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1,2-Dioctanoylglycerol accelerates or retards mitotic progression in Tradescantia stamen hair cells as a function of the time of its addition.

We have treated living, intact stamen hair cells from the spiderwort plant, Tradescantia virginiana, with 0.5 microgram/ml or 60 micrograms/ml 1,2-dioctanoylglycerol, a potent and permeant activator of protein kinase C, and have observed the rates of progression of mitosis from prophase through anaphase. We have found that in addition to the concentration used, the time of initial treatment with 1,2-dioctanoylglycerol defines the response by the cells. The cells rapidly undergo nuclear envelope breakdown when this diglyceride is added in very late prophase, 0 to approximately 8 min prior to the time of normal nuclear envelope breakdown. Anaphase onset occurs 28 min after nuclear envelope breakdown, rather than after the 33 min interval observed in untreated cells. Rapid progression through metaphase is also observed if cells are treated with 0.5 microgram/ml 1,2-dioctanoylglycerol during prometaphase, up to 15 min after nuclear envelope breakdown. The addition of 0.5 microgram/ml 1,2-dioctanoylglycerol in late metaphase, approximately 26 min after nuclear envelope breakdown, results in sister chromatid separation slightly ahead of its normal time, 33 min after nuclear envelope breakdown, and in precocious cell plate vesicle aggregation, 3-5 min earlier than that observed in untreated cells. Treatment of cells with 60 micrograms/ml of 1,2-dioctanoylglycerol at any point during the interval from 0 to approximately 5 min prior to nuclear envelope breakdown results in precocious entry into anaphase. If cells are treated with either 0.5 microgram/ml or 60 micrograms/ml 1,2-dioctanoylglycerol earlier than 20 min before nuclear envelope breakdown, they do not enter mitosis, but instead revert to interphase without dividing. When 1,2-dioctanoylglycerol is added at other times during mitosis, the rate of subsequent mitotic progression is dramatically slowed; the cells require greater than 55 min to progress from nuclear envelope breakdown to anaphase onset, though once in anaphase, the cells progress onward to cytokinesis at normal rates. Treatments o of cells with 1,3-dioctanoylglycerol at any point during prophase, prometaphase, or metaphase are without effect on the rate of subsequent mitotic progression. The shifts in response by cells treated at specific times with 1,2-dioctanoylglycerol during mid- and late metaphase may be indicative of the existence of one or more regulatory switch points (i.e., checkpoints) just prior to anaphase onset.

Anaphase↗

Heat shock alters centrosome organization leading to mitotic dysfunction and cell death.

To identify the cellular target(s) responsible for thermal killing in the G1 phase of the cell cycle, synchronous cultures of Chinese hamster ovary cells (CHO) were heat shocked and studied for one cell cycle by time-lapse videomicroscopy and immunocytochemistry. At the first mitosis post-heating, the fraction of cells giving rise to multinucleated progeny approximately equaled the nonclonogenic fraction. In addition, the cells yielding multinucleated progeny were delayed in prophase-metaphase relative to the cells yielding two uninucleated progeny (clonogenic cells). To study the basis for the delay in prophase-metaphase and subsequent formation of multinucleated cells, cells in mitosis were examined by immunofluorescence for spindle abnormalities. Multipolar mitotic spindles and chromosome misalignment were induced by heat. All multiple spindle poles induced by heat stained for pericentriolar material (PCM), the microtubule nucleating material of centrosomes. Heated cells in mitosis also contained additional foci of PCM which were not associated with the spindle. Cells made thermotolerant by a nonlethal heat shock were resistant to both thermal killing and the induction of multiple foci of PCM. Quantitative analysis revealed a good correlation between the fraction of cells with multipolar spindles, the fraction with more than two foci of PCM, and the nonclonogenic fraction. These data indicate that heat-induced alterations to the PCM of centrosomes resulted in multipolar mitotic spindles, delay in prophase-metaphase, and formation of multinucleated cells which were nonclonogenic. These results identify the centrosome as a G1 target for cell killing.

