reorganization of actin cytoskeleton at the growing …second, actin dynamics in the f-actin...

INTRODUCTION

Cytokinesis in animal cells begins at late anaphase to telophasewith formation of the contractile ring, a ring-shaped bundle ofactin filaments in the cleavage furrow (CF) cortex (Schroeder,1975; Mabuchi, 1986). It has been demonstrated that geometryof furrowing is determined by the astral microtubules inechinoderm eggs (Rappaport, 1965; Hiramoto, 1971), or thecentral spindle in mammalian cultured cells (Wheatley andWang, 1996; Wheatley et al., 1998), which may transmit asignal to induce the formation of the CF. It has beendemonstrated that the contractile force of the ring is generatedby the actin-myosin interaction (Mabuchi and Okuno, 1977;Knecht and Loomis, 1987; DeLozanne and Spudich, 1987).Even though the framework of the mechanism of cytokinesiswas established by these studies, many questions still remainto be answered. The most important questions are (1) what isthe entity of the cleavage signal or signaling pathway?; and (2)

how is the contractile ring assembled from its constituents at amolecular level?

Xenopusegg is a good system for studying the mechanismof furrow formation to answer the above questions for thefollowing reasons. In amphibian eggs, the CF first appears atthe animal pole and advances toward the vegetal hemisphere,being formed continuously at the growing ends. Therefore, thesequences of CF formation can be investigated intensively atthe growing end of the CF, while the central region of the CFis suitable for investigating the mechanism of the contraction.Furthermore, the egg is large enough for microinjection atspecified regions of the cell.

In this report, reorganization of actin and myosin into thecontractile apparatus during furrow formation of Xenopuseggwas first highlighted to answer Question (2). In budding yeastcells (Epp and Chant, 1997; Lippincott and Li, 1998), seaurchin eggs (Mabuchi, 1994) and cultured mammalian cells(Mittal et al., 1987; Cao and Wang, 1990a; Cao and Wang,

401

We studied reorganization of actin-myosin cytoskeleton atthe growing ends of the cleavage furrow of Xenopuseggs inorder to understand how the contractile ring is formedduring cytokinesis.

Reorganization of F-actin structures during the furrowformation was demonstrated by rhodamine-phalloidinstaining of the cleavage furrow and by time-lapse scanningwith laser scanning microscopy of F-actin structures in thecleavage furrow of live eggs to which rhodamine-G-actinhad been injected. Actin filaments assemble to form smallclusters that we call ‘F-actin patches’ at the growing end ofthe furrow. In live recordings, we observed emergence andrapid growth of F-actin patches in the furrow region. Thesepatches then align in tandem, elongate and fuse with eachother to form short F-actin bundles. The short bundles thenform long F-actin bundles that compose the contractilering.

During the furrow formation, a cortical movementtowards the division plane occurs at the growing ends ofthe furrow, as shown by monitoring wheatgerm agglutinin-conjugated fluorescent beads attached to the egg surface.As a result, wheatgerm agglutinin-binding sites accumulate

and form ‘bleb-like’ structures on the surface of the furrowregion. The F-actin patch forms and grows underneath thisstructure. The slope of F-actin accumulation in the interiorregion of the furrow exceeds that of accumulation of thecortex transported by the cortical movement. In addition,rhodamine-G-actin microinjected at the growing end isimmediately incorporated into the F-actin patches. Thesedata, together with the rapid growth of F-actin patches inthe live image, suggest that actin polymerization occurs inthe contractile ring formation.

Distribution of myosin II in the cleavage furrow was alsoexamined by immunofluorescence microscopy. Myosin IIassembles as spots at the growing end underneath the bleb-like structure. It was suggested that myosin is transportedand accumulates as spots by way of the cortical movement.F-actin accumulates at the position of the myosin spot alittle later as the F-actin patches. The myosin spots and theF-actin patches are then simultaneously reorganized toform the contractile ring bundles

Key words: Cytokinesis, Actin, Myosin, Xenopusegg, Contractilering, Cleavage furrow

SUMMARY

Reorganization of actin cytoskeleton at the growingend of the cleavage furrow of Xenopus egg duringcytokinesisTatsuhiko Noguchi 1,* and Issei Mabuchi 1,2

1Division of Biology, School of Arts and Sciences, University of Tokyo, 3-8-1, Komaba, Meguro-ku, Tokyo, 153-8902, Japan2Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585, Japan*Author for correspondence (e-mail: [email protected])

Accepted 31 October 2000Journal of Cell Science 114, 401-412 © The Company of Biologists Ltd

RESEARCH ARTICLE

402

1990b; Fishkind and Wang, 1993), the sequential changes inlocalization of actin and myosin during the CF formation havebeen studied. However, information is limited, since these cellsare either too small to see the cytoskeletal changes in detail, orthey form the contractile ring too quickly. On the other hand,in cells that naturally undergo unilateral cleavage, such asamphibian eggs, the reorganization of the actin-myosincytoskeleton has not yet been well studied. We took theadvantage of Xenopus eggs to analyze contractile ringformation by examining the cytoskeletal changes at thegrowing end of the CF. Second, actin dynamics in the F-actinstructures in the CF was examined. Although it has been knownthat the contractile ring is formed very quickly, little is knownhow actin molecules are incorporated into it. It has beenreported in cultured mammalian cells that F-actin in thecontractile ring may be recruited from pre-existing cortical F-actin (Cao and Wang, 1990a; Cao and Wang, 1990b). However,it has recently been suggested that actin polymerization maybe involved in the contractile ring formation, since profilin,which is capable of accelerating actin polymerization, is foundin the CF of fission yeast (Balasubramanian et al., 1994),Tetrahymena(Edamatsu et al., 1992) and cultured mammaliancells (Watanabe et al., 1996). Moreover, disruption of thefunction of actin-depolymerizing factor ADF/cofilin inhibitscytokinesis in some organisms (Abe et al., 1996; Gunsalus etal., 1995), suggesting that turnover of actin may play a role incytokinesis. To this end, we extensively examined the actindynamics of contractile ring.

We carried out following experiments in order to elucidatethese points. We first examined the F-actin organization in theCF of Xenopuseggs both by fluorescent staining of F-actin inthe CFs isolated at various stages, and by observation of liveeggs into which rhodamine-labeled G-actin had been injected.In addition, rapid incorporation of G-actin into the contractilering both during and after its formation was demonstrated.Second, we examined localization of myosin II at the growingend of the CF by immunofluorescence microscopy. Third, wecompared the distribution of F-actin, myosin II and surfacewheatgerm agglutinin (WGA)-binding sites, in order todetermine the sequence of appearance of these components atthe growing end.

MATERIALS AND METHODS

Handling of animals and eggsFemale Xenopus laeviswere induced to ovulate by injection of 400units of human chorionic gonadotropin (Denka Seiyaku, Tokyo,Japan) a day before use. Mature eggs were inseminated with a spermsuspension obtained by macerating the testes in DeBoer’s solution(110 mM NaCl, 1.3 mM KCl, 0.44 mM CaCl2 with addition ofNaHCO3 to pH 7.3), and activated in a tap water. Five minutes later,the eggs were dejellied with 2% cysteine, pH 8.1. Vitelline membraneswere removed manually with watchmaker’s forceps. The eggs werecultured in modified Steinberg’s solution (MSS; 60 mM NaCl, 0.67mM KCl, 0.34 mM Ca (NO3)2, 0.83 mM MgSO4, 10 mM N-2-hydroxyethylpiperazine-N’-ethanesulfonic acid (Hepes), pH 7.4) atroom temperature.

