PREMATURE CONDENSATION OF SPERMATOGENIC CELL CHROMOSOMES

The technique of premature chromosome condensation (PCC) has been used primarily to study interphase chromosomes of somatic cells. In this study, mitotic cells were fused to cells from the mouse testes to examine the chromosomes of germ cells. The testes contain various types of cells, both germinal and nongerminal. In these initial studies, four types of PCC morphologies were observed. Chromosome morphology of the PCC and labeling experiments demonstrated the mouse cell origin of various PCC. Attempts were next made to determine the cell types producing the PCC. Spermatogonia, diplotene spermatocytes, secondary spermatocytes and round spermatids are proposed to be the origin of the PCC morphologies. Some PCC could be banded by G and C banding techniques and the mouse chromosomes identified.^ Studies were subsequently undertaken to evaluate this technique as a method of evaluating damage to germ cells. Testicular cells from irradiated mice were fused to mitotic cells and the PCC examined. Both round spermatids and secondary spermatocytes exhibited chromosome damage in the form of chromatid breaks. A linear correlation was found between the dose of irradiation and the number of breaks per cell. This technique may develop into a useful method for evaluating the clastogenic effect of agents on the germ cells. ^

CYTOLOGICAL, ULTRASTRUCTURAL, AND BIOCHEMICAL STUDIES OF THE STRUCTURE OF PREMATURELY CONDENSED CHROMOSOMES

The phenomenon of premature chromosome condensation, resulting from fusion between mitotic and interphase cells, includes dissolution of the interphase nuclear framework, thus allowing a direct visualization of interphase chromosomes. Light microscope morphology of prematurely condensed chromosomes (PCC) from synchronized HeLa cells supports the model of an interphase "chromosome condensation cycle". PCC are increasingly attenuated as cells progress through G(,1). A maximum degree of decondensation is observed at active sites of DNA replication during S phase, and a condensed morphology is rapidly resumed following completion of replication of a chromosome segment.^ To permit ultrastructural and biochemical studies of PCC, a procedure was developed to induce premature chromosome condensation at high frequency. This was achieved by polyethylene glycol (PEG)-mediated fusion of a dense monolayer of mitotic and interphase cells induced by centrifugation onto lectin-coated culture dishes. Using this method, PCC induction frequencies of 60-90% are routinely obtained.^ Scanning electron microscope analysis of PCC spreads revealed that the extension of PCC during progression through G(,1) is accompanied by a transition of the basic 30 nm chromatin fiber from tightly packed looping fibers to extended longitudinal fibers. Sites of active DNA replication is S-PCC were indicated to be organized a single longitudinal fibers. Following replication of a chromosome segment, a rapid reorganization from the extended longitudinal fiber to packed looping fibers occurs. The postreplication maturation process appears to include the assembly of a chromosome core consisting of multiple longitudinal fibers.^ The role of histone H1 phosphorylation in PCC formation was investigated by acidurea polyacrylamide gel electrophoresis of total histone extracted from metaphase chromosomes and PCC following high frequency fusion. This investigation failed to demonstrate an extensive phosphorylation of H1 associated with PCC formation. However, significant dephosphorylation of superphosphorylated metaphase chromosome H1 was observed, indicating that interphase H1-phosphatase activity is dominant over metaphase H1 kinase activity. These observations provide evidence against models suggesting a role for H1 superphosphorylation in triggering mitotic condensation of chromosomes. ^

