Reduction of cortical pulling at mitotic entry facilitates aster centration.

Equal cell division relies upon astral microtubule-based centering mechanisms, yet how the interplay between mitotic entry, cortical force generation and long astral microtubules leads to symmetric cell division is not resolved. We report that a cortically located sperm aster displaying long astral microtubules that penetrate the whole zygote does not undergo centration until mitotic entry. At mitotic entry, we find that microtubule-based cortical pulling is lost. Quantitative measurements of cortical pulling and cytoplasmic pulling together with physical simulations suggested that a wavelike loss of cortical pulling at mitotic entry leads to aster centration based on cytoplasmic pulling. Cortical actin is lost from the cortex at mitotic entry coincident with a fall in cortical tension from ∼300pN/µm to ∼100pN/µm. Following the loss of cortical force generators at mitotic entry, long microtubule-based cytoplasmic pulling is sufficient to displace the aster towards the cell center. These data reveal how mitotic aster centration is coordinated with mitotic entry in chordate zygotes.

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.

Microtubule-Based Mechanisms of Pronuclear Positioning.

The zygote is defined as a diploid cell resulting from the fusion of two haploid gametes. Union of haploid male and female pronuclei in many animals occurs through rearrangements of the microtubule cytoskeleton into a radial array of microtubules known as the sperm aster. The sperm aster nucleates from paternally-derived centrioles attached to the male pronucleus after fertilization. Nematode, echinoderm, and amphibian eggs have proven as invaluable models to investigate the biophysical principles for how the sperm aster unites male and female pronuclei with precise spatial and temporal regulation. In this review, we compare these model organisms, discussing the dynamics of sperm aster formation and the different force generating mechanism for sperm aster and pronuclear migration. Finally, we provide new mechanistic insights for how sperm aster growth may influence sperm aster positioning.

Echinoderm eggs as a model for discoveries in cell biology.

I happen to have been trained in cell and developmental biology in the early 1970s, which was near the beginning of the explosive growth of the field of cell biology. The American Society for Cell Biology had been founded in 1960 and so the field was in its early days. Cell biology research was dominated by the use of the electron microscope and by protein biochemistry. Molecular biology and the use of genetics were in their infancy. When we track the path of discoveries in cell biology contributed by research using echinoderm eggs, we follow the development of new technologies in genetics, molecular biology, biochemistry and biophysics, bioengineering, and imaging. The changes in approaches and methods have led to many key discoveries in cell biology through the use of sea urchin, sand dollar and sea star eggs. These include the discovery of cyclin, cytoplasmic dynein, rho activation for cytokinesis, new membrane addition as a late event in cytokinesis, multiple kinesins playing multiple roles, how flagella beat, the dynamics of microtubules in the mitotic apparatus, control over centrosomes and cell cycle checkpoints, the process of nuclear envelope breakdown for cell division, the discovery of 1-methyl adenine (hormones) as the trigger for meiotic maturation, Ca++ transients controlling cell activation and exocytosis among others. What I hope to provide in this perspective is to highlight some of those wonderful discoveries as my own career evolved to contribute to the field.

Influence of cell polarity on early development of the sea urchin embryo.

BACKGROUND: Establishment and maintenance of cell polarity is critical for normal embryonic development. Previously, it was thought that the echinoderm embryo remained relatively unpolarized until the first asymmetric division at the 16-cell stage. Here, we analyzed roles of the cell polarity regulators, the PAR complex proteins, and how their disruption in early development affects later developmental milestones. RESULTS: We found that PAR6, aPKC, and CDC42 localize to the apical cortex as early as the 2-cell stage and that this localization is retained through the gastrula stage. Of interest, PAR1 also colocalizes with these apical markers through the gastrula stage. Additionally, PAR1 was found to be in complex with aPKC, but not PAR6. PAR6, aPKC, and CDC42 are anchored in the cortical actin cytoskeleton by assembled myosin. Furthermore, assembled myosin was found to be necessary to maintain proper PAR6 localization through subsequent cleavage divisions. Interference with myosin assembly prevented the embryos from reaching the blastula stage, while transient disruptions of either actin or microtubules did not have this effect. CONCLUSIONS: These observations suggest that disruptions of the polarity in the early embryo can have a significant impact on the ability of the embryo to reach later critical stages in development.

How to be at the right place at the right time: the importance of spindle positioning in embryos.

