Equatorial Cortex Research Articles

Liver tetraploidization is controlled by a new process of incomplete cytokinesis

Cytokinesis is precisely controlled in both time and space to ensure equal distribution of the genetic material between daughter cells. Incomplete cytokinesis can be associated with developmental or pathological cell division programs leading to tetraploid progenies. In this study we decipher a new mechanism of incomplete cytokinesis taking place in hepatocytes during post-natal liver growth. This process is initiated in vivo after weaning and is associated with an absence of anaphase cell elongation. In this process, formation of a functional contractile actomyosin ring was never observed; indeed, actin filaments spread out along the cortex were not concentrated to the putative site of furrowing. Recruitment of myosin II to the cortex, controlled by Rho-kinase, was impaired. Astral microtubules failed to contact the equatorial cortex and to deliver their molecular signal, preventing activation of the RhoA pathway. These findings reveal a new developmental cell division program in the liver that prevents cleavage-plane specification.

The Localization of Inner Centromeric Protein (INCENP) at the Cleavage Furrow Is Dependent on Kif12 and Involves Interactions of the N Terminus of INCENP with the Actin Cytoskeleton

The inner centromeric protein (INCENP) and other chromosomal passenger proteins are known to localize on the cleavage furrow and to play a role in cytokinesis. However, it is not known how INCENP localizes on the furrow or whether this localization is separable from that at the midbody. Here, we show that the association of Dictyostelium INCENP (DdINCENP) with the cortex of the cleavage furrow involves interactions with the actin cytoskeleton and depends on the presence of the kinesin-6-related protein Kif12. We found that Kif12 is found on the central spindle and the cleavage furrow during cytokinesis. Kif12 is not required for the redistribution of DdINCENP from centromeres to the central spindle. However, in the absence of Kif12, DdINCENP fails to localize on the cleavage furrow. Domain analysis indicates that the N terminus of DdINCENP is necessary and sufficient for furrow localization and that it binds directly to the actin cytoskeleton. Our data suggest that INCENP moves from the central spindle to the furrow of a dividing cell by a Kif12-dependent pathway. Once INCENP reaches the equatorial cortex, it associates with the actin cytoskeleton where it then concentrates toward the end of cytokinesis.

GEF-H1 Modulates Localized RhoA Activation during Cytokinesis under the Control of Mitotic Kinases

Formation of the mitotic cleavage furrow is dependent upon both microtubules and activity of the small GTPase RhoA. GEF-H1 is a microtubule-regulated exchange factor that couples microtubule dynamics to RhoA activation. GEF-H1 localized to the mitotic apparatus in HeLa cells, particularly at the tips of cortical microtubules and the midbody, and perturbation of GEF-H1 function induced mitotic aberrations, including asymmetric furrowing, membrane blebbing, and impaired cytokinesis. The mitotic kinases Aurora A/B and Cdk1/Cyclin B phosphorylate GEF-H1, thereby inhibiting GEF-H1 catalytic activity. Dephosphorylation of GEF-H1 occurs just prior to cytokinesis, accompanied by GEF-H1-dependent GTP loading on RhoA. Using a live cell biosensor, we demonstrate distinct roles for GEF-H1 and Ect2 in regulating Rho activity in the cleavage furrow, with GEF-H1 catalyzing Rho activation in response to Ect2-dependent localization and initiation of cell cleavage. Our results identify a GEF-H1-dependent mechanism to modulate localized RhoA activation during cytokinesis under the control of mitotic kinases.

Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells

Polo-like kinases (Plks) play crucial roles in mitosis and cell division. Whereas lower eukaryotes typically contain a single Plk, mammalian cells express several closely related but functionally distinct Plks. We describe here a chemical genetic system in which a single Plk family member, Plk1, can be inactivated with high selectivity and temporal resolution by using an allele-specific, small-molecule inhibitor, as well as the application of this system to dissect Plk1's role in cytokinesis. To do this, we disrupted both copies of the PLK1 locus in human cells through homologous recombination and then reconstituted Plk1 activity by using either the wild-type kinase (Plk1(wt)) or a mutant version whose catalytic pocket has been enlarged to accommodate bulky purine analogs (Plk1(as)). When cultured in the presence of these analogs, Plk1(as) cells accumulate in prometaphase with defects that parallel those found in PLK1(Delta/Delta) cells. In addition, acute treatment of Plk1(as) cells during anaphase prevents recruitment of both Plk1 itself and the Rho guanine nucleotide exchange factor (RhoGEF) Ect2 to the central spindle, abolishes RhoA GTPase localization to the equatorial cortex, and suppresses cleavage furrow formation and cell division. Our studies define and illuminate a late mitotic function of Plk1 that, although difficult or impossible to detect in Plk1-depleted cells, is readily revealed with chemical genetics.