Animals↗

S6 phosphorylation is regulated at multiple levels in Xenopus laevis oocytes.

It is known that the 40s ribosomal protein S6 undergoes a dramatic increase in its level of phosphorylation during Xenopus oocyte meiotic maturation in response to progesterone stimulation. During prophase arrest, the majority of S6 has 0 moles phosphate per mole protein; this increases to 4-5 moles phosphate per mole protein by the time of germinal vesicle breakdown (GVBD). Our in vitro and in vivo studies indicate that the accumulation of phosphate on S6 is the net result of a 4-5-fold increase in S6 kinase activity and a 30-50% decrease in the rate of dephosphorylation and/or turnover of phosphate groups on S6 in maturing oocytes. In addition, the level of phosphorylation of S6 on 80s monosomes injected into non-hormone-stimulated oocytes was unexpectedly high. This indicates that the S6 kinase/phosphatase ratio in prophase arrested oocytes is higher than anticipated from previous studies. This observation implies that the majority of the oocyte ribosomes may be sequestered from any S6 kinase during meiotic prophase. Furthermore, these observations suggest that a portion of the increased accumulation of phosphate on S6 may be the result of increased accessibility of the ribosomes to S6 kinase during oocyte meiotic maturation.

Animals↗

Calcium dynamics during starfish oocyte maturation and fertilization.

Intracellular free calcium levels in starfish oocytes have been monitored during meiotic maturation and fertilization using calcium-sensitive fluorescent dyes combined with confocal laser scanning microscopy or fura ratioing techniques. In time-lapse analyses of prophase-arrested and maturing oocytes, calcium transients were elicited by inositol 1,4,5-trisphosphate (IP3), ryanodine, or caffeine, indicating that both the IP3-sensitive and IP3-insensitive receptors of the oocyte's calcium release channels could be stimulated to mobilize calcium ions. Fertilization also triggered a global calcium wave that appeared to travel faster around the cortex than through the center of the oocyte, and maturing oocytes developed normally after their fertilization-induced calcium waves had been imaged. Prophase-arrested specimens, on the other hand, did not undergo germinal vesicle breakdown or cleavage after displaying a fertilization-induced calcium transient throughout their cytoplasm and nucleus, confirming previous observations that calcium spikes are not sufficient to induce development in immature oocytes. In addition, although the calcium spikes triggered by sperm or caffeine reached similar normalized peak heights, fertilization-induced calcium waves in maturing oocytes tended to be more prolonged than the fertilization waves observed in prophase-arrested oocytes or the caffeine-triggered spikes elicited at any stage of maturation. Collectively, such findings suggest that the total amount of releasable calcium does not vary appreciably during maturation, but the patterns of the calcium transients can differ depending on the stage of maturation and/or the type of calcium-releasing agent. Possible artifacts affecting these findings are assessed, and the results are discussed relative to the functioning of calcium release pathways during starfish oocyte maturation and fertilization.

Animals↗

p53 localizes to the centrosomes and spindles of mitotic cells in the embryonic chick epiblast, human cell lines, and a human primary culture: An immunofluorescence study.

Immunofluorescent staining of mitotic centrosomes and spindles by anti-p53 antibodies was observed in the embryonic chick epiblast by epifluorescence microscopy and in three human cancer cell lines, an SV40-immortalized cell line, and a normal human fibroblast culture by confocal microscopy. In the chick epiblast, the centrosomes stained from early prophase through to the formation of the G1 nuclei and the spindle fibers stained from prophase through to telophase. In the human cells, the staining was observed from late prophase to telophase. The epiblast was stained by the anti-p53 antibodies DO-1, Ab-6, and Bp53-12. The human cells were also stained by these antibodies as well as by other anti-p53 antibodies. Preabsorption of DO-1 and Bp53-12 with purified tubulin did not diminish the immunostaining, showing that the antibodies were not reacting with tubulin in the mitotic centrosomes and spindles. The immunostaining in the chick epiblast was very clearly localized to the mitotic centrosomes and spindles, revealing a cytoplasmic location for p53 during mitosis and accounting for earlier reports of an association between p53, tubulin, and centrosomes. The localization of p53 to the spindle supports an involvement of p53 in spindle function.