Anti- Xenopus myosin II serumXenopuscytoplasmic myosin II was prepared from oocytes accordingto Satterwhite et al. with modification (Satterwhite et al., 1992). TheKI-ATP gel filtration step was omitted because the exclusion of actin

was not necessary in the present study. The crude myosin fractionobtained after two cycles of polymerization and depolymerization wasfractionated successively by hydroxylapatite column chromatographyand Sephadex G-200 gel filtration column chromatography. The peakfractions obtained after the gel filtration column chromatography wereconcentrated by precipitation with 5% (w/v) trichloroacetic acid, andmyosin II heavy chain was separated by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE). After CoomassieBrilliant Blue staining, the band was dissected, homogenized inFreund’s complete adjuvant, and injected subcutaneously into twomale rabbits. About a month after the first immunization, the boostinjections were carried out every two weeks followed byimmunoblotting to check the production of antibodies.

Fixation and staining of isolated CFEggs were fixed in F-buffer (0.1 M KCl, 5 mM MgCl2, 0.2 mM CaCl2,50 mM Hepes, pH 7.5) containing 3.7% formaldehyde for 30 to 50minutes at room temperature at various stages during the firstcleavage. Then they were treated with 0.5% Triton X-100 in F-bufferfor 15 minutes. The cortices gradually became peelable after thetreatment with Triton X-100. After three washes with F-buffer, F-actinwas stained with fluorescently labeled phalloidin (Molecular Probes,Eugene, OR) for 1 hour, and the cortices containing CF were isolatedmanually from the eggs by using a glass needle. Forimmunofluorescence staining, the eggs were fixed 30-35 minutes asdescribed above, and then transferred into 0.5% Triton X-100 in F-buffer. The CFs were carefully isolated from the eggs within 15minutes of the transfer. Both the times for the fixation and for theTriton X-100 treatment were critical for excluding the yolk granulesfrom the preparation, which was important because fluorescentlylabeled secondary antibodies strongly bound to yolk granules in anonspecific manner. The isolated cortices were post-fixed with 3.7%formaldehyde for 10 minutes to preserve the actin-myosincytoskeleton. After three washes with a phosphate buffered saline(PBS), the isolated cortices were incubated in 2% bovine serumalbumin (BSA) dissolved in PBS for 1 hour. The cortices were thenincubated with anti-Xenopusmyosin II serum (1/100 dilution) for 90minutes, washed with PBS three times, and incubated withrhodamine-conjugated anti-rabbit IgG antibodies (Organon Teknika,Cappel Research Products, Durham, NC) for 90 minutes, followed bythree washes. For counterstaining of F-actin, Bodipy-phallacidin(Molecular Probes) in PBS was added to the cortices and incubatedfor 30 minutes. WGA-binding sites were stained with 10 µg/mlfluorescein isothiocyanate (FITC)-WGA (Vector Labs, Burlingame,CA) for more than 30 minutes. The fluorescently stained cortices wererinsed with PBS, mounted in Mowiol, and examined immediately byfluorescence microscopy from the surface side, as described below.

Preparation of fluorescently labeled G-actinRabbit skeletal muscle actin was prepared according to Spudich andWatt (Spudich and Watt, 1971) and gel-filtered through a SephadexG-100 column. Both actin and BSA were labeled with 5-carboxytetramethylrhodamine succinimidyl ester (Molecular Probes),as described by Kellogg et al. (Kellogg et al., 1988). The labeled G-actin (rhodamine-actin) and the labeled BSA (rhodamine-BSA) (both4 mg/ml) in G-buffer (1 mM Hepes, pH 7.5, 0.1 mM ATP, 0.1 mMCaCl2) were frozen in liquid nitrogen. The labeled proteins werecentrifuged at 100,000 g for 1 hour just prior to use.

Microinjection of rhodamine-actin at the growing endVitelline membranes of fertilized and dejellied eggs were manuallyremoved and the eggs were transferred into MSS. The volume ofinjectant was determined by measuring the diameter of dropletsinjected into a salad oil droplet. The eggs were injected with less than0.1 nl of labeled protein solution near the growing end of the CFduring stage 2 (see Fig. 1H) or a middle region of the CF during stage3. To improve the reproducibility and to avoid possible formation of

JOURNAL OF CELL SCIENCE 114 (2)

403Actin reorganization during cytokinesis

aggregates of rhodamine-actin in the CF region, the injection site wasrestricted to about 50 µm away from the growing end of the CF,avoiding the division plane. Under these conditions, the injectionneither interfered with the advancing of the CF nor with theorganization of F-actin structures in the CF. The eggs were fixed 30to 60 seconds after the injection, and counterstained with Bodipy-phallacidin to reveal total F-actin organization as described above.

Preparation of lectin-conjugated beads and visualizationof cortical movementsIt has previously been shown that WGA-binding sites accumulate inthe CF region of Xenopusegg (Tencer, 1978). In order to demonstratethe cortical movement at the growing end of the CF, WGA wasconjugated to carboxylated fluorescent polystyrene beads(FluoSphere, Molecular Probes) according to previously describedmethods (Wang et al., 1994; Tompson et al., 1996) with modifications.A 2% aqueous suspension of FluoSphere was mixed with an equalvolume of 8% electron microscope (EM) grade glutaraldehyde (WakoPure Chemical Industries, Osaka) and incubated at 25oC for 2 hourswith gentle agitation. The beads were gently pelleted and rinsed threetimes with 25 mM Na-phosphate buffer, pH 7.0. An equal volumeof 2 mg/ml WGA was added to 2% (w/v) suspension of thegultaraldehyde-activated beads and the suspension was gently agitatedovernight at 4oC. The WGA-conjugated beads were then pelleted andrinsed three times with 50 mM Tris (hydroxymethyl) aminomethanebuffer, pH 8.0 to stop the reaction. The beads were stored in MSScontaining 0.02% NaN3 at 4oC before use. They were rinsed threetimes with MSS prior to use.

The WGA-beads were applied to fertilized eggs withoutfertilization membranes and incubated for 15 minutes. The eggs werethen washed gently to remove unadhered beads. In most cases, anumber of beads adhered on the surface of the eggs, which wereenough for real-time recording of the cortical movement.Subsequently, residual WGA-binding sites on the living egg surfacewere labeled with rhodamine-WGA to visualize the CF. The real-timemovement of each bead was recorded as described below. Thismethod allowed us to reveal spatial and temporal relationship betweenthe growing end of the CF and the cortical movement around it at ahigh resolution.

Immunoblot analysisDejellied oocytes were washed three times with Marc’s ModifiedRinger’s solution (0.1 M NaCl, 2 mM KCl, 2 mM CaCl2, 1 mMMgCl2, 5 mM Hepes, pH 7.4) and then three times with X-buffer (0.1M KCl, 2 mM MgCl2, 0.2 mM CaCl2, 50 mM sucrose, 10 mM Hepes,pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10µg/ml leupeptin, 10 µg/ml pepstatin A). The oocytes were gentlycentrifuged at 100 g and the buffer was discarded. The oocytes wereresuspended in five volumes of X-buffer containing 0.5% Triton X-100, and the suspension was homogenized with a hand-made Eponpestle-Eppendorf tube homogenizer. The homogenate was centrifugedat 10,000 g for 20 minutes and transparent supernatant was collectedavoiding contamination by floating lipid and yolk pellets. The lysatewas diluted in the Laemmli’s sample buffer (Laemmli, 1970) andsubjected to SDS-PAGE, followed by transfer onto a polyvinylidenedifluoride membrane. The membrane was blocked with 2% skim milkin PBS for 1 hour, and then incubated with anti-myosin II serum orpreimmune serum diluted to 1/10,000 with the blocking solution. Thesignal was detected using a chemiluminescence system (AmershamInternational, UK).

