RESEARCH ARTICLE 401 Reorganization of actin cytoskeleton at the growing end of the cleavage furrow of Xenopus egg during cytokinesis

Tatsuhiko Noguchi1,* and Issei Mabuchi1,2 1Division of Biology, School of Arts and Sciences, University of Tokyo, 3-8-1, Komaba, Meguro-ku, Tokyo, 153-8902, Japan 2Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585, Japan *Author for correspondence (e-mail: [email protected])

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

SUMMARY

We studied reorganization of actin-myosin cytoskeleton at and form ‘bleb-like’ structures on the surface of the furrow the growing ends of the cleavage furrow of Xenopus eggs in region. The F-actin patch forms and grows underneath this order to understand how the contractile ring is formed structure. The slope of F-actin accumulation in the interior during cytokinesis. region of the furrow exceeds that of accumulation of the Reorganization of F-actin structures during the furrow cortex transported by the cortical movement. In addition, formation was demonstrated by rhodamine-phalloidin rhodamine-G-actin microinjected at the growing end is staining of the cleavage furrow and by time-lapse scanning immediately incorporated into the F-actin patches. These with laser scanning microscopy of F-actin structures in the data, together with the rapid growth of F-actin patches in cleavage furrow of live eggs to which rhodamine-G-actin the live image, suggest that actin polymerization occurs in had been injected. Actin filaments assemble to form small the contractile ring formation. clusters that we call ‘F-actin patches’ at the growing end of Distribution of myosin II in the cleavage furrow was also the furrow. In live recordings, we observed emergence and examined by immunofluorescence microscopy. Myosin II rapid growth of F-actin patches in the furrow region. These assembles as spots at the growing end underneath the bleb- patches then align in tandem, elongate and fuse with each like structure. It was suggested that myosin is transported other to form short F-actin bundles. The short bundles then and accumulates as spots by way of the cortical movement. form long F-actin bundles that compose the contractile F-actin accumulates at the position of the myosin spot a ring. little later as the F-actin patches. The myosin spots and the During the furrow formation, a cortical movement F-actin patches are then simultaneously reorganized to towards the division plane occurs at the growing ends of form the contractile ring bundles the furrow, as shown by monitoring wheatgerm agglutinin- conjugated fluorescent beads attached to the egg surface. Key words: Cytokinesis, Actin, Myosin, Xenopus egg, Contractile As a result, wheatgerm agglutinin-binding sites accumulate ring, Cleavage furrow

INTRODUCTION how is the contractile ring assembled from its constituents at a molecular level? Cytokinesis in animal cells begins at late anaphase to telophase Xenopus egg is a good system for studying the mechanism with formation of the contractile ring, a ring-shaped bundle of of furrow formation to answer the above questions for the actin filaments in the cleavage furrow (CF) cortex (Schroeder, following reasons. In amphibian eggs, the CF first appears at 1975; Mabuchi, 1986). It has been demonstrated that geometry the animal pole and advances toward the vegetal hemisphere, of furrowing is determined by the astral microtubules in being formed continuously at the growing ends. Therefore, the echinoderm eggs (Rappaport, 1965; Hiramoto, 1971), or the sequences of CF formation can be investigated intensively at central spindle in mammalian cultured cells (Wheatley and the growing end of the CF, while the central region of the CF Wang, 1996; Wheatley et al., 1998), which may transmit a is suitable for investigating the mechanism of the contraction. signal to induce the formation of the CF. It has been Furthermore, the egg is large enough for microinjection at demonstrated that the contractile force of the ring is generated specified regions of the cell. by the actin-myosin interaction (Mabuchi and Okuno, 1977; In this report, reorganization of actin and myosin into the Knecht and Loomis, 1987; DeLozanne and Spudich, 1987). contractile apparatus during furrow formation of Xenopus egg Even though the framework of the mechanism of cytokinesis was first highlighted to answer Question (2). In budding yeast was established by these studies, many questions still remain cells (Epp and Chant, 1997; Lippincott and Li, 1998), sea to be answered. The most important questions are (1) what is urchin eggs (Mabuchi, 1994) and cultured mammalian cells the entity of the cleavage signal or signaling pathway?; and (2) (Mittal et al., 1987; Cao and Wang, 1990a; Cao and Wang, 402 JOURNAL OF CELL SCIENCE 114 (2)

