Asymmetric distribution of mitochondria and of spindle microtubules in opposite directions in differential mitosis of germ line cells in Acricotopus

Additional chromosomes present only in the germ line are a specific feature of the Orthocladiinae, a subfamily of the Chironomidae. During the complex chromosome cycle in the orthocladiid Acricotopus lucidus, about half of the germ-line-limited chromosomes (Ks) are eliminated in the first division of the primary germ cells. Following normal gonial mitoses, the reduction in the number of Ks is compensated for, in the last mitosis prior to meiosis, by a monopolar movement of the unseparated Ks, while the somatic chromosomes (Ss) segregate equally. This differential mitosis produces daughter cells with different chromosome constitutions and diverse developmental fates. A preferential segregation of mitochondria occurs to one pole associated with an asymmetric formation of the mitotic spindle. This has been detected in living gonial cells in both sexes by using MitoTracker probes and fluorochrome-labelled paclitaxel (taxol). In males, the resulting unequal partitioning of mitochondria to the daughter cells is equalised by the transport of mitochondria through a permanent cytoplasmic bridge from the aberrant spermatocyte to the primary spermatocyte. This asymmetry in the distribution and in the segregation of cytoplasmic components in differential gonial mitosis in Acricotopus may be involved in the process of cell-fate determination. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

X-ray induced rearrangements between germ-line limited and soma chromosomes of Acricotopus lucidus (Diptera,Chironomidae)

The germ-line limited chromosomes (Ks) [K being derived from Keimbahn (Bauer, 1970)] of Acricotopus lucidus were studied in gonial and differential mitosis. After C-banding the soma chromosomes (Ss) are stained only at their centromeric regions whereas the Ks exhibit centromeric, intercalary and terminal heterochromatin. By X-raying sperms it was attempted to transfer K sections on or into Ss in order to bring finally S-linked K sections to polytenization in the salivary glands, and to obtain more knowledge about the structure of Ks. Seven F₁-larvae were detected with K-S-rearrangements: four with insertions of heterochromatic segments, two with insertions of sections with S-homologous banding pattern and one with a translocated K chromosome part, which consists of S-homologous euchromatic sections as well as of an intercalary and a terminal heterochromatic segment. The present results strongly suggest that the Ks of A. lucidus are derived from the Ss by rearrangements and by formation and accumulation of repetitive sequences.     

Action de l'hydrate de chloral sur les mitoses de segmentation de l'œuf de pleurodèle

In eggs of Pleurodeles treated with chloralhydrate (0.1 M) spindle and astral fibers are progressively destroyed after 4 hours, leading to apolar nuclei, apolar mitoses and “monopolar” mitoses, the so-called star metaphases. After 1 hour the spindle is shortened, but not narrowed and separated from the poles and asters. Its microtubules, grown before metaphase, are first inhibited at their ends near the centrospheres. After 4 hours, defibrillated achromatic material, stained by methyl blue, surrounds a clearer zone originating from the nucleoplasm, in which chromosomes are embedded. At the EM level the treatment induces the formation of unusual tubular bodies connected with the centrospheres and of similar bodies related to kinetochores and chromosomes. These bodies are formed of tubular residues, parallel or in concentric systems, the latter embedded in a matrix containing tightly packed filaments of 170 Å diameter. The star metaphase is characterized by homogeneous centrospheres formed only of filaments and completely independent from kinetochores and chromosomes. Chromosomes are radially distributed around a central common mass, which keeps the chromosomes together; it is formed of a finely fibrous matrix containing disordered microtubular residues; kinetochores are embedded in the common mass. Fuzziness and alteration of chromosomes proceed as a direct action of the chloralhydrate. The star metaphase is not a real “monopolar” mitosis.     

