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  • 1
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Anaphase lagging of autosomes was observed in 6.1±5.4% of the primary spermatocytes in untreated larvae of the crane fly, Nephrotoma suturalis. Lagging was induced by exposure of larvae to 6 ° C and during recovery at 22 ° C from exposure to 0.2, 2, and 6 ° C. The incidence of anaphase lag was maximal at 80 to 90 min of recovery. Induced lagging was observed at that recovery time after exposures of only 2.5 h to 2 or 0.2 ° C, but its incidence increased with longer exposures. As many as 85% of the cells in anaphase contained autosomal laggards after 61 h at 2 ° C and 80 to 90 min of recovery. At 2 ° C, cells reached the prophase-prometaphase transition, but spindles did not appear to form. Those cells proceeded through prometaphase during recovery, reaching mid-anaphase after 80 to 90 min of recovery. Chromosomes that lagged at anaphase during recovery from 2 ° C were observed in living cells to be half-bivalents derived from bivalents that congressed to the metaphase plate. One or both half-bivalents of any bivalent could lag. In some cells, one half-spindle had more half-bivalents than the other. Cells with autosomal laggards often did not cleave, and in uncleaved cells the second division employed spindles having two, three, or four poles. The basis of induced lagging might be a lapse in spindle attachment or motive force application at the start of anaphase or a failure of chromosomes to achieve proper orientation before the onset of anaphase.
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  • 2
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Mitotic micronuclei were isolated from Tetrahymena thermophila in a medium containing hexylene glycol and their ultrastructure was analyzed using thin section techniques. The two stages selected for analysis were early prometaphase and metaphase. A comparison of data from these two stages revealed several differences in nuclear morphology. Metaphase nuclei were longer, they contained more microtubules, and the distribution of microtubules at metaphase was different from that at early prometaphase. Increases in microtubule number and length were clearly evident in peripheral sheath microtubules, which are a unique class of microtubules that can be distinguished from other classes on the basis of their close association to the nuclear membrane. Growth of peripheral sheath microtubules is thought to be significant because it could be the mechanical basis of nuclear elongation. Crossbridges were observed throughout the spindle between all classes of microtubules, but the exact function of these elements remains to be determined.
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  • 3
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Chromosome segregation in primary spermatocytes of the crane fly Nephrotoma suturalis was studied after exposure to Colcemid at doses that did not completely inhibit spindle formation. Colcemid was added either to the medium in which larvae were cultured or to Tricine buffer in which isolated testes were incubated. Patterns of chromosome segregation were analyzed in fixed, Feulgen-stained smears of testes from Colcemid-treated larvae and in living cell preparations. Anomalies observed during the first meiotic division at higher than normal frequencies in Colcemid-treated spermatocytes included anaphase lagging of autosomes, chromosomal strands, tripolar and tetrapolar divisions, and unequal distribution of chromosomes to secondary cells. Following those doses of Colcemid that induced the above anomalies, the length of the birefringent spindle in primary spermatocytes was shorter than normal. This effect on spindle length also was apparent in Giemsastained preparations of fixed cells, in which the two centrosomes at the spindle poles were differentiated from the rest of the cytoplasm. The results indicate a correlation between the inhibition of spindle formation and the induction of anomalous patterns of chromosome segregation.
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  • 4
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Addition of Colcemid to the medium in which larvae of the crane fly Nephrotoma suturalis are cultivated induces a number of anomalous patterns of chromosome segregation. One of these is the anaphase lagging of autosomal half-bivalents. To investigate the cause of anaphase lagging, the orientation of sister kinetochores in Colcemidtreated spermatocytes having lagging half-bivalents was analyzed in serial sections. In contrast to nonlaggard halfbivalents that had pure syntelic orientation (sister kinetochores having all of their kinetochores microtubules (KMTs) extending to the same pole), six of the seven autosomal laggards that were selected for analysis had kinetochores with either amphitelic orientation (sister kinetochores each with a bundle of KMTs extending to opposite poles) or merotelic orientation (a single kinetochore having KMTs extending toward both poles). An additional laggard had syntelic orientation but two of the microtubules that were in its kinetochore fiber passed through the kinetochore and extended beyond it toward the equator. The bipolar malorientations observed in anaphase half-bivalents are interpreted to be a cause of the anaphase lagging induced by Colcemid treatment. Furthermore, it is hypothesized that such bipolar malorientations also may be stabilized at metaphase and thus explain the unusual tilting of metaphase bivalents commonly observed in Colcemid-treated cells.