1990b; Fishkind and Wang, 1993), the sequential changes in was not necessary in the present study. The crude myosin fraction localization of actin and myosin during the CF formation have obtained after two cycles of polymerization and depolymerization was been studied. However, information is limited, since these cells fractionated successively by hydroxylapatite column chromatography are either too small to see the cytoskeletal changes in detail, or and Sephadex G-200 gel filtration column chromatography. The peak they form the contractile ring too quickly. On the other hand, fractions obtained after the gel filtration column chromatography were in cells that naturally undergo unilateral cleavage, such as concentrated by precipitation with 5% (w/v) trichloroacetic acid, and myosin II heavy chain was separated by sodium dodecyl sulfate amphibian eggs, the reorganization of the actin-myosin (SDS)-polyacrylamide gel electrophoresis (PAGE). After Coomassie cytoskeleton has not yet been well studied. We took the Brilliant Blue staining, the band was dissected, homogenized in advantage of Xenopus eggs to analyze contractile ring Freund’s complete adjuvant, and injected subcutaneously into two formation by examining the cytoskeletal changes at the male rabbits. About a month after the first immunization, the boost growing end of the CF. Second, actin dynamics in the F-actin injections were carried out every two weeks followed by structures in the CF was examined. Although it has been known immunoblotting to check the production of antibodies. that the contractile ring is formed very quickly, little is known how actin molecules are incorporated into it. It has been Fixation and staining of isolated CF reported in cultured mammalian cells that F-actin in the Eggs were fixed in F-buffer (0.1 M KCl, 5 mM MgCl2, 0.2 mM CaCl2, contractile ring may be recruited from pre-existing cortical F- 50 mM Hepes, pH 7.5) containing 3.7% formaldehyde for 30 to 50 minutes at room temperature at various stages during the first actin (Cao and Wang, 1990a; Cao and Wang, 1990b). However, cleavage. Then they were treated with 0.5% Triton X-100 in F-buffer it has recently been suggested that actin polymerization may for 15 minutes. The cortices gradually became peelable after the be involved in the contractile ring formation, since profilin, treatment with Triton X-100. After three washes with F-buffer, F-actin which is capable of accelerating actin polymerization, is found was stained with fluorescently labeled phalloidin (Molecular Probes, in the CF of fission yeast (Balasubramanian et al., 1994), Eugene, OR) for 1 hour, and the cortices containing CF were isolated Tetrahymena (Edamatsu et al., 1992) and cultured mammalian manually from the eggs by using a glass needle. For cells (Watanabe et al., 1996). Moreover, disruption of the immunofluorescence staining, the eggs were fixed 30-35 minutes as function of actin-depolymerizing factor ADF/cofilin inhibits described above, and then transferred into 0.5% Triton X-100 in F- cytokinesis in some organisms (Abe et al., 1996; Gunsalus et buffer. The CFs were carefully isolated from the eggs within 15 al., 1995), suggesting that turnover of actin may play a role in minutes of the transfer. Both the times for the fixation and for the Triton X-100 treatment were critical for excluding the yolk granules cytokinesis. To this end, we extensively examined the actin from the preparation, which was important because fluorescently dynamics of contractile ring. labeled secondary antibodies strongly bound to yolk granules in a We carried out following experiments in order to elucidate nonspecific manner. The isolated cortices were post-fixed with 3.7% these points. We first examined the F-actin organization in