Cyclic regulation of cytokinesis in amphibian eggs

The effects of amphibian egg cytoplasm extracted at different times after activation and during the first four cleavages on cytokinesis were examined. Extracts of artificially activated or fertilized Xenopus or Pleurodeles eggs taken at the time of activation (T = 0) provoked precocious cleavage furrows in Pleurodeles eggs. Between T = 0.25 and T = 0.75 of the first cell cycle, the period corresponding to interphase, an inhibitory effect was found, and the division of injected eggs was delayed up to 30%. After T = 0.75, that is during mitosis, the cleavage induction effect was observed again. These enhancing and inhibitory effects were also found in the two fractions obtained following gel filtration of the cytoplasmic extracts. These experiments support the hypothesis that two antagonistic factors control cytokinesis. The inhibitory factor is active only during interphase, while the positive factor is present during mitosis and appears to regulate cytokinesis.     

Localization and activation of the ARF6 GTPase during cleavage furrow ingression and cytokinesis

The ARF6 GTPase mediates cell shape changes in interphase cells through its effects on membrane cycling and actin remodeling. In this study, we focus our attention on the dynamics of cell division and present evidence supporting a novel role for ARF6 during cleavage furrow ingression and cytokinesis. We demonstrate that endogenous ARF6 redistributes during mitosis and concentrates near the cleavage furrow during telophase. Constitutively activated ARF6 localizes to the plasma membrane at the site of cleavage furrow ingression and midbody formation, and dominant negative ARF6 remains cytoplasmic. By using a novel pull-down assay for ARF6-GTP, we find an abrupt, but transient, increase in ARF6-GTP levels as cells progress through cytokinesis. Whereas high levels of expression of a GTPase-defective ARF6 mutant induce aberrant phenotypes in cells at cytokinesis, cells expressing low levels of ARF6 mutants do not display a significant mitotic delay or cytokinesis defect, presumably due to compensatory or redundant mechanisms that allow cytokinesis to proceed when the ARF6 GTPase cycle is disrupted. Finally, actin accumulation and phospholipid metabolism at the cleavage furrow are unchanged in cells expressing ARF6 mutants, suggesting that ARF6 may be involved in membrane remodeling during cytokinesis via effector pathways that are distinct from those operative in interphase cells.     

Cortical and cytoplasmic flow polarity in early embryonic cells of Caenorhabditis elegans

We have examined the cortex of Caenorhabditis elegans eggs during pseudocleavage (PC), a period of the first cell cycle which is important for the generation of asymmetry at first cleavage (Strome, S. 1989. Int. Rev. Cytol. 114: 81-123). We have found that directed, actin dependent, cytoplasmic, and cortical flow occurs during this period coincident with a rearrangement of the cortical actin cytoskeleton (Strome, S. 1986. J. Cell Biol. 103: 2241-2252). The flow velocity (4-7 microns/min) is similar to previously determined particle movements driven by cortical actin flows in motile cells. We show that directed flows occur in one of the daughters of the first division that itself divides asymmetrically, but not in its sister that divides symmetrically. The cortical and cytoplasmic events of PC can be mimicked in other cells during cytokinesis by displacing the mitotic apparatus with the microtubule polymerization inhibitor nocodazole. In all cases, the polarity of the resulting cortical and cytoplasmic flows correlates with the position of the attenuated mitotic spindle formed. These cortical flows are also accompanied by a change in the distribution of the cortical actin network. The polarity of this redistribution is similarly correlated with the location of the attenuated spindle. These observations suggest a mechanism for generating polarized flows of cytoplasmic and cortical material during embryonic cleavages. We present a model for the events of PC and suggest how the poles of the mitotic spindle mediate the formation of the contractile ring during cytokinesis in C. elegans.     

A set of proteins showing cell cycle dependent modification in the early mouse embryo

The pattern of protein synthesis in the mouse egg shows several changes at fertilization and during first mitosis. Three groups of newly synthesized proteins, with molecular weights of about 30,000, 35,000, and 46,000, show variations in mobility on one- and two-dimensional gels that correlate with the cell cycle. Each group is composed of a polypeptide that is synthesized in unmodified form during interphase but is modified reversibly during meiosis or mitosis, by a process involving phosphorylation. Although these proteins cease to be synthesized during the second cell cycle, those made earlier persist and continue to show the same modifications during the next cell cycle. Like other eggs, fertilized mouse eggs show a requirement for protein synthesis in order to enter mitosis.     