Fluorescence microscopy and image-processingSpecimens were examined using a Zeiss Axioscope microscopeequipped with a Neo-Fluar 10×/numerical aperture (NA) 0.3, a Neo-Fluar 20×/NA 0.7 and a Plan-Apochromat 63×/NA 1.4 oil immersionobjective lenses. Microphotographs were taken on Kodak T-MAXASA 400 films. Live recordings of WGA-conjugated beads on the

surface of living eggs were performed using an Achroplan water10×/NA 0.3 or a 40×/NA 0.75 lens. Fluorescent images were detectedusing a color chilled CCD camera (C5810, Hamamatsu Photonics,Hamamatsu, Japan). The digital images were processed by Photoshop4.0j and by FISH imaging software (Hamamatsu Photonics). Z-sections of fluorescently stained egg surface were performed using aZeiss 510 confocal laser scanning microscope (LSM) and a DeltaVision system (Applied Precision, Issaquah, WA) attached to anOlympus IX-70-SIF microscope.

Time-lapse confocal microscopy of living eggs20 nl of 4 mg/ml rhodamine-actin was injected into an egg within 30minutes of fertilization. The rhodamine-actin diffused evenly in thecytoplasm before the first cleavage. The growing ends of the CF weremarked by FITC-WGA staining. The egg was settled in a handmadechamber in which it was compressed by a coverslip in order to flattenthe cortex (see Fig. 2F). The growing end of the CF in the flattenedanimal cortex was examined by LSM equipped with 63× Plan-apochromat oil immersion lens at an optical section of 1.5 µm.Serially scanned images were taken to demonstrate changes in the F-actin orgaization during the contractile ring formation.

RESULTS

F-actin organization in the cleavage furrow atvarious stages during the first cleavage of Xenopusegg F-actin structures in the CF cortices isolated from Xenopuseggs at various stages were revealed by rhodamine-phalloidinstaining in order to investigate the reorganization of F-actincytoskeleton during the 1st cleavage (see Fig. 1H for thestages). At the beginning of furrow formation, when a blacksingle stripe appeared at the animal pole (stage 1), numerousclusters of F-actin, the diameter of which was 0.5 to 1 µm, wereformed in the CF region (Fig. 1A,B). We call this structure the‘F-actin patch’. Besides the F-actin patches, large F-actinaggregates of several micrometers in diameter were alsorecognized in this region. At this stage, no contraction of theCF along its longitudinal axis was detected. In the followingstage (stage 2, Fig. 1C-E), the CF initiated contraction alongits longitudinal axis. We found three distinct F-actin structuresat this stage. At the growing end of the CF, the F-actin patchesand radially oriented short and thick F-actin bundles (shortactin bundles) were observed (Fig. 1C,E). The mean length ofthe short F-actin bundles was 2.8±1.1 µm. These bundlesseemed to consist of several small F-actin patches that hadfused with each other in a linear fashion. However, in thecentral region of the CF, a number of long F-actin bundles, thewidth of which varied from 0.1 to 1 µm, were observed (Fig.1D). The long F-actin bundles were aligned in a parallelfashion to the longitudinal axis of the CF. We could notdetermine their exact length, but they were more than severalmicrometers long. We assumed that the long F-actin bundlewas a unit of the contractile ring. It seemed that at the growingend of the CF, the F-actin patches are formed at first, then somepopulation of the patches are aligned into the short F-actinbundles, and finally the short actin bundles are reorganized intothe long F-actin bundles. As the furrow region became widerand wider during stages 2 and 3, the number of the long F-actinbundles increased. The width of a belt of the long F-actinbundles reached 100 µm at stage 3 (Fig. 1F). Suddenly, in thenext stage (stage 4, Fig. 1G), the belt became tightly packed.

404

The width of the belt in this stage was less than 20 µm. Thischange seemed to coincide with deepening of the furrow andwith addition of a large amount of new cell membrane in theCF. Finally, at stage 5, two growing ends merged at the vegetalpole to form a complete ring structure that encircles this largecell (not shown).

Time-lapse imaging of the reorganization of F-actinpatches into F-actin bundles in stage 2 CFIn order to demonstrate the reorganization of F-actin patchesinto F-actin bundles at the growing end of the CF in stage 2 asdescribed above, we visualized the F-actin structures in the CFof living eggs under a confocal LSM, into which rhodamine-actin had been injected. First, a rhodamine-actin-injected eggwas fixed, and the cortex was isolated and counterstained withBodipy-phallacidin. The rhodamine fluorescence pattern wassimilar to the Bodipy fluorescence pattern (Fig. 2A-C). Thus,we concluded that rhodamine-actin was faithfully incorporatedinto the CF F-actin structures as an intact monomer. Next, toverify further that the signal detected in the thin optical sectionobtained with the LSM is attributed to rhodamine-actinincorporated into the cortical F-actin but not to free G-actindiffused in the cytoplasm, we carried out a z-axis sectioningfrom outside through surface to inside the egg (Fig. 2D). Wefound a clear fluorescence peak at the cortex, of the rhodamine-actin-injected egg. However, no apparent peak but a diffusedfluorescence was observed in the cytoplasm of rhodamine-BSA-injected egg (Fig. 2E). The width of the rhodamine-actinpeak along z-axis was less than 2 µm. Therefore, we fixed thedepth (focal plane) to be scanned at the peak of rhodamine-actin and set the thickness of the optical section at 1.5 µmin the following examinations. At these settings, we were able to obtain a live image (Fig. 2G) of rhodamine-actin in theCF.

The sequential imaging demonstrated the reorganization ofthe F-actin structures at the growing end of the CF in stage 2.In this region, numerous F-actin patches were observed. As theCF advanced, the number of F-actin bundles graduallyincreased in the same area (Fig. 2H,I). At a highermagnification, we could pursue destination of each patch (Fig.2J). The patches seemed to emerge at random positions.Timing of emergence seemed to be different among the patcheseven in a small area of the cortex. It took 30 to 40 seconds forthe full growth of the patch from the emergence to reachingmaximum in fluorescence (see arrowhead 3 in Fig. 2J). Then,several neighbouring patches, which were close to a lineparallel to the long axis of the furrow, interconnected with eachother, and arranged to line up in the direction of the line toform a short bundle (Fig. 2J). A little later, the short bundlesfused with each other at their ends to form longer bundles (Fig.2K). These observations directly demonstrate that the F-actinpatches are reorganized into the F-actin bundles.

Rapid incorporation of G-actin into F-actin patchesand F-actin bundlesWe investigated how fast G-actin is incorporated into thecontractile ring structure by injecting rhodamine-actin near thecontractile ring. The injections were made at sites 50 µm awayfrom the CF. Whole F-actin organization was visualized bycounterstaining with Bodipy-phallacidin after fixation of thecell. Most of the injected eggs cleaved normally under the

experimental conditions. No difference was observed betweenthe F-actin organization in the CF of the injected eggs and thatof uninjected eggs. Results are summarized in Table 1.

First, rhodamine-actin was injected near the growing end ofa CF during stage 2 and the egg was fixed at 30 seconds afterthe injection. In most cases, rhodamine-actin was incorporatedinto all the F-actin patches near the injection site and also someshort actin bundles (Fig. 3A-E).

In late stage 2 or stage 3, the contractile ring, which wascomposed of numerous long F-actin bundles, was well formed(see Fig. 1F). To investigate whether established contractilering can incorporate G-actin, rhodamine-actin wasmicroinjected near the central region of the CF and the cell wasfixed at 60 to 90 seconds after the injection. As shown in Fig.3F-J, rhodamine-actin was incorporated into the long F-actinbundles and there was no difference in the fluorescence patternbetween the incorporated rhodamine-actin and Bodipy-phallacidin staining of the whole F-actin structures. However,only weak signals were detected when the fixation wasperformed at 30 seconds after the injection (not shown). Thus,the rate of incorporation of rhodamine-actin into the long F-actin bundles was apparently slower than that into the F-actinpatches at the growing end. As a control, rhodamine-BSA wasmicroinjected. No particular signal of rhodamine-BSA wasdetected in the actin cytoskeleton (not shown).

