Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin, Eg5, arrests cells in mitosis with monoastral spindles. Here, we use monastrol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplication. The mitotic arrest due to monastrol is also rapidly reversible. Chromosomes in monastrol-treated cells frequently have both sister kinetochores attached to microtubules extending to the center of the monoaster (syntelic orientation). Mitotic arrest–deficient protein 2 (Mad2) localizes to a subset of kinetochores, suggesting the activation of the spindle assembly checkpoint in these cells. Mad2 localizes to some kinetochores that have attached microtubules in monastrol-treated cells, indicating that kinetochore microtubule attachment alone may not satisfy the spindle assembly checkpoint. Monastrol also inhibits bipolar spindle formation in Xenopus egg extracts. However, it does not prevent the targeting of Eg5 to the monoastral spindles that form. Imaging bipolar spindles disassembling in the presence of monastrol allowed direct observations of outward directed forces in the spindle, orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to study mitotic processes, detection and correction of chromosome malorientation, and contributions of Eg5 to spindle assembly and maintenance.

/pdf/probing-spindle-assembly-mechanisms-with-monastrol-a-small-7mau3egu9o.pdf

Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5.

The assembly, rearrangement and disassembly of the synaptonemal complex during the stages from leptotene to anaphase I of meiosis are described. The assembly of the lateral component of the synaptonemal complex between the sister chromatids of the leptotene chromosome causes the undivided appearance of the leptotene chromosome in the light microscope. During zygotene the lateral component mediates the assembly of the synaptonemal complex between homologous segments of homologous as well as homoeologous chromosomes. Chromosome and bivalent interlockings are a regular feature during chromosome pairing at zygotene. They are resolved by breakage and precise repair of the chromosomes. The phase of synaptonemal complex formation between homologous chromosome segments is succeeded by a phase of rearrangements. In this phase, chromosome homology is no longer required for synaptonemal complex assembly. This reduces the number of univalent chromosome sections. Rearrangement of chromosome pairing with the synaptonemal complex is not expected to take place at positions where a crossover has occurred. Disassembly of the synaptonemal complex at diplotene is initiated at specific regions along the bivalent arms. Chiasmata are formed from retained pieces of the synaptonemal complex in which a recombination nodule is or had been present. A limit to the number of available recombination nodules and a mechanism which ensures that at least one nodule is placed in each bivalent arm are sources for the positive interference observed in linkage studies employing three-point tests. The time of crossing over relative to the zygotene assembly and pachytene rearrangement phases of the synaptonemal complex determines if multivalents or exclusively bivalents are found at metaphase I in amphidiploids and autotetraploids. The synaptonemal complex provides both the structural basis for the regular disjunction of chromosomes at meiosis and gene recombination between homologous or homoeologous chromosome arms.

The synaptonemal complex in genetic segregation.

In Situ Localization of Parental Genomes in a Wide Hybrid

The leptotene/zygotene transition of meiosis, as defined by classical cytological studies, is the period when homologous chromosomes, already being discernible individualized entities, begin to be close together or touching over portions of their lengths. This period also includes the bouquet stage: Chromosome ends, which have already become integral components of the inner nuclear membrane, move into a polarized configuration, along with other nuclear envelope components. Chromosome movements, active or passive, also occur. The detailed nature of interhomologue interactions during this period, with special emphasis on the involvement of chromosome ends, and the overall role for meiosis and recombination of chromosome movement and, especially, the bouquet stage are discussed.

The leptotene-zygotene transition of meiosis

With the assumption that a portion that comprises some 10 percent of the genomes in higher organisms cannot be without a raison d'etre, an extensive review led us to conclude that a certain amount of constitutive heterochromatin is essential in multicellular organisms at two levels of organization, chromosomal and nuclear. At the chromosomal level, constitutive heterochromatin is present around vital areas within the chromosomes. Around the centromeres, for example, heterochromatin is believed to confer protection and strength to the centromeric chromatin. Around secondary constrictions, heterochromatic blocks may ensure against evolutionary change of ribosomal cistrons by decreasing the frequency of crossing-over in these cistrons in meiosis and absorbing the effects of mutagenic agents. During meiosis heterochromatin may aid in the initial alignment of chromosomes prior to synapsis and may facilitate speciation by allowing chromosomal rearrangement and providing, through the species specificity of its DNA, barriers against cross-fertilization. At the nuclear level of organization, constitutive heterochromatin may help maintain the proper spatial relationships necessary for the efficient operation of the cell through the stages of mitosis and meiosis. In the unicellular procaryotes, the presence of a small amount of genetic information in one chromosome obviates the need for constitutive heterochromatin and a nuclear membrane. At higher levels of organization, with an increase in the size of the genome and with evolution of cellular and sexual differentiation, the need for compartmentalization and structural components in the nucleus became imminent. The portion of the genome that was concerned with synthesis of ribosomal RNA was enlarged and localized in specific chromosomes, and the centromere became part of each chromosome when the mitotic spindle was developed in evolution. Concomitant with these changes in the genome, repetitive sequences in the form of constitutive heterochromatin appeared, probably as a result of large-scale duplication. The repetitive DNA's were kept through natural selection because of their importance in preserving these vital regions and in maintaining the structural and functional integrity of the nucleus. The association of satellite (or highly repetitive) DNA with constitutive heterochromatin is understandable, since it stresses the importance of the structural rather than transcriptional roles of these entities. Nuclear satellite DNA's have one property in common despite their species specificity, namely heterochromatization. In this sense the apparent species specificity of satellite DNA may be the result of natural selection for duplicated short polynucleotide segments that are nontranscriptional and can be utilized in specific structural roles.

https://science.sciencemag.org/content/sci/174/4015/1200.full.pdf

Heterochromatin, satellite DNA, and cell function. Structural DNA of eucaryotes may support and protect genes and aid in speciation.