CORTICAL F‐ACTIN REORGANIZATION AND A CONTRACTILE RING‐LIKE STRUCTURE FOUND DURING THE CELL CYCLE IN THE RED CRYPTOMONAD,PYRENOMONAS HELGOLANDII1

Cortical F‐actin reorganization during the cell cycle was observed in Pyrenomonas helgolandii U. J. Santore (SAG 28.87) for the first time in Cryptophyta using fluorescein‐isothiocyanate (FITC)–phalloidin staining. In interphase, a number of F‐actin bundles were observed as straight lines running parallel to the long axis of the cell on the cell cortical region. They extended from an F‐actin bundle that runs along the margin of the vestibulum. Although the F‐actin bundles running parallel to the long axis of the cell disappeared during anaphase, they gradually reappeared in telophase. By contrast, the F‐actin bundle along the vestibulum margin remained visible during cytokinesis and dynamically changed following the enlargement of the vestibulum, suggesting that F‐actin was involved in the mechanism of vestibulum enlargement. F‐actins were not found in the cytoplasmic and nucleoplasmic regions throughout the cell cycle. In addition, a contractile ring‐like structure appeared at the cleavage furrow during cytokinesis. Treatment with cytochalasin B and latrunculin B significantly inhibited the formation of cleavage furrow, resulting in forming an abnormal cell with two nuclei, suggesting that cytokinesis in P. helgolandii is controlled by the contractile ring‐like structure constituted of F‐actin.     

Cell cycle-associated changes in Slingshot phosphatase activity and roles in cytokinesis in animal cells

During cytokinesis the actomyosin-based contractile ring is formed at the equator, constricted, and then disassembled prior to cell abscission. Cofilin stimulates actin filament disassembly and is implicated in the regulation of contractile ring dynamics. However, little is known about the mechanism controlling cofilin activity during cytokinesis. Cofilin is inactivated by phosphorylation on Ser-3 by LIM-kinase-1 (LIMK1) and is reactivated by a protein phosphatase Slingshot-1 (SSH1). Here we show that the phosphatase activity of SSH1 decreases in the early stages of mitosis and is elevated in telophase and cytokinesis in HeLa cells, a time course correlating with that of cofilin dephosphorylation. SSH1 co-localizes with F-actin and accumulates onto the cleavage furrow and the midbody. Expression of a phosphatase-inactive SSH1 induces aberrant accumulation of F-actin and phospho-cofilin near the midbody in the final stage of cytokinesis and frequently leads to the regression of the cleavage furrow and the formation of multinucleate cells. Co-expression of cofilin rescued the inhibitory effect of phosphatase-inactive SSH1 on cytokinesis. These results suggest that SSH1 plays a critical role in cytokinesis by dephosphorylating and reactivating cofilin in later stages of mitosis.     

Microtubule cycles in oocytes of the surf clam, Spisula solidissima: an immunofluorescence study

Oocytes of the surf clam, Spisula solidissima, underwent germinal vesicle breakdown and two meiotic divisions to give off polar bodies when they were fertilized or parthenogenetically activated with KCl. Fertilized eggs further proceeded to mitosis and cleaved, while parthenogenetically activated eggs remained uncleaved. We examined changes in microtubule-containing structures during meiotic divisions and subsequent mitotic processes by immunofluorescence. A monoclonal anti-tubulin antibody was applied to alcohol-fixed eggs from which the vitelline membrane had been removed by protease digestion. Up to the stage of second polar body formation, the pattern of microtubule organization in the first and second meiotic spindles was identical in both fertilized and parthenogenetically activated eggs. However, while fertilized eggs formed a sperm aster and mitotic spindles later, activated eggs formed only monaster- or ring-shaped microtubule-containing structures which underwent cycles of alternating formation and breakdown. Lactoorecin staining of parthenogenetically activated eggs revealed that the chromosome cycle could occur in these eggs, in phase with this microtubule cycle.     

