The telomere bouquet controls the meiotic spindle

Bouquet formation, in which telomeres gather to a small region of the nuclear membrane in early meiosis, has been observed in diverse eukaryotes, but the function of the bouquet has remained a mystery. Here, we demonstrate that the telomere bouquet plays a crucial role in controlling the behavior of the fission yeast microtubule-organizing center (known as the spindle pole body or SPB) and the meiotic spindle. Using mutations that specifically disrupt the bouquet, we analyze chromosome, SPB, and spindle dynamics throughout meiosis. If the bouquet fails to form, the SPB becomes fragmented at meiosis I, leading to monopolar, multiple, and mislocalized spindles. Correct SPB and spindle behavior require not only the SPB recruitment of telomere proteins but also that the proteins are properly bound to telomeric DNA. This discovery illuminates an unanticipated level of communication between chromosomes and the spindle apparatus that may be widely conserved among eukaryotes.     

Centrosome dynamics during mammalian oocyte maturation with a focus on meiotic spindle formation

Oocyte maturation is an important process required to achieve optimal oocyte quality, and later affects fertilization potential and subsequent embryo development. The maturation process includes synchronized nuclear and cytoplasmic remodeling, in which cytoskeletal and centrosome dynamics play an important role and significantly participate in cellular signaling. Centrosome remodeling within the maturing oocyte is essential for accurate meioisis I and II spindle formation, specifically to separate chromosomes accurately during the two successive, highly asymmetric meiotic cell divisions. Centrosomal abnormalities result in inaccurate microtubule organization and inaccurate chromosome alignment, with failures in chromosome segregation leading to aneuploidy and chromosomal abnormalities. The present review is focused on cytoskeletal and centrosome remodeling during oocyte maturation, with specific attention to γ-tubulin, pericentrin, the Nuclear Mitotic Apparatus (NuMA) protein, and microtubule organization. Species-specific differences will be discussed for rodent (mouse) and non-rodent (bovine, porcine) species, and for human oocytes.     

Colchicine action at meiotic prophase revealed by SC-spreading

In the present paper earlier findings on the interference of colchicine with presynaptic alignment and synaptonemal complex formation in Allium ursinum are corroborated and several other direct or indirect consequences of colchicine treatment, like deformations and decomposition of lateral elements and a high incidence of non-homologous associations are reported. Mechanisms of meiotic alignment and pairing are discussed with respect to their susceptibility to the known cytological and biochemical activities of colchicine.     

Kinesin-1 prevents capture of the oocyte meiotic spindle by the sperm aster

Centrioles are lost during oogenesis and inherited from the sperm at fertilization. In the zygote, the centrioles recruit pericentriolar proteins from the egg to form a mature centrosome that nucleates a sperm aster. The sperm aster then captures the female pronucleus to join the maternal and paternal genomes. Because fertilization occurs before completion of female meiosis, some mechanism must prevent capture of the meiotic spindle by the sperm aster. Here we show that in wild-type Caenorhabditis elegans zygotes, maternal pericentriolar proteins are not recruited to the sperm centrioles until after completion of meiosis. Depletion of kinesin-1 heavy chain or its binding partner resulted in premature centrosome maturation during meiosis and growth of a sperm aster that could capture the oocyte meiotic spindle. Kinesin prevents recruitment of pericentriolar proteins by coating the sperm DNA and centrioles and thus prevents triploidy by a nonmotor mechanism.     

Rotation of meiotic spindle is controlled by microfilaments in mouse oocytes

The completion of meiosis requires the spatial and temporal coordination of cytokinesis and karyokinesis. During meiotic maturation, many events, such as formation, location, and rotation of the meiotic spindle as well as chromosomal movement, polar body extrusion, and pronuclear migration, are dependent on regulation of the cytoskeleton system. To study functions of microfilaments in meiosis, we induced metaphase II (MII) mouse oocytes to resume meiosis by in vitro fertilization or parthenogenetic activation, and we treated such oocytes with cytochalasin B (CB). The changes of the meiotic spindle, as visualized in preparations stained for beta-tubulin and chromatin, were observed by fluorescent confocal microscopy. The meiotic spindle of MII oocytes was observed to be parallel to the plasmalemma. After meiosis had resumed, the spindle rotated to the vertical position so that the second polar body could be extruded into the perivitelline space. When meiosis resumed and oocytes were treated with 10 micro g/ml of CB, the spindle rotation was inhibited. Consequently, the oocyte formed an extra pronucleus instead of extruding a second polar body. These results indicate that spindle rotation is essential for polar body extrusion; it is the microfilaments that play a crucial role in regulating rotation of the meiotic spindle.     