Cortical movement at the growing end of the CFIt has been reported in cultured mammalian cells that F-actinin the contractile ring is accumulated by transportation fromsurrounding cortex rather than new actin polymerizationduring the formation of the contractile ring (Cao and Wang,1990a; Cao and Wang, 1990b). However, above resultssuggest that the actin polymerization occurs in order to formthe F-actin patches at the growing end. To further investigatewhether the F-actin accumulated in the CF is derived fromcortical actin or newly polymerized F-actin, we carried outtwo experiments. First, we examined movement of cortexaround the CF during the furrow formation, which maytransport the cortical F-actin to the division plane. Second,we compared the amounts of cortex and F-actin accumulatedin the furrow region in stage 1.

WGA is known to bind to a carbohydrate moiety ofproteins on the cell surface. When a dividing egg at stage 1was stained with FITC-WGA, the CF appeared as afluorescent spindle-shaped region surrounded by a dark zoneabout 20-30 µm wide on both sides of the furrow where somestress wrinkles were recognized (Fig. 4A), indicating that theWGA-binding sites were accumulated in the CF. The darkzone will be called peri-CF zone hereafter. Simultaneouslabeling with rhodamine-WGA and WGA-fluorescent beadsof the WGA-binding sites on the surface of living eggsenabled us to observe the furrow position and its progressionin real time. A movement of the WGA beads fromsurrounding cortex towards the CF was detected (Fig. 4B).The mean speed of the movement during the stage 1 was15.8±3.5 µm/min (n=11). We call this movement the ‘corticalmovement’. This movement actively occurred in the peri-CFzone. It started at the beginning of the stage 1, before theformation of the short F-actin bundles or the long F-actinbundles, indicating that the cortical movement wasindependent of the contraction of the contractile ring.



Accumulation of F-actin in the early CFThe dominant F-actin structure in the CF in the stage 1 was theF-actin patch as described above (Fig. 1). The double stainingusing rhodamine-phalloidin and FITC-WGA enabled us tocompare the accumulation of F-actin and that of WGA-bindingsites during CF formation (Fig. 5A,B). Line profiling analysisof the fluorescence intensity of both rhodamine-phalloidin andFITC-WGA along the furrow axis was carried out to quantifyF-actin and the WGA-binding sites, respectively, of the earlyfurrow. It revealed that the amount of the WGA-binding sitesincreased gradually from the end to the middle of the CF up to1.5±0.3-fold (n=9) (Fig. 5D). However, the amount of F-actinincreased gradually at first and then steeply to reach a plateaunear the middle. The total increase was 3.3±1.2-fold (2.3- to5.2-fold, n=12). In other words, the slope of the F-actinaccumulation exceeded that of the accumulation of the cortexin the interior region of the CF. This suggests that a significantpart of F-actin in the CF accumulates by a mechanism that isdifferent from the cortical movement. In addition, it wasestimated that the steeply increasing phase of F-actincorresponded to about 1 minute.

At a higher magnification, the FITC-WGA staining revealednumerous spherical structures on the surface of the CF (Fig. 5).These structures were extruded from the surface, which wasapparent by changing the focus of the microscope. We call thisstructure ‘WGA-binding bleb-like structure’ (WGA-bleb). TheF-actin patch seemed to localize inside the WGA-bleb especiallynear the neck region (Fig. 5E-J). At the end of the furrow markedby the FITC-WGA staining, the WGA-blebs colocalized with F-actin patches containing a very low level of F-actin (Fig. 5K,M).However, in the middle region of the CF, they colocalized withF-actin patches containing a high level of F-actin (Fig. 5L,N),although the blebs themselves did not seem to change both size

and brightness, indicating that amount of F-actin in the patchesincreased from the end to the middle region of the early CF,being consistent with the result of the line profiling analysis.

Effects of cytoskeleton inhibitors on the formationof WGA-blebs 5 mM 2, 3-butanedione monoxyme (BDM), which has beenknown to inhibit myosin II ATPase activity (Cramer andMitchison, 1995), was microinjected into eggs near thegrowing end of the CF during stage 1 or stage 2. As judged bythe FITC-WGA staining, the WGA-bleb formation at thegrowing end was markedly inhibited by the injection (12/14CFs). During stage 2 when the contraction of the contractilering had started, the furrow partially regressed and thecontractile activity was significantly depressed by the BDMinjection (7/16 CFs). The second and third cleavages occurredalmost normally, suggesting that the inhibitory effect of BDMwas reversed; the drug may have diffused out from the eggs.

When eggs were incubated either in 0.1 mM cytochalasin Bfor 40 minutes (n=25) or 2.3 µM latrunculin A for 20 minutes(n=10), before formation of the CF to disrupt cortical actinfilaments, the WGA-bleb formation was significantly disturbedin all the eggs examined. These results suggest that bothmyosin and cortical F-actin are required in the formation of theWGA-blebs.

Immunofluorescent staining of myosin II in the CFIn order to examine the distribution of myosin II and to compareit with that of F-actin structures, antibodies were raised againstmyosin II heavy chains purified from Xenopusoocytes. A whole-cell lysate excluding yolk granules was subjected to immunoblotanalysis using the anti-myosin II serum to check the specificityof the antibody. An intense signal was detected with the

Fig. 1.F-actin-containing structuresin the CFs of Xenopuseggs. F-actinstructures in isolated CFs werestained with rhodamine-phalloidin.(B,D,E) Selected parts of A,C, at ahigher magnification. (A,B) Stage 1CF. Numerous F-actin patches andaggregates are seen. (C-E), Stage 2CF. F-actin patches (0.5 ~ 1 µm indiameter) are emerging at thegrowing end (small arrows in E).Large F-actin aggregates (large arrowin E) are also seen. Short F-actinbundles, which are radially orientedare formed in addition to the F-actinpatches at the growing end (E,arrowheads). Long F-actin bundles,which are aligned parallel to the axisof the CF, are formed in the centralregion (D, arrowheads). (F) Stage 3CF. The furrow region becomeswider, and the long F-actin bundles(arrow) increase in number. (G) Stage4 CF. The long F-actin bundles aresuddenly packed to form the thinnercontractile ring. (H) Stages of the 1stcleavage of Xenopusegg. Scale bar inA: 20 µm for A,C,F,G. Scale bar inB: 10 µm for B,D,E.

406

antiserum as a single band at a position of 200 kDa, while nosignal was detected with pre-immune serum (Fig. 6A).

Immunofluorescent staining with the anti-myosin II serumrevealed that, at the growing end of the CF, myosin II assemblyoccurs to form dotty clusters (Fig. 6B,F). We call this structure‘myosin spot’. The size of the myosin spots throughout thegrowing end region was quite homogeneous with a meandiameter of 0.5±0.1 µm. At a higher magnification of thegrowing end of the CF (Fig. 6F,G), the myosin spotscolocalized with newly emerging F-actin patches. Somemyosin spots were arranged into tandem arrays along the shortF-actin bundles. Interestingly, at the periphery of the growingend of the CF, the myosin spots were not accompanied by theF-actin staining, suggesting that myosin spot formationprecedes the F-actin accumulation at F-actin patch. In aninterior region of the CF, the tandemly aligned myosin spots

seemed to form a fibrous structure along the long F-actinbundles (Fig. 6H,I). In contrast, neither spot-like structure norfibrous structure was observed when the furrow was stainedwith the pre-immune serum as a control.

Appearance of myosin II, F-actin and WGA-bindingsite at the growing end of CFThe relationship between the WGA-binding sites, the F-actinpatches, and the myosin spots described above was investigatedby staining the CF at stage 1 with FITC-WGA, Bodipy-phallacidin and anti-myosin II antibodies (Fig. 7).