A Cytological Analysis of Differentiation Without Cleavage in Cytochalasin B- and Colchicine-Treated Embryos of Chaetopterus pergamentaceus

Chaetopterus eggs undergo characteristic ooplasmic rearrangements during development. Ooplasmic rearrangement in the absence of cell division is called differentiation without cleavage. Treatment of fertilized eggs with cytochalasin B allowed the continuation of nuclear divisions in the absence of cytoplasmic division. The ooplasmic rearrangements in uncleaved cytochalasin B-treated fertilized eggs closely paralleled those of normal development. Colchicine treatment, which blocks mitosis, arrested ooplasmic movements at a stage comparable to that of normal embryos at first cleavage. Neither drug eliminated the segregation between hyaloplasm and endoplasm, even though colchicine prevented the later rearrangements. Localizing movements are therefore dependent upon normal microtubule function, but not on microfilament function. The maintenance of localized materials does not seem to depend exclusively on either of these organelles.     

Protein synthesis in mouse embryos with experimentally produced asynchrony between chromosome replication and cell division

Summary The cleavage of fertilized mouse eggs was prevented during cytochalasin B incubation and consequently these eggs became tetraploid the following day during in vitro culture. When the eggs were cultured further in normal medium, they cleaved and gave rise to tetraploid blastocysts. Protein synthesis was analysed in these embryos at different developmental stages using two-dimensional polyacrylamide gel electrophoresis. The protein synthesis pattern of one-cell tetraploid eggs was intermediate between those of normal one- and two-cell embryos. Tetraploid two-cell embryos expressed protein sets equivalent to those of untreated four-cell embryos, and tetraploid four-cell embryos synthesized proteins similar to those of four- to eight-cell controls. At subsequent pre-implantation stages the asynchrony was no longer detectable. When fertilized eggs were cultured continuously in the presence of cytochalasin B, they became tetraploid, octoploid and more and more polyploid without cleavage occurring. The protein synthesis patterns expressed by these one-cell polyploid eggs did not resemble that of normal fertilized eggs, but were similar to those of cleaving control embryos and blastocysts of equivalent age and nuclear division. These results strongly suggest that in early mouse embryos stage-specific translation is temporally correlated with chromosome replication (karyokinesis) and independent of cell division (cytokinesis) or cell interaction.Some of these results were presented at the IX Congress of the International Society of Developmental Biologists in Basle, Switzerland, August 28–September 1, 1981     

Actin dynamics during the cell cycle in Chlamydomonas reinhardtii.

We have used two monoclonal antibodies to demonstrate the presence and localization of actin in interphase and mitotic vegetative cells of the green alga Chlamydomonas reinhardtii. Commercially available monoclonal antibodies raised against smooth muscle actin (Lessard: Cell Motil. Cytoskeleton 10:349-362, 1988; Lin: Proc. Natl. Acad. Sci. USA 78:2335-2339, 1981) identify Chlamydomonas actin as a approximately 43,000-M(r) protein by Western immunoblot procedures. In an earlier study, Detmers and coworkers (Cell Motil. 5:415-430, 1985) first identified Chlamydomonas actin using NBD-phallacidin and an antibody raised against Dictyostelium actin; they demonstrated that F-actin is localized in the fertilization tubule of mating gametes. Here, we show by immunofluorescence that vegetative Chlamydomonas cells have an array of actin that surrounds the nucleus in interphase cells and undergoes dramatic reorganization during mitosis and cytokinesis. This includes the following: reorganization of actin to the anterior of the cell during preprophase; the formation of a cruciate actin band in prophase; reorganization to a single anterior actin band in metaphase; rearrangement forming a focus of actin anterior to the metaphase plate; reextension of the actin band in anaphase; presence of actin in the forming cleavage furrow during telophase and cytokinesis; and finally reestablishment of the interphase actin array. The studies presented here do not allow us to discriminate between G and F-actin. None the less, our observations, demonstrating dynamic reorganization of actin during the cell cycle, suggest a role for actin that may include the movement of basal bodies toward the spindle poles in mitosis and the formation of the cleavage furrow during cytokinesis.     