Animals↗

Microtubule-driven nuclear movements and linear elements as meiosis-specific characteristics of the fission yeasts Schizosaccharomyces versatilis and Schizosaccharomyces pombe.

Meiotic prophase in Schizosaccharomyces pombe is characterized by striking nuclear movements and the formation of linear elements along chromosomes instead of tripartite synaptonemal complexes. We analysed the organization of nuclei and microtubules in cells of fission yeasts undergoing sexual differentiation. S. japonicus var. versatilis and S. pombe cells were studied in parallel, taking advantage of the better cytology in S. versatilis. During conjugation, microtubules were directed towards the mating projection. These microtubules seem to lead the haploid nuclei together in the zygote by interaction with the spindle pole bodies at the nuclear periphery. After karyogamy, arrays of microtubules emanating from the spindle pole body of the diploid nucleus extended to both cell poles. The same differentiated microtubule configuration was elaborated upon induction of azygotic meiosis in S. pombe. The cyclic movements of the elongated nuclei between the cell poles is reflected by a dynamic and coordinated shortening and lengthening of the two microtubule arrays. When the nucleus was at a cell end, one array was short while the other bridged the whole cell length. Experiments with inhibitors showed that microtubules are required for karyogamy and for the elongated shape and movement of nuclei during meiotic prophase. In both fission yeasts the SPBs and nucleoli are at the leading ends of the moving nuclei. Astral and cytoplasmic microtubules were also prominent during meiotic divisions and sporulation. We further show that in S. versatilis the linear elements formed during meiotic prophase are similar to those in S. pombe. Tripartite synaptonemal complexes were never detected. Taken together, these findings suggest that S. pombe and S. versatilis share basic characteristics in the organization of microtubules and the structure and behaviour of nuclei during their meiotic cell cycle. The prominent differentiations of microtubules and nuclei may be involved in the pairing, recombination, and segregation of meiotic chromosomes.

Benomyl↗

The perinuclear microtubule-organizing center and the synaptonemal complex of higher plants share a common antigen: its putative transfer and role in meiotic chromosomal ordering.

Recognition of homologous chromosomes during meiotic prophase is associated in most cases with the formation of the synaptonemal complex along the length of the chromosome. Telomeres, located at the nuclear periphery, are preferential initiation sites for the assembly of the synaptonemal complex. In most eukaryotic cells, telomeres cluster in a restricted area, leading to the "bouquet" configuration in leptotene-zygotene, while this typical organization progressively disappears in late zygotene-pachytene. We wondered whether such striking changes in the intranuclear ordering and pairing of meiotic chromosomes during the progression of prophase I could be correlated with activity of the centrosome and/or microtubule-organizing center (MTOC). Plant cells may be used as a model of special interest for this study as the whole nuclear surface acts as an MTOC, unlike other cell types where MTOCs are restricted to centrosomes or spindle pole bodies. Using a monoclonal antibody (mAb 6C6) raised against isolated calf centrosomes we found that the 6C6 antigen is present over the entire surface of the plant meiotic nucleus, in early prophase I, before chromosomal pairing. At zygotene, short fragments of chromosomes become stained near the nuclear envelope and within the nucleus. At pachytene, after complete synapsis, the labeling specifically concentrates within the synaptonemal complexes, although the nuclear surface is no longer reactive. Ultrastructural localization using immunogold labeling indicates that the 6C6 antigen is colocalized with the synaptonemal complex structures. Later in metaphase I, the antigen is found at the kinetochores. Our data favor the idea that the 6C6 antigen may function as a particular "chromosomal passenger-like" protein. These observations shed new light on the molecular organization of the plant synaptonemal complex and on the redistribution of cytoskeleton-related antigens during initiation of meiosis. They suggest that antigens of MTOCs are relocated to chromosomes during the synapsis process starting at telomeres and contribute to the spatial arrangement of meiotic chromosomes. Such cytoskeleton-related antigens may acquire different functions depending on their localization, which is cell-cycle regulated.