It seemed from the double-staining using anti-myosin


Fig. 2.Reorganization of the F-actinpatches into F-actin bundles at thegrowing end of the stage 2 CF in aliving egg. Rhodamine-actin wasinjected to visualize the F-actinstructures at the growing end of a CF ina living egg. (A-C) a CF-containingcortex of the rhodamine-actin-injectedegg was isolated and examined for therhodamine fluorescence in order toverify the incorporation of rhodamine-actin into F-actin structures in the CF

(A). Whole F-actin in the cortex wascounterstained with Bodipy-phallacidin (B).(C) Merged image of (A,B). (D,E) z-axissections from outside, through surface, toinside of eggs (from the top to the bottom ofthe pictures). Twelve optical sections of 1 µmthick were piled up and the z-axis-sectionedimages were reconstructed. In a rhodamine-actin-injected egg (D), a sharp peak offluorescence (arrowhead) was recognized thatwe attributed to the signal from cortical F-actin. However, there was only diffused signal

of fluorescence in cytoplasm without obvious peak in arhodamine-BSA-injected egg (E). (F) The handmadechamber for scanning the cortex of a rhodamine-actin-injected egg. (G) A live image of a stage 2 CF ofrhodamine-actin injected egg at low magnification.(H) The area of the scanning (blue rectangles) at thegrowing end of the CF shown in I. The dots, the brokenlines and the black lines represent F-actin patches, thepatches linked with each other and F-actin bundles,respectively. The blue arrow indicates the direction of thegrowth of the CF. (I) Sequential images of the F-actinstructures at the growing end of the CF of a living egg at ahigher magnification. The F-actin patches (small arrows)and the F-actin bundles (arrowheads) were clearlyobserved. In the left-hand picture (0 seconds), thedominant F-actin structures are the F-actin patches. As thefurrow advanced, the number of F-actin bundles gradually

increased in this area (pictures 23.6 and 43,3 seconds).(J) Further magnified pictures showing an area of I. The F-actinpatches move to line up and fuse with each other to form shortF-actin bundles. Arrowheads indicate individual F-actinpatches. Among them, some are newly developing (arrowheads1 and 3). (K) Another magnified area of I. The short F-actinbundles further fuse with each other at their ends to form longerand thinner F-actin bundles. Arrowheads indicate the individualshort F-actin bundles. The times (seconds) of the recordings

from 0 seconds are indicated in each image. Scale bar: 40 µm inA-C; 10 µm in D,E; 30 µm in G; 10 µm in I; 2 µm in J,K.


antibodies and Bodipy-phallacidin that accumulation ofmyosin II precedes that of F-actin at the growing end of theCF in all CFs examined (n=16, Fig. 7A,B). Line profilinganalysis revealed that myosin II accumulation occurs graduallyfrom the end to the middle of the CF (Fig. 7D, average extentof increased myosin; 1.7±0.4 fold, n=13). This contrasted withthe accumulation of F-actin, which was first gradual and thensteep as described above. This suggests that myosin spotformation precedes the accumulation of F-actin at the F-actinpatches in the CF, and the F-actin accumulation continues afterthe completion of the myosin spot formation.

The double staining of stage 1 CF using anti-myosin IIantibodies and FITC-WGA showed that assembly of theWGA-blebs and that of the myosin spots occurred in a similarmanner (Fig. 7E,F). Both extents and slopes of increase ofmyosin and WGA-binding sites from the end to the middle ofthe early furrow were comparable (not shown for Fig. 7E,F;see Figs 5D, 7D). The myosin spot and the WGA-blebcolocalized with each other at the end of CF (Fig. 7G,H). Inthe bleb, myosin was concentrated at the neck region.

DISCUSSION

Xenopusegg has been a good system for studying factorsinvolved in cytokinesis, because of the ease with which it canbe micromanipulated and microinjected. However, little isknown about the organization of F-actin structures in the CFof this cell both during and after its formation. Here, wedemonstrated process of organization of actin and myosin into

the contractile ring of Xenopuseggs. The Methods and Resultspresented in this report contribute to determining the steps atwhich various factors are involved in cytokinesis function.

F-actin reorganization and relationship betweeneach F-actin structuresOur observations demonstrate that there are three distinct F-actinstructures in the growing end of the CF of Xenopuseggs duringearly stages of cytokinesis. The first sign of reorganization of theactin cytoskeleton is the F-actin patch formation which initiatesat stage 1. During stage 2, some of the F-actin patches are fusedwith each other and obviously organized into a radial array ofshort F-actin bundles. Finally, in the central region of the furrow,the short F-actin bundles seem to become reorganized into thelong F-actin bundles. The long bundles may correspond to theF-actin bundles in the CF of newt eggs, previously demonstratedby EM to compose the contractile ring (Perry et al., 1971;Mabuchi et al., 1988). This sequence was confirmed by the liverecordings of rhodamine-actin incorporated in the F-actinstructures in the CF. The long F-actin bundles are finally tightlypacked into the thin contractile ring at stage 4.

In case of the sea urchin egg, F-actin becomes accumulatedat the equator at the anaphase-telophase transition. The F-actinthen forms bundles, and the bundles are arranged to form thetightly packed contractile ring (Mabuchi, 1994; Yonemura andKinoshita, 1986). The transient region at the growing end ofthe CF of Xenopuseggs, where the F-actin patches and theshort F-actin bundles are formed, may correspond to the F-actin accumulation in the equatorial cortex of the sea urchinegg. The long F-actin bundle formation and its packing at stage

Fig. 3.Fluorescent images of CFs isolated from eggs into which rhodamine-actin had been microinjected. (A,C,F,H) Rhodamine fluorescenceimages. (B,D,G,I) Staining of total F-actin with Bodipy-phallacidin. (E,J) Merged images of C,D and H,I, respectively. The sites ofmicroinjection are indicated by large arrows. (A,B) Rhodamine-actin was microinjected near the growing end of a stage 2 CF and the egg wasfixed within 30 seconds. (C,D) Magnified micrographs of A,B, respectively. Rhodamine-actin incorporation occurred into both F-actin patches(arrowhead) and short F-actin bundles (small arrows). (F,G) Rhodamine-actin was microinjected near the central region of a stage 3 CF and theegg was fixed within 90 seconds. (H,I) Magnified micrographs of F,G, respectively. Rhodamine-actin was incorporated homogeneously intolong F-actin bundles (arrows). Scale bars in B,F: 20 µm for A,B,F,G. Scale bars in D,I: 5 µm for C-E,H-J.

408

4 in the Xenopusegg CF may correspond to the formation ofwide parallel arrays of F-actin bundles at the equator and theirpacking at later stages in the sea urchin egg, respectively (seeFig. 8).

New actin polymerization at the growing end The steep increase of F-actin occurred during the earlyfurrowing in the CF region between the growing end and themiddle. The slope of the increase exceeded that of the cortex,which was monitored by the WGA-binding site. This resultindicates that a significant portion of F-actin accumulated asthe patches cannot be accounted for by transportation ofcortical F-actin by the cortical movement. A high-resolutionanalysis revealed that the accumulation occurred underneaththe WGA-bleb. Moreover, we directly visualized a rapidincorporation of microinjected rhodamine-actin into the F-actin patches and the short F-actin bundles at the growing end.


Fig. 4.Cortical movement around the CF. (A) A stage 2 CF of an FITC-WGA-labeled egg. Note that a slightly dark region (peri-CF zone, arrow)surrounds the brightly stained furrow. (B) Time-lapse images of a growingend surface of a stage 1-2 CF double-stained with rhodamine-WGA (parts1 and 3) and WGA-fluorescent beads (parts 2 and 4). Yellow lines indicatethe division plane. An arrow in part 3 indicates the position of the CF. Redspots with numbers in parts 2 and 4 indicate some individual WGA-beads.Numbers in the bottom right-hand corner indicate the time (seconds) ofthe recording. At 0 seconds, the growing end has not appeared in themicroscopic area, while it reaches the upper right-hand corner by 30seconds. The WGA-beads gradually move towards the cleavage plane asthe growing end advances. (C) The cortical movement at the growing end.The cortical movement towards the division plane initiates at thebeginning of stage 1. Scale bars: 100 µm in A; 10 µm in B.

