Single Aster Research Articles

Defect dynamics in active polar fluids vs. active nematics.

Topological defects play a key role in two-dimensional active nematics, and a transient role in two-dimensional active polar fluids. Using a variational method, we study both the transient and long-time behavior of defects in two-dimensional active polar fluids in the limit of strong order and overdamped, compressible flow, and compare the defect dynamics with the corresponding active nematics model studied recently. One result is non-central interactions between defect pairs for active polar fluids, and by extending our analysis to allow orientation dynamics of defects, we find that the orientation of +1 defects, unlike that of ±1/2 defects in active nematics, is not locked to defect positions and relaxes to asters. Moreover, using a scaling argument, we explain the transient feature of active polar defects and show that in the steady state, active polar fluids are either devoid of defects or consist of a single aster. We argue that for contractile (extensile) active nematic systems, +1 vortices (asters) should emerge as bound states of a pair of +1/2 defects, which has been recently observed. Moreover, unlike the polar case, we show that for active nematics, a linear chain of equally spaced bound states of pairs of +1/2 defects can screen the activity term. A common feature in both models is the appearance of +1 defects (elementary in polar and composite in nematic) in the steady state.

High revivability of vitrified-warmed bovine mature oocytes after recovery culture with α-tocopherol.

The objective of this study was to investigate whether developmental competence of vitrified-warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either l-ascorbic acid or α-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to α-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1-36.3% vs 19.2-25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between α-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in α-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with α-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that α-tocopherol treatment of vitrified-warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.

Aster formation and rupture transition in semi-flexible fiber networks with mobile cross-linkers

Fibrous active network structures whose properties are regulated by motor proteins, or simply motors, are fundamental to life. Here, a full elastic and three dimensional model for such networks and motors is presented. The effects of surface anchoring are accounted for and we demonstrate that for unidirectional motors two basic contractile phases emerge in these systems. The transition is governed by a single parameter (τb/τc) which is the ratio of the breaking strain (τb) and the motility limiting strain (τc) of the motors. For τb/τc ≲ 2 and clamped boundaries, the network ruptures and formation of local asters occurs with a high density of motors at the centre and the fibers radially spanning out. This phase displays contraction strain during the formation of asters but the network stress is relaxed once the asters have emerged, demonstrating that the formation of aster-like structures provides a mechanism for stress relaxation. For 2.7 ≲ τb/τc the network remains intact, but reaches a force equilibrium with a high contraction strain in the case of clamped boundaries. Between these two limits the network is partly ruptured. Experimental measurements (e.g. J. T. Nishizaka, H. Miyata, H. Yoshikawa, S. Ishiwata and K. Kinosita Jr., Nature, 1995, 377, 251 and J. F. Finer, R. M. Simmons, J. A. Spudich, Nature, 1994, 368, 113) indicate that actin filament and myosin motors interact with τb/τc ≈ 2.7 which is right at the limit of motor induced fracture for a random network, indicating that e.g. a cytoskeleton with active myosin is susceptible to rupture. This is perhaps not a coincidence and may well be an important factor contributing to cellular dynamics. In the case of free boundaries the network collapses onto one single aster. We also show that the distribution of energy on the motors is a power-law, below the motility limit energy, with the exponent −0.5.

Three-dimensional preparation and imaging reveal intrinsic microtubule properties

We present an experimental investigation of microtubule dynamic instability in three dimensions, based on laser light-sheet fluorescence microscopy. We introduce three-dimensional (3D) preparation of Xenopus laevis egg extracts in Teflon-based cylinders and provide algorithms for 3D image processing. Our approach gives experimental access to the intrinsic dynamic properties of microtubules and to microtubule population statistics in single asters. We obtain evidence for a stochastic nature of microtubule pausing.

Computer Simulations and Image Processing Reveal Length-Dependent Pulling Force as the Primary Mechanism for C. elegans Male Pronuclear Migration

A male pronucleus migrates toward the center of an egg to reach the female pronucleus for zygote formation. This migration depends on microtubules growing from two centrosomes associated with the male pronucleus. Two mechanisms were previously proposed for this migration: a "pushing mechanism," which uses the pushing force resulting from microtubule polymerization, and a "pulling mechanism," which uses the length-dependent pulling force generated by minus-end-directed motors anchored throughout the cytoplasm. We combined two computer-assisted analyses to examine the relative contribution of these mechanisms to male pronuclear migration. Computer simulation revealed an intrinsic difference in migration behavior of the male pronucleus between the pushing and pulling mechanisms. In vivo measurements using image processing showed that the actual migration behavior in Caenorhabditis elegans confirms the pulling mechanism. A male pronucleus having a single centrosome migrated toward the single aster. We propose that the pulling mechanism is the primary mechanism for male pronuclear migration.