IDENTIFICATION AND ANALYSIS OF A NOVEL ROLE FOR THE TOUSLED-LIKE KINASE IN REGULATING MITOTIC SPINDLE DYNAMICS

Deregulation of kinase activity is one example of how cells become cancerous by evading evolutionary constraints. The Tousled kinase (Tsl) was initially identified in Arabidopsis thaliana as a developmentally important kinase. There are two mammalian orthologues of Tsl and one orthologue in C. elegans, TLK-1, which is essential for embryonic viability and germ cell development. Depletion of TLK-1 leads to embryonic arrest large, distended nuclei, and ultimately embryonic lethality. Prior to terminal arrest, TLK-1-depleted embryos undergo aberrant mitoses characterized by poor metaphase chromosome alignment, delayed mitotic progression, lagging chromosomes, and supernumerary centrosomes. I discovered an unanticipated requirement for TLK-1 in mitotic spindle assembly and positioning. Normally, in the newly-fertilized zygote (P0) the maternal pronucleus migrates toward the paternal pronucleus at the posterior end of the embryo. After pronuclear meeting, the pronuclear-centrosome complex rotates 90° during centration to align on the anteroposterior axis followed by nuclear envelope breakdown (NEBD). However, in TLK-1-depleted P0 embryos, the centrosome-pronuclear complex rotation is significantly delayed with respect to NEBD and chromosome congression, Additionally, centrosome positions over time in tlk-1(RNAi) early embryos revealed a defect in posterior centrosome positioning during spindle-pronuclear centration, and 4D analysis of centrosome positions and movement in newly fertilized embryos showed aberrant centrosome dynamics in TLK-1-depleted embryos. Several mechanisms contribute to spindle rotation, one of which is the anchoring of astral microtubules to the cell cortex. Attachment of these microtubules to the cortices is thought to confer the necessary stability and forces in order to rotate the centrosome-pronuclear complex in a timely fashion. Analysis of a microtubule end-binding protein revealed that TLK-1-depleted embryos exhibit a more stochastic distribution of microtubule growth toward the cell cortices, and the types of microtubule attachments appear to differ from wild-type embryos. Additionally, fewer astral microtubules are in the vicinity of the cell cortex, thus suggesting that the delayed spindle rotation could be in part due to a lack of appropriate microtubule attachments to the cell cortex. Together with recently published biochemical data revealing the Tousled-like kinases associate with components of the dynein microtubule motor complex in humans, these data suggest that Tousled-like kinases play an important role in mitotic spindle assembly and positioning.

Identification of two distinct human SMC protein complexes involved in mitotic chromosome dynamics

The structural maintenance of chromosomes (SMC) family member proteins previously were shown to play a critical role in mitotic chromosome condensation and segregation in yeast and Xenopus. Other family members were demonstrated to be required for DNA repair in yeast and mammals. Although several different SMC proteins were identified in different organisms, little is known about the SMC proteins in humans. Here, we report the identification of four human SMC proteins that form two distinct heterodimeric complexes in the cell, the human chromosome-associated protein (hCAP)-C and hCAP-E protein complex (hCAP-C/hCAP-E), and the human SMC1 (hSMC1) and hSMC3 protein complex (hSMC1/hSMC3). The hCAP-C/hCAP-E complex is the human ortholog of the Xenopus chromosome-associated protein (XCAP)-C/XCAP-E complex required for mitotic chromosome condensation. We found that a second complex, hSMC1/hSMC3, is required for metaphase progression in mitotic cells. Punctate vs. diffuse distribution patterns of the hCAP-C/hCAP-E and hSMC1/hSMC3 complexes in the interphase nucleus indicate independent behaviors of the two complexes during the cell cycle. These results suggest that two distinct classes of SMC protein complexes are involved in different aspects of mitotic chromosome organization in human cells.

Cleavage Furrows Formed between Centrosomes Lacking an Intervening Spindle and Chromosomes Contain Microtubule Bundles, INCENP, and CHO1 but Not CENP-E

PtK1 cells containing two independent mitotic spindles can cleave between neighboring centrosomes, in the absence of an intervening spindle, as well as at the spindle equators. We used same-cell video, immunofluorescence, and electron microscopy to compare the structure and composition of normal equatorial furrows with that of ectopic furrows formed between spindles. As in controls, ectopic furrows contained midbodies composed of microtubule bundles and an electron-opaque matrix. Despite the absence of an intervening spindle and chromosomes, the midbodies associated with ectopic furrows also contained the microtubule-bundling protein CHO1 and the chromosomal passenger protein INCENP. However, CENP-E, another passenger protein, was not found in ectopic furrows but was always present in controls. We also examined cells in which the ectopic furrow initiated but relaxed. Although relaxing furrows contained overlapping microtubules from opposing centrosomes, they lacked microtubule bundles as well as INCENP and CHO1. Together these data suggest that the mechanism defining the site of furrow formation during mitosis in vertebrates does not depend on the presence of underlying microtubule bundles and chromosomes or on the stable association of INCENP or CHO1. The data also suggest that the completion of cytokinesis requires the presence of microtubule bundles and specific proteins (e.g., INCENP, CHO1, etc.) that do not include CENP-E.