Fig. 5.Distributions of F-actin patches andWGA-binding sites in stage 1 CFs.(A-D) Comparison of distribution of WGA-binding sites on the surface as revealed byFITC-WGA staining (A) and F-actindistribution as revealed by rhodamine-phalloidin staining of a stage 1 CF (B).(C) Merged image of A,B. White rectangleindicates the area where the line profilinganalysis was performed. (D) Fluorescenceintensities of rhodamine-phalloidin (red line)and FITC-WGA (green line). Right-handarrows indicate the tip of the FITC signal,while left-hand arrows indicate the middle ofthe CF. (E-J) z-axis sectioned micrographs ofWGA-blebs and F-actin patches taken atdifferent focal planes by the Delta Visionsystem. The optical sections were 0.2 µmthick. At the middle level of the WGA blebs

(E), weak signal of rhodamine-phalloidin was observed inside of the bleb (F), whileat the bottom level of the blebs (H), strong signal of rhodamine-phalloidin wasobserved inside of the bleb (I). (G,J) Merged images of E-F and H-I, respectively.(K-N) Magnified z-section images of FITC-WGA (K,L) and rhodamine-phalloidin(M,N) staining, acquired by using the LSM. The optical sections are 0.7 µm thick. Atthe tip of the furrow (K,M), WGA-blebs colocalized with F-actin patches, whichwere only dimly stained. In an interior region of the CF (L,N), WGA-blebscolocalized with brightly stained F-actin patches which increased in number(arrowheads). Scale bar in B: 50 µm for A,B. Scale bar in J: 1.5 µm for E-J. Scale barin N: 2.5 µm for K-N.


Fig. 6.Distribution of myosin II in the CFs ofXenopuseggs. (A) Specificity of anti-Xenopusoocyte myosin II antibodies. Immunoblot analysisagainst a total lysate of Xenopusoocytesexcluding yolk granules with pre-immune serum(lane 1; diluted to 1/10,000) and with anti-myosinII serum (lane 2; diluted to 1/10,000), respectively.(B-I) Immunofluorescence microscopy of myosinII in the stage 2 CFs. (B,F,H) Staining with anti-Xenopusmyosin II serum. (D) Staining with pre-immune serum. (C,E,G,I) Staining with Bodipy-phallacidin. (F-I) Micrographs at highermagnifications. (B) Numerous myosin spots areformed and aligned into radial arrays (smallarrows in B) at the growing end. (D) These spotsare not observed in a CF stained with pre-immuneserum. (F,G) A number of the myosin spotscolocalize with emerging F-actin patches (smallarrows) near the growing end of the CF, whilethose in periphery (arrowheads) have nocorresponding F-actin patches. Some myosinspots are arranged into tandem arrays along theshort F-actin bundles (F,G, large arrows). (H,I) Aninterior region of the CF. The myosin spots (smallarrowheads) are aligned on the long F-actinbundles (arrows) showing a fibrous appearance.Scale bar in D: 20 µm for B-E. Scale bar in G: 10µm for F,G. Scale bar in I: 5 µm for H,I.

Fig. 7.Distribution of F-actin patches, WGA-binding sites and myosin II in stage 1 CFs.(A-D) Comparison of myosin II distribution (A)with F-actin distribution (B) in a CF. The myosinspot formation apparently precedes the F-actinaccumulation at F-actin patches. (C) Merged imageof A,B. White rectangle indicates the area wherethe line profiling analysis was performed.(D) Fluorescence intensities of myosin II (red line)and Bodipy-phallacidin (green line). Right-handarrows indicate the tip of the myosin staining, whileleft-hand arrows indicate the middle of the CF.(E,F) Comparison of myosin II distribution (E) withdistribution of WGA-binding sites (F) in a CF. Themyosin spot formation and the WGA-blebformation start in the same region. Arrows on theright indicate the tip of the staining. (G,H) Myosinspots and the WGA-blebs, respectively, at the verytip of the CF taken at a higher magnification at asame focal plane. At the tip of the CF, myosin spotscolocalize with the WGA-blebs at their neck region(G,H, arrows). Scale bar in B: 50 µm for A-C,E,F;Scale bar in G: 2.5 µm for G,H.

410

The incorporation took place within 30seconds of the injection, at a position 50µm from furrow region. We estimated thetime required for rhodamine-actin to travelthis distance in the Xenopusegg cytoplasmby observing the injected protein to be lessthan 20 seconds (not shown). This is ingood agreement with an estimate of 22seconds (Berg, 1983), using the diffusioncoefficient of G-actin in cytoplasm of seaurchin eggs (Wang et al., 1982; Salmon etal., 1984; Hiramoto and Kaneda, 1988).However, it would take more than 3minutes by cortical movement. Thus, the speed of theincorporation is explained by diffusion followed bypolymerization, but not by transportation by corticalmovement. We also observed emergence and growth of theactin patches by live recording of the rhodamine-actin-injectedegg in a CF region where no cortical movement occurs. Thetiming of the emergence was different between patches. Thismay be explained by spontaneous polymerization occurring inindividual patch, but may not be explained by accumulation ofF-actin by cortical movement, which would cause asynchronized formation of the patches. By the line profilinganalysis, the extent of the F-actin increase in the middle CFwas double that of the increase of the WGA-binding sites.These results suggest that a half amount of F-actin in the CFmay be derived from the new actin polymerization. The rest ofF-actin in the CF might be transported from the surroundingcortex through the cortical movement. F-actin in the dimlystained patches at the tip of the CF could represent thispopulation of F-actin.

Rhodamine-actin incorporation into the contractileringWe found that rhodamine-actin is evenly incorporated into thelong actin bundles at the middle region of the furrow at aconsiderable speed, although it was a little slower than the oneat the growing end. This result suggests that the turnover ofactin in the contractile ring actively occurs in vivo, and theactin incorporation sites distribute homogeneously along thecontractile ring. These features are distinct from those of stressfibers in cultured cells (Turnacioglu et al., 1998): it takes

several minutes after microinjection to label the incorporationsite with fluorescently labeled G-actin, and the incorporationsites are distributed in a punctuate manner along the stressfiber. The whole stress fiber is labeled at about 40 minutes afterthe injection, while the whole contractile ring was labeled inless than two minutes. Therefore, the contractile ring is thestructure much more dynamic than the stress fiber. It has beenshown that actin-depolymerizing factor/cofilin, which canaccelerate the turnover rate of actin (Carlier et al., 1997;Rosenblatt et al., 1997; Theriot, 1997), is present in the CF ofXenopuseggs (Abe et al., 1996). This protein may facilitate theactin turnover in the contractile ring.

The sequential relationship of the myosin spotformation and F-actin patch formationMyosin II first formed spots at the growing end. The size ofthe myosin spots was close to that of the cytoplasmic ‘myosinspots’, seen in the active lamella undergoing protrusion,previously reported for fibroblastic cells (Verkhovsky andBorisy, 1993; Verkhovsky et al., 1995). These spots have beenfound to be clusters of short myosin filaments called‘minifilaments’. Fishkind et al. have recently reported that theminifilaments are localized to the CF of tissue cultured cells(Fishkind et al., 1996). The myosin spots in the XenopuseggCF may also consist of the minifilaments. It is of interest thatmyosin II in fission yeast also forms spots around the divisionsite, and then the spots are interconnected and packed into thecontractile ring structure (Motegi et al., 2000). The assemblyof myosin II as spots prior to the formation of the contractilering may be common in cytokinesis in animal and fungal cells.