Raman spectroscopic evidence for nuclear disulfide in isolated lenses of hyperbaric oxygen-treated guinea pigs

Our laboratory treats guinea pigs with hyperbaric oxygen (HBO) as a model for investigating the formation of nuclear cataract. Previous analyses of lens supernatants using this model have shown an increase in disulfide (–SS–) and loss of sulfhydryl (–SH) in the lens nucleus of O 2-treated animals. In this paper, we have used the non-invasive technique of Raman spectroscopy to confirm these findings in intact, freshly-excised lenses. Guinea pigs were treated 3 times per week with HBO for a total of 50 (4 months of treatment) or 85 (7 months of treatment) times to induce an increased level of lens nuclear light scattering. Intact lenses were analyzed by Raman spectroscopy using a 514.5 nm laser and collecting the scattered light in a 90° geometry. The laser beam was focused either in the lens nucleus or equatorial cortex. Changes in the levels of –SS– (503 cm −1) and –SH (2577 cm −1) vibrations were measured. Raman spectra were analyzed by fitting Lorentzian profiles to the observed data in the –SS– and –SH regions. –SS– levels in the O 2-treated nucleus were found to have increased by a factor of 2.1 ( p = 0.0001) and 2.5 ( p = 0.001) after 50 and 85 HBO treatments, respectively, compared to age-matched controls. Based on previous biochemical analyses, the –SS– increase was due mainly to the formation of protein disulfide (PSSP) with contribution also from protein/thiol mixed disulfides, but not from oxidized glutathione. –SH levels in the O 2-treated nucleus decreased by 13% ( p = 0.007) and 35% ( p = 0.001) after 50 and 85 HBO treatments, respectively, compared to age-matched controls. No significant increase in –SS– or loss of –SH was observed in the lens cortex of the O 2-treated guinea pigs. The Raman spectroscopy results rule out the possibility that artifactual production of –SS– and loss of –SH occurred during homogenization of lenses in previous studies. The data provide additional evidence to support a link between O 2, disulfide-crosslinking of lens crystallins in the nucleus, and nuclear cataract.

Rho Kinase's Role in Myosin Recruitment to the Equatorial Cortex of Mitotic Drosophila S2 Cells Is for Myosin Regulatory Light Chain Phosphorylation

BackgroundMyosin II recruitment to the equatorial cortex is one of the earliest events in establishment of the cytokinetic contractile ring. In Drosophila S2 cells, we previously showed that myosin II is recruited to the furrow independently of F-actin, and that Rho1 and Rok are essential for this recruitment [1]. Rok phosphorylates several cellular proteins, including the myosin regulatory light chain (RLC).Methodology/Principal FindingsHere we express phosphorylation state mimic constructs of the RLC in S2 cells to examine the role of RLC phosphorylation involving Rok in the localization of myosin. Phosphorylation of the RLC is required for myosin localization to the equatorial cortex during mitosis, and the essential role of Rok in this localization and for cytokinesis is to maintain phosphorylation of the RLC. The ability to regulate the RLC phosphorylation state spatio-temporally is not essential for the myosin localization. Furthermore, the essential role of Citron in cytokinesis is not phosphorylation of the RLC.Conclusions/SignificanceWe conclude that the Rho1 pathway leading to myosin localization to the future cytokinetic furrow is relatively straightforward, where only Rok is needed, and it is only needed to maintain phosphorylation of the myosin RLC.