G-banding of germ line limited chromosomes in Acricotopus lucidus (Diptera,Chironomidae)

The germ line limited (K) chromosomes of Acricotopus lucidus (Diptera, Chironomidae) were stained for G-banding on gonial mitoses, along with the somatic (S) chromosomes. Nine different types of K chromosomes could be distinguished by the G-banding pattern and other cytological criteria. Various combinations of K chromosomes were found in the complements of different individuals and cells: some Ks were missing and others were present up to as many as five times. No two animals were completely alike in the composition of their gonial chromosome complement. Thus none of the different K types can be essential. These results are discussed in view of the complex chromosome cycle of the Orthocladiinae.     

The first mitosis of the mouse embryo is prolonged by transitional metaphase arrest

The first mitosis of the mouse embryo is almost twice as long as the second. The mechanism of the prolongation of the first mitosis remains unknown, and it is not clear whether prometaphase or metaphase or both are prolonged. Prometaphase is characterized by dynamic chromosome movements and spindle assembly checkpoint activity, which prevents anaphase until establishment of stable kinetochore-microtubule connections. The end of prometaphase is correlated with checkpoint inactivation and disappearance of MAD2L1 (MAD2) and RSN (CLIP-170) proteins from kinetochores. Spindle assembly checkpoint operates during the early mouse mitoses, but it is not clear whether it influences their duration. Here, we determine the length of prometaphases and metaphases during the first two embryonic mitoses by time-lapse video recording of chromosomes and by immunolocalization of MAD2L1 and RSN proteins. We show that the duration of the two prometaphases does not differ and that MAD2L1 and RSN disappear from kinetochores very early during each mitosis. The first metaphase is significantly longer than the second one. Therefore, the prolongation of the first embryonic mitosis is due to a prolonged metaphase, and the spindle assembly checkpoint cannot be involved in this process. We show also that MAD2L1 staining disappears gradually from kinetochores of oocytes arrested at metaphase of the second meiotic division. This shows a striking similarity between the first embryonic mitosis and metaphase arrest in oocytes. We postulate that the first embryonic mitosis is prolonged by a transient metaphase arrest that is independent of the spindle assembly checkpoint and is similar to metaphase II arrest. The molecular mechanism of this transient arrest remains to be elucidated.     

Isolation and chromosomal localization of a germ line-specific highly repetitive DNA family in Acricotopus lucidus (Diptera, Chironomidae)

. In the chironomid Acricotopus lucidus, parts of the genome, the germ line-limited chromosomes, are eliminated from the future soma cells during early cleavage divisions. A highly repetitive, germ line-specific DNA sequence family was isolated, cloned and sequenced. The monomers of the tandemly repeated sequences range in size from 175 to 184 bp. Analysis of sequence variation allowed the further classification of the germ line-restricted repetitive DNA into two related subfamilies, A and B. Fluorescence in situ hybridization to gonial metaphases demonstrated that the sequence family is highly specific for the paracentromeric heterochromatin of the germ line-limited chromosomes. Restriction analysis of genomic soma DNA of A. lucidus revealed another tandem repetitive DNA sequence family with monomers of about 175 bp in length. These DNA elements are found only in the centromeric regions of all soma chromosomes and one exceptional germ line-limited chromosome by in situ hybridization to polytene soma chromosomes and gonial metaphase chromosomes. The sequences described here may be involved in recognition, distinction and behavior of soma and germ line-limited chromosomes during the complex chromosome cycle in A. lucidus and may be useful for the genetic and cytological analysis of the processes of elimination of the germ line-limited chromosomes in the soma and germ line. Received: 12 April 1997; in revised form 26 June 1997 / Accepted: 29 June 1997     

Centrosome hyperamplification with the formation of multiple asters and programmed chromosome inactivation in aberrant spermatocytes during male meiosis in Acricotopus