the formaldehyde for 10 minutes to preserve the actin-myosin CF of Xenopus eggs both by fluorescent staining of F-actin in cytoskeleton. After three washes with a phosphate buffered saline the CFs isolated at various stages, and by observation of live (PBS), the isolated cortices were incubated in 2% bovine serum eggs into which rhodamine-labeled G-actin had been injected. albumin (BSA) dissolved in PBS for 1 hour. The cortices were then In addition, rapid incorporation of G-actin into the contractile incubated with anti-Xenopus myosin II serum (1/100 dilution) for 90 ring both during and after its formation was demonstrated. minutes, washed with PBS three times, and incubated with Second, we examined localization of myosin II at the growing rhodamine-conjugated anti-rabbit IgG antibodies (Organon Teknika, Cappel Research Products, Durham, NC) for 90 minutes, followed by end of the CF by immunofluorescence microscopy. Third, we three washes. For counterstaining of F-actin, Bodipy-phallacidin compared the distribution of F-actin, myosin II and surface (Molecular Probes) in PBS was added to the cortices and incubated wheatgerm agglutinin (WGA)-binding sites, in order to for 30 minutes. WGA-binding sites were stained with 10 µg/ml determine the sequence of appearance of these components at fluorescein isothiocyanate (FITC)-WGA (Vector Labs, Burlingame, the growing end. CA) for more than 30 minutes. The fluorescently stained cortices were rinsed with PBS, mounted in Mowiol, and examined immediately by fluorescence microscopy from the surface side, as described below. MATERIALS AND METHODS Preparation of fluorescently labeled G-actin Handling of animals and eggs Rabbit skeletal muscle actin was prepared according to Spudich and Female Xenopus laevis were induced to ovulate by injection of 400 Watt (Spudich and Watt, 1971) and gel-filtered through a Sephadex units of human chorionic gonadotropin (Denka Seiyaku, Tokyo, G-100 column. Both actin and BSA were labeled with 5- Japan) a day before use. Mature eggs were inseminated with a sperm carboxytetramethylrhodamine succinimidyl ester (Molecular Probes), suspension obtained by macerating the testes in DeBoer’s solution as described by Kellogg et al. (Kellogg et al., 1988). The labeled G- (110 mM NaCl, 1.3 mM KCl, 0.44 mM CaCl2 with addition of actin (rhodamine-actin) and the labeled BSA (rhodamine-BSA) (both NaHCO3 to pH 7.3), and activated in a tap water. Five minutes later, 4 mg/ml) in G-buffer (1 mM Hepes, pH 7.5, 0.1 mM ATP, 0.1 mM the eggs were dejellied with 2% cysteine, pH 8.1. Vitelline membranes CaCl2) were frozen in liquid nitrogen. The labeled proteins were were removed manually with watchmaker’s forceps. The eggs were centrifuged at 100,000 g for 1 hour just prior to use. cultured in modified Steinberg’s solution (MSS; 60 mM NaCl, 0.67 mM KCl, 0.34 mM Ca (NO3)2, 0.83 mM MgSO4, 10 mM N-2- Microinjection of rhodamine-actin at the growing end hydroxyethylpiperazine-N’-ethanesulfonic acid (Hepes), pH 7.4) at Vitelline membranes of fertilized and dejellied eggs were manually room temperature. removed and the eggs were transferred into MSS. The volume of injectant was determined by measuring the diameter of droplets Anti-Xenopus myosin II serum injected into a salad oil droplet. The eggs were injected with less than Xenopus cytoplasmic myosin II was prepared from oocytes according 0.1 nl of labeled protein solution near the growing end of the CF to Satterwhite et al. with modification (Satterwhite et al., 1992). The during stage 2 (see Fig. 1H) or a middle region of the CF during stage KI-ATP gel filtration step was omitted because the exclusion of actin 3. To improve the reproducibility and to avoid possible formation of Actin reorganization during cytokinesis 403 aggregates of rhodamine-actin in the CF region, the injection site was surface of living eggs were performed using an Achroplan water restricted to about 50 µm away from the growing end of the CF, 10×/NA 0.3 or a 40×/NA 0.75 lens. Fluorescent images were detected avoiding the division plane. Under these conditions, the injection using a color chilled CCD camera (C5810, Hamamatsu Photonics, neither interfered with the advancing of the CF nor with the Hamamatsu, Japan). The digital images were processed by Photoshop