Cortical Contraction Research Articles

Loss of PTEN induces microtentacles through PI3K-independent activation of cofilin

Loss of PTEN tumor suppressor enhances metastatic risk in breast cancer, although the underlying mechanisms are poorly defined. We report that homozygous deletion of PTEN in mammary epithelial cells induces tubulin-based microtentacles (McTNs) that facilitate cell reattachment and homotypic aggregation. Treatment with contractility-modulating drugs showed that McTNs in PTEN(-/-) cells are suppressible by controlling the actin cytoskeleton. Because outward microtubule extension is counteracted by actin cortical contraction, increased activity of actin-severing proteins could release constraints on McTN formation in PTEN(-/-) cells. One such actin-severing protein, cofilin, is activated in detached PTEN(-/-) cells that could weaken the actin cortex to promote McTNs. Expression of wild-type cofilin, an activated mutant (S3A), and an inactive mutant (S3E) demonstrated that altering cofilin phosphorylation directly affects McTNs formation. Chemical inhibition of PI3K did not reduce McTNs or inactivate cofilin in PTEN(-/-) cells. Additionally, knock-in expression of the two most common PI3K-activating mutations observed in human cancer patients did not increase McTNs or activate cofilin. PTEN loss and PI3K activation also caused differential activation of the cofilin regulators, LIM-kinase1 (LIMK) and Slingshot-1L (SSH). Furthermore, McTNs were suppressed and cofilin was inactivated by restoration of PTEN in the PTEN(-/-) cells, indicating that both the elevation of McTNs and the activation of cofilin are specific results arising from PTEN loss. These data identify a novel mechanism by which PTEN loss could remodel the cortical actin network to facilitate McTNs that promote tumor cell reattachment and aggregation. Using isogenic MCF-10A PTEN(-/-) and PIK3CA mutants, we have further demonstrated that there are clear differences in activation of cofilin, LIMK and SSH between PTEN loss and PI3K activation, providing a new evidence that these mutations yield distinct cytoskeletal phenotypes, which could have an impact on tumor biology.

The septin cytoskeleton facilitates membrane retraction during motility and blebbing

Increasing evidence supports a critical role for the septin cytoskeleton at the plasma membrane during physiological processes including motility, formation of dendritic spines or cilia, and phagocytosis. We sought to determine how septins regulate the plasma membrane, focusing on this cytoskeletal element's role during effective amoeboid motility. Surprisingly, septins play a reactive rather than proactive role, as demonstrated during the response to increasing hydrostatic pressure and subsequent regulatory volume decrease. In these settings, septins were required for rapid cortical contraction, and SEPT6-GFP was recruited into filaments and circular patches during global cortical contraction and also specifically during actin filament depletion. Recruitment of septins was also evident during excessive blebbing initiated by blocking membrane trafficking with a dynamin inhibitor, providing further evidence that septins are recruited to facilitate retraction of membranes during dynamic shape change. This function of septins in assembling on an unstable cortex and retracting aberrantly protruding membranes explains the excessive blebbing and protrusion observed in septin-deficient T cells.

Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes

MaturemammalianoocytesarepoisedforcompletingmeiosisII (MII) on fertilization by positioning the spindle close to an actomyosin-rich cortical cap. Here, we show that the Arp2/3 complex localizes to the cortical cap in a Ran-GTPase-dependent manner and nucleates actin filaments in the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 activity leads to rapid dissociation of the spindle from the cortex. Live-cell imaging and spatiotemporal image correlation spectroscopy analysis reveal that actin filaments flow continuously away from the Arp2/3-rich cortex, driving a cytoplasmic streaming expected to exert a net pushing force on the spindle towards the cortex. Arp2/3 inhibition not only diminishes this actin flow and cytoplasmic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, moving the spindle away from the cortex. Thus, the asymmetric MII spindle position is dynamically maintained as a result of balanced forces governed by the Arp2/3 complex.