Changes in levels of polymeric tubulin associated with activation and dorsoventral polarization of the frog egg

The level of polymeric tubulin was measured during the first cell cycle of the electrically activated and the fertilized egg of Xenopus laevis. Eggs were homogenized in a microtubule-stabilizing medium, and the amount of tubulin pelleted by centrifugation was determined by quantitative Western blots. The pelleted tubulin (polymer) was in the form of microtubules based on the presence of microtubules in the pellet and on the effects of cold, nocodazole, and D2O. Unactivated eggs had a high level of polymer (greater than 0.1 microgram/egg) which disappeared within minutes of activation. The level of polymer stayed low (less than 0.02 microgram/egg) until halfway through the cell cycle (0.5 on a normalized time scale) when the level rose to the preactivation value. There was a decrease associated with metaphase (0.85 normalized time) and a return to a high level at first cleavage (1.0 normalized time). Fertilized eggs showed a similar pattern although the amount of polymer increased earlier (0.3-0.5 normalized time), presumably due to the spermaster. The depolymerization of microtubules at activation indicates that there is a dramatic change of the cytoskeleton at this time. The polymerization at 0.5 normalized time coincides with the start of the cytoplasmic shift leading to dorsoventral polarity. This result, together with previous inhibitor studies, shows that microtubules are involved in dorsoventral polarization of the embryo.     

Mip1 associates with both the Mps1 kinase and actin,and is required for cell cortex stability and anaphase spindle positioning

The Mps1 family of protein kinases contributes to cell cycle control by regulating multiple microtubule cytoskeleton activities. We have uncovered a new Mps1 substrate that provides a novel link between Mps1 and the actin cytoskeleton. We have identified a conserved human Mps1 (hMps1) interacting protein and have termed Mps1 interacting protein-1 (Mip1). Mip1 defines an uncharacterized family of conserved proteins that contain coiled-coil and calponin homology domains. We demonstrate that Mip1 is a phosphoprotein that interacts with hMps1 in vitro and in vivo and is a hMps1 substrate. Mip1 exhibits dynamic localization during the cell cycle; Mip1 localizes to the actin cytoskeleton during interphase, the spindle in early mitosis and the cleavage furrow during cytokinesis. Mip1 function is required to ensure proper spindle positioning at the onset of anaphase after cells begin furrow ingression. Cells depleted of Mip1 exhibit aberrant mitotic actin filament organization, excessive membrane blebbing, dramatic spindle rocking and chromosome distribution errors during early cytokinesis producing high numbers of binucleate cells. Our data indicate that Mip1 is a newly recognized component of the actin cytoskeleton that interacts with hMps1 and that it is essential to ensure proper segregation of the genome during cell cleavage.Key words: Mps1 kinase, actin, Mip1, cytokinesis     

Targeting of dystroglycan to the cleavage furrow and midbody in cytokinesis

Dystroglycan is a cell adhesion molecule that interacts with ezrin family proteins and also components of the extracellular signal-regulated kinase pathway. Ezrin and extracellular signal-regulated kinase are both involved in aspects of the cell division cycle. We therefore examined the role of dystroglycan during cytokinesis. Endogenous dystroglycan colocalised with ezrin at the cleavage furrow and midbody during cytokinesis in REF52 cells. Live cell imaging of green fluorescent protein-tagged dystroglycan in Swiss 3T3 and Hela cells revealed a similar localisation. Live cell imaging of a dystroglycan lacking its cytoplasmic domain revealed an even membrane localisation but no cleavage furrow or midbody localisation. Deletion of a previously identified ezrin-binding site in the dystroglycan cytoplasmic domain however only resulted in a slight reduction in cleavage furrow localisation but loss of midbody staining. There was no apparent cytokinetic defect in cells depleted for dystroglycan, however apoptosis levels were considerably higher in dystroglycan knockdown cells. Cell cycle analysis showed a delay in G2/M transition, possibly caused by a more than 50% reduction in extracellular signal-regulated kinase levels in the knockdown cells. Dystroglycan may therefore not only have a role in organising the contractile ring through direct or indirect associations with actin, but can also modulate the cell cycle by affecting extracellular signal-regulated kinase levels.     