Spindle positioning is an imperative cellular process that regulates a number of different developmental events throughout embryogenesis. The spindle must be properly positioned in embryos not only for the segregation of chromosomes, but also to segregate developmental determinants into different daughter blastomeres. In this review, the role of spindle positioning is explored in several different developmental model systems, which have revealed the diversity of factors that regulate spindle positioning. The C. elegans embryo, the Drosophila neuroblast, and ascidian embryos have all been utilized for the study of polarity-dependent spindle positioning, and exploration of the proteins that are required for asymmetric cell division. Work in the sea urchin embryo has examined the influence of cell shape and factors that affect secondary furrow formation. The issue of size scaling in extremely large cells, as well as the requirement for spindle positioning in developmental fate decisions in vertebrates, has been addressed by work in the Xenopus embryo. Further work in mouse oocytes has examined the roles of actin and myosin in spindle positioning. The data generated from these model organisms have made unique contributions to our knowledge of spindle positioning. Future work will address how all of these different factors work together to regulate the position of the spindle.

Polar expansion during cytokinesis.

Vesicle trafficking and new membrane addition at the cleavage furrow have been extensively documented. However, less clear is the old idea that expansion at the cell poles occurs during cytokinesis. We find that new membrane is added to the cell poles during anaphase, causing the plasma membrane to expand coincident with the constriction of the contractile ring and may provide a pushing force for membrane ingression at the furrow. This membrane addition occurs earlier during mitosis than membrane addition at the furrow and is dependent on actin and astral microtubules. The membrane that is added at the polar regions is compositionally distinct from the original cell membrane in that it is devoid of GM(1) , a component of lipid rafts. These findings suggest that the growth of the plasma membrane at the cell poles during cell division is not due to stretching as previously thought, but due to the addition of compositionally unique new membrane.

'Life is a highway': membrane trafficking during cytokinesis.

Cytokinesis, the final stage of the cell cycle, is an essential step toward the formation of two viable daughter cells. In recent years, membrane trafficking has been shown to be important for the completion of cytokinesis. Vesicles originating from both the endocytic and secretory pathways are known to be shuttled to the plasma membrane of the ingressing cleavage furrow, delivering membrane and proteins to this dynamic region. Advances in cell imaging have led to exciting new discoveries regarding vesicle movement in living cells. Recent work has revealed a significant role for membrane trafficking, as controlled by regulatory proteins, during cytokinesis in animal cells. The endocytic and secretory pathways as well as motor proteins are revealed to be essential in the delivery of vesicles to the cleavage furrow during cytokinesis.

Cell polarity emerges at first cleavage in sea urchin embryos.

In protostomes, cell polarity is present after fertilization whereas most deuterostome embryos show minimal polarity during the early cleavages. We now show establishment of cell polarity as early as the first cleavage division in sea urchin embryos. We find, using the apical markers G(M1), integrins, and the aPKC-PAR6 complex, that cells are polarized upon insertion of distinct basolateral membrane at the first division. This early apical-basolateral polarity, similar to that found in much larger cleaving amphibian zygotes, reflects precocious functional epithelial cell polarity. Isolated cleavage blastomeres exhibit polarized actin-dependent fluid phase endocytosis only on the G(M1), integrin, microvillus-containing apical surface. A role for a functional PAR complex in cleavage plane determination was shown with experiments interfering with aPKC activity, which results in several spindle defects and compromised blastula development. These studies suggest that cell and embryonic polarity is established at the first cleavage, mediated in part by the Par complex of proteins, and is achieved by directed insertion of basolateral membrane in the cleavage furrow.

Cytokinesis: a new lipid aboard the raft.

Several components of membrane rafts play a critical role in cytokinesis. A recent paper reports a new lipid component of these rafts required for proper cell division.

Cytokinesis and the establishment of early embryonic cell polarity.

Cleavage divisions in many animals form a blastula made up of a simple polarized epithelium. This simple embryonic epithelium possesses an apical surface covered with microvilli and primary cilia separated from the basolateral surfaces by cell-cell junctions. The apical membrane proteins and lipids differ from those of the basolateral on these embryonic epithelial cells, as is found in adult epithelial cells. Formation of cell polarity in embryos at fertilization, including those from both protostomes and deuterostomes, uses the same molecules and signalling machinery as do polarizing epithelial cells that polarize upon cell-cell contact. In addition, the actin-myosin cytoskeleton plays an integral role in establishment and maintenance of this early cell polarity. However, early cleaving blastomeres from higher organisms including echinoderms and vertebrates have not been considered to exhibit cell polarity until formation of junctions at the third through to the fifth cleavage divisions. The role of new membrane addition into the late cleavage furrow during the early rounds of cytokinesis may play a key role in the early establishment of cell polarity in all animal embryos.

Cytokinesis: LET-ting the asters signal.

Cytokinesis is regulated by both astral microtubules and the midzone microtubules of the mitotic apparatus. A new study in Caenorhabditis elegans has identified the polarity factor LET-99 and its heterotrimeric G-protein regulators as components of the signaling pathway downstream of astral microtubules.

The genome of the sea urchin Strongylocentrotus purpuratus.

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.