Initial diameter of the polar body contractile ring is minimized by the centralspindlin complex

Polar body formation is an essential step in forming haploid eggs from diploid oocytes. This process involves completion of a highly asymmetric cytokinesis that results in a large egg and two small polar bodies. Unlike mitotic contractile rings, polar body contractile rings assemble over one spindle pole so that the spindle must move through the contractile ring before cytokinesis. During time-lapse imaging of C. elegans meiosis, the contractile ring moved downward along the length of the spindle and completed scission at the midpoint of the spindle, even when spindle length or rate of ring movement was increased. Patches of myosin heavy chain and dynamic furrowing of the plasma membrane over the entire embryo suggested that global cortical contraction forces the meiotic spindle and overlying membrane out through the contractile ring center. Consistent with this model, depletion of myosin phosphatase increased the velocity of ring movement along the length of the spindle. Global dynamic furrowing, which was restricted to anaphase I and II, was dependent on myosin II, the anaphase promoting complex and separase, but did not require cortical contact by the spindle. Large cortical patches of myosin during metaphase I and II indicated that myosin was already in the active form before activation of separase. To identify the signal at the midpoint of the anaphase spindle that induces scission, we depleted two proteins that mark the exact midpoint of the spindle during late anaphase, CYK-4 and ZEN-4. Depletion of either protein resulted in the unexpected phenotype of initial ingression of a polar body ring with twice the diameter of wild type. This phenotype revealed a novel mechanism for minimizing polar body size. Proteins at the spindle midpoint are required for initial ring ingression to occur close to the membrane-proximal spindle pole.

The c-Myc target glycoprotein1balpha links cytokinesis failure to oncogenic signal transduction pathways in cultured human cells.

An increase in chromosome number, or polyploidization, is associated with a variety of biological changes including breeding of cereal crops and flowers, terminal differentiation of specialized cells such as megakaryocytes, cellular stress and oncogenic transformation. Yet it remains unclear how cells tolerate the major changes in gene expression, chromatin organization and chromosome segregation that invariably accompany polyploidization. We show here that cancer cells can initiate increases in chromosome number by inhibiting cell division through activation of glycoprotein1b alpha (GpIbα), a component of the c-Myc signaling pathway. We are able to recapitulate cytokinesis failure in primary cells by overexpression of GpIbα in a p53-deficient background. GpIbα was found to localize to the cleavage furrow by microscopy analysis and, when overexpressed, to interfere with assembly of the cellular cortical contraction apparatus and normal division. These results indicate that cytokinesis failure and tetraploidy in cancer cells are directly linked to cellular hyperproliferation via c-Myc induced overexpression of GpIbα.

A mitotic kinesin-6, Pav-KLP, mediates interdependent cortical reorganization and spindle dynamics in Drosophila embryos

Introduction Dynamic interactions between the mitotic spindle, the cell cortex and the plasma membrane underlie many crucial cellular phenomena. For example, during cell division, the localization of the cleavage furrow is specified by the mitotic spindle (Glotzer, 2001; Scholey et al., 2003; Odell and Foe, 2008), whereas actomyosin-based cortical contractions contribute to spindle morphogenesis (Rosenblatt et al., 2004). In addition, membrane transport to the furrow is crucial for cell-cell abscission and the completion of cytokinesis (Finger and White, 2002). Recently, it has been shown that vesicles associated with F-actin are transported on furrow microtubules (MTs) (Albertson et al., 2008). However, the underlying molecular mechanisms remain largely obscure. Drosophila embryogenesis is amenable to the study of interactions between the spindle, cortex and membrane using genetic analysis, inhibitor microinjection and microscopy (Glover, 2005). In the Drosophila syncytial embryo, nuclei share the same cytoplasm and undergo 13 synchronous divisions without any intervening cytokinesis; however, following the migration of the nuclei to the embryonic surface during cycle 9, mitotic furrows form transiently around each spindle (Mazumdar and Mazumdar, 2002). The mitotic furrows grow longer during each metaphase, serving as barriers to maintain the spacing between adjacent spindles, and then regress during late anaphase and telophase. Subsequently, during interphase of cycle 14, cellularization furrows descend from the blastoderm surface to partition the nuclei into individual cells. Many studies suggest that spindle morphogenesis, as monitored by the separation of the spindle poles, depends upon a balance of forces generated by multiple molecular motors that guide the spindle through a sequence of quiescent steady states interspersed with phases of rapid spindle-pole separation (Sharp et al., 2000; Brust-Mascher et al., 2004; Cytrynbaum et al., 2005). The mitotic furrows maintain a constant distance to the poles throughout mitosis, and perturbations of mitotic molecules can cause both spindle and cortical defects during the early mitotic cycles (Cytrynbaum et al., 2005). To test the hypothesis that this force balance reflects a dynamic relationship between the spindle and the cortex, we need to know the identity of the crucial molecules that influence the structure and dynamics of both the spindle and the cortex. It is unknown how the spindle affects cortical reorganization and, conversely, how the cortex affects spindle morphogenesis. Both cellularization and metaphase furrow formation in the early Drosophila embryo are driven by the MT-dependent delivery of membrane vesicles to the furrow region (Strickland and Burgess, 2004; Riggs et al., 2003). As in conventional cytokinesis, new furrow membrane does not seem to be derived from the expansion of the pre-existing surface membrane, but rather forms through the insertion of membrane from internal stores, such as the recycling endosome (RE), Golgi or both (Lecuit and Weichaus, 2000; Sisson A mitotic kinesin-6, Pav-KLP, mediates interdependent cortical reorganization and spindle dynamics in Drosophila embryos

