the envelope is persistent and the chromosomes are always compact. During early prophase, when chromosomes are beginning to split, the nucleoli remain functional, whereas in late prophase they contain only a NOR and the granular component, and the chromosomes are surrounded by many protein masses. In early telophase, the nucleolar material coating the chromosomes migrates along with the chromosomes. Nucleologenesis occurs through the formation of prenucleolar bodies around lateral or telomeric nucleofilaments extruding from the chromosomes. Several chromosomes can contribute to the formation of one nucleolus. The behaviour of these ‘persistent nucleoli’ in a closed-nucleus model such as that of the dinoflagellates is discussed with regard to the higher eukaryotes.

scholarly journals Human Cyclin a Is Required for Mitosis until Mid Prophase

1999 ◽  
Vol 147 (2) ◽  
pp. 295-306 ◽  
Author(s):  
Nobuaki Furuno ◽  
Nicole den Elzen ◽  
Jonathon Pines
Keyword(s):  
Cyclin A ◽  
Time Lapse ◽  
S Phase ◽  
Early Prophase ◽  
Cyclin Dependent Kinase ◽  
Late Prophase ◽  
Daughter Cells ◽  
G2 Phase ◽  
Rate Limiting

We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21Cip1/Waf1/Sdi1 (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.

scholarly journals Entry into Mitosis in Vertebrate Somatic Cells Is Guarded by a Chromosome Damage Checkpoint That Reverses the Cell Cycle When Triggered during Early but Not Late Prophase

1998 ◽  
Vol 142 (4) ◽  
pp. 1013-1022 ◽  
Author(s):  
Conly L. Rieder ◽  
Richard W. Cole
Keyword(s):  
Cell Cycle ◽  
Somatic Cells ◽  
Early Prophase ◽  
Checkpoint Control ◽  
Late Prophase ◽  
Cytoplasmic Microtubule ◽  
Nuclear Envelope Breakdown ◽  
Prophase Nucleus ◽  
Nuclear Events ◽  
A Cell

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G2 checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.

scholarly journals Chromosome misalignment is associated with PLK1 activity at cenexin-positive mitotic centrosomes

2019 ◽  
Vol 30 (13) ◽  
pp. 1598-1609 ◽  
Author(s):  
Erica G. Colicino ◽  
Katrina Stevens ◽  
Erin Curtis ◽  
Lindsay Rathbun ◽  
Michael Bates ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole ◽  
Dependent Manner ◽  
Binding Partner ◽  
Mitotic Kinase ◽  
Daughter Cells ◽  
Chromosome Distribution ◽  
Spindle Poles ◽  
Mother Centriole

The mitotic kinase, polo-like kinase 1 (PLK1), facilitates the assembly of the two mitotic spindle poles, which are required for the formation of the microtubule-based spindle that ensures appropriate chromosome distribution into the two forming daughter cells. Spindle poles are asymmetric in composition. One spindle pole contains the oldest mitotic centriole, the mother centriole, where the majority of cenexin, the mother centriole appendage protein and PLK1 binding partner, resides. We hypothesized that PLK1 activity is greater at the cenexin-positive older spindle pole. Our studies found that PLK1 asymmetrically localizes between spindle poles under conditions of chromosome misalignment, and chromosomes tend to misalign toward the oldest spindle pole in a cenexin- and PLK1-dependent manner. During chromosome misalignment, PLK1 activity is increased specifically at the oldest spindle pole, and this increase in activity is lost in cenexin-depleted cells. We propose a model where PLK1 activity elevates in response to misaligned chromosomes at the oldest spindle pole during metaphase.

Changes in the metaphase transit times and the pattern of sister chromatid separation in stamen hair cells of Tradescantia after treatment with protein phosphatase inhibitors

1992 ◽  
Vol 102 (4) ◽  
pp. 691-715 ◽  
Author(s):  
S.M. Wolniak ◽  
P.M. Larsen
Keyword(s):  
Nuclear Envelope ◽  
Transit Time ◽  
Okadaic Acid ◽  
Hair Cells ◽  
Protein Phosphatase ◽  
Metaphase Plate ◽  
Spindle Pole ◽  
Phosphatase Inhibitors ◽  
Nuclear Envelope Breakdown ◽  
Transit Times

Stamen hair cells from the spiderwort plant, Tradescantia virginiana, exhibit remarkably predictable metaphase transit times, making them uniquely suitable for temporal studies on mitotic regulation. In this study, we describe two kinds of experiments that test whether protein phosphatase activity is a necessary prerequisite for entry into anaphase in living, mitotic cells. We treated cells at specific points during prophase, prometaphase and metaphase with the broad-spectrum protein phosphatase inhibitor, alpha-naphthyl phosphate (administered by microinjection), or with the naturally occurring, potent phosphatase inhibitors okadaic acid, microcystin-LR or microcystin-RR (administered by perfusion), and we have observed changes in the metaphase transit time that are primarily dependent on the time of initial exposure to the inhibitor. Maximal extensions of the metaphase transit time result from alpha-naphthyl phosphate microinjections initiated in mid-metaphase, 10–20 min after nuclear envelope breakdown. Perfusions with okadaic acid started during a specific interval in mid-metaphase, 15–20 min after nuclear envelope breakdown, resulted in a statistically significant extension of the metaphase transit time. Perfusions with either microcystin-LR or microcystin-RR initiated 15–26 min after nuclear envelope breakdown extended the metaphase transit times significantly. Treatments of cells with okadaic acid or with either of the microcystins initiated outside this mid-metaphase interval either were without effect or, alternatively, resulted in a significant shortening of the metaphase transit time. In addition to their effects on the timing of anaphase onset, treatments with these protein phosphatase inhibitors also resulted in a remarkable change in the way in which these cells enter anaphase. Sister chromatid separation in stamen hair cells typically requires only 5 seconds, but after treatment with any of these inhibitors some, but not all, of the chromatids split apart at anaphase onset. Those that split begin to migrate toward the spindle pole regions, while those that fail to split remain at the metaphase plate. Later, more of the paired chromatids split apart and begin moving toward the spindle pole regions. Those that fail to separate remain at the metaphase plate. This process can be repeated several times before all of the chromatids have separated. Thus, entry into anaphase becomes extremely asynchronous, and as much as 30 min can transpire between the centromeric separation of the first and last chromosomes. Some of the chromosomes complete their anaphase movements before others have even split apart at the metaphase plate. Asynchronous separation did not result in a permanent segregation anomaly.(ABSTRACT TRUNCATED AT 400 WORDS)

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