Sea Urchin Zygotes Research Articles

A Pushing Mechanism for Microtubule Aster Positioning in a Large Cell Type

After fertilization, microtubule (MT) sperm asters undergo long-range migration to accurately position pronuclei. Due to the large sizes of zygotes, the forces driving aster migration are considered to be from pulling on the astral MTs by dynein, with no significant contribution from pushing forces. Here, we re-investigate the forces responsible for sperm aster centration in sea urchin zygotes. Our quantifications of aster geometry and MT density preclude a pulling mechanism. Manipulation of aster radial lengths and growth rates, combined with quantitative tracking of aster migration dynamics, indicates that aster migration is equal to the length of rear aster radii, supporting a pushing model for centration. We find that dynein inhibition causes an increase in aster migration rates. Finally, ablation of rear astral MTs halts migration, whereas front and side ablations do not. Collectively, our data indicate that a pushing mechanism can drive the migration of asters in a large cell type.

Resistance is futile: Centering forces yield for asymmetric cell division.

Asymmetric cell division relies on microtubule-based forces to asymmetrically position the mitotic apparatus. In this issue, Sallé et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201807102) use magnetic tweezers to induce asymmetric division in sea urchin zygotes, demonstrating that asymmetry could arise from a time-dependent weakening of centering forces.

Asymmetric division through a reduction of microtubule centering forces.

Asymmetric divisions are essential for the generation of cell fate and size diversity. They implicate cortical domains where minus end-directed motors, such as dynein, are activated to pull on microtubules to decenter asters attached to centrosomes, nuclei, or spindles. In asymmetrically dividing cells, aster decentration typically follows a centering phase, suggesting a time-dependent regulation in the competition between microtubule centering and decentering forces. Using symmetrically dividing sea urchin zygotes, we generated cortical domains of magnetic particles that spontaneously cluster endogenous dynein activity. These domains efficiently attract asters and nuclei, yielding marked asymmetric divisions. Remarkably, aster decentration only occurred after asters had first reached the cell center. Using intracellular force measurement and models, we demonstrate that this time-regulated imbalance results from a global reduction of centering forces rather than a local maturation of dynein activity at the domain. Those findings define a novel paradigm for the regulation of division asymmetry.

Antibodies to sea urchin zygote particles and sperm vesicles reveal unexpected structures during cleavage and localize in known fate regions

Antibodies to sea urchin zygote particles and sperm vesicles reveal unexpected structures during cleavage and localize in known fate regions

Using sea urchin gametes and zygotes to investigate centrosome duplication.

Centriole structure and function in the sea urchin zygote parallel those in mammalian somatic cells. Here, I briefly introduce the properties and attributes of the sea urchin system that make it an attractive platform for the study of centrosome and centriole duplication. These attributes apply to all echinoderms readily available from commercial suppliers: sea urchins, sand dollars, and starfish. I list some of the practical aspects of the system that make it a cost- and time-effective system for experimental work and then list properties that are a “tool kit” that can be used to conduct studies that would not be practical, or in some cases not possible, with mammalian somatic cells. Since centrioles organize and localize the pericentriolar material that nucleates the astral arrays of microtubules (Bobinnec et al. in J Cell Biol 143(6):1575–1589, 1998), the pattern of aster duplication over several cell cycles can be used as a reliable measure for centriole duplication (Sluder and Rieder in J Cell Biol 100(3):887–896, 1985). Descriptions of the methods my laboratory has used to handle and image echinoderm zygotes are reviewed in Sluder et al. (Methods Cell Biol 61:439–472, 1999). Also included is a bibliography of papers that describe additional methods.

High throughput microinjections of sea urchin zygotes.

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

High Throughput Microinjections of Sea Urchin Zygotes

High Throughput Microinjections of Sea Urchin Zygotes

Localization of cytokinesis factors to the future cell division site by microtubule‐dependent transport

The mechanism by which spindle microtubules (MTs) determine the site of cell division in animal cells is still highly controversial. Putative cytokinesis "signals" have been proposed to be positioned by spindle MTs at equatorial cortical regions to increase cortical contractility and/or at polar regions to decrease contractility [Rappaport, 1986; von Dassow, 2009]. Given the relative paucity of MTs at the future division site, it has not been clear how MTs localize cytokinesis factors there. Here, we test cytokinesis models using computational and experimental approaches. We present a simple lattice-based model in which signal-kinesin complexes move by transient plus-end directed movements on MTs interspersed with occasions of uniform diffusion in the cytoplasm. In simulations, complexes distribute themselves initially at the spindle midzone and then move on astral MTs to accumulate with time at the equatorial cortex. Simulations accurately predict cleavage patterns of cells with different geometries and MT arrangements and elucidate several experimental observations that have defied easy explanation by previous models. We verify this model with experiments on indented sea urchin zygotes showing that cells often divide perpendicular to the spindle at sites distinct from the indentations. These studies support an equatorial stimulation model and provide a simple mechanism explaining how cytokinesis factors localize to the future division site.