Cortical movement

F-actin patch formation(Actin polymerization?)

Myosin spot formation

Reorganization of the F-actin patchesinto F-actin bundles

(Elongation and fusion of F-actin patches)

Top viewSide view

WGA-bleb

Fig. 8.Schematic representation of sequentialreorganization of actin-myosin cytoskeleton atthe growing end of the CF in the Xenopusegg.

Table 1. Incorporation of rhodamine-actin into the F-actin patches, the short actin bundles and the long actin bundles inthe cleavage furrow

Incorporation into CFF-actin (number incorporated/

Injection site Injectant structure stained total number examined)

Growing end 4 mg/ml rhodamine-actin F-actin patches 26/28Short actin bundles 23/28

4 mg/ml rhodamine-BSA None 0/11Central region 4 mg/ml rhodamine-actin Long actin bundles 13/21

4 mg/ml rhodamine-BSA None 0/7

The volume of each injectant was adjusted to between 0.1 and 0.2 nl.Experiments were carried out using eggs from more than four individual females.


The double staining experiments lead us to two importantfeatures of the myosin assembly. First, myosin colocalizes withthe WGA-bleb at the tip of the CF. The WGA-blebs wereformed by the accumulation of WGA-binding proteinstransported by the cortical movement. These observationssuggest that myosin is also transported from outside of the CFby the cortical movement. Second, the formation of the myosinspots precedes the F-actin accumulation at F-actin patches.This suggests that the actin polymerization takes place after theassembly of myosin at the same site. In budding yeast andfission yeast, myosin II accumulates in the division site earlierthan does F-actin (Lippincott and Li, 1998; Motegi et al.,2000). In addition, myosin assembles faster than F-actin in anartificial wound in Xenopusoocyte (Bement et al., 1999).However, in the sea urchin egg, we have reported that theassembly of myosin and F-actin occurs at the same time in theprocess of the formation of the contractile ring (Mabuchi,1994). This difference could be due to the difference of thespecies, or to resolution of the microscopic systems used.

Although the myosin spot formation preceded the growth ofthe F-actin patch, it is still not clear whether the myosin spotformation is independent of F-actin or not. As discussed above,a part of F-actin could be transported to the CF through thecortical movement, and there were actually dimly stained F-actin patches before the accumulation of F-actin at the growingend of the CF (Fig. 5). Thus, it is possible that a ‘precursor’ ofthe F-actin patch may simultaneously be formed on the myosinspot through the cortical movement. Structural investigation byEM is required to understand how myosin and F-actin interactto construct the substructure of the contractile ring at thegrowing end.

The mechanism of the reorganization of F-actinpatches into F-actin bundlesHow are the F-actin patches reorganized into F-actin bundlesat the growing end of the CF? The live recording of the F-actinmovement made it possible to reveal this sequence in detail;several neighboring longitudinal F-actin patches areinterconnected with each other and aligned lengthways, and thepatches elongate and fuse with each other to form a bundle.Both ultrastructural organization and biochemical compositionof the F-actin patch must be clarified in order to understand themechanism of the conversion of F-actin patches into bundles.It must be noted that organization of myosin also changes inparallel to that of F-actin: the myosin spots also elongate andfuse with each other. It may be that F-actin-myosin interactionparticipates somehow in this dynamic cytoskeletal change.Again in fission yeast, organization of myosin spots into thecontractile ring requires interaction between F-actin andmyosin (Naqvi et al., 1999; Motegi et al., 2000). Thisinteraction may commonly contribute to the final step of thecontractile ring formation.

Role of the cortical movement The role of the cortical movement seems to recruit andaccumulate cortical components, which are necessary in theformation of the contractile ring, into the presumptive CFregion. Myosin II was strongly suggested to be one of suchcomponents. The cortical movement occurs only in the peri-CF zone, which is 20 to 30 µm wide. Since the corticalmovement is one of the earliest events in the CF formation, the

peri-CF zone could be the cortical region that is responding tothe signal of the CF formation. It is tempting to speculate thata role of the cleavage signal is to stimulate the equatorial areaof the cortex to induce the cortical movement in order toaccumulate further signaling molecules and materialsnecessary to construct the contractile ring. Similar movementsof the cortex around the CF have been observed in sea urchineggs by means of attachment of carbon particles on the surface(Dan, 1943), and in cultured mammalian cells by attachmentof concanavalin A-beads (Wang et al., 1994). A traction forcein the equatorial cortex of dividing cultured cells demonstratedby a silicone-rubber method (Burton and Taylor, 1997) mayalso reflect the cortical movement.

The WGA-bleb formation was inhibited by application ofBDM, cytochalasin B or latrunculin A. This result suggests thatboth a myosin family motor protein and F-actin are involvedin the cortical movement. It is tempting to speculate thatmyosin II binds to the membrane, generates the force for thecortical movement, and translocates itself to the CF along theactin cytoskeleton. However, other types of myosin cannot beexcluded from the candidates of the motor protein for thecortical movement. The mechanism by which the corticalmovement and the myosin II assembly are induced needsclarification, in order to understand how the CF is inducedduring cytokinesis.

We thank Dr Hiroshi Kubota of the Kyoto University for help withkeeping frogs in the laboratory; the late Mr Masao Shinoda for adviceand help in constructing the frog aquarium; and Dr Richard Elinsonof the University of Toronto for suggesting the staining of F-actin inthe cortex of Xenopuseggs.

REFERENCES

Abe, H., Obinata, T., Minamide, L. S. and Bamburg, J. R.(1996). Xenopuslaevis actin-depolymerizing factor/cofilin: A phosphorylation-regulatedprotein essential for development. J. Cell Biol.132, 871-885.

Balasubramanian, M. K., Hirani, B. R., Burke, J. D. and Gould, K. L.(1994). The Schizosaccharomyces pombecdc3+ gene encodes a profilinessential for cytokinesis. J. Cell Biol.125, 1289-1301.

Bement, W. M., Mandato, C. A. and Kirsch, M. N.(1999). Wound-inducedassembly and closure of an actomyosin purse string in Xenopusoocytes.Curr. Biol. 9, 579-587.

Berg, H. C. (1983). In Random Walks in Biology, pp. 10-14. Princeton:Princeton University Press.

Burton, K. and Taylor, D. L. (1997). Traction forces of cytokinesis measuredwith optically modified elastic substrate. Nature385, 450-454.

Cao, L. G. and Wang, Y. L. (1990a). Mechanism of the formation ofcontractile ring in dividing cultured animal cells: I. Recruitment ofpreexisting actin filaments into the cleavage furrow. J. Cell Biol.110, 1089-1096.

Cao, L. G. and Wang, Y. L. (1990b). Mechanism of the formation ofcontractile ring in dividing cultured animal cells: II. Cortical movement ofmicroinjected actin filaments. J. Cell Biol.111, 1905-1912.

Carlier, M.-F., Laurent, V., Santolini, J., Melki, R., Didry, D., Xia, G.-X.,Hong, Y., Chua, N.-H. and Pantaloni, D.(1997). Actin depolymerizingfactor (ADF/cofilin) enhances the rate of filament turnover: implication inactin-based motility. J. Cell Biol.136, 1307-1323.

Cramer, L. P. and Mitchison, T. J.(1995). Myosin is involved in postmitoticcell spreading.J. Cell Biol.131, 179-189.

Dan, K. (1943). Behavior of the cell surface during cleavage VI. On themechanism of cell division. J. Fac. Sci. Tokyo Imp. Univ.6, 323-368.

DeLozanne, A. and Spudich, J. A.(1987). Disruption of the Dictyosteliummyosin heavy-chain gene by homologous recombination. Science236,1086-1091.