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the Yeast Saccharomyces cerevisiaeV⃞

The spindle pole body (SPB) is the major microtubule-organizing center of budding yeast and is the functional equivalent of the centrosome in higher eukaryotic cells. We used fast-frozen, freeze-substituted cells in conjunction with high-voltage electron tomography to study the fine structure of the SPB and the events of early spindle formation. Individual structures were imaged at 5–10 nm resolution in three dimensions, significantly better than can be achieved by serial section electron microscopy. The SPB is organized in distinct but coupled layers, two of which show ordered two-dimensional packing. The SPB central plaque is anchored in the nuclear envelope with hook-like structures. The minus ends of nuclear microtubules (MTs) are capped and are tethered to the SPB inner plaque, whereas the majority of MT plus ends show a distinct flaring. Unbudded cells containing a single SPB retain 16 MTs, enough to attach to each of the expected 16 chromosomes. Their median length is ∼150 nm. MTs growing from duplicated but not separated SPBs have a median length of ∼130 nm and interdigitate over the bridge that connects the SPBs. As a bipolar spindle is formed, the median MT length increases to ∼300 nm and then decreases to ∼30 nm in late anaphase. Three-dimensional models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complement and extend what is known about the three-dimensional structure of the yeast mitotic spindle and further our understanding of the organization of the SPB in intact cells.

The Dynamic Behavior of Individual Microtubules Associated with Chromosomes In Vitro

Mitotic movements of chromosomes are usually coupled to the elongation and shortening of the microtubules to which they are bound. The lengths of kinetochore-associated microtubules change by incorporation or loss of tubulin subunits, principally at their chromosome-bound ends. We have reproduced aspects of this phenomenon in vitro, using a real-time assay that displays directly the movements of individual chromosome-associated microtubules as they elongate and shorten. Chromosomes isolated from cultured Chinese hamster ovary cells were adhered to coverslips and then allowed to bind labeled microtubules. In the presence of tubulin and GTP, these microtubules could grow at their chromosome-bound ends, causing the labeled segments to move away from the chromosomes, even in the absence of ATP. Sometimes a microtubule would switch to shortening, causing the direction of movement to change abruptly. The link between a microtubule and a chromosome was mechanically strong; 15 pN of tension was generally insufficient to detach a microtubule, even though it could add subunits at the kinetochore–microtubule junction. The behavior of the microtubules in vitro was regulated by the chromosomes to which they were bound; the frequency of transitions from polymerization to depolymerization was decreased, and the speed of depolymerization-coupled movement toward chromosomes was only one-fifth the rate of shortening for microtubules free in solution. Our results are consistent with a model in which each microtubule interacts with an increasing number of chromosome-associated binding sites as it approaches the kinetochore.

The Three-dimensional Study of Chromosomes and Upstream Binding Factor-immunolabeled Nucleolar Organizer Regions Demonstrates Their Nonrandom Spatial Arrangement during Mitosis

The volumic rearrangement of both chromosomes and immunolabeled upstream binding factor in entire well-preserved mitotic cells was studied by confocal microscopy. By using high-quality three-dimensional visualization and tomography, it was possible to investigate interactively the volumic organization of chromosome sets and to focus on their internal characteristics. More particularly, this study demonstrates the nonrandom positioning of metaphase chromosomes bearing nucleolar organizer regions as revealed by their positive upstream binding factor immunolabeling. During the complex morphogenesis of the progeny nuclei from anaphase to late telophase, the equal partitioning of the nucleolar organizer regions is demonstrated by quantification, and their typical nonrandom central positioning within the chromosome sets is revealed.

Cell Cycle–regulated Proteolysis of Mitotic Target Proteins

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase–anaphase transition and continues through G1 phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C–dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, we show that addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we report that injection of dominant negative Ubc3/Cdc34, whose role in G1–S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans.

Mini-chromosomes derived from the human Y chromosome by telomere directed chromosome breakage.

We have used telomeric DNA to break two acrocentric derivatives of the human Y chromosome into mini-chromosomes that are small enough to be size- fractionated by pulsed-field gel electrophoresis. One of the mini-chromosomes is about 7 Mb in size and sequence-tagged site analysis of this molecule suggests that it corresponds to a simple truncation of the short arm of the Y chromosome. Five of the mini-chromosomes are derived from the long arm, are all rearranged by more than a simple truncation, and range in size from 4.0 Mb to 9 Mb. We have studied the mitotic stabilities of these mini-chromosomes and shown that they are stably maintained by cells proliferating in culture for about 100 cell divisions.

Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination.