Cyclin E in centrosome duplication and reduplication in sea urchin zygotes

When protein synthesis is completely blocked from before fertilization, the sea urchin zygote arrests in first S phase and the paternal centrosome reduplicates multiple times. However, when protein synthesis is blocked starting in prophase of first mitosis, the zygote divides and the blastomeres arrest in a G1-like state. The centrosome inherited from this mitosis duplicates only once in each blastomere for reasons that are not understood. The late G1 rise in cyclin E/cdk2 kinase activity initiates centrosome duplication in mammalian cells and its activity is needed for centrosome duplication in Xenopus egg extracts. Since the half-time for cyclin E turnover is normally approximately 1 h in sea urchin zygotes, the different behaviors of centrosomes during G1 and S phase arrests could be due to differential losses of cyclin E and its associated kinase activities at these two arrest points. To better understand the mechanisms that limit centrosome duplication, we characterize the levels of cyclin E and its associated kinase activity at the S phase and G1 arrest points. We first demonstrate that cyclin E/cdk2 kinase activity is required for centrosome duplication and reduplication in sea urchin zygotes. Next we find that cyclin E levels and cyclin E/cdk2 kinase activities are both constitutively and equivalently elevated during both the S phase and G1 arrests. This indicates that centrosome duplication during the G1 arrest is limited by a block to reduplication under conditions permissive for duplication. The cytoplasmic conditions of S phase, however, abrogate this block to reduplication.

Cyclin E in centrosome duplication and reduplication in sea urchin zygotes

Cyclin E in centrosome duplication and reduplication in sea urchin zygotes

Cyclin E/Cdk2 is required for sperm maturation, but not DNA replication, in early sea urchin embryos

The cell cycle is driven by the activity of cyclin/cdk complexes. In somatic cells, cyclin E/cdk2 oscillates throughout the cell cycle and has been shown to promote S-phase entry and initiation of DNA replication. In contrast, cyclin E/cdk2 activity remains constant throughout the early embryonic development of the sea urchin and localizes to the sperm nucleus following fertilization. We now show that cyclin E localization to the sperm nucleus following fertilization is not unique to the sea urchin, but also occurs in the surf clam, and inhibition of cyclin E/cdk2 activity by roscovitine inhibits the morphological changes indicative of male pronuclear maturation in sea urchin zygotes. Finally, we show that inhibition of cyclin E/cdk2 activity does not block DNA replication in the early cleavage cycles of the sea urchin. We conclude that cyclin E/cdk2 activity is required for male pronuclear maturation, but not for initiation of DNA replication in early sea urchin development.

“Birth of the cool”: Using sea urchin zygotes to study centrosome duplication, cell division, and cytokinesis

AbstractThe successful division of the cell depends on several morphological events that must be coordinated in a cell cycle dependent fashion. Crucial to this process is the remodeling of the cytoskeleton to both establish the bipolar spindle during mitosis, and cleave the cell into two daughters during cytokinesis. From the standpoint of the cytoskeleton, this process begins during interphase with the duplication of the centrosome; it is the two daughter centrosomes that will assemble the poles of the mitotic spindle and establish its necessary bipolarity. Following the cell cycle transition into mitosis, the spindle must assemble in order to properly align the sister chromatids at the center of the cell, and release the “wait anaphase checkpoint”. As the spindle transports the disjoined sister chromatids to the spindle poles, the cell must rapidly undergo cytokinesis, cleaving the cell into two – the plane of cleavage being established by the spindle itself. Understanding the temporal regulation and molecular basis for these events has come from extensive experiments using a variety of model systems, and has benefited from cell biological, molecular genetic, and biophysical approaches. One of the earliest and most important model systems for studying mitosis is the sea urchin zygote. With their large size, rapid/synchronous cell cycles, and advantages for conduct of biochemical and cytological investigations on the same system, fertilized sea urchin eggs have revealed many of the fundamental properties of centrosome duplication, cell division and cytokinesis. Here we review several key studies that have utilized the sea urchin zygote to explore mechanisms that coordinate and drive two of the major cytoskeletal events of mitotic division – centrosome duplication and cytokinesis.