Edamatsu, M., Hirono, M. and Watanabe, Y.(1992). Tetrahymenaprofilinis localized in the division furrow. J. Biochem.112, 637-642.

412

Epp, J. A. and Chant, J.(1997). An IQGAP-related protein controls actin-ring formation and cytokinesis in yeast. Curr. Biol. 7, 921-929.

Fishkind, D. J. and Wang, Y.-L. (1993). Orientation and three-dimensionalorganization of actin filaments in dividing cultured cells. J. Cell Biol.123,837-848.

Fishkind, D. J., Herman, I. M., Manubay, C. M. and Wang, Y.-L.(1996).Myosin II organization in cell division. Mol. Biol. Cell.7, Suppl. 3259a.

Gunsalus, K. C., Bonaccorsi, S., Williams, E., Verni, F., Gatti, M. andGoldberg, M. L. (1995). Mutations in twinstar, a Drosophilla geneencoding a coffilin/ADF homologue, result in defects in centrosomemigration and cytokinesis. J. Cell Biol.131, 1243-1259.

Hiramoto, Y. (1971). Analysis of cleavage stimulus by means ofmicromanipulation of sea urchin eggs. Exp. Cell Res.68, 291-298.

Hiramoto, Y. and Kaneda, I. (1988). Diffusion of Substances in thecytoplasm and across the nuclear envelope in egg cells. Protoplasma2, 88-94.

Kellogg, D. R., Mitchison, T. J. and Alberts, B. M.(1988). Behavior ofmicrotubules and actin filaments in living Drosophila embryos.Development103, 675-686.

Knecht, D. A. and Loomis, W. F.(1987). Antisense RNA inactivation ofmyosin heavy-chain gene-expression in Dictyostelium discoideum. Science236, 1081-1086.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assemblyof the head of bacteriophage T4. Nature227, 680-685.

Lippincott, J. and Li, R. (1998). Sequential assembly of myosin II, anIQGAP-like protein, and filamentous actin to a ring structure involved inbudding yeast cytokinesis. J. Cell Biol.140, 355-66.

Mabuchi, I. (1986). Biochemical aspects of cytokinesis. Int. Rev. Cytol.101,245-281.

Mabuchi, I. (1994). Cleavage furrow: timing of emergence of contractile ringactin filaments and establishment of the contractile ring by filament bundlingin sea urchin eggs. J. Cell Sci.107, 1853-1862.

Mabuchi, I. and Okuno, M. (1977). The effect of myosin antibody on thedivision of starfish blastomeres. J. Cell Biol.74, 251-263.

Mabuchi, I., Tsukita, S., Tsukita, S. and Sawai, T.(1988). Cleavage furrowisolated from newt eggs: Contraction, organization of the actin filaments andprotein components of the furrow. Proc. Natl. Acad. Sci. USA85, 5966-5970.

Mittal, B., Sanger, J. M. and Sanger, J. W.(1987). Visualization of myosinin the cytoplasm, cleavage furrow and mitotic spindle of human cells. J. CellBiol. 105, 1753-1760.

Motegi, F., Nakano, K. and Mabuchi, I. (2000). Molecular mechanism ofmyosin-II assembly at the division site in Schizosaccharomyces pombe. J.Cell. Sci.113, 1813-1825.

Naqvi, N. I., Eng, K., Gould, K. L. and Balasubramanian, M. K.(1999).Evidence for F-actin-dependent and –independent mechanisms involved inassembly and stability of the medial actomyosin ring in fission yeast. EMBOJ. 15, 854-862.

Perry, M. M., John, H. A. and Thomas, N. S. T.(1971). Actin-like filamentsin the cleavage furrow of newt egg. Exptl. Cell Res.65, 249-253.

Rappaport, R. (1965). Geometrical relations of the cleavage stimulus ininvertebrate eggs. J. Theor. Biol.9, 51-66.

Rosenblatt, J., Agnew, B. J., Abe, H., Bamburg, J. R. and Mitchison T. J.(1997). Xenopusactin depolymerizing factor/cofilin (XAC) is responsible

for the turnover of actin filaments in Listeria monocytogenestails. J. CellBiol. 136, 1323-1332.

Salmon, E. D., Saxton, W. M., Leslie, R. J., Karow, M. L. and McIntosh,J. R. (1984). Diffusion coefficient of fluorescein-labeled tubulin in thecytoplasm of embryonic cells of a sea urchin: video image analysis offluorescence redistribution after photobleaching. J. Cell Biol.99, 2157-2164.

Satterwhite, L. L., Lohka, M. J., Wilson, K. L., Scherson, T. Y., Cisek, L.J., Corden, J. L. and Pollard, T. D.(1992). Phosphorylation of myosin-IIregulatory light chain by cyclin-p34cdc2: A mechanism for the timing ofcytokinesis. J. Cell Biol.118, 595-605.

Schroeder, T. E.(1975). Dynamics of the contractile ring. InMolecules andCell Movement(ed. S. Inoue and R. E. Stephens), pp. 305-334. New York:Raven Press.

Spudich, J. A. and Watt, S.(1971). The regulation of rabbit skeletal musclecontraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J. BiolChem.246, 4866-4871.

Tencer, R.(1978). Transmembrane effects of lectins. Exp. Cell Res.116, 253-260.

Theriot, J. A. (1997). Acceleration on a treadmill: ADF/cofilin promotes rapidactin filament turnover in the dynamic cytoskeleton. J. Cell Biol.136, 1165-1168.

Tompson, C., Lin, C-H. and Forscher, P.(1996). An Aplysiacell adhesionmolecule associated with site-directed actin filament assembly in neuronalgrowth cones. J. Cell Sci.109, 2843-2854.

Turnacioglu, K. K., Sanger, J. W. and Sanger, J. M.(1998). Sites ofmonomeric actin incorporation in living PTK2 and REF-52 cells. Cell Motil.Cytoskeleton40, 59-70.

Verkhovsky, A. B. and Borisy, G. G. (1993). Non-sarcomeric mode ofmyosin-II organization in the fibroblast lamellum. J. Cell Biol. 123, 637-652.

Verkhovsky, A. B., Svitkina, T. M. and Borisy, G. G.(1995). Myosin IIfilament assemblies in the active lamella of fibroblast: their morphogenesisand role in the formation of actin filament bundles. J. Cell Biol.131, 989-1002.

Wang, Y-L., Lanni, F., McNeil, P. L., Ware, B. R. and Taylor, D. L.(1982).Mobility of cytoplasmic and membrane-associated actin in living cells.Proc. Natl. Acad. Sci. USA79, 4660-4664.

Wang, Y-L., Silverman, J. D. and Cao, L. G.(1994). Single particle trackingof surface receptor movement during cell division. J. Cell Biol.127, 963-971.

Watanabe, N., Madaule, P., Reid, T., Ishizaki, T., Watanabe, G., Kakizuka,A., Saito, Y., Nakao, K., Jockusch, B. M. and Narumiya, S.(1996).p140mDia, a mammalian homologue of Drosophila diaphanous, is a targetprotein for Rho small GTPase and is a ligand for profilin. EMBO J.16, 3044-3056.

Wheatley, S. P., O’Connell, C. B. and Wang, Y.-L.(1998). Inhibition ofchromosomal separation provides insights into cleavage furrow stimulationin cultured epithelial cells. Mol. Biol. Cell8, 2173-2184.

Wheatley, S. P. and Wang, Y.-L.(1996). Midzone microtubule bundles arecontinuously required for cytokinesis in cultured epithelial cells. J. Cell Biol.135, 981-989.

Yonemura, S. and Kinoshita, S.(1986). Actin filament organization in thesand dollar egg cortex. Dev. Biol.115, 171-183.


reorganization of actin cytoskeleton at the growing …second, actin dynamics in the f-actin...

Documents