Centromeres at premeiotic interphase are clustered and situated in a small area of the nucleus opposite to the nuclear envelope associated heterochromatic masses. The centromeres may occur singly or they may associate to form a structure composed of 2 or more centromeres. Many centromere associations are nonhomologous. Interphase centromeres are not attached to the nuclear envelope. — At zygotene and pachytene centromeres are no longer clustered at one pole of the nucleus but rather are distributed throughout the nucleus. Premeiotic associations appear to be resolved prior to meiotic pairing. Only homologous centromere associations occur during zygotene and pachytene. There is no indication that premeiotic centromere associations are involved in prezygotene alignment of homologous chromosomes.

Centromere behavior during interphase and meiotic prophase in Allium fistulosum from 3-D, E.M. reconstruction

Individual bivalents or chromosomes have been identified in Drosophila melanogaster spermatocytes at metaphase I, anaphase I, metaphase II and anaphase II in electron micrographs of serial sections. Identification was based on a combination of chromosome volume analysis, bivalent topology, and kinetochore position. — Kinetochore microtubule numbers have been obtained for the identified chromosomes at all four meiotic stages. Average numbers in D. melanogaster are relatively low compared to reported numbers of other higher eukaryotes. There are no differences in kinetochore microtubule numbers within a stage despite a large (approximately tenfold) difference in chromosome volume between the largest and the smallest chromosome. A comparison between the two meiotic metaphases (metaphase I and metaphase II) reveals that metaphase I kinetochores possess twice as many microtubules as metaphase II kinetochores. — Other microtubules in addition to those that end on or penetrate the kinetochore are found in the vicinity of the kinetochore. These microtubules penetrate the chromosome rather than the kinetochore proper and are more numerous at metaphase I than at the other division stages.

Meiosis in Drosophila melanogaster. I. Chromosome identification and kinetochore microtubule numbers during the first and second meiotic divisions in males.

Prometaphase I chromosome behavior was examined in wild-type Drosophila melanogaster primary spermatocytes. Cine analysis of live cells reveals that bivalents exhibit complex motions that include (1) transient bipolar orientations, (2) simultaneous reorientation of homologous kinetochores, (3) movements not parallel to the spindle axis, and (4) movement along the nuclear membrane. --Kinetochores and kinetochore microtubule have been analyzed for bivalents previously studied in life. The results suggest that most chromosome motions (complex though they may be) can be explained by poleward forces acting on or through kinetochore microtubules that span the distance between the kinetochore and the vicinity of a pole. The results also suggest that the majority of short kinetochore microtubules may be remnants of previous microtubule-mediated associations between a kinetochore and a pole.

Kinetochore microtubules and chromosome movement during prometaphase in Drosophila melanogaster spermatocytes studied in life and with the electron microscope

At metaphase I synaptonemal complex (SC) material is located in a continuous but irregularly shaped bundle between sister chromatids. Only at the site of a chiasma is it present between homologous chromosomes. When the chromosomes pull apart at anaphase I the SC material becomes rearranged into poly-SCs which dissociate from the chromosomes. The observations agree with previous reports that modified SCs may function in meiotic chromosome disjunction.

The distribution of synaptonemal complex material in metaphase I bivalents of Locusta and Chloealtis (Orthoptera: Acrididae)

Orientation disruptor (ord), a meiotic mutant that is recombination defective in females and disjunction defective in males and females, has been analyzed using serial section electron and light microscopy. From analysis of primary spermatocytes we have confirmed that ord males are defective in some aspect of the mechanism(s) that holds sister chromatids together during meiosis. In addition, we have determined that ord causes high frequencies of nondisjunction during spermatogonial mitotic divisions, as well as during the meiotic divisions. Mitotic nondisjunction involves the large autosomes more frequently than the sex chromosomes or chromosome 4 and results in high frequencies of primary spermatocytes that are either monosomic or trisomic for chromosome 2 or 3. Abnormalities in spermatocyte cyst formation are also observed in males homozygous for ord. These abnormalities include loss of regulation of meiotic synchrony and the number of gonial cell divisions.

https://www.genetics.org/content/genetics/102/4/751.full.pdf

Kathleen Church

Papers

Centromere behavior during interphase and meiotic prophase in Allium fistulosum from 3-D, E.M. reconstruction

Meiosis in Drosophila melanogaster. I. Chromosome identification and kinetochore microtubule numbers during the first and second meiotic divisions in males.

Kinetochore microtubules and chromosome movement during prometaphase in Drosophila melanogaster spermatocytes studied in life and with the electron microscope

The distribution of synaptonemal complex material in metaphase I bivalents of Locusta and Chloealtis (Orthoptera: Acrididae)

Meiosis in Drosophila melanogaster, III. The effect of orientation disruptor (ord) on gonial mitotic and the meiotic divisions in males.