Specialization in the behaviour of chromosomes on the meiotic spindle

The kinetic activity of chromosomes is either distributed evenly along the length of the chromosome or localized in a single centromere. Within the group of organisms with evenly distributed (holokinetic) activity great variation occurs, which has its consequences for chromatid segregation. Three major types, with some overlap, can be distinguished. They form a gradient of increasing specialization. Arguments are presented for the hypothesis that the monokinetic chromosomes are nothing but the end point in the same gradient and do not form a fundamentally different category. These arguments include: concentration of kinetic activity at meiosis in some types of holokinetic chromosomes, and atavistic traits in monokinetic chromosomes pointing to a holokinetic origin. Possible reasons for the wide distribution of monokinetic chromosomes in the more evolved taxa are given.     

Compartmentalization of the yeast meiotic nucleus revealed by analysis of ectopic recombination

As yeast cells enter meiosis, chromosomes move from a centromere-clustered (Rabl) to a telomere-clustered (bouquet) configuration and then to states of progressive homolog pairing where telomeres are more dispersed. It is uncertain at which stage of this process sequences commit to recombine with each other. Previous analyses using recombination between dispersed homologous sequences (ectopic recombination) support the view that, on average, homologs are aligned end to end by the time of commitment to recombination. We have undertaken further analyses incorporating new inserts, chromosome rearrangements, an alternate mode of recombination initiation, and mutants that disrupt nuclear structure or telomere metabolism. Our findings support previous conclusions and reveal that distance from the nearest telomere is an important parameter influencing recombination between dispersed sequences. In general, the farther dispersed sequences are from their nearest telomere, the less likely they are to engage in ectopic recombination. Neither the mode of initiating recombination nor the formation of the bouquet appears to affect this relationship. We suggest that aspects of telomere localization and behavior influence the organization and mobility of chromosomes along their entire length, during a critical period of meiosis I prophase that encompasses the homology search.     

Complex patterns of mitochondrial dynamics in human pancreatic cells revealed by fluorescent confocal imaging

Mitochondrial morphology and intracellular organization are tightly controlled by the processes of mitochondrial fission–fusion. Moreover, mitochondrial movement and redistribution provide a local ATP supply at cellular sites of particular demands. Here we analysed mitochondrial dynamics in isolated primary human pancreatic cells. Using real time confocal microscopy and mitochondria-specific fluorescent probes tetramethylrhodamine methyl ester and MitoTracker Green we documented complex and novel patterns of spatial and temporal organization of mitochondria, mitochondrial morphology and motility. The most commonly observed types of mitochondrial dynamics were ( i ) fast fission and fusion; ( ii ) small oscillating movements of the mitochondrial network; ( iii ) larger movements, including filament extension, retraction, fast (0.1–0.3 μm/sec.) and frequent oscillating (back and forth) branching in the mitochondrial network; ( iv ) as well as combinations of these actions and ( v ) long-distance intracellular translocation of single spherical mitochondria or separated mitochondrial filaments with velocity up to 0.5 μm/sec. Moreover, we show here for the first time, a formation of unusual mitochondrial shapes like rings, loops, and astonishingly even knots created from one or more mitochondrial filaments. These data demonstrate the presence of extensive heterogeneity in mitochondrial morphology and dynamics in living cells under primary culture conditions. In summary, this study reports new patterns of morphological changes and dynamic motion of mitochondria in human pancreatic cells, suggesting an important role of integrations of mitochondria with other intracellular structures and systems.     

Taxol affects meiotic spindle function in locust spermatocytes

Summary Addition of 0.1 to 10 M taxol to meiotic spindles in locust spermatocytes leads to a concentration dependent promotion of MT assembly at the centrosomes and depletion of MTs at the kinetochores, leading to the formation of prominent asters. In anaphase spindles, the equatorial region of the interzone becomes partly depleted of MTs, too. Microcinematographically, cytostatic effects are highly concentration/time dependent, being most rapid and nearly complete at 10 M taxol, but even in 0.1 M and 1 M taxol anaphase A movement is clearly affected. The drug strongly reduces the rate of chromosome-to-pole movement (anaphase A), leading to an insufficient separation of the chromosomes which indirectly hampers cytokinesis. Obviously, the chromosomal movement seems to be ratelimited by the compactness of the centrosomal asters reaching the equatorial plane in meta- and anaphase. Although the interzonal MT-number has become strongly reduced, anaphase B is not seriously affected but appears even slightly accelerated. Together with an occasional broadening of the cell equator (transverse elongation) instead of normal elongation, these results could be taken as an indication of the previously suggested active role of the cell's cortex in spindle pole separation during anaphase B (Daub andHauser 1986).Prof. Dr. K.-E.Wohlfarth-Bottermann on the occasion of his 65th birthday.     