Self-organized pattern formation in motor-microtubule mixtures

We model the stable self-organized patterns obtained in the nonequilibrium steady states of mixtures of molecular motors and microtubules. In experiments [Nédélec et al., Nature (London) 389, 305 (1997); Surrey et al., Science 292, 1167 (2001)] performed in a quasi-two-dimensional geometry, microtubules are oriented by complexes of motor proteins. This interaction yields a variety of patterns, including arrangements of asters, vortices, and disordered configurations. We model this system via a two-dimensional vector field describing the local coarse-grained microtubule orientation and two scalar density fields associated to molecular motors. These scalar fields describe motors which either attach to and move along microtubules or diffuse freely within the solvent. Transitions between single aster, spiral, and vortex states are obtained as a consequence of confinement, as parameters in our model are varied. For systems in which the effects of confinement can be neglected, we present a map of nonequilibrium steady states, which includes arrangements of asters and vortices separately as well as aster-vortex mixtures and fully disordered states. We calculate the steady state distribution of bound and free motors in aster and vortex configurations of microtubules and compare these to our simulation results, providing qualitative arguments for the stability of different patterns in various regimes of parameter space. We study the role of crowding or "saturation" effects on the density profiles of motors in asters, discussing the role of such effects in stabilizing single asters. We also comment on the implications of our results for experiments.

All meiotic chromosomes and both centrosomes at spindle pole in the zygotes discarded as two polar bodies in clam Corbicula leana: unusual polar body formation observed by antitubulin immunofluorescence.

To understand the unusual polar body formation in the androgenetic clam, Corbicula leana, whole-mount eggs stained with monoclonal antibodies against alpha-tubulin, gamma-tubulin, and 4'-6'-diamidino-2-phenylindole were examined. The meiotic spindle was located at the peripheral region of the egg at metaphase I, and its axis was parallel to the egg surface. After segregation of chromosomes at anaphase I, cytoplasmic bulges formed at both meiotic spindle pole sites. Centrosomes were located at the apical portion of the each bulge. From the apical portion of the bulge a bundle of astral microtubules radiated toward the bulge base in late anaphase resembling a half spindle. Maternal chromosomes and both centrosomes were all distributed in two "first polar bodies" and were eventually discarded. After the polar body formation only one male pronucleus existed in the egg cytoplasm. The present study showed that the anaphase microtubules originating from a single aster can induce the polar body formation without overlapping of microtubules from the opposing aster.

Initiation and Organization of Events During the First Cell Cycle in Mammals: Applications in Cloning

The technology of cloning involves transplanting a diploid nucleus into a mature oocyte cytoplast. The cytoplast is then activated to initiate the first cell cycle of development as a nuclear transplant embryo. Initiation and regulation of events during the first cell cycle are, therefore, critical for proper reprogramming of the donor nucleus and development as a cloned embryo. Activation is normally induced by the sperm and is mediated by a series of intracellular free calcium ([Ca(2+)](i)) oscillations that last for several hours. Although it is not known precisely how the sperm induces activation, current evidence favors the delivery, by the sperm, of a soluble protein factor that causes the production of IP3. IP3 acts to open a Ca(2+) channel in the endoplasmic reticulum and release Ca(2+) into the cytosol. A variety of methods have been used to duplicate or replace the sperm-induced [Ca(2+)](i) increase to cause activation in nuclear transplant embryos. It has been found that treatments that cause a single transient [Ca(2+)](i) activate some oocytes with the level of activation increasing as the oocyte ages. Attempts have been made to extend the period of time over which [Ca(2+)](i) oscillations occur. This has been successful in increasing activation rates of less mature oocytes but the techniques are still cumbersome. An alternative method, that has been very successful, is the combination of a treatment that elevates [Ca(2+)](i) and a treatment that maintains low levels of maturation promoting factor for several hours after the initial [Ca(2+)](i) elevation. The sperm also contributes the centrosome that organizes microtubules during the first cell cycle. One current hypothesis for regulation of sperm centrosomal activity consists of a dephosphorylation of sperm connecting piece proteins following sperm entry into the oocyte and activation of the oocyte. Dephosphorylation of these proteins results in the disassembly of the connecting piece and assembly of a functional centrosome. In nuclear transfer, centrosomal components are contributed by the donor cell. If the cell is fused to the cytoplast before centriole replication then a single aster forms. If the cell is fused after centriole replication then two asters form. In either case and even in parthenogenetic oocytes, which do not have centrioles, the first cell cycle progresses to metaphase. However, progress is slow and some defects are observed in the assembly of chromosomes into a metaphase plate.