Antibodies, Monoclonal↗

Organization of heterologous DNA inserts on the mouse meiotic chromosome core.

With simultaneous immunofluorescence and fluorescent in situ hybridization, we have determined the organization of native and heterologous DNA sequences relative to the cores of meiotic prophase chromosomes. The normal chromatin organization is demonstrated with probes of mouse sequences: a cosmid probe that identifies unique sequences and a 720 kb yeast artificial chromosome (YAC) probe that recognizes a specific region of the chromatin domain. The heterologous DNA consists of a 1.8 Mb insertion of 40 tandem head-to-tail phage lambda LIZ vectors and of 11.4 Mb of bacterial/mouse DNA repeats. The lengthy lambda insert is unusual in that it is not contained in the chromatin domain of chromosome 4 and in that it fails to form direct attachments to the chromosome core. The ends are attached indirectly, probably by means of the flanking mouse sequences. At late stages of meiotic prophase, while the terminal attachments remain the same, the lambda DNA becomes highly compacted. Apparently, higher order condensation and core attachment are independent processes. The condensed inserts relax precociously at metaphase I. In the mouse heterozygous for the insert, the two sister inserts are usually merged, as are all four inserts in the homozygous mouse. Evidently chromatin loops with identical sequences can become associated during meiotic prophase. Mouse sequences within a heterologous DNA insert (repeats of bacterial plasmid pBR322 with a mouse beta-globin insert) were observed to restore some degree of core attachment.

Animals↗

The distribution of male meiotic pairing sites on chromosome 2 of Drosophila melanogaster: meiotic pairing and segregation of 2-Y transpositions.

The distribution of meiotic pairing sites on a Drosophila melanogaster autosome was studied by characterizing patterns of prophase pairing and anaphase segregation in males heterozygous for a number of 2-Y transpositions, collectively covering all of chromosome arm 2R and one-fourth of chromosome arm 2L. It was found that all transpositions involving euchromatin from chromosome 2, even short stretches, increased the frequency of prophase I quadrivalents involving the sex and second chromosome bivalents above background levels. Quadrivalent frequencies were the same whether the males carried both elements of the transposition or just the Dp(2:Y) element along with two normal chromosome 2s, indicating that pairing is non-competitive. The frequency of quadrivalents was proportional to the size of the transposed region, suggesting that pairing sites are widely distributed on chromosome 2. Moreover, all but the smallest transpositions caused a detectable bias in the segregation ratio, in favor of alternate segregations, indicating that the prophase associations were effective in orienting centromeres to opposite poles. One transposition involving only heterochromatin of chromosome 2 had no effect on quadrivalent frequency, consistent with previous evidence that autosomal heterochromatin lacks meiotic pairing ability in males. One region at the base of chromosome arm 2L proved to be especially effective in stimulating quadrivalent formation and anaphase segregation, indicating the presence of a strong pairing site in this region. It is concluded that autosomal pairing in D. melanogaster males is based on general homology, despite the lack of homologous recombination.

Animals↗

The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein acts predominately from leptotene to pachytene and represses homologous recombination in male meiosis.

Normal progression of genetic recombination requires timely degradation of many proteins, but little is known about the proteolytic mechanism. The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein is a component of the Skp1-Cullin-F-box-protein (SCF) ubiquitin ligases that target a variety of proteins for degradation via the 26S proteasome pathway. Previous studies indicate that the early defects of the mutant ask1-1 occur in a prophase-I period overlapping with the period of homologous recombination. We provide evidence in this report that ASK1 is predominately expressed from leptotene to pachytene, and negatively regulates recombination. First, the ASK1 transcript was found not to co-exist with that of its closest homolog ASK2 only during prophase I of male meiosis, suggesting that ASK1 is functionally non-redundant only in prophase I. Second, the peak level of an ASK1-green fluorescence protein (GFP) fusion protein expressed by an ASK1 promoter region occurred only from leptotene to pachytene. The ASK1-GFP in a dominant negative fashion resulted in abnormal tetrads resembling those of the ask1-1 mutant, supporting that the expression timing of the ASK1-GFP in male meiocytes reflects the expression timing of the endogenous ASK1. Lastly, using a marker for recombination events, a significant increase in recombination frequency was detected in plants heterozygous for ask1-1. These results indicate that ASK1 normally plays a repressive role in male recombination in Arabidopsis.