DNA molecules undergoing transformation into yeast are highly recombinogenic, even when diverged. We reasoned that transformation-associated recombination (TAR) could be employed to clone large DNAs containing repeat sequences, thereby eliminating the need for in vitro enzymatic reactions such as restriction and ligation and reducing the amount of DNA handling. Gently isolated human DNA was transformed directly into yeast spheroplasts along with two genetically marked (M1 and M2) linearized vectors that contained a human Alu sequence at one end and a telomere sequence at the other end (Alu-CEN-M1-TEL and Alu-M2-TEL). Nearly all the M1-selected transformants had yeast artificial chromosomes (YACs) containing human DNA inserts that varied in size from 70 kb to > 600 kb. Approximately half of these had also acquired the unselected M2 marker. The mitotic segregational stability of YACs generated from one (M1) or two (M1 and M2) vector(s) was comparable, suggesting de novo generation of telomeric ends. Since no YACs were isolated when rodent DNAs or a vector lacking an Alu sequence was used, the YACs were most likely the consequence of TAR between the repeat elements on the vector(s) and the human DNA. Using the BLUR13 Alu-containing vector, we demonstrated that human DNA could be efficiently cloned from mouse cells that contained a single human chromosome 16. The distribution of cloned DNAs on chromosome 16 was determined by fluorescence in situ hybridization. We propose that TAR cloning can provide an efficient means for generating YACs from specific chromosomes and subchromosome fragments and that TAR cloning may be useful for isolating families of genes and specific genes from total genome DNA.

Inhibition of S/G(2) phase CDK4 reduces mitotic fidelity

Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G(1) into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G(2) phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G(2) phase and mitosis. The G(2) phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G(2) phase that is critical for G(2)/M progression and the fidelity of mitosis.

Regulation of mitotic BubR1 phosphorylation by the BubR1 pseudokinase domain

BubR1 est une protéine importante dans le point de contrôle de la mitose pour la stabilisation des interactions entre kinétochores et microtubules (KT-MT). Ces fonctions protègent de la ségrégation anormale des chromosomes et de l’instabilité du génome. BubR1 possède des sites de phosphorylation mitotique hautement conservés dans le domaine régulant l’attachement des kinétochores (KARD), où S676 et S670 sont phosphorylées respectivement par la kinase polo-like 1 (Plk1) et par la kinase cycline-dépendante 1 (Cdk1). Ces sites de phosphorylation sont essentiels pour le recrutement de la phosphatase PP2A-B56, qui stabilise les interactions KT-MT. Nos résultats montrent que la délétion entière ou des mutations qui déstabilisent le domaine pseudokinase de BubR1, causent la perte de phosphorylation des résidus S676 et S670 en mitose. Notre hypothèse est que le domaine pseudokinase de BubR1 peut jouer un rôle essentiel dans la régulation de la phosphorylation du KARD et donc dans la stabilisation des interactions KT-MT.

Novel insights into mitotic chromosome condensation

The fidelity of mitosis is essential for life, and successful completion of this process relies on drastic changes in chromosome organization at the onset of nuclear division. The mechanisms that govern chromosome compaction at every cell division cycle are still far from full comprehension, yet recent studies provide novel insights into this problem, challenging classical views on mitotic chromosome assembly. Here, we briefly introduce various models for chromosome assembly and known factors involved in the condensation process (e.g. condensin complexes and topoisomerase II). We will then focus on a few selected studies that have recently brought novel insights into the mysterious way chromosomes are condensed during nuclear division.

Synteny of human chromosomes 14 and 15 in the platyrrhines (Primates, Platyrrhini)

In order to study the intra- and interspecific variability of the 14/15 association in Platyrrhini, we analyzed 15 species from 13 genera, including species that had not been described yet. The DNA libraries of human chromosomes 14 and 15 were hybridized to metaphases of Alouatta guariba clamitans, A. caraya, A. sara, Ateles paniscus chamek, Lagothrix lagothricha, Brachyteles arachnoides, Saguinus midas midas, Leontopithecus chrysomelas, Callimico goeldii, Callithrix sp., Cebus apella, Aotus nigriceps, Cacajao melanocephalus, Chiropotes satanas and Callicebus caligatus. The 14/15 hybridization pattern was present in 13 species, but not in Alouatta sara that showed a 14/15/14 pattern and Aotus nigriceps that showed a 15/14/15/14 pattern. In the majority of the species, the HSA 14 homologue retained synteny for the entire chromosome, whereas the HSA 15 homologue displayed fragmented segments. Within primates, the New World monkeys represent the taxon with the highest variability in chromosome number (2n = 16 to 62). The presence of the HSA 14/15 association in all species and subspecies studied herein confirms that this association is the ancestral condition for platyrrhines and that this association has been retained in most platyrrhines, despite the occurrence of extensive inter- and intrachromosomal rearrangements in this infraorder of Primates.