Deficiency in myosin light-chain phosphorylation causes cytokinesis failure and multipolarity in cancer cells

Cancer cells often have unstable genomes and increased centrosome and chromosome numbers, which play an important part of malignant transformation in the most recent models tumorigenesis. However, very little is known about divisional failures in cancer cells that may lead to chromosomal and centrosomal amplifications. We show here that cancer cells often failed at cytokinesis due to decreased phosphorylation of the myosin regulatory light chain (MLC), a key regulatory component of cortical contraction during division. Reduced MLC phosphorylation was associated with high expression of myosin phosphatase and/or reduced myosin light chain kinase levels. Furthermore, expression of phosphomimetic MLC largely prevented cytokinesis failure in the tested cancer cells. When myosin light chain phosphorylation was restored to normal levels by phosphatase knockdown multinucleation, and multipolar mitosis were both markedly reduced, resulting in enhanced genome stabilization. Furthermore, both overexpression of myosin phosphatase or inhibition of the myosin light chain kinase (MLCK) in nonmalignant cells can recapitulate some of the mitotic defects of cancer cells, including multinucleation and multipolar spindles, indicating these changes are sufficient to reproduce the cytokinesis failures we see in cancer cells. These results for the first time define the molecular defects leading to divisional failure in cancer cells.

Bazooka is required for polarisation of the Drosophila anterior-posterior axis

The Drosophila anterior-posterior (AP) axis is determined by the polarisation of the stage 9 oocyte and the subsequent localisation of bicoid and oskar mRNAs to opposite poles of the cell. Oocyte polarity has been proposed to depend on the same PAR proteins that generate AP polarity in C. elegans, with a complex of Bazooka (Baz; Par-3), Par-6 and aPKC marking the anterior and lateral cortex, and Par-1 defining the posterior. The function of the Baz complex in oocyte polarity has remained unclear, however, because although baz-null mutants block oocyte determination, egg chambers that escape this early arrest usually develop normal polarity at stage 9. Here, we characterise a baz allele that produces a penetrant polarity phenotype at stage 9 without affecting oocyte determination, demonstrating that Baz is essential for axis formation. The dynamics of Baz, Par-6 and Par-1 localisation in the oocyte indicate that the axis is not polarised by a cortical contraction as in C. elegans, and instead suggest that repolarisation of the oocyte is triggered by posterior inactivation of aPKC or activation of Par-1. This initial asymmetry is then reinforced by mutual inhibition between the anterior Baz complex and posterior Par-1 and Lgl. Finally, we show that mutation of the aPKC phosphorylation site in Par-1 results in the uniform cortical localisation of Par-1 and the loss of cortical microtubules. Since non-phosphorylatable Par-1 is epistatic to uninhibitable Baz, Par-1 seems to function downstream of the other PAR proteins to polarize the oocyte microtubule cytoskeleton.