RacGAP50C is sufficient to signal cleavage furrow formation during cytokinesis

Several studies indicate that spindle microtubules determine the position of the cleavage plane at the end of cell division, but their exact role in triggering the formation and ingression of the cleavage furrow is still unclear. Here we show that in Drosophila depletion of either the GAP (GTPase-activating protein) or the kinesin-like subunit of the evolutionary conserved centralspindlin complex prevents furrowing without affecting the association of astral microtubules with the cell cortex. Moreover, time-lapse imaging indicates that astral microtubules serve to deliver the centralspindlin complex to the equatorial cortex just before furrow formation. However, when the GAP-signaling component was mislocalized around the entire cortex using a membrane-tethering motif, this caused ectopic furrowing even in the absence of its motor partner. Thus, the GAP component of centralspindlin is both necessary and sufficient for furrow formation and ingression and astral microtubules provide a route for its delivery to the cleavage site.

Centralspindlin regulates ECT2 and RhoA accumulation at the equatorial cortex during cytokinesis

During determination of the cell division plane, an actomyosin contractile ring is induced at the equatorial cell cortex by signals from the mitotic apparatus and contracts to cause cleavage furrow progression. Although the small GTPase RhoA is known to regulate the progression, probably by controlling actin filament assembly and enhancing actomyosin interaction, any involvement of RhoA in division plane determination is unknown. In this study, using a trichloroacetic acid (TCA) fixation protocol we recently developed, we show that RhoA accumulates at the equatorial cortex before furrow initiation and continues to concentrate at the cleavage furrow during cytokinesis. We also demonstrate that both Rho activity and microtubule organization are required for RhoA localization and proper furrowing. Selective disruption of microtubule organization revealed that both astral and central spindle microtubules can recruit RhoA at the equatorial cortex. We find that centralspindlin and ECT2 are required for RhoA localization and furrowing. Centralspindlin is localized both to central spindle microtubules and at the tips of astral microtubules near the equatorial cortex and recruits ECT2. Positional information for division plane determination from microtubules is transmitted to the cell cortex to organize actin cytoskeleton through a mechanism involving these proteins.

Distinct pathways control recruitment and maintenance of myosin II at the cleavage furrow during cytokinesis

The correct localization of myosin II to the equatorial cortex is crucial for proper cell division. Here, we examine a collection of genes that cause defects in cytokinesis and reveal with live cell imaging two distinct phases of myosin II localization. Three genes in the rho1 signaling pathway, pebble (a Rho guanidine nucleotide exchange factor), rho1, and rho kinase, are required for the initial recruitment of myosin II to the equatorial cortex. This initial localization mechanism does not require F-actin or the two components of the centralspindlin complex, the mitotic kinesin pavarotti/MKLP1 and racGAP50c/CYK-4. However, F-actin, the centralspindlin complex, formin (diaphanous), and profilin (chickadee) are required to stably maintain myosin II at the furrow. In the absence of these latter genes, myosin II delocalizes from the equatorial cortex and undergoes highly dynamic appearances and disappearances around the entire cell cortex, sometimes associated with abnormal contractions or blebbing. Our findings support a model in which a rho kinase-dependent event, possibly myosin II regulatory light chain phosphorylation, is required for the initial recruitment to the furrow, whereas the assembly of parallel, unbranched actin filaments, generated by formin-mediated actin nucleation, is required for maintaining myosin II exclusively at the equatorial cortex.

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.

Myosin-II-Dependent Localization and Dynamics of F-Actin during Cytokinesis

The assembly of an F-actin- and myosin-II-containing contractile ring (CR) is required for cytokinesis in eukaryotic cells. Interactions between myosin II and actin in the ring are believed to generate the force that constricts the cell into two daughters. The mechanism(s) that contribute to the spatially and temporally regulated assembly and disassembly of the CR at the cell equator are poorly understood. We generated an LLCPK1 epithelial cell line that stably expresses GFP-actin. Live confocal imaging showed accumulation of GFP-actin in the equatorial cortex from late anaphase through cytokinesis. Fluorescence recovery after photobleaching (FRAP) experiments showed that actin in the CR is highly dynamic (t(1/2) = 26 s). In some cells, movement of GFP-actin toward the equatorial region was observed and contributed to FRAP. Blocking actin dynamic turnover with jasplakinolide demonstrates that dynamic actin is required for CR formation and cytokinesis. To test the role of myosin II in actin turnover and transport during CR formation, we inhibited myosin light-chain kinase with ML7 and myosin II ATPase activity with blebbistatin. Inhibition of myosin light-chain phosphorylation resulted in clearance of GFP-actin from the equatorial region, a reduction in myosin II in the furrow, and inhibition of cytokinesis. Treatment with blebbistatin did not block CR formation but reduced FRAP of GFP-actin and prevented completion of cytokinesis. These results demonstrate that the majority of actin in the CR is highly dynamic and establish novel roles for myosin II in the retention and dynamic turnover of actin in the CR.