Surface behavior in artificially constricted sand dollar eggs and egg fragments

AbstractIn mathematical and computer models of cytokinesis in animal cells, it is sometimes assumed that the nature of the surface is such that it will develop furrowing behavior independently of the mitotic apparatus in response to folding and distortion. Similarly, it has been postulated that after the dividing cell has achieved a moderate degree of constriction, the process is completed with little or no additional energy or preorganized structure. The purpose of these experiments was to test those assumptions by artificially constricting the surface of Echinarachnius parma eggs with a pair of hooks at the time of cytokinesis.When the surface of unnucleated egg fragments or those containing single asters were constricted to 27–30 μm in diameter, no furrowing occurred in the constriction plane, and the fragments immediately rounded up when the hooks were removed. When fragments containing a single aster were reshaped into cylinders, the surface developed astral constrictions. When intact cells were constricted so that the constriction plane did not coincide with the plane of an astral center, no furrowing occurred. When the constriction plane and that of the astral center coincided or when they were very close, furrowing activity developed and then regressed.These results indicate that the sand dollar egg surface does not tend to develop furrowing activity in response to imposed folds and distortion independently of the mitotic apparatus. They also suggest that single asters can elicit contractile activity in nearby surface.

Surface contractile activity associated with isolated asters in cylindrical sand dollar eggs

AbstractThe purpose of this investigation was to explore the capacity of isolated asters of the mitotic apparatus of elicit contractile activity in adjacent cell surface. When a fertilized Echinarachnius parma egg with an intact mitotic apparatus was confined in an 82‐μm‐i.d. silicone rubber capillary, the furrow developed at the midpoint between the asters. The mitotic apparatus could be broken by inserting and ejecting the egg from the capillary. The asters of a broken mitotic apparatus were usually abnormally far apart. When asters were as far as 68 μm apart, furrowing occurred; when the intervening distance was greater, there was no furrowing. When the asters were too far apart to establish a furrow between them, constrictions appeared adjacent to one of the asters. The constrictions could shift or could cut through the asters that appeared to elicit them. Cylindrical cells in 0.015 M ethyl urethane formed furrows when the mitotic apparatus was intact but formed neither furrows nor astral constrictions when the asters were far apart. Astral constrictions did not form in the presence of an active furrow. They were frequently temporary and they appeared to be slower and less acute than normal furrows. Single asters remaining after half of the amphiaster was removed had the same effect as widely separated asters. The form of the constriction was related to the position of the aster in the cylinder. When the aster was centered, the constriction resembled a furrow; but when it was near one of the poles, the adjacent surface pulled away from the capillary wall and the radius of curvature at the pole decreased. When initially distant asters were pushed together, a furrow formed between them. These results contribute to a better understanding of the interaction between the mitotic apparatus and the egg surface and the formation of the cleavage mechanism.

Ultrastructural analysis of the initiation and development of cytasters in sea-urchin eggs.

Two approaches were used to study the origin and overall development of cytasters in relation to the emergence and maturation of new centrioles in sea-urchin eggs. A continuous hypertonic treatment was used to gather information on cytoplasmic areas of potential cytaster formation and the subsequent development of these cytastral areas. A two-step parthenogenetic stimulation procedure was used to analyse various cytastral changes during and after mitosis. Potential cytastral areas are associated with extensive Golgi complexes and astral formation occurs only about newly induced centrioles. The construction of a single aster involves a large redistribution of local cytoplasmic structures, concentrating some components, excluding others and orienting more and more microtubules in an increasingly focussed arrangement. These events are correlated with an extensive accumulation of astral endoplasmic reticulum, an increase in the size of the astral area, and a gradual acquisition of a more typical radial configuration. The astral shape becomes more pronounced during mitosis, after which the cytasters regress, but retain the mature centrioles. The data reveal that cytaster formation is initiated after the activation and appearance of centriolar precursor bodies, and that both centrioles and associated cytasters are complementary structures, which develop in unison. The results suggest that developing centrioles, from the moment of their emergence, control and direct the events of cytastral formation.

Cytasters induced within unfertilized sea-urchin eggs.

Conditions that induce the formation of asters in unfertilized sea-urchin eggs have been investigated. Monasters were formed by treatment of eggs with acidic or basic sea-water, or procaine- or thymol-containing sea-water. A second treatment step, incubation with D2O-containing, ethanol-containing or hypertonic sea-water induced multiple cytasters. The number and size of cytasters varied according to the concentration of agents and duration of the first and second treatments, and also upon the species of eggs and the season in which the eggs were obtained. Generally, a longer second treatment or a higher concentration of the second medium resulted in a higher number of cytasters per egg. Asters were isolated and then examined by light and electron microscopy. Isolated monasters apparently lacked centrioles, whereas cytasters obtained from eggs undergoing the two-step treatment contained one or more centrioles. Up to eight centrioles were seen in a single aster; the centrioles appeared to have been produced during the second incubation. Centrospheres prepared from isolated asters retained the capacity to nucleate the formation of microtubules in vitro as assayed by light and electron microscopy. Many microtubules radiated from the centre of isolated asters, whether they contained centrioles or not. This observation is consistent with many other reports that microtubule-organizing centres need not contain centrioles.