In the germ line of the midge Acricotopus lucidus, an unequal chromosome segregation occurs in the last gonial mitosis prior to meiosis. This results in one daughter cell receiving only somatic chromosomes (Ss), whereas the other cell is given all the so-called germ line limited chromosomes (Ks) in addition to the Ss. The cytokinesis following this differential mitosis is incomplete and the daughter cells remain connected by a permanent cytoplasmic bridge. The cell with the Ss and Ks develops into a primary oocyte or spermatocyte, whereas the cell containing only Ss differentiates as a nurse cell in the female or as an aberrant spermatocyte in the male. When the primary spermatocyte enters meiosis, the Ss in the connected aberrant spermatocyte undergo chromosome condensation but the aberrant spermatocyte remains undivided, with the condensed metaphase status and inactivation of the Ss persisting during both meiotic divisions. These events indicate a programmed inactivation of all chromosomes in the aberrant spermatocyte at the beginning of meiosis. The alterations in the microtubule arrangements and of the distribution of mitochondria in the spermatocytes during meiosis have been followed via live-cell fluorescence labelling with the TubulinTracker and MitoTracker reagents and by transmission electron microscopy. The observations reveal a hyperamplification of the centrosomes and the formation of tetrapolar asters in the non-dividing aberrant spermatocytes containing the condensed Ss. The programmed inactivation of the Ss in the aberrant spermatocyte is suggested to have developed during evolution to inhibit the entry of the aberrant spermatocytes into meiosis, thereby preventing the formation of sperms containing only Ss but no Ks.     

Chromosome elimination in germ line-soma differentiation of Acricotopus lucidus (Diptera, Chironomidae).

During germ line-soma differentiation in early syncytial embryonic development of the chironomid Acricotopus lucidus, a complement of supernumerary chromosomes, the so-called germ line limited chromosomes (Ks), is excluded from the future somatic nuclei in the course of elimination mitoses. The Ks lag behind in the equatorial plane, while the somatic chromosomes (Ss) segregate equally. After elimination mitoses, the Ks are only present in the pole cells, the primary germ cells. In the divisions before their elimination, the Ks frequently showed delayed separation of sister chromatids with high-frequency formation of anaphasic bridges and lagging in pole movement as detected in 4',6-diamidino-2-phenylindole (DAPI)-stained squash preparations of early embryos. To determine if all of the Ks are eliminated in one step during a single mitosis, a fluorescence in situ hybridization (FISH) analysis of early embryonic divisions was performed using probes of germ line specific repetitive DNA sequences, which specifically label the Ks in their centromeric regions. In most cases, all of the Ks are lost in one mitosis; however, occasionally one or several of the Ks can escape their elimination by segregating and moving poleward together with the Ss. The escaping Ks will then be eliminated in one of the following mitoses. This clearly indicates that the specific conditions to eliminate Ks are not restricted to only one division. Possible mechanisms of elimination of Ks are discussed.     

Fission Yeast Bub1 Is a Mitotic Centromere Protein Essential for the Spindle Checkpoint and the Preservation of Correct Ploidy through Mitosis

The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. We investigated the role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses. We find that bub1 ⁺ is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function. Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis. We show that Bub1 also has a crucial role in normal, unperturbed mitoses. Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss. Such genomic instability is even more dramatic in Δbub1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that bub1 ⁺ function is essential to maintain correct ploidy through mitosis. As in larger eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis. However, unlike its vertebrate counterpart, a pool of Bub1 remains centromere-associated at metaphase and even until telophase. We discuss the possibility of a role for the Bub1 kinase after the metaphase–anaphase transition.     

Kinetochore appearance during meiosis,fertilization and mitosis in mouse oocytes and zygotes