Cortical Actin Turnover during Cytokinesis Requires Myosin II

The involvement of myosin II in cytokinesis has been demonstrated with microinjection, genetic, and pharmacological approaches; however, the exact role of myosin II in cell division remains poorly understood. To address this question, we treated dividing normal rat kidney (NRK) cells with blebbistatin, a potent inhibitor of the nonmuscle myosin II ATPase. Blebbistatin caused a strong inhibition of cytokinesis but no detectable effect on the equatorial localization of actin or myosin. However, whereas these filaments dissociated from the equator in control cells during late cytokinesis, they persisted in blebbistatin-treated cells over an extended period of time. The accumulation of equatorial actin was caused by the inhibition of actin filament turnover, as suggested by a 2-fold increase in recovery half-time after fluorescence photobleaching. Local release of blebbistatin at the equator caused localized accumulation of equatorial actin and inhibition of cytokinesis, consistent with the function of myosin II along the furrow. However, treatment of the polar region also caused a high frequency of abnormal cytokinesis, suggesting that myosin II may play a second, global role. Our observations indicate that myosin II ATPase is not required for the assembly of equatorial cortex during cytokinesis but is essential for its subsequent turnover and remodeling.

Both midzone and astral microtubules are involved in the delivery of cytokinesis signals: insights from the mobility of aurora B.

To address the mechanism that coordinates cytokinesis with mitosis, we have studied the dynamics of aurora B, a chromosomal passenger protein involved in signaling cytokinesis. Photobleaching analyses indicated dynamic exchange of aurora B between a centromeric and a cytoplasmic pool before anaphase onset, and stable associations with microtubules after anaphase onset. Bleaching near centromeres upon anaphase onset affected the subsequent appearance of fluorescence along midzone microtubules, but not that near the lateral equatorial cortex, suggesting that there were centromeric-dependent and -independent pathways that transported aurora B to the equator. The former delivered centromeric aurora B along midzone microtubules, whereas the latter delivered cytoplasmic aurora B along astral microtubules. We suggest that cultured cells use midzone microtubules as the primary signaling pathway for cytokinesis, whereas embryos, with their stockpile of cytoplasmic proteins and large sizes, rely primarily on astral microtubules.

Forceps-guided nuclear cleavage cataract extraction

We introduce a manual nuclear fragmentation technique, forceps-guided nuclear cleavage. A 5.5 to 7.0 mm superior scleral incision is started 1.5 mm posterior to the limbus. Two additional 1.0 mm paracenteses are made at 3 and 9 o’clock in clear cornea close to the limbus. A continuous curvilinear capsulorhexis (CCC) is created; in most cases, 4 to 5 radial relaxing incisions are made in the CCC. The anterior and equatorial cortex and epinucleus are removed with 2-handed irrigation/aspiration via the 2 paracenteses with the nucleus in the capsular bag. The nucleus is prolapsed into the anterior chamber. A nucleus hook is inserted via the 3 o’clock paracentesis and applied to the 6 o’clock nuclear equator to hold the nucleus. A nucleus cleaving forceps is inserted through the upper incision to the 12 o’clock equator of the nucleus and advanced to one-third depth of the nucleus. The forceps is relaxed while the nucleus is cleaved in half.

Water diffusion in the rabbit lens in vivo.