Arabidopsis↗

Nuclear ploidy is contingent on the microtubular cycle responsible for plant cytokinesis.

Division of the plant cell relies on the preprophase band of microtubules (PPB)-phragmoplast system. Cells of onion (Allium cepa L.) root meristems were rendered binucleate by preventing the consolidation of cell plate formation in telophase with 5 mM caffeine. These binucleates developed either a single PPB around one of their two nuclei or two PPBs, one per nucleus, in the prophase of the ensuing mitosis. Prophase cells developing one single PPB were shorter in length (42.3 +/- 4.1 microm) than those developing 2 PPBs (49.8 +/- 4.1 microm), and interphase duration was inversely related to cell length. Cells whose length was less than or equal to 42 microm, i.e., which had not even reached the mean size of the small binucleates in prophase, were followed throughout mitosis. In metaphase, they always assembled two mitotic spindles (one per nucleus). However, the cells that had assembled a single PPB also developed a single phragmoplast in telophase, leading to polyploidization. As these meristematic cells were not wide enough to accommodate the midzones of both mitotic spindles in any single plane transversal to the cell elongation axis, the spindles tilted until their midzones formed a continuum where the single common phragmoplast assembled. Its position was thereby uncoupled from that of the preceding PPB. Subsequently, the chromosomes from two different half-spindles were included, by a common nuclear envelope, in a single tetraploid nucleus. Finally, the cytokinetic plate segregated the two tetraploid nuclei formed at each side of the phragmoplast into two independent sister cells.

Cytokinesis↗

Activation of p34cdc2 kinase by cyclin is negatively regulated by cyclic amp-dependent protein kinase in Xenopus oocytes.

Microinjection of a bacterially expressed stable delta 90 sea urchin cyclin B into Xenopus prophase oocytes, in absence or presence of cycloheximide, provokes the activation of histone H1 kinase and the tyrosine dephosphorylation of p34cdc2. Unexpectedly, when prophase oocytes are submitted to a treatment known to elevate the intracellular cAMP level (3-isobutyl-1-methylxanthine and cholera toxin), delta 90 cyclin has no effect and the oocytes remain blocked in prophase. This inhibition is reverted by the microinjection of the inhibitor of cAMP-dependent protein kinase. When delta 90 cyclin is microinjected into oocytes depleted of endogenous cyclins (cycloheximide-treated metaphase I) and in the presence of a high intracellular concentration of cAMP, p34cdc2 kinase is tyrosine rephosphorylated. Altogether, our results indicate that in Xenopus oocyte, cAMP-dependent protein kinase (A-kinase) controls the formation of the cyclin B/p34cdc2 complex which remains inactive and tyrosine phosphorylated.

Animals↗

Cyclic AMP binding to the amphibian oocyte plasma membrane: possible interrelationship between meiotic arrest and membrane fluidity.