Movement of membrane domains and requirement of membrane signaling molecules for cytokinesis.

Plasma membrane subdomains enriched in sphingolipids, cholesterol, and signaling proteins are critical for organization of actin, membrane trafficking, and cell polarity, but the role of such domains in cytokinesis in animal cells is unknown. Here, we show that eggs form a plasma membrane domain enriched in ganglioside G(M1) and cholesterol where tyrosine phosphorylated proteins occur at late anaphase at the contractile ring. The equatorial membrane domain forms by movement-specific lipids and proteins and is dependent on anaphase onset, myosin light chain phosphorylation, actin, and microtubules. Isolated detergent-resistant membranes contain Src and PLCgamma, which become tyrosine phosphorylated at cytokinesis, and whose activation is required for furrow progression. These studies suggest that membrane domains at the cleavage furrow possess a signaling pathway that contributes to cytokinesis.

Induction of cytokinesis is independent of precisely regulated microtubule dynamics.

Astral microtubules (MTs) emanating from the mitotic apparatus (MA) during anaphase are required for stimulation of cytokinesis in eggs. We have used green fluorescent protein-labeled EB1 to observe MT dynamics during mitosis and cytokinesis in normal sea urchin eggs. Analysis of astral MT growth rates during anaphase shows that MTs contact the polar cortex earlier than the equatorial cortex after anaphase onset but that a normal cleavage furrow is not induced until contact with MTs has been achieved throughout the cortex. To assess the role of MT dynamics in initiation of cytokinesis, we used a collection of small molecule drugs to affect dynamics. Hexylene glycol resulted in rapid astral elongation due to decreased MT catastrophe and precocious furrowing. Taxol suppressed MT dynamics but did not inhibit furrow induction when the MA was manipulated toward the cortex. Urethane resulted in short, highly dynamic astral MTs with increased catastrophe that also stimulated furrowing upon being brought into proximity to the cortex. Our findings indicate that astral MT contact with the cortex is necessary for furrow initiation but that the dynamic state of astral MTs does not affect their competency to stimulate furrowing.

Cytokinesis: new roles for myosin.

Myosin II is the motor for cytokinesis, an event at the end of cell division during which the animal cell uses a contractile ring to pinch itself in half. New and surprising research shows that myosin, either through light chain phosphorylation or through its ATPase activity, also plays an important role in both the assembly and disassembly of the actin contractile ring.

Site selection for the cleavage furrow at cytokinesis.

The question of how the site for division of the cytoplasm is determined at the end of mitosis has been studied for over a century, and it remains an active, controversial and fascinating problem in cell biology. This problem draws on the use of several model cell types, with the goal of understanding and identifying how the cell cycle regulates signals between the mitotic apparatus and the cell cortex. Studies in different cell types and using a vast array of techniques reveal different answers: these might reflect differences in experimental approaches, multiple and redundant mechanisms and, importantly, diversity in biology. In this article (which is part of the Cytokinesis series), we present a summary and critique of the major models for the roles of the mitotic apparatus microtubules in stimulating furrow formation at cytokinesis.

Pathways for membrane trafficking during cytokinesis.

The molecular mechanisms underlying targeted deposition of new membrane at the advancing furrow of a dividing cell have long been intriguing to cell biologists. Three recent studies have made use of Drosophila cellularization to explore current questions in this field. These findings indicate that both the secretory pathway and endosomal recycling contribute membrane to the advancing furrow. Furthermore, new work reveals that vesicles derived from the Rab11 recycling endosome (RE) promote actin remodeling at the furrow.

Transitions regulating the timing of cytokinesis in embryonic cells.

Anaphase, mitotic exit, and cytokinesis proceed in rapid succession, and while mitotic exit is a requirement for cytokinesis in yeast, it may not be a direct requirement for furrow initiation in animal cells. In this report, we physically manipulated the proximity of the mitotic apparatus (MA) to the cell cortex in combination with microinjection of effectors of the spindle checkpoint and CDK1 activity to determine how the initiation of cytokinesis is coupled to the onset of anaphase and mitotic exit. Whereas precocious contact between the MA and the cell surface advanced the onset of cytokinesis into early anaphase A, furrowing could not be advanced prior to the metaphase-anaphase transition. Additionally, while cells arrested in anaphase could be induced to initiate cleavage furrows, cells arrested in metaphase could not. Finally, activation of the mitotic checkpoint in one spindle of a binucleate cell failed to arrest cytokinesis induced by the control spindle but did inhibit the formation of furrows between the arrested MA and the control, nonarrested MA. Our experiments suggest that the competence of the mitotic apparatus to initiate cytokinesis is not dependent on cyclin degradation but does require anaphase-promoting complex (APC) activity and, thus, inactivation of the mitotic checkpoint.