Mip1 associates with both the Mps1 kinase and actin,and is required for cell cortex stability and anaphase spindle positioning

The Mps1 family of protein kinases contributes to cell cycle control by regulating multiple microtubule cytoskeleton activities. We have uncovered a new Mps1 substrate that provides a novel link between Mps1 and the actin cytoskeleton. We have identified a conserved human Mps1 (hMps1) interacting protein we have termed Mps1 interacting protein-1 (Mip1). Mip1 defines an uncharacterized family of conserved proteins that contain coiled-coil and calponin homology domains. We demonstrate that Mip1 is a phosphoprotein that interacts with hMps1 in vitro and in vivo and is a hMps1 substrate. Mip1 exhibits dynamic localization during the cell cycle; Mip1 localizes to the actin cytoskeleton during interphase, the spindle in early mitosis, and the cleavage furrow during cytokinesis. Mip1 function is required to ensure proper spindle positioning at the onset of anaphase after cells begin furrow ingression. Cells depleted of Mip1 exhibit aberrant mitotic actin filament organization, excessive membrane blebbing, dramatic spindle rocking, and chromosome distribution errors during early cytokinesis producing high numbers of binucleate cells. Our data indicate that Mip1 is a newly recognized component of the actin cytoskeleton that interacts with hMps1 and that it is essential to ensure proper segregation of the genome during cell cleavage.     

Microfilaments during sea urchin fertilization: Fluorescence detection with rhodaminyl phalloidin

Rhodaminyl-labeled phalloidin is used to demonstrate the distribution of microfilaments during fertilization and early development in eggs of the sea urchins Arbacia punctulata and Lytechinus variegatus. The surface of unfertilized eggs have numerous punctate fluorescence sites at which rhodaminyl phalloidin binds, indicating the presence of actin oligomers or polymers. During fertilization this punctate pattern of fluorescence begins to change. Within thirty seconds of insemination, the fertilization cone is first detectable with this technique as an erect structure on the surface of the egg. The fertilization cone grows to a maximum size by 8–9 minutes, and is resorbed by 16 minutes after insemination. The surface of the fertilized egg displays numerous fluorescent fibers by 10 minutes after insemination rather than the punctate fluorescence observed in unfertilized eggs, indicative of the burst of microfilament assembly resulting in microvillar elongation. The elongated microfilaments persist through cytokinesis. Staining is also detected throughout the cortices of unfertilized, fertilized, and cleaving eggs. Cytochalasin E (10 μM, 30 min) prevents microfilament elongation and cytokinesis and reduces the cortical staining intensity after fertilization. At cleavage, contractile rings, appearing as narrow equatorial bundles of fibers, have been detected in Lytechinus variegatus as transient structures.     

LIN-5 is a novel component of the spindle apparatus required for chromosome segregation and cleavage plane specification in Caenorhabditis elegans

Successful divisions of eukaryotic cells require accurate and coordinated cycles of DNA replication, spindle formation, chromosome segregation, and cytoplasmic cleavage. The Caenorhabditis elegans gene lin-5 is essential for multiple aspects of cell division. Cells in lin-5 null mutants enter mitosis at the normal time and form bipolar spindles, but fail chromosome alignment at the metaphase plate, sister chromatid separation, and cytokinesis. Despite these defects, cells exit from mitosis without delay and progress through subsequent rounds of DNA replication, centrosome duplication, and abortive mitoses. In addition, early embryos that lack lin-5 function show defects in spindle positioning and cleavage plane specification. The lin-5 gene encodes a novel protein with a central coiled-coil domain. This protein localizes to the spindle apparatus in a cell cycle- and microtubule-dependent manner. The LIN-5 protein is located at the centrosomes throughout mitosis, at the kinetochore microtubules in metaphase cells, and at the spindle during meiosis. Our results show that LIN-5 is a novel component of the spindle apparatus required for chromosome and spindle movements, cytoplasmic cleavage, and correct alternation of the S and M phases of the cell cycle.     