organization of F-actin structures in the CF. The eggs were fixed 30 4.0j and by FISH imaging software (Hamamatsu Photonics). Z- to 60 seconds after the injection, and counterstained with Bodipy- sections of fluorescently stained egg surface were performed using a phallacidin to reveal total F-actin organization as described above. Zeiss 510 confocal laser scanning microscope (LSM) and a Delta Vision system (Applied Precision, Issaquah, WA) attached to an Preparation of lectin-conjugated beads and visualization Olympus IX-70-SIF microscope. of cortical movements It has previously been shown that WGA-binding sites accumulate in Time-lapse confocal microscopy of living eggs the CF region of Xenopus egg (Tencer, 1978). In order to demonstrate 20 nl of 4 mg/ml rhodamine-actin was injected into an egg within 30 the cortical movement at the growing end of the CF, WGA was minutes of fertilization. The rhodamine-actin diffused evenly in the conjugated to carboxylated fluorescent polystyrene beads cytoplasm before the first cleavage. The growing ends of the CF were (FluoSphere, Molecular Probes) according to previously described marked by FITC-WGA staining. The egg was settled in a handmade methods (Wang et al., 1994; Tompson et al., 1996) with modifications. chamber in which it was compressed by a coverslip in order to flatten A 2% aqueous suspension of FluoSphere was mixed with an equal the cortex (see Fig. 2F). The growing end of the CF in the flattened volume of 8% electron microscope (EM) grade glutaraldehyde (Wako animal cortex was examined by LSM equipped with 63× Plan- Pure Chemical Industries, Osaka) and incubated at 25oC for 2 hours apochromat oil immersion lens at an optical section of 1.5 µm. with gentle agitation. The beads were gently pelleted and rinsed three Serially scanned images were taken to demonstrate changes in the F- times with 25 mM Na-phosphate buffer, pH 7.0. An equal volume actin orgaization during the contractile ring formation. of 2 mg/ml WGA was added to 2% (w/v) suspension of the gultaraldehyde-activated beads and the suspension was gently agitated overnight at 4oC. The WGA-conjugated beads were then pelleted and RESULTS rinsed three times with 50 mM Tris (hydroxymethyl) aminomethane buffer, pH 8.0 to stop the reaction. The beads were stored in MSS F-actin organization in the cleavage furrow at containing 0.02% NaN3 at 4oC before use. They were rinsed three various stages during the first cleavage of Xenopus times with MSS prior to use. egg The WGA-beads were applied to fertilized eggs without fertilization membranes and incubated for 15 minutes. The eggs were F-actin structures in the CF cortices isolated from Xenopus then washed gently to remove unadhered beads. In most cases, a eggs at various stages were revealed by rhodamine-phalloidin number of beads adhered on the surface of the eggs, which were staining in order to investigate the reorganization of F-actin enough for real-time recording of the cortical movement. cytoskeleton during the 1st cleavage (see Fig. 1H for the Subsequently, residual WGA-binding sites on the living egg surface stages). At the beginning of furrow formation, when a black were labeled with rhodamine-WGA to visualize the CF. The real-time single stripe appeared at the animal pole (stage 1), numerous movement of each bead was recorded as described below. This clusters of F-actin, the diameter of which was 0.5 to 1 µm, were method allowed us to reveal spatial and temporal relationship between formed in the CF region (Fig. 1A,B). We call this structure the the growing end of the CF and the cortical movement around it at a ‘F-actin patch’. Besides the F-actin patches, large F-actin high resolution. aggregates of several micrometers in diameter were also Immunoblot analysis recognized in this region. At this stage, no contraction of the Dejellied oocytes were washed three times with Marc’s Modified CF along its longitudinal axis was detected. In the following Ringer’s solution (0.1 M NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM stage (stage 2, Fig. 1C-E), the CF initiated contraction along MgCl2, 5 mM Hepes, pH 7.4) and then three times with X-buffer (0.1 its longitudinal axis. We found three distinct F-actin structures M KCl, 2 mM MgCl2, 0.2 mM CaCl2, 50 mM sucrose, 