The events of mammalian fertilization overlap with the completion of meiosis and first mitosis; the pronuclei never fuse, instead the parental genomes first intermix at the mitotic spindle equator at metaphase. Since kinetochores are essential for the attachment of chromosomes to spindle microtubules, this study explores their appearance and behavior in mouse oocytes, zygotes and embryos undergoing the completion of meiosis, fertilization and mitoses. Kinetochores are traced with immunofluorescence microscopy using autoimmune sera from patients with CREST (CREST = calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) scleroderma. These sera cross-react with the 17 kDa centromere protein (CENP-A) and the 80 kDa centromere protein (CENP-B) found at the kinetochores in human cell cultures. The unfertilized oocyte is ovulated arrested at second meiotic metaphase and kinetochores are detectable as paired structures aligned at the spindle equator. At meiotic anaphase, the kinetochores separate and remain aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular to the spindle axis is lost. The female pronucleus and the second polar body nucleus each receive a detectable complement of kinetochores. Mature sperm have neither detectable centrosomes nor detectable kinetochores, and shortly after sperm incorporation kinetochores become detectable in the decondensing male pronucleus. In pronuclei, the kinetochores are initially distributed randomly and later found in apposition with nucleoli. At mitosis, the kinetochores behave in a pattern similar to that observed at meiosis or mitosis in somatic cells: irregular distribution at prophase, alignment at metaphase, separation at anaphase and redistribution at telophase. They are also detectable in later stage embryos. Colcemid treatment disrupts the meiotic spindle and results in the dispersion of the meiotic chromosomes along the oocyte cortex; the chromosomes remain condensed with detectable kinetochores. Fertilization of Colcemid-treated oocytes results in the incorporation of a sperm which is unable to decondense into a male pronucleus. Remarkably kinetochores become detectable at 5 h post-insemination, suggesting that the emergence of the paternal kinetochores is not strictly dependent on male pronuclear decondensation.     

Structural evolution of the germ line-limited chromosomes in Acricotopus

The elimination of chromatin or whole chromosomes from the future somatic nuclei during germ line-soma differentiation in early embryogenesis is a genetic phenomenon found in a wide variety of animal species. Less is known about the origin, structure, and function of the germ line-limited chromosomes. In the chironomid Acricotopus lucidus fluorescence in situ hybridization (FISH) with labeled soma DNA to "Keimbahn" chromosomes (Ks) and soma chromosomes (Ss) of spermatogonial mitoses revealed that each of the nine different K types possesses large S-homologous sections, mostly in the distal parts of both chromosome arms. Painting probes of the three Ss and of each of their chromosome arms were generated by microdissection of polytene salivary gland chromosomes and subsequent amplification by the degenerate oligonucleotide-primed polymerase chain reaction. Multicolor FISH demonstrated that each of the Ks, with the exception of one K type, was painted by only one of the three S probes. Furthermore, in seven Ks, one chromosome arm was painted by the long-arm probe and the other by the short-arm probe of the S concerned. The hybridization pattern strongly suggests that each of these K types is derived from a specific S. One function of the S-homologous K sections is thought to be determination of the regular occurrence of crossover events, with the resulting chiasmata in these sections ensuring correct segregation of the K homologs during meiosis. Reverse chromosome painting on polytene S sets with a probe generated from metaphase Ks corroborates the above results and produces conclusive evidence for the hypothesis that during evolution the Ks have developed from the Ss by endopolyploidization and rearrangements followed by the accumulation of germ line-specific repetitive DNA sequences in the centromeric regions.     

Localization and function of mSpindly during mouse oocyte meiotic maturation

Spindly was first identified in Drosophila; its homologues are termed SPDL-1 in Caenorhabditis elegans and Hs Spindly/hSpindly in humans. In all species, Spindly and its homologues function by recruiting dynein to kinetochores and silencing SAC in mitosis of somatic cells. Depletion of Spindly causes an extensive metaphase arrest during somatic mitoses in Drosophila, C. elegans and humans. In Drosophila, Spindly is required for shedding of Rod and Mad2 from the kinetochores in metaphase; in C. elegans, SPDL-1 presides over the recruitment of dynein and MDF-1 to the kinetochores; in humans, Hs Spindly is required for recruiting both dynein and dynactin to kinetochores but it is dispensable for removal of checkpoint proteins from kinetochores. The present study was designed to investigate the localization and function of the Spindly homologue (mSpindly) during mouse oocyte meiotic maturation by immunofluorescent analysis, and by overexpression and knockdown of mSpindly. We found that mSpindly was typically localized to kinetochores when chromatin condensed into chromosomes after GVBD. In metaphase of both first meiosis and second meiosis, mSpindly was localized not only to kinetochores but also to the spindle poles. Overexpression of mSpindly did not affect meiotic progression, but its depletion resulted in an arrest of the pro-MI/MI stage, failure of anaphase entry and subsequent polar body emission, and in abnormal spindle morphology and misaligned chromosomes. Our data suggest that mSpindly participates in SAC silencing and in spindle formation as a recruiter and/or a transporter of kinetochore proteins in mouse oocytes, but that it needs to cooperate with other factors to fulfill its function.     