The Drosophila Calcipressin Sarah Is Required for Several Aspects of Egg Activation

Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract ; it is unknown whether calcium signaling is involved. We report here that mutations in sarah, which encodes an inhibitor of the calcium-dependent phosphatase calcineurin, disrupt several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. We also find the conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation.

Targeted new membrane addition in the cleavage furrow is a late, separate event in cytokinesis.

Cytokinesis in animal cells is accomplished in part by an actomyosin contractile ring. Recent work on amphibian, Drosophila, and Caenorhabditis elegans embryos implicates membrane trafficking and delivery as essential for cytokinesis. However, the relative contributions of contractile ring constriction versus membrane insertion to cytokinesis and the temporal relationship between these processes are largely unexplored. Here we monitor secretion of the extracellular matrix protein, hyalin, as a marker for new plasma membrane addition in dividing sea urchin zygotes. We find that new membrane addition occurs specifically in the cleavage furrow late in telophase independent of contractile ring constriction. The directed equatorial deposition of new furrow membrane requires astral microtubules and release of internal stores of Ca(2+), but not the presence of a central spindle. Further, cells arrested in M phase do not secrete hyalin, suggesting that mitotic exit is required for new membrane addition. These results demonstrate that astral overlap in equilaterally dividing cells not only serves to specify positioning and contraction of the contractile ring, but also to direct the delivery of new membrane to the furrow as a late, independent event during cytokinesis.

Nucleo-cytoplasmic interactions that control nuclear envelope breakdown and entry into mitosis in the sea urchin zygote.

In sea urchin zygotes and mammalian cells nuclear envelope breakdown (NEB) is not driven simply by a rise in cytoplasmic cyclin dependent kinase 1-cyclin B (Cdk1-B) activity; the checkpoint monitoring DNA synthesis can prevent NEB in the face of mitotic levels of Cdk1-B. Using sea urchin zygotes we investigated whether this checkpoint prevents NEB by restricting import of regulatory proteins into the nucleus. We find that cyclin B1-GFP accumulates in nuclei that cannot complete DNA synthesis and do not break down. Thus, this checkpoint limits NEB downstream of both the cytoplasmic activation and nuclear accumulation of Cdk1-B1. In separate experiments we fertilize sea urchin eggs with sperm whose DNA has been covalently cross-linked to inhibit replication. When the pronuclei fuse, the resulting zygote nucleus does not break down for >180 minutes (equivalent to three cell cycles), even though Cdk1-B activity rises to greater than mitotic levels. If pronuclear fusion is prevented, then the female pronucleus breaks down at the normal time (average 68 minutes) and the male pronucleus with cross-linked DNA breaks down 16 minutes later. This male pronucleus has a functional checkpoint because it does not break down for >120 minutes if the female pronucleus is removed just prior to NEB. These results reveal the existence of an activity released by the female pronucleus upon its breakdown, that overrides the checkpoint in the male pronucleus and induces NEB. Microinjecting wheat germ agglutinin into binucleate zygotes reveals that this activity involves molecules that must be actively translocated into the male pronucleus.

Licensing centrosome duplication

HINCHCLIFFE, E. H., CASSELS, G. O., RIEDER, C. L. and SLUDER, G. (1998) The coordination of centrosome reproduction with nuclear events of the cell cycle in the sea urchin zygote J Cell Biol. 140, 1417–1426

The coordination of centrosome reproduction with nuclear events of the cell cycle in the sea urchin zygote.

Centrosomes repeatedly reproduce in sea urchin zygotes arrested in S phase, whether cyclin-dependent kinase 1-cyclin B (Cdk1-B) activity remains at prefertilization levels or rises to mitotic values. In contrast, when zygotes are arrested in mitosis using cyclin B Delta-90, anaphase occurs at the normal time, yet centrosomes do not reproduce. Together, these results reveal the cell cycle stage specificity for centrosome reproduction and demonstrate that neither the level nor the cycling of Cdk1-B activity coordinate centrosome reproduction with nuclear events. In addition, the proteolytic events of the metaphase-anaphase transition do not control when centrosomes duplicate. When we block protein synthesis at first prophase, the zygotes divide and arrest before second S phase. Both blastomeres contain just two complete centrosomes, which indicates that the cytoplasmic conditions between mitosis and S phase support centrosome reproduction. However, the fact that these daughter centrosomes do not reproduce again under such supportive conditions suggests that they are lacking a component required for reproduction. The repeated reproduction of centrosomes during S phase arrest points to the existence of a necessary "licensing" event that restores this component to daughter centrosomes during S phase, preparing them to reproduce in the next cell cycle.