Local Cortical Tension by Myosin II Guides 3D Endothelial Cell Branching

A key feature of angiogenesis is directional control of endothelial cell (EC) morphogenesis and movement [1]. During angiogenic sprouting, endothelial "tip cells" directionally branch from existing vessels in response to biochemical cues such as VEGF or hypoxia and migrate and invade the surrounding extracellular matrix (ECM) in a process that requires ECM remodeling by matrix metalloproteases (MMPs) [2-4]. Tip EC branching is mediated by directional protrusion of subcellular pseudopodial branches [5, 6]. Here, we seek to understand how EC pseudopodial branching is locally regulated to directionally guide angiogenesis. We develop an in vitro 3D EC model system in which migrating ECs display branched pseudopodia morphodynamics similar to those in living zebrafish. Using this system, we find that ECM stiffness and ROCK-mediated myosin II activity inhibit EC pseudopodial branch initiation. Myosin II is dynamically localized to the EC cortex and is partially released under conditions that promote branching. Local depletion of cortical myosin II precedes branch initiation, and initiation can be induced by local inhibition of myosin II activity. Thus, local downregulation of myosin II cortical contraction allows pseudopodium initiation to mediate EC branching and hence guide directional migration and angiogenesis.

Lipid levels in sperm, eggs, and during fertilization in Xenopus laevis

Critical developmental periods, such as fertilization, involve metabolic activation, membrane fusion events such as sperm-egg or plasma membrane-cortical granule merger, and production and hydrolysis of phospholipids. However, there has been no large-scale quantification of phospholipid changes during fertilization. Using an enzymatic assay, traditional FA analysis by TLC and gas chromatography, along with a new method of phospholipid measurement involving HPLC separation and evaporative light-scattering detection, we report lipid levels in eggs, sperm, and during fertilization in Xenopus laevis. Sperm were found to contain different amounts of phospholipids as compared with eggs. During fertilization, total phosphatidylinositol, lysophosphatidylcholine, sphingomyelin, and phosphatidylserine decreased, and ceramide increased, whereas there was no change in phosphatidylcholine, cardiolipin, or phosphatidylethanolamine. FA analysis of phospholipids found numerous changes during fertilization. Because there is an increase in sn-1,2-diacylglycerol at fertilization, the FAs associated with this increase and the source of the increase in this neutral lipid were examined. Finally, activation of phospholipase C, phospholipase D, phospholipase A2, autotoxin, and sphingomyelinase at fertilization is discussed.

Redundant Mechanisms Recruit Actin into the Contractile Ring in Silkworm Spermatocytes

Cytokinesis is powered by the contraction of actomyosin filaments within the newly assembled contractile ring. Microtubules are a spindle component that is essential for the induction of cytokinesis. This induction could use central spindle and/or astral microtubules to stimulate cortical contraction around the spindle equator (equatorial stimulation). Alternatively, or in addition, induction could rely on astral microtubules to relax the polar cortex (polar relaxation). To investigate the relationship between microtubules, cortical stiffness, and contractile ring assembly, we used different configurations of microtubules to manipulate the distribution of actin in living silkworm spermatocytes. Mechanically repositioned, noninterdigitating microtubules can induce redistribution of actin at any region of the cortex by locally excluding cortical actin filaments. This cortical flow of actin promotes regional relaxation while increasing tension elsewhere (normally at the equatorial cortex). In contrast, repositioned interdigitating microtubule bundles use a novel mechanism to induce local stimulation of contractility anywhere within the cortex; at the antiparallel plus ends of central spindle microtubules, actin aggregates are rapidly assembled de novo and transported laterally to the equatorial cortex. Relaxation depends on microtubule dynamics but not on RhoA activity, whereas stimulation depends on RhoA activity but is largely independent of microtubule dynamics. We conclude that polar relaxation and equatorial stimulation mechanisms redundantly supply actin for contractile ring assembly, thus increasing the fidelity of cleavage.