MARCKS actin-binding capacity mediates actin filament assembly during mitosis in human hepatic stellate cells

Cross-linking between the actin cytoskeleton and plasma membrane actin-binding proteins is a key interaction responsible for the mechanical properties of the mitotic cell. Little is known about the identity, the localization, and the function of actin filament-binding proteins during mitosis in human hepatic stellate cells (hHSC). The aim of the present study was to identify and analyze the cross talk between actin and myristoylated alanine-rich kinase C substrate (MARCKS), an important PKC substrate and actin filament-binding protein, during mitosis in primary hHSC. Confocal analysis and chromosomal fraction analysis of mitotic hHSC demonstrated that phosphorylated (P)-MARCKS displays distinct phase-dependent localizations, accumulates at the perichromosomal layer, and is a centrosomal protein belonging to the chromosomal cytosolic fraction. Aurora B kinase (AUBK), an important mitotic regulator, β-actin, and P-MARCKS concentrate at the cytokinetic midbody during cleavage furrow formation. This localization is critical since MARCKS-depletion in hHSC is characterized by a significant loss in cytosolic actin filaments and cortical β-actin that induces cell cycle inhibition and dislocation of AUBK. A depletion of AUBK in hHSC affects cell cycle, resulting in multinucleation. Quantitative live cell imaging demonstrates that the actin filament-binding capacity of MARCKS is key to regulate mitosis since the cell cycle inhibitory effect in MARCKS-depleted cells caused abnormal cell morphology and an aberrant cytokinesis, resulting in a significant increase in cell cycle time. These findings implicate that MARCKS, an important PKC substrate, is essential for proper cytokinesis and that MARCKS and its partner actin are key mitotic regulators during cell cycle in hHSC.     

The Effects of Cytoskeleton Inhibitors on Cytoplasmic Localization in Chaetopterus pergamentaceus

We have treated fertilized and KCl-activated eggs of Chaetopterus pergamentaceus with microfilament and microtubule inhibitors to test the relationship of these cytoskeletal components to cytoplasmic localization. Low doses of cytochalasin B inhibited cleavage in fertilized eggs. Such embryos underwent differentiation without cleavage, a process characterized by relocalization of the yolky endoplassm to the center of the uncleaved egg and by the formation of cilia. Similar treatment of KCl-activated eggs inhibited ciliation, but not endoplasmic relocalization. Reversible inhibition of the first cleavage resulted in equal cleavage and differentiation of a larva lacking an apical organ. Inhibition of the first two cleavages resulted in differentiation without cleavage. At all concentrations high enough to block mitosis, colchicine prevented ciliation and endoplasmic relocalization. Thus microtubule organization, but not microfilament organization, is required for ooplasmic reorganization and differentiation without cleavage.     

Influence of the polyamine spermine on the organization of cortical filaments in isolated cortices of Xenopus laevis eggs

Microinjection of spermine into Xenopus laevis eggs induces precocious furrowing, and together with known spermine-actin interactions, suggests that spermine may be affecting cortical microfilaments involved in cytokinesis. An electron microscopic study of injected eggs revealed that the ultrastructure of the induced furrows was similar to that of both artificially activated eggs and fertilized eggs. In isolated egg cortices, increasing spermine concentrations (1, 3 and 10 mM) resulted in marked changes in cortical microfilament organization. At low concentrations, spermine appeared to stabilize microfilaments and at higher concentrations induced lateral associations between filaments and formation of bundles. The actin nature of these cortical microfilaments was confirmed by immunocytochemistry. The electrophoretic profiles of proteins from control and spermine-treated isolated cortices were similar. Although the total protein content of isolates in 3 and 10 mM spermine was elevated, the relative actin content remained constant. The results are in agreement with previous in vitro studies of polyamine interactions with actin and support the hypothesis that a polyamine-actin interaction may be important in the regulation of cytokinesis.     

Microtuble organization in Xenopus eggs during the first cleavage and its role in cytokinesis

It has been suggested that the organization of microtubules during mitosis plays an important role in cytokinesis in animal cells. We studied the organization of microtubules during the first cleavage and its role in cytokinesis of Xenopus eggs. First, we examined the immunofluorescent localization of microtubules in Xenopus eggs at various stages during the first cleavage. The astral microtubules that extend from each of the two centrosomes towards the division plane meet and connect with each other at the division plane as cytokinesis proceeds. The microtubular connection thus advances from the animal pole to the vegetal pole, and its leading edge is located approximately beneath the leading edge of the cleavage furrow. Furthermore, an experiment using nocodazole suggests that microtubules have an essential role in advancement of the cleavage furrow, but neither in contraction nor maintenance of the already formed contractile ring which underlies the cleavage furrow membrane. These results suggest that the astral microtubules play an important role in controlling the formation of the contractile ring in Xenopus eggs.