Two proteins isolated from sea urchin sperm flagella: structural components common to the stable microtubules of axonemes and centrioles.

Biochemical fractionation of axonemal microtubules yields the protofilament ribbon (pf-ribbon), an insoluble structure of 3-4 longitudinal protofilaments composed primarily of alpha/beta tubulin, tektins A, B and C, and two previously uncharacterized polypeptides of 77 kDa and 83 kDa. We have isolated the 77/83 kDa polypeptides (termed Sp77 and Sp83) from sperm flagella of the sea urchin Stronglyocentrotus purpuratus and raised polyclonal antibodies against them. Sp77 and Sp83 copurify exclusively with the pf-ribbon. Both the anti-Sp77 and anti-Sp83 antibodies detected the nine outer doublets and the basal bodies of sea urchin sperm by immunofluorescence microscopy. In addition, the anti-Sp83 antibody, but not the anti-Sp77 antibody, detected a single 83 kDa polypeptide on immunoblots of unfertilized sea urchin egg cytoplasm, and a single polypeptide of 80 kDa on blots of isolated mitotic spindles from Chinese hamster ovary (CHO) cells. Previous studies have shown that tektins are present in the basal bodies and centrosomes/centrioles of cells ranging from clam to human. We found that anti-Sp83 decorates the spindle poles in sea urchin zygotes, and the interphase centrosome and spindle poles in CHO cells. In CHO cells arrested in S phase with aphidicolin, anti-Sp83 detects multiple centrosomes. The staining of the centrosome was not disrupted by prolonged nocodazole treatment, suggesting that the 80 kDa polypeptide is associated with the centrioles themselves. Our observations demonstrate that, like tektins, Sp77 and Sp83 are structural proteins associated with stable doublet microtubules, and may be components of basal bodies and centrioles of sea urchins and mammalian cells.

Chapter 22 Using Sea Urchin Gametes for the Study of Mitosis

Publisher Summary This chapter describes the methods found useful for the study of cell division in early cleavage stage sea urchin zygotes. Successful and reliable fertilization of sea urchin eggs is simple if a few precautions are kept in mind. First, use only clean glassware that has not been exposed to fixatives or metal ions. If the glassware is washed in a central facility, be certain that there is no residual detergent present. There have been good results with the use of “conditioned” glassware that is dedicated to use with live cells. At the end of the day rinse the glassware with distilled water and store it filled with distilled water. The glassware should be thoroughly washed if it is exposed to drugs. To conduct routine fertilizations prepare a sperm solution adding just enough sperm to the test tube to make the water barely opalescent. If the sperms are too concentrated, there is the risk of polyspermy. A dilute solution of eggs is prepared and a small amount of sperm solution is added. There is no precise ratio that needs to be attained, but the water around the eggs should appear clear to the eye.

Identification of a cysteine protease responsible for degradation of sperm histones during male pronucleus remodeling in sea urchins

We have identified a 60-kDa cysteine protease that is associated with chromatin in sea urchin zygotes. This enzyme was found to be present as a proenzyme in unfertilized eggs and was activated shortly after fertilization. At a pH of 7.8-8.0, found after fertilization, the enzyme degraded the five sperm-specific histones (SpH), while the native cleavage-stage (CS) histone variants remained unaffected. Based on its requirements for reducing agents, its inhibition by sulfhydryl blocking compounds and its sensitivity to the cysteine-type protease inhibitors (2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane-ethyl-es ter (E-64 d), cystatin and leupeptin, this protease can be defined as a cysteine protease. Consistently, this protease was not affected by serine-type protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and pepstatin. The substrate selectivity and pH modulation of the protease activity strongly suggest its role in the removal of sperm-specific histones, which determines sperm chromatin remodeling after fertilization. This suggestion was further substantiated by the inhibition of sperm histones degradation in vivo by E-64 d. Based on these three lines of evidence, we postulate that this cysteine protease is responsible for the degradation of sperm-specific histones which occurs during male pronucleus formation.