10 mM Hepes, at this stage. At the growing end of the CF, the F-actin patches pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 and radially oriented short and thick F-actin bundles (short µg/ml leupeptin, 10 µg/ml pepstatin A). The oocytes were gently actin bundles) were observed (Fig. 1C,E). The mean length of centrifuged at 100 g and the buffer was discarded. The oocytes were the short F-actin bundles was 2.8±1.1 µm. These bundles resuspended in five volumes of X-buffer containing 0.5% Triton X- seemed to consist of several small F-actin patches that had 100, and the suspension was homogenized with a hand-made Epon fused with each other in a linear fashion. However, in the pestle-Eppendorf tube homogenizer. The homogenate was centrifuged central region of the CF, a number of long F-actin bundles, the at 10,000 g for 20 minutes and transparent supernatant was collected µ avoiding contamination by floating lipid and yolk pellets. The lysate width of which varied from 0.1 to 1 m, were observed (Fig. was diluted in the Laemmli’s sample buffer (Laemmli, 1970) and 1D). The long F-actin bundles were aligned in a parallel subjected to SDS-PAGE, followed by transfer onto a polyvinylidene fashion to the longitudinal axis of the CF. We could not difluoride membrane. The membrane was blocked with 2% skim milk determine their exact length, but they were more than several in PBS for 1 hour, and then incubated with anti-myosin II serum or micrometers long. We assumed that the long F-actin bundle preimmune serum diluted to 1/10,000 with the blocking solution. The was a unit of the contractile ring. It seemed that at the growing signal was detected using a chemiluminescence system (Amersham end of the CF, the F-actin patches are formed at first, then some International, UK). population of the patches are aligned into the short F-actin Fluorescence microscopy and image-processing bundles, and finally the short actin bundles are reorganized into Specimens were examined using a Zeiss Axioscope microscope the long F-actin bundles. As the furrow region became wider equipped with a Neo-Fluar 10×/numerical aperture (NA) 0.3, a Neo- and wider during stages 2 and 3, the number of the long F-actin Fluar 20×/NA 0.7 and a Plan-Apochromat 63×/NA 1.4 oil immersion bundles increased. The width of a belt of the long F-actin objective lenses. Microphotographs were taken on Kodak T-MAX bundles reached 100 µm at stage 3 (Fig. 1F). Suddenly, in the ASA 400 films. Live recordings of WGA-conjugated beads on the next stage (stage 4, Fig. 1G), the belt became tightly packed. 404 JOURNAL OF CELL SCIENCE 114 (2) The width of the belt in this stage was less than 20 µm. This experimental conditions. No difference was observed between change seemed to coincide with deepening of the furrow and the F-actin organization in the CF of the injected eggs and that with addition of a large amount of new cell membrane in the of uninjected eggs. Results are summarized in Table 1. CF. Finally, at stage 5, two growing ends merged at the vegetal First, rhodamine-actin was injected near the growing end of pole to form a complete ring structure that encircles this large a CF during stage 2 and the egg was fixed at 30 seconds after cell (not shown). the injection. In most cases, rhodamine-actin was incorporated into all the F-actin patches near the injection site and also some Time-lapse imaging of the reorganization of F-actin short actin bundles (Fig. 3A-E). patches into F-actin bundles in stage 2 CF In late stage 2 or stage 3, the contractile ring, which was In order to demonstrate the reorganization of F-actin patches composed of numerous long F-actin bundles, was well formed into F-actin bundles at the growing end of the CF in stage 2 as (see Fig. 1F). To investigate whether established contractile described above, we visualized the F-actin structures in the CF ring can incorporate G-actin, rhodamine-actin was of living eggs under a confocal LSM, into which rhodamine- microinjected near the central region of the CF and the cell was actin had been injected. First, a rhodamine-actin-injected egg fixed at 60 to 90 seconds after the injection. As shown in Fig. was fixed, and the cortex was isolated and counterstained with 3F-J, rhodamine-actin was incorporated into the