Cyclic AMP, which maintains the vertebrate oocyte in prophase arrest under physiological conditions, exhibits specific and saturable binding to the cytoplasmic face of the prophase-arrested Rana pipiens oocyte plasma membrane. Scatchard type analyses of [3H]cAMP binding to isolated plasma membranes indicate a single class of binding sites with a Kd = 19.3 +/- 7.0 nM at cAMP concentrations below 10(-6) M and additional low affinity site(s) and/or non-specific binding at concentrations above 10(-6) M. Photoaffinity labeling of prophase oocyte plasma membranes with [32P]-8-N3cAMP demonstrates cAMP/cGMP-displacable binding of 8-N3[32P]cAMP to a 100-110 kDa peptide doublet. Plasma membrane fluidity was monitored by electron spin resonance in isolated plasma-vitelline membranes using a 5-doxyl stearic acid probe. Exogenous dibutyryl cAMP (dbcAMP) produces an increase in membrane fluidity within minutes and blocks and/or reverses the progesterone-induced decrease in plasma membrane fluidity. The dbcAMP concentration that produced half-maximal fluidity increase (10 microM) corresponds to the half-maximal inhibiting dose of dbcAMP for progesterone induction of meiosis. Cholera toxin, which elevates intracellular cAMP and blocks meiosis, also increases membrane fluidity and inhibits progesterone-induced decrease in membrane fluidity. Elevated levels of intracellular cAMP thus appear to maintain meiotic arrest by binding to specific plasma membrane site(s) and maintaining the plasma membrane in a relatively fluid state. The progesterone-induced fall in intracellular cAMP first reported in Rana thus appears to be responsible for the progesterone-induced increase in membrane fluidity and further suggests that the change in membrane order is essential for the resumption of the meiotic divisions.

Affinity Labels↗

Cyclin-dependent kinase directly regulates initiation of meiotic recombination.

Meiosis is a specialized cell division that halves the genome complement, producing haploid gametes/spores from diploid cells. Proper separation of homologous chromosomes at the first meiotic division requires the production of physical connections (chiasmata) between homologs through recombinational exchange of chromosome arms after sister-chromatid cohesion is established but before chromosome segregation takes place. The events of meiotic prophase must thus occur in a strictly temporal order, but the molecular controls coordinating these events have not been well elucidated. Here, we demonstrate that the budding yeast cyclin-dependent kinase Cdc28 directly regulates the formation of the DNA double-strand breaks that initiate recombination by phosphorylating the Mer2/Rec107 protein and thereby modulating interactions of Mer2 with other proteins required for break formation. We propose that this function of Cdc28 helps to coordinate the events of meiotic prophase with each other and with progression through prophase.

Amino Acid Sequence↗

A component of C. elegans meiotic chromosome axes at the interface of homolog alignment, synapsis, nuclear reorganization, and recombination.

A universal feature of meiotic prophase is the pairing of homologous chromosomes, a fundamental prerequisite for the successful completion of all subsequent meiotic events. HIM-3 is a Caenorhabditis elegans meiosis-specific non-cohesin component of chromosome axes that is required for synapsis. Our characterization of new him-3 alleles reveals previously unknown functions for the protein. HIM-3 is required for the establishment of initial contacts between homologs, for the nuclear reorganization characteristic of early meiotic prophase, and for the coordination of these events with synaptonemal complex (SC) assembly. Despite the absence of homolog alignment, we find that recombination is initiated efficiently, indicating that initial pairing is not a prerequisite for early steps of the recombination pathway. Surprisingly, RAD-51-marked recombination intermediates disappear with apparent wild-type kinetics in him-3 null mutants in which homologs are spatially unavailable for recombination, raising the possibility that HIM-3's presence at chromosome axes inhibits the use of sister chromatids as templates for repair. We propose that HIM-3 is a molecular link between multiple landmark events of meiotic prophase; it is critical for establishing chromosome identity by configuring homologs to facilitate their recognition while simultaneously imposing structural constraints that later promote the formation of the crossover essential for proper segregation.

Alleles↗

Dissociation of DNA polymerase alpha-primase complex during meiosis in Coprinus cinereus.

Previously, the activity of DNA polymerase alpha was found in the meiotic prophase I including non-S phase stages, in the basidiomycetes, Coprinus cinereus. To study DNA polymerase alpha during meiosis, we cloned cDNAs for the C. cinereus DNA polymerase alpha catalytic subunit (p140) and C. cinereus primase small subunit (p48). Northern analysis indicated that both p140 and p48 are expressed not only at S phase but also during the leptotene/zygotene stages of meiotic prophase I. In situ immuno-staining of cells at meiotic prophase I revealed a sub population of p48 that does not colocalize with p140 in nuclei. We also purified the pol alpha-primase complex from meiotic cells by column chromatography and characterized its biochemical properties. We found a subpopulation of primase that was separated from the pol alpha-primase complex by phosphocellulose column chromatography. Glycerol gradient density sedimentation results indicated that the amount of intact pol alpha-primase complex in crude extract is reduced, and that a smaller complex appears upon meiotic development. These results suggest that the form of the DNA polymerase alpha-primase complex is altered during meiotic development.