Cytoplasmic Force Gradient in Migrating Adhesive Cells

Amoeboid movement is believed to involve a pressure gradient along the cell length, with contractions in the posterior region driving cytoplasmic streaming forward. However, a parallel mechanism has yet to be demonstrated in migrating adhesive cells. To probe the distribution of intracellular forces, we microinjected high molecular weight linear polyacrylamide (PAA) as a passive force sensor into migrating NIH3T3 fibroblasts. Injected PAA appeared as amorphous aggregates that underwent shape change and directional movement in response to differential forces exerted by the surrounding environment. PAA injected into the posterior region moved toward the front, whereas PAA in the anterior region never moved to the posterior region. This preferential forward movement was observed only in migrating cells with a defined polarity. Disruption of myosin II activity by blebbistatin inhibited the forward translocation of PAA while cell migration persisted in a disorganized fashion. These results suggest a myosin II-dependent force gradient in migrating cells, possibly as a result of differential cortical contractions between the anterior and posterior regions. This gradient may be responsible for the forward transport of cellular components and for maintaining the directionality during cell migration.

α-Actinin Is Required for Tightly Regulated Remodeling of the Actin Cortical Network during Cytokinesis

Localization of the actin crosslinking protein, alpha-actinin, to the cleavage furrow has been previously reported. However, its functions during cytokinesis remain poorly understood. We have analyzed the functions of alpha-actinin during cytokinesis by a combination of molecular manipulations and imaging-based techniques. alpha-actinin gradually dissipated from the cleavage furrow as cytokinesis progressed. Overexpression of alpha-actinin caused increased accumulation of actin filaments because of inhibition of actin turnover, leading to cytokinesis failure. Global depletion of alpha-actinin by siRNA caused a decrease in the density of actin filaments throughout the cell cortex, surprisingly inducing accelerated cytokinesis and ectopic furrows. Local ablation of alpha-actinin induced accelerated cytokinesis specifically at the site of irradiation. Neither overexpression nor depletion of alpha-actinin had an apparent effect on myosin II organization. We conclude that cytokinesis in mammalian cells requires tightly regulated remodeling of the cortical actin network mediated by alpha-actinin in coordination with actomyosin-based cortical contractions.

Endomitotic MKs Undergo Cortical Contraction during Anaphase but Do Not Complete Furrow Ingression.

Megakaryocytes (MKs) develop high degrees of polyploidy in the course of normal maturation through a process called endomitosis. Previous studies have shown that endomitotic MKs abort anaphase after sister chromatid separation, and studies using fixed cells have shown that a midzone is formed in anaphase but that a well-developed cleavage furrow is not. However, studies in fixed cells may miss transient dynamic events that are critical for evaluating potential mechanisms of failed cytokinesis in endomitosis. Therefore we have used live cell imaging as a tool to further define the ability of primary endomitotic MKs to furrow during anaphase. We imaged mouse marrow MKs expressing tubulin fused to YFP, acquiring brightfield and fluorescent images every 2 minutes. Endomitotic cells were identified by the formation of a multipolar spindle, and anaphase was defined as the point at which separation of spindle poles was visible. The extent of cell shape change following anaphase was determined visually and also by measuring the circularity of the cell, with a perfectly round cell having a circularity value of 1. We found that 56% of endomitotic MKs underwent some shape change (n=25), visible as localized indentation, protrusion or elongation of the cell cortex, with an average reduction in circularity of 0.043 occurring a mean of 14.6 minutes after the initiation of anaphase. After an average of 7.8 minutes the cells generally reverted to a round shape without completing furrow ingression. In contrast, a typical mitotic control cell showed a reduction in circularity of 0.443 occurring 8 minutes after the start of anaphase and resulting in the formation of two cells (see Figure 1, representative cells). To determine if the shape change seen in endomitosis after anaphase is dependent on RhoA, we imaged MKs in the presence of the RhoA inhibitor C3 toxin. Although 2 μg/ml C3 toxin blocked cytokinesis in diploid control cells, it did not measurably alter the frequency (44%, n=16) or degree of shape change (0.039) in endomitotic MKs following anaphase, although treatment did appear to delay shape change (onset occurring a mean of 24.8 min after anaphase) and prolong the period of cortical contractility. Therefore although endomitotic MKs exhibit a phase of increased cortical contractility following anaphase, it is not solely dependent on the activity of RhoA and it does not result in sustained furrow ingression. Live cell imaging represents a powerful new tool for dissecting the mechanisms of endomitosis. [Display omitted]