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  • 5
    ISSN: 0886-1544
    Keywords: chromosome movement ; meiosis ; spermatocytes ; prophase ; nuclear envelope ; aster ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Association of bivalent chromosomes with the astral centers and nuclear envelope was analyzed in crane-fly spermatocytes during the final hours of diakinesis. In contrast to other systems in which movement of chromosomes during diakinesis correlates with the clustering of bivalents near the astral centers, such clustering is not prevalent in crane-fly spermatocytes. Polarization indices of bivalents calculated 5 to 10 minutes before the end of diakinesis provided evidence for polarization of only a fraction of all bivalents. Similar results were obtained in a large number of fixed cells in which asters and chromosomes were preferentially stained. Ultrastructural analysis of cells in late diakinesis revealed significant contact between bivalents and the nuclear envelope in all 46 cells that were analyzed. The extent of contact in some cells was greater than in others. Sites of contact included the telomeric ends of bivalents, and in some cases the distribution of contact sites suggested the possible involvement of centromeres in chromosome-nuclear envelope association. The results are consistent with the hypothesis that a dynamic interaction between chromosomes and nuclear envelope may exist during late prophase, when the movement of chromosomes is known to occur.
    Additional Material: 6 Ill.
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  • 6
    ISSN: 0886-1544
    Keywords: crane flies ; meiosis ; spermatocytes ; chromosome movement ; nuclear envelope ; prophase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Meiotic prophase in spermatocytes of the crane fly, Nephrotoma suturalis, involves both the condensation and the movement of bivalent chromosomes. Since crane flies have only four bivalents that appear highly condensed during late prophase, changes of position and orientation of those bivalents relative to one another can be seen easily in living cells. Chromosome movement during the final 1 to 2 hr of diakinesis was analyzed in detail. Maximal velocities of prophase movements were between 0.1 and 1 μM/min. Metakinetic movements during prometaphase have similar velocities. To assess the physiological basis of prophase movements, experiments employing cyanide and cold treatment were performed. Prophase movements were abolished completely by cyanide, and, for the most part, the velocities of chromosomes in the cold at 2°C and 6°C were less than that of untreated cells at 22°C. The results suggest that prophase movements are energy dependent and may involve an enzyme-catalyzed process occurring in close association with the nuclear envelope.
    Additional Material: 7 Ill.
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  • 7
    ISSN: 0886-1544
    Keywords: mitosis ; mitotic spindle ; kinetochore ; microtubule ; micronucleus ; Tetrahymena ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Mitotic micronuclei were isolated from Tetrahymena thermophila and data on spindle ultrastructure were obtained from serial, transverse sections. Comparison of data from nuclei at meta- and early anaphase with data from nuclei at late anaphase showed that during anaphase, sister kinetochores move from the equator to the spindle poles, but kinetochore translocation occurs without any apparent change in either the number or length of kinetochore microtubules. This unprecedented result is ascribed significance with regard to the mechanism of kinetochore transport since there are only a limited number of ways that result could be achieved. The organization of the peripheral sheath changes during anaphase as evidenced by gaps in the sheath at late anaphase. Numerous kinetochore and non-kinetochore microtubules are located in polar regions of the spindle at late anaphase, whereas those regions contained only peripherally arranged microtubules at earlier stages. Tracking of individual kinetochore microtubules in late anaphase nuclei showed that some of them appeared to become incorporated into the peripheral sheath near the pole. At early and late anaphase, crossbridges connect adjacent microtubules throughout the spindle poleward to the kinetochores, as well as in the interzone.