Blotting, Northern↗

Centriole replication. A study of spermatogenesis in the snail Viviparus.

This paper describes the replication of centrioles during spermatogenesis in the Prosobranch snail, Viviparus malleatus Reeve. Sections for electron microscopy were cut from pieces of testis fixed in OsO(4) and embedded in the polyester resin Vestopal W. Two kinds of spermatocytes are present. These give rise to typical uniflagellate sperm carrying the haploid number of 9 chromosomes, and atypical multiflagellate sperm with only one chromosome. Two centrioles are present in the youngest typical spermatocyte. Each is a hollow cylinder about 160 mmicro in diameter and 330 mmicro long. The wall consists of 9 sets of triplet fibers arranged in a characteristic pattern. Sometime before pachytene an immature centriole, or procentriole as it will be called, appears next to each of the mature centrioles. The procentriole resembles a mature centriole in most respects except length: it is more annular than tubular. The daughter procentriole lies with its axis perpendicular to that of its parent. It presumably grows to full size during the late prophase, although the maturation stages have not been observed with the electron microscope. It is suggested that centrioles possess a constant polarization. The distal end forms the flagellum or other centriole products, while the proximal end represents the procentriole and is concerned with replication. The four centrioles of prophase (two parents and two daughters) are distributed by the two meiotic divisions to the four typical spermatids, in which they function as the basal bodies of the flagella. Atypical spermatocytes at first contain two normal centrioles. Each of these becomes surrounded by a cluster of procentrioles, which progressively elongate during the late prophase. After two aberrant meiotic divisions the centriole clusters give rise to the basal bodies of the multiflagellate sperm. These facts are discussed in the light of the theory, first proposed by Pollister, that the supernumerary centrioles in the atypical cells are derived from the centromeres of degenerating chromosomes.

Animals↗

Orientation of spindle axis and distribution of plasma membrane proteins during cell division in polarized MDCKII cells.

MDCKII cells differentiate into a simple columnar epithelium when grown on a permeable support; the monolayer is polarized for transport and secretion. Individual cells within the monolayer continue to divide at a low rate without disturbing the function of the epithelium as a barrier to solutes. This presents an interesting model for the study of mitosis in a differentiated epithelium which we have investigated by confocal immunofluorescence microscopy. We monitored the distribution of microtubules, centrioles, nucleus, tight junctions, and plasma membrane proteins that are specifically targeted to the apical and basolateral domains. The stable interphase microtubule cytoskeleton was rapidly disassembled at prophase onset and reassembled at cytokinesis. As the interphase microtubules disassembled at prophase, the centrioles moved from their interphase position at the apical membrane to the nucleus and acquired the ability to organize microtubule asters. Orientation of the spindle parallel to the plane of the monolayer occurred between late prophase and metaphase and persisted through cytokinesis. The cleavage furrow formed asymmetrically perpendicular to the plane of the monolayer initiating at the basolateral side and proceeding to the apical domain. The interphase microtubule network reformed after the centrioles migrated from the spindle poles to resume their interphase apical position. Tight junctions (ZO-1), which separate the apical from the basolateral domains, remained assembled throughout all phases of mitosis. E-cadherin and a 58-kD antigen maintained their basolateral plasma membrane distributions, and a 114-kD antigen remained polarized to the apical domain. These proteins were useful for monitoring the changes in shape of the mitotic cells relative to neighboring cells, especially during telophase when the cell shape changes dramatically. We discuss the changes in centriole position during the cell cycle, mechanisms of spindle orientation, and how the maintenance of polarized plasma membrane domains through mitosis may facilitate the rapid reformation of the polarized interphase cytoplasm.

Animals↗