Blebbing of Dictyostelium cells in response to chemoattractant

Stimulation of Dictyostelium cells with a high uniform concentration of the chemoattractant cyclic-AMP induces a series of morphological changes, including cell rounding and subsequent extension of pseudopodia in random directions. Here we report that cyclic-AMP also elicits blebs and analyse their mechanism of formation. The surface area and volume of cells remain constant during blebbing indicating that blebs form by the redistribution of cytoplasm and plasma membrane rather than the exocytosis of internal membrane coupled to a swelling of the cell. Blebbing occurs immediately after a rapid rise and fall in submembraneous F-actin, but the blebs themselves contain little F-actin as they expand. A mutant with a partially inactivated Arp2/3 complex has a greatly reduced rise in F-actin content, yet shows a large increase in blebbing. This suggests that bleb formation is not enhanced by the preceding actin dynamics, but is actually inhibited by them. In contrast, cells that lack myosin-II completely fail to bleb. We conclude that bleb expansion is likely to be driven by hydrostatic pressure produced by cortical contraction involving myosin-II. As blebs are induced by chemoattractant, we speculate that hydrostatic pressure is one of the forces driving pseudopod extension during movement up a gradient of cyclic-AMP.

Two Phases of Astral Microtubule Activity during Cytokinesis in C. elegans Embryos

Microtubules of the mitotic spindle are believed to provide positional cues for the assembly of the actin-based contractile ring and the formation of the subsequent cleavage furrow during cytokinesis. In Caenorhabditis elegans, astral microtubules have been thought to inhibit cortical contraction outside the cleavage furrow. Here, we demonstrate by live imaging and RNA interference (RNAi) that astral microtubules play two distinct roles in initiating cleavage furrow formation. In early anaphase, microtubules are required for contractile ring assembly; in late anaphase, microtubules show different cortical behavior and seem to suppress cortical contraction at the poles, as suggested in previous studies. These two distinct phases of microtubule behavior depend on distinct regulatory pathways, one involving the gamma-tubulin complex and the other requiring aurora-A kinase. We propose that temporal and spatial regulation of two distinct phases of astral microtubule behavior is crucial in specifying the position and timing of furrowing.

The mechanism of cortical ingression during early cytokinesis: thinking beyond the contractile ring hypothesis

Owing to the rapid advances in genomic, proteomic and imaging technologies, the field of cytokinesis has seen rapid advances during the past decade. However, the basic model for the early stage of ingression, known as the contractile ring hypothesis, remains largely unchanged. From recent observations, it is becoming clear that early cytokinesis of animal cells involves a more extensive set of events, both temporally and spatially, than what is encompassed by the original contractile ring hypothesis. Activities relevant to cytokinesis, such as cortical contraction, can initiate well before onset of anaphase. Furthermore, equatorial ingression can involve multiple events in different regions of the cortex, including the establishment of anterior-posterior polarity, the modulation of cortical deformability, the expansion and compression of the cell cortex, and forces directed towards the interior of the cell or away from the equator. In this article (which is part of the Cytokinesis series), I evaluate critically key observations on when, where and how early ingression of animal cells takes place.

Polarity of the ascidian egg cortex and relocalization of cER and mRNAs in the early embryo

The mature ascidian oocyte is a large cell containing cytoplasmic and cortical domains polarized along a primary animal-vegetal (a-v) axis. The oocyte cortex is characterized by a gradient distribution of a submembrane monolayer of cortical rough endoplasmic reticulum (cER) and associated maternal postplasmic/PEM mRNAs (cER-mRNA domain). Between fertilization and first cleavage, this cER-mRNA domain is first concentrated vegetally and then relocated towards the posterior pole via microfilament-driven cortical contractions and spermaster-microtubule-driven translocations. The cER-mRNA domain further concentrates in a macroscopic cortical structure called the centrosome attracting body (CAB), which mediates a series of asymmetric divisions starting at the eight-cell stage. This results in the segregation of determinant mRNAs and their products in posterior cells of the embryo precursors of the muscle and germ line. Using two species of ascidians (Ciona intestinalis and Phallusia mammillata), we have pursued and amplified the work initiated in Halocynthia roretzi. We have analysed the cortical reorganizations in whole cells and in cortical fragments isolated from oocytes and from synchronously developing zygotes and embryos. After fertilization, we observe that a cortical patch rich in microfilaments encircles the cER-mRNA domain, concentrated into a cortical cap at the vegetal/contraction pole (indicating the future dorsal pole). Isolated cortices also retain microtubule asters rich in cER (indicating the future posterior pole). Before mitosis, parts of the cER-mRNA domain are detected, together with short microtubules, in isolated posterior (but not anterior) cortices. At the eight-cell stage, the posteriorly located cER-mRNA domain undergoes a cell-cycle-dependant compaction into the CAB. The CAB with embedded centrosomal microtubules can be isolated with cortical fragments from eight-cell-stage embryos. These and previous observations indicate that cytoskeleton-driven repositioning and compaction of a polarized cortical domain made of rough ER is a conserved mechanism used for polarization and segregation of cortical maternal mRNAs in embryos of evolutionarily distant species of ascidians.