Sea urchin growth dynamics at microstructural length scale revealed by Mn-labeling and cathodoluminescence imaging

Background

Fluorochrome staining is among the most widely used techniques to study growth dynamics of echinoderms. However, it fails to detect fine-scale increments because produced marks are commonly diffusely distributed within the skeleton. In this paper we investigated the potential of trace element (manganese) labeling and subsequent cathodoluminescence (CL) imaging in fine-scale growth studies of echinoderms.

Results

Three species of sea urchins (Paracentrotus lividus, Echinometra sp. and Prionocidaris baculosa) were incubated for different periods of time in seawater enriched in different Mn²⁺ concentrations (1 mg/L; 3 mg/L; 61.6 mg/L). Labeling with low Mn²⁺ concentrations (at 1 mg/L and 3 mg/L) had no effect on behavior, growth and survival of sea urchins in contrast to the high Mn²⁺ dosage (at 61.6 mg/L) that resulted in lack of skeleton growth. Under CL, manganese produced clearly visible luminescent growth fronts in these specimens (observed in sectioned skeletal parts), which allowed for a determination of the average extension rates and provided direct insights into the morphogenesis of different types of ossicles. The three species tend to follow the same patterns of growth. Spine growth starts with the formation of microspines which are simultaneously becoming reinforced by addition of thickening layers. Spine septa develop via deposition of porous stereom that is rapidly (within less than 2 days) filled by secondary calcite. Development of the inner cortex in cidaroids begins with the formation of microspines which grow at ~3.5 μm/day. Later on, deposition of the outer polycrystalline cortex with spinules and protuberances proceeds at ~12 μm/day. The growth of tooth can be rapid (up to ~1.8 mm/day) and starts with the formation of primary plates (pp) in plumula. Later on, during the further growth of pp in aboral and lateral directions, secondary extensions develop inside (in chronological order: lamellae, needles, secondary plate, prisms and carinar processes), which are increasingly being solidified towards the incisal end. Interradial growth in the ambital interambulacral test plates exceeds meridional growth and inner thickening.

Conclusions

Mn²⁺ labeling coupled with CL imaging is a promising, low-cost and easily applicable method to study growth dynamics of echinoderms at the micro-length scale. The method allowed us to evaluate and refine models of echinoid skeleton morphogenesis.

     

Differentiation of cytoplasmic and meiotic spindle assembly MCAK functions by Aurora B-dependent phosphorylation

The KinI kinesin MCAK is a microtubule depolymerase important for governing spindle microtubule dynamics during chromosome segregation. The dynamic nature of spindle assembly and chromosome-microtubule interactions suggest that mechanisms must exist that modulate the activity of MCAK, both spatially and temporally. In Xenopus extracts, MCAK associates with and is stimulated by the inner centromere protein ICIS. The inner centromere kinase Aurora B also interacts with ICIS and MCAK raising the possibility that Aurora B may regulate MCAK activity as well. Herein, we demonstrate that recombinant Aurora B-INCENP inhibits Xenopus MCAK activity in vitro in a phosphorylation-dependent manner. Substituting endogenous MCAK in Xenopus extracts with the alanine mutant XMCAK-4A, which is resistant to inhibition by Aurora B-INCENP, led to assembly of mono-astral and monopolar structures instead of bipolar spindles. The size of these structures and extent of tubulin polymerization in XMCAK-4A extracts indicate that XM-CAK-4A is not defective for microtubule dynamics regulation throughout the cytoplasm. We further demonstrate that the ability of XMCAK-4A to localize to inner centromeres is abolished. Our results show that MCAK regulation of cytoplasmic and spindle-associated microtubules can be differentiated by Aurora B-dependent phosphorylation, and they further demonstrate that this regulation is required for bipolar meiotic spindle assembly.     

Radial spindle and the phenotype of the maize meiotic mutant, dv

The morphological phenotype of the maize meiotic mutant dv (divergent spindle) has been further analysed by visualization of the division spindle and examination of its fine structure in mother cells of pollen. Previous research showed that dv blocks convergence of spindle fibres at the poles. New observations reveal abnormalities caused by this mutation, with dv showing disturbances in nuclear envelope breakdown during vesiculation, preventing the spindle fibres from adopting a bipolar orientation (with convergence on the poles). The anomalies result in radial spindles which are similar to monoastral spindles in animal cells.     

Peculiar spindle configuration in the pollen tube revealed by the anti-tubulin immunofluorescence method

The spindles in generative cell divisions within the pollen tubes ofCalanthe andImpatiens were revealed by anti-α-tubulin immunofluorescence methods. They were peculiar configurations in which the metaphase chromosomes lay tandemly in some lines along the spindle axis and the sister chromosomes separated anti-parallelly by the spindle elongation during anaphase.