    Additional Material: 14 Ill.
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  • 8
    ISSN: 0886-1544
    Keywords: chromosome movement ; Colcemid ; nocodazole ; meiotic prophase ; microtubules ; vinblastine ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effects of Colcemid, nocodazole, and vinblastine on microtubules and on the movement of chromosomes during late diakinesis were investigated in spermatocytes of the crane fly Nephrotoma suturalis. The kinds of movements observed in untreated cells - sex bivalent rotations, sex bivalent excursions, and rotations and positional changes of autosomal bivalents - also were observed in drug-treated cells. These results were obtained in living cells in which the disruption and inhibition of microtubule assembly had been confirmed with polarized light microscopy. Effects of Colcemid and nocodazole also were assessed in fixed cells using electron microscopy. The results are in agreement with a hypothesis that microtubules are not a force-generating component of the molecular machinery that brings about prophase movements.
    Additional Material: 15 Ill.
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  • 9
    ISSN: 0886-1544
    Keywords: kinetochores ; spindle apparatus ; anaphase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We investigated the involvement of kinetochore microtubules (kMTs) in mediating chromosome-to-pole connections in crane-fly (Nephrotoma suturalis and Nephrotoma ferruginea) spermatocytes. Two experimental treatments were used to yield spindles with reduced numbers of nonkinetochore microtubules (nkMTs). Short-term (10-15 min) exposure of spermatocytes to 2°C caused depolymerization of the majority of nkMTs, resulting in a kMT:(kMT + nkMT) ratio of 0.76. Long-term (24h) exposure to 2°C followed by recovery at 6°C resulted in a kMT:(kMT + nkMT) ratio of 0.55, the spindle having more nkMTs than a 2°C-treated spindle but fewer than an untreated spindle, in which the kMT:(kMT + nkMT) ratio was 0.27. The numbers and lengths of kMTs in 6°C-grown spindles were similar to those in untreated cells, suggesting that the overall inhibition of MT assembly at 6°C apparently did not affect the mechanism by which kMTs are formed. We observed most kMTs of early anaphase spindles to be long (〉3 μm), and many extended to the polar regions of the spindle. Thus, the crane-fly spindle appears not to be as atypical as it was previously suggested to be.
    Additional Material: 5 Ill.
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  • 10
    ISSN: 0886-1544
    Keywords: chromosome orientation ; prometaphase ; meiosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: At metaphase in crane-fly primary spermatocytes, the two sister kinetochores at the centromere of each homologue in a bivalent normally are adjacent and face the same pole; one homologue has all its kinetochore microtubules (kMTs) extending toward one pole and its partner has all its kMTs extending toward the opposite pole. In contrast, during recovery from exposure to 2°C, one or both homologues in many metaphase bivalents had bipolar malorientations: all kMTs of one kinetochore extended toward one pole and some or all those of its sister extended toward the other. Metaphase sister kinetochores that had most of their kMTs extending toward the same pole were adjacent, and those with most extending toward opposite poles were separated from each other. Distances between homologous centromeres were similar to those in properly oriented bivalents. Maloriented bivalents were tilted relative to the spindle axis, and analysis of living cells showed that tilted configurations were rare during prometaphase in untreated cells but frequently arose in cold-recovering cells as initial configurations, then persisted through metaphase. This was in contrast to unipolar configurations of bivalents (configurations suggesting orientation of both homologous centromeres toward the same pole), which always reoriented shortly after the configuration arose. We conclude that in cold-recovering cells, bipolar malorientations are more stable than unipolar malorientations, and the orientation process is affected such that bipolar malorientations arise in bivalents upon initial interaction with the spindle and persist through metaphase.
    Additional Material: 6 Ill.
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