No abstract

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 degrees C caused depolymerization of the majority of nkMTs, resulting in a kMT:(kMT + nkMT) ratio of 0.76. Long-term (24h) exposure to 2 degrees C followed by recovery at 6 degrees C resulted in a kMT:(kMT + nkMT) ratio of 0.55, the spindle having more nkMTs than a 2 degrees 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 degrees C-grown spindles were similar to those in untreated cells, suggesting that the overall inhibition of MT assembly at 6 degrees C apparently did not affect the mechanism by which kMTs are formed. We observed most kMTs of early anaphase spindles to be long (greater than 3 microns), 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.

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 degrees C and during recovery at 22 degrees C from exposure to 0.2, 2, and 6 degrees 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 degrees 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 degrees C and 80 to 90 min of recovery. At 2 degrees 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 degrees 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.

Exposing crane fly larvae to 6°C or returning them to 22°C after exposure to 6, 2, or 0 .2°C can induce any number of autosomes in their primary spermatocytes to lag near the spindle equator at anaphase. Autosomal laggards in cold-recovering cells are contained in bivalents until anaphase (Janicke, M. A., and J . R. LaFountain, 1982, Chromosoma, 85 :619-631) . We report here documentation that lagging autosomes in cold-treated and coldrecovering cells are maloriented . During meiosis I, half-bivalents usually associate with only one pole via kinetochore fibers, with sister chromatids being oriented to the same pole. In contrast, laggards had kinetochore microtubules (kMTs) extending from them toward both poles : one sister was oriented to one pole and the other had some or all of its kMTs extending toward the opposite pole. Bipolar malorientation of autosomal laggards also was observed in one untreated cell. The number of kMTs per half-bivalent was similar in lagging and nonlagging autosomes, and those kMTs were contained in long birefringent kinetochore fibers. The overall spindle structure in cold-recovering cells was similar to that observed in untreated anaphase cells . Giemsa-stained centromeric dots of sister chromatids were contiguous in nonlaggards and separated in laggards at anaphase . We conclude that bipolar malorientations can exist at anaphase in chromosomes that remain paired until anaphase, that cold recovery increases the frequency of that anomaly, and that such malorientations may be one cause of anaphase lag.During the reduction division of meiosis, homologues usually pair to form bivalents . Sister chromatids of one homologue (half-bivalent) become oriented (via a kinetochore fiber) to one spindle pole, and the sister chromatids of its partner homologue both become oriented to the opposite pole. This orientation is thought to cause the bivalent to be subjected to bipolar forces that (a) result in its congression to the metaphase plate, and (b) ensure that upon its separation into halfbivalents at anaphase onset, the coupled sister chromatids of one half-bivalent move together to one pole while the partner half-bivalent moves to the opposite pole (13,29) . In reductiondivision chromosomes that do not pair or become unpaired before anaphase (univalents), sister chromatids may become oriented to opposite poles (25,29) . Univalents with such orientations may congress and then "lag" (see Materials and Methods for terminology) near the spindle equator at anaphase . Laggards may or may not eventually move poleward, and several routes exist by which lagging may lead to the THE fOURNAL OF CELL BIOLOGY " VOLUME 98 MARCH 1984 859-869 ® The Rockefeller University Press " 0021-9525/84/03/0869/11 $1 .00

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 degrees 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.