To examine water diffusion in the crystalline lens and sugar cataracts in the rabbits in vivo. Water self-diffusion in the lens cortices of alloxan-diabetic and galactosemic rabbits was examined with magnetic resonance imaging (MRI). The animals were positioned in a 4.7-tesla animal system in conjunction with a 1-inch surface coil for the eye. Diffusion-weighted MRI was conducted using a pulsed-gradient spin-echo sequence with a gradient strength of 0-6 Gs/cm in the primary and secondary coordinates. Other MRI parameters included TR (repetition time)/TE (echo time) = 2,000/10 ms, a field of view of 4 cm, and a 256 x 128 matrix. There appeared an increase in water relaxation resulting in an increase of % (equatorial cortex depth)/(lens long axis) from 18 in the lenses of normal rabbits to 30.4 and 39.9 in the lenses of galactosemic and diabetic rabbits, respectively. In addition, water diffusion changed in the lens of the diabetic rabbit with an increasing intracellular fluidity along the long axis of the cortical fibers, for example, the diffusion coefficient changed from a normal of 0.48 to 0.96 x 10(-5) cm2 s-1 in the lens of the diabetic rabbit. These results showed altered water mobility due to subcellular disturbances occurring before any apparent lens opacities. Further, there also was an increase in the water diffusivity in the aqueous humor from a normal of 1.77 to 2.67 x 10(-5) cm2 s-1 in the galactosemic rabbit eye suggesting an increase in either free water proportion or thermal convection. Resistance to water self-diffusion appeared to relate to lens fiber orientation and intracellular protein order. Diffusion imaging therefore can be used to examine water self-diffusion to detect early osmotic alteration of lens fibers.

Morphological differences between lens fibers in albino and pigmented rats.

The purpose of this study was to investigate the morphological characteristics of lens fibers in albino and pigmented rats by scanning electron microscopy. In addition to the ubiquitous interdigitating edge protrusions many ball-and-socket junctions were found on the lateral surfaces of lens fibers in pigmented rats. Notable differences in density, shape and size between superficial and deep cortical layers were observed. Especially, in the intermediate equatorial cortex large ball-and-socket junctions were found. In contrast, only few and small ball-and-socket junctions were observed in albino rats and many ruptures of lens fiber membranes were present in the anterior, superficial and intermediate equatorial cortex. The present observations show that different strains of rats have a different morphology of lens fibers. In view of a postulated role of ball-and-socket junctions in calcium homeostasis in the lens this may account for differences in cataractogenesis between albino and pigmented rats.

Chromosomal passengers

What are they? Chromosomal passengers are proteins associated with chromosomes during the early stages of mitosis, then with the spindle midzone and equatorial cortex during anaphase and telophase, and finally with the midbody/spindle remnant at the exit from mitosis. It was this unusual dynamic behaviour that first brought INCENP to notice in 1985. Also known as… INCENP, Aurora-B, BIR1/survivin and TD-60. Aliases for INCENP include Sli15p in budding yeast and ICP-1 in C. elegans. Aurora-B is also known in mammals as AIM-1 and Aurora-1, in budding yeast as Ipl1p and as AIR-2 in C. elegans. Survivin is known as BIR1 in both yeasts and C. elegans, and as Deterin in Drosophila.Fig. 1 Most mysterious… TD-60 is a passenger protein, but next to nothing is known about its function. Its localization has been described as a ‘telophase disk’, a distribution seen for all passengers during anaphase/telophase. Whether the disk is a discrete structure or simply a manifestation of the spindle midzone remains a matter of some debate. Not to be confused with… CENP-E, chromokinesin, Polo kinase... A number of proteins are localised a bit like chromosomal passengers. However these proteins occupy slightly different locations on the chromosomes: the kinetochore for CENP-E and Polo; all along the chromosome arms for chromokinesin. Some of these proteins may interact functionally or physically with the passenger proteins. Strangest behaviour… Canonical passenger proteins accumulate in the inner centromeres during metaphase prior to transferring to the spindle midzone at anaphase. If chromosomal passengers do not first concentrate at centromeres, it seems they are not able to move on to the central spindle and midbody later in mitosis. No one knows what is so special about the inner centromere. Who needs them? Chromosomal passengers are essential in eukaryotes from yeast to mouse. INCENP and BIR1/survivin have been knocked out in mouse and budding yeast: result-dead mice and dead yeast. RNAi has shown that INCENP, Aurora-B and Survivin are essential in C. elegans and that the first two are also essential in Drosophila cultured cells. What do they do? The active ingredient in the passenger complex is probably Aurora-B, which apparently phosphorylates a number of important substrates required for chromosome structure, kinetochore function, chromosome bi-orientation at metaphase, and the completion of cytokinesis. INCENP is required for the correct targeting of Aurora-B at all times in mitosis and for Survivin movement to the centromere and all of its spindle and midbody locations late in mitosis. BIR1/Survivin targets Aurora-B during mitosis in C. elegans. Most controversial… BIR1/Survivin seems to love to confound researchers. BIR1/Survivin looks like an inhibitor of apoptosis protein, and dominant-negative mutants can kill cells like nobody's business. In fact, a number of clear and well controlled studies seem to suggest that Survivin may help prevent mitotic cells from apoptosis. However, both yeast and mouse knockouts show pretty convincingly that BIR1/Survivin mutants die, not because of programmed cell death, but because they make a hash of chromosome segregation or cytokinesis. The jury is still out on this one, but at the moment the apoptosis camp looks like they have the most to prove. Where can I find out more BIR1/Survivin-GFP (green) localised to the spindle midzone at anaphase. Image courtesy of Dr. Sally P. Wheatley.