Meiosis in Drosophila melanogaster

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.     

Chromosome Fragments Possessing Only One Kinetochore Can Congress to the Spindle Equator

We used laser microsurgery to cut between the two sister kinetochores on bioriented prometaphase chromosomes to produce two chromosome fragments containing one kinetochore (CF1K). Each of these CF1Ks then always moved toward the spindle pole to which their kinetochores were attached before initiating the poleward and away-from-the-pole oscillatory motions characteristic of monooriented chromosomes. CF1Ks then either: (a) remained closely associated with this pole until anaphase (50%), (b) moved (i.e., congressed) to the spindle equator (38%), where they usually (13/19 cells) remained stably positioned throughout the ensuing anaphase, or (c) reoriented and moved to the other pole (12%). Behavior of congressing CF1Ks was indistinguishable from that of congressing chromosomes containing two sister kinetochores. Three-dimensional electron microscopic tomographic reconstructions of CF1Ks stably positioned on the spindle equator during anaphase revealed that the single kinetochore was highly stretched and/or fragmented and that numerous microtubules derived from the opposing spindle poles terminated in its structure. These observations reveal that a single kinetochore is capable of simultaneously supporting the function of two sister kinetochores during chromosome congression and imply that vertebrate kinetochores consist of multiple domains whose motility states can be regulated independently.     

Activation of the MKK/ERK Pathway during Somatic Cell Mitosis: Direct Interactions of Active ERK with Kinetochores and Regulation of the Mitotic 3F3/2 Phosphoantigen

The mitogen-activated protein (MAP) kinase pathway, which includes extracellular signal–regulated protein kinases 1 and 2 (ERK1, ERK2) and MAP kinase kinases 1 and 2 (MKK1, MKK2), is well-known to be required for cell cycle progression from G1 to S phase, but its role in somatic cell mitosis has not been clearly established. We have examined the regulation of ERK and MKK in mammalian cells during mitosis using antibodies selective for active phosphorylated forms of these enzymes. In NIH 3T3 cells, both ERK and MKK are activated within the nucleus during early prophase; they localize to spindle poles between prophase and anaphase, and to the midbody during cytokinesis. During metaphase, active ERK is localized in the chromosome periphery, in contrast to active MKK, which shows clear chromosome exclusion. Prophase activation and spindle pole localization of active ERK and MKK are also observed in PtK₁ cells. Discrete localization of active ERK at kinetochores is apparent by early prophase and during prometaphase with decreased staining on chromosomes aligned at the metaphase plate. The kinetochores of chromosomes displaced from the metaphase plate, or in microtubule-disrupted cells, still react strongly with the active ERK antibody. This pattern resembles that reported for the 3F3/2 monoclonal antibody, which recognizes a phosphoepitope that disappears with kinetochore attachment to the spindles, and has been implicated in the mitotic checkpoint for anaphase onset (Gorbsky and Ricketts, 1993. J. Cell Biol. 122:1311–1321). The 3F3/2 reactivity of kinetochores on isolated chromosomes decreases after dephosphorylation with protein phosphatase, and then increases after subsequent phosphorylation by purified active ERK or active MKK. These results suggest that the MAP kinase pathway has multiple functions during mitosis, helping to promote mitotic entry as well as targeting proteins that mediate mitotic progression in response to kinetochore attachment.     