Regulation of cell cycle by the anaphase spindle midzone

BackgroundA number of proteins accumulate in the spindle midzone and midbody of dividing animal cells. Besides proteins essential for cytokinesis, there are also components essential for interphase functions, suggesting that the spindle midzone and/or midbody may play a role in regulating the following cell cycle.ResultsWe microsurgically severed NRK epithelial cells during anaphase or telophase, such that the spindle midzone/midbody was associated with only one of the daughter cells. Time-lapse recording of cells severed during early anaphase indicated that the cell with midzone underwent cytokinesis-like cortical contractions and progressed normally through the interphase, whereas the cell without midzone showed no cortical contraction and an arrest or substantial delay in the progression of interphase. Similar microsurgery during telophase showed a normal progression of interphase for both daughter cells with or without the midbody. Microsurgery of anaphase cells treated with cytochalasin D or nocodazole indicated that interphase progression was independent of cortical ingression but dependent on microtubules.ConclusionsWe conclude that the mitotic spindle is involved in not only the separation of chromosomes but also the regulation of cell cycle. The process may involve activation of components in the spindle midzone that are required for the cell cycle, and/or degradation of components that are required for cytokinesis but may interfere with the cell cycle.

Exploring the mechanism of action of the sperm-triggered calcium-wave pacemaker in ascidian zygotes.

In ascidians, as in mammals, sperm trigger repetitive Ca2+-waves that originate from cortical pacemakers situated in the vegetal hemisphere of the zygotes. In ascidians, a vegetal protrusion termed the contraction pole (CP) acts as the Ca2+-wave pacemaker, but the mechanism that underlies the generation of a Ca2+-wave pacemaker is not known. Here, we tested four hypotheses to determine which factors at the CP are involved in setting the pace of the ascidian Ca2+-wave pacemaker: (1) localized Ca2+ influx; (2) accumulation of phosphatidylinositol (4,5)bisphosphate [PtdIns(4,5)P2]; (3) accumulation of cortical endoplasmic reticulum (cER); and (4) enrichment of the sperm activating factor. We developed a method of dynamically monitoring the location of the CP during fertilization using a plekstrin homology (PH) domain from phospholipase Cdelta1 coupled to green fluorescent protein (GFP) that binds PtdIns(4,5)P2. We found that eggs in Ca2+-free sea water displayed Ca2+ waves that originated from the CP, showing that enhanced CP Ca2+ influx does not determine the origin of the pacemaker. Also, disruption of the PH::GFP-labelled CP once it had formed did not dislodge the Ca2+-wave pacemaker from that site. Next, when we prevented the accumulation of cER at the CP, all of the Ca2+ waves came from the site of sperm-egg fusion and the frequency of Ca2+ oscillations was unaltered. These data show that local Ca2+ influx, the accumulation of PtdIns(4,5)P2 and cER at the CP are not required for Ca2+-wave pacemaker function and instead suggest that a factor associated with the sperm determines the site of the Ca2+-wave pacemaker. Finally, when we injected ascidian sperm extract into the centre of unfertilized ascidian eggs that had been treated with microfilament- and microtubule-disrupting drugs, all the Ca2+ waves still originated from near the plasma membrane, showing that the sperm factor does not require an intact cortex if it is enriched near the plasma membrane (PM). We suggest that the Ca2+-releasing sperm factor might be tethered near or on the PM and that following the cortical contraction, it is translocated to the vegetal CP, thus making that site act as a Ca2+-wave pacemaker.