The mechanism of cytokinesis: reconsideration and reconciliation.

The widely held models of cytokinesis contend that signals for cleavage are transmitted by astral microtubules, and that such signals elicit the assembly and contraction of an equatorial band of actin-myosin II filaments. However, experiments during the past decade have painted an increasingly complex picture, including strong evidence for the involvement of chromosomal passenger proteins and interzonal microtubules, and the involvement of not only cortical contraction but also cytoskeletal disintegration. The purpose of this article is to consider alternative models that might better accommodate both old and new observations. It is proposed that chromosomal passenger proteins undergo dynamic associations at centromeres during metaphase and are recruited from the cytoplasm to both astral and interzonal microtubules during anaphase. In addition, cytokinesis may be driven by global inward contractions coupled to a localized collapse of the equatorial cortex.

INCENP Binds Directly to Tubulin and Requires Dynamic Microtubules to Target to the Cleavage Furrow

Inner centromere protein (INCENP) is a chromosomal passenger protein with an essential role in mitosis. At the metaphase/anaphase transition, some INCENP transfers from the centromeres to the central spindle; the remainder then transfers to the equatorial cortex prior to cleavage furrow formation. The molecular associations dictating INCENP behavior during mitosis are currently unknown. Here we show that targeting INCENP to the cleavage plane requires dynamic microtubules, but not F-actin. When microtubules are eliminated, INCENP is dispersed across the entire cell cortex. Yeast two-hybrid and in vitro binding data demonstrate that INCENP binds directly to β-tubulin via a conserved domain encompassing residues 48–85. Furthermore, INCENP binds to microtubules polymerized from purified tubulin in vitro and appears to bundle microtubules when expressed in the interphase cytoplasm. These data indicate that INCENP is a microtubule-binding protein that targets to the equatorial cortex through interactions requiring microtubules.

Dynamic changes in microtubule organization during division of the primitive dinoflagellate Oxyrrhis marina

The marine dinoflagellate Oxyrrhis marina has three major microtubular systems: the flagellar apparatus made of one transverse and one longitudinal flagella and their appendages, cortical microtubules, and intranuclear microtubules. We investigated the dynamic changes of these microtubular systems during cell division by transmission and scanning electron microscopy, and confocal fluorescent laser microscopy. During prophase, basal bodies, both flagella and their appendages were duplicated. In the round nucleus situated in the cell centre, intranuclear microtubules appeared radiating toward the centre of the nucleus from densities located in some nuclear pores. During metaphase, both daughter flagellar apparatus separated and moved apart along the main cell axis. Microtubules of ventral cortex were also duplicated and moved with the flagellar apparatus. The nucleus flattened in the longitudinal direction and became discoid-shaped close to the equatorial plane. Many bundles of microtubules ran parallel to the short axis of the nucleus (cell long axis), between which chromosomes were arranged in the same direction. During ana–telophase, the nucleus elongated along the longitudinal axis and took a dumbbell shape. At this stage a contractile ring containing actin was clearly observed in the equatorial cortex. The cortical microtubule network seemed to be cut into two halves at the position of the actin bundle. Shortly after, the nucleus divided into two nuclei, then the cell body was constricted at its equator and divided into one anterior and one posterior halves which were soon rebuilt to produce two cells with two full sets of cortical microtubules. From our observations, several mechanisms for the duplication of the microtubule networks during mitosis in O. marina are discussed.