Molecular evolution of homologous gene sequences in germline-limited and somatic chromosomes of Acricotopus.

The origin of germline-limited chromosomes (Ks) as descendants of somatic chromosomes (Ss) and their structural evolution was recently elucidated in the chironomid Acricotopus. The Ks consist of large S-homologous sections and of heterochromatic segments containing germline-specific, highly repetitive DNA sequences. Less is known about the molecular evolution and features of the sequences in the S-homologous K sections. More information about this was received by comparing homologous gene sequences of Ks and Ss. Genes for 5.8S, 18S, 28S, and 5S ribosomal RNA were choosen for the comparison and therefore isolated first by PCR from somatic DNA of Acricotopus and sequenced. Specific K DNA was collected by microdissection of monopolar moving K complements from differential gonial mitoses and was then amplified by degenerate oligonucleotide primer (DOP)-PCR. With the sequence data of the somatic rDNAs, the homologous 5.8S and 5S rDNA sequences were isolated by PCR from the DOP-PCR sequence pool of the Ks. In addition, a number of K DOP-PCR sequences were directly cloned and analysed. One K clone contained a section of a putative N-acetyltransferase gene. Compared with its homolog from the Ss, the sequence exhibited few nucleotide substitutions (99.2% sequence identity). The same was true for the 5.8S and 5S sequences from Ss and Ks (97.5%-100% identity). This supports the idea that the S-homologous K sequences may be conserved and do not evolve independently from their somatic homologs. Possible mechanisms effecting such conservation of S-derived sequences in the Ks are discussed.     

Kinetochore rearrangement in meiosis II requires attachment to the spindle

The distinctive behaviors of chromosomes in mitosis and meiosis depend upon differences in kinetochore position. Kinetochore position is well established except for a critical transition between meiosis I and meiosis II. We examined kinetochore position during the transition and compared it with the position of kinetochores in mitosis. Immunofluorescence staining using the 3F3/2 antibody showed that in mitosis in grasshopper cells, as in other organisms, kinetochores are positioned on opposite sides of the two sister chromatids. In meiosis I, sister kinetochores are positioned side by side. At nuclear envelope breakdown in meiosis II, sister kinetochores are still side by side, but are separated by the time all chromosomes have fully attached in metaphase II. Micromanipulation experiments reveal that this switch from side-by-side to separated sister kinetochores requires attachment to the spindle. Moreover, it is irreversible, as chromosomes detached from a metaphase II spindle retain separate kinetochores. How this critical separation of sister kinetochores occurs in meiosis is uncertain, but clearly it is not built into the chromosome before nuclear envelope breakdown, as it is in mitosis.     

Analysis of Scc1-deficient cells defines a key metaphase role of vertebrate cohesin in linking sister kinetochores

Cleavage of the cohesin subunit Scc1p/Mcd1p/Rad21 permits sister chromatid separation and is considered to trigger anaphase onset. It has also been suggested that the cohesin complex is essential for chromosome condensation and for assembling fully functional kinetochores. Here, we used vertebrate cells conditionally deficient in Scc1 to probe cohesin function in mitosis. Cells lacking cohesin arrest in prometaphase, with many chromosomes failing to align at a metaphase plate and high levels of the spindle assembly checkpoint protein, BubR1, at all kinetochores. We show that the structural integrity of chromosomes is normal in the absence of Scc1. Furthermore, specific inhibition of topoisomerase II, which is required for decatenation of replicated chromosomes, can bypass the cohesin requirement for metaphase chromosome alignment and spindle checkpoint silencing. Since the kinetochore effects of Scc1 deficiency can be compensated for by topoisomerase II inhibition, we conclude that Scc1 is not absolutely required for kinetochore assembly or function, and that its principal role in allowing the onset of anaphase is the establishment of sufficient inter-sister tension to allow biorientation.