long F-actin Bodipy-phallacidin. The rhodamine fluorescence pattern was bundles and there was no difference in the fluorescence pattern similar to the Bodipy fluorescence pattern (Fig. 2A-C). Thus, between the incorporated rhodamine-actin and Bodipy- we concluded that rhodamine-actin was faithfully incorporated phallacidin staining of the whole F-actin structures. However, into the CF F-actin structures as an intact monomer. Next, to only weak signals were detected when the fixation was verify further that the signal detected in the thin optical section performed at 30 seconds after the injection (not shown). Thus, obtained with the LSM is attributed to rhodamine-actin the rate of incorporation of rhodamine-actin into the long F- incorporated into the cortical F-actin but not to free G-actin actin bundles was apparently slower than that into the F-actin diffused in the cytoplasm, we carried out a z-axis sectioning patches at the growing end. As a control, rhodamine-BSA was from outside through surface to inside the egg (Fig. 2D). We microinjected. No particular signal of rhodamine-BSA was found a clear fluorescence peak at the cortex, of the rhodamine- detected in the actin cytoskeleton (not shown). actin-injected egg. However, no apparent peak but a diffused fluorescence was observed in the cytoplasm of rhodamine- Cortical movement at the growing end of the CF BSA-injected egg (Fig. 2E). The width of the rhodamine-actin It has been reported in cultured mammalian cells that F-actin peak along z-axis was less than 2 µm. Therefore, we fixed the in the contractile ring is accumulated by transportation from depth (focal plane) to be scanned at the peak of rhodami surrounding cortex rather than new actin polymerization ne-actin and set the thickness of the optical section at 1.5 µm during the formation of the contractile ring (Cao and Wang, in the following examinations. At these settings, we were 1990a; Cao and Wang, 1990b). However, above results able to obtain a live image (Fig. 2G) of rhodamine-actin in the suggest that the actin polymerization occurs in order to form CF. the F-actin patches at the growing end. To further investigate The sequential imaging demonstrated the reorganization of whether the F-actin accumulated in the CF is derived from the F-actin structures at the growing end of the CF in stage 2. cortical actin or newly polymerized F-actin, we carried out In this region, numerous F-actin patches were observed. As the two experiments. First, we examined movement of cortex CF advanced, the number of F-actin bundles gradually around the CF during the furrow formation, which may increased in the same area (Fig. 2H,I). At a higher transport the cortical F-actin to the division plane. Second, magnification, we could pursue destination of each patch (Fig. we compared the amounts of cortex and F-actin accumulated 2J). The patches seemed to emerge at random positions. in the furrow region in stage 1. Timing of emergence seemed to be different among the patches WGA is known to bind to a carbohydrate moiety of even in a small area of the cortex. It took 30 to 40 seconds for proteins on the cell surface. When a dividing egg at stage 1 the full growth of the patch from the emergence to reaching was stained with FITC-WGA, the CF appeared as a maximum in fluorescence (see arrowhead 3 in Fig. 2J). Then, fluorescent spindle-shaped region surrounded by a dark zone several neighbouring patches, which were close to a line about 20-30 µm wide on both sides of the furrow where some parallel to the long axis of the furrow, interconnected with each stress wrinkles were recognized (Fig. 4A), indicating that the other, and arranged to line up in the direction of the line to WGA-binding sites were accumulated in the CF. The dark form a short bundle (Fig. 2J). A little later, the short bundles zone will be called peri-CF zone hereafter. Simultaneous fused with each other at their ends to form longer bundles (Fig. labeling with rhodamine-WGA and WGA-fluorescent beads 2K). These observations directly demonstrate that the F-actin of the WGA-binding sites on the surface of living eggs patches are reorganized into the F-actin bundles. enabled us to observe the furrow position and its progression in real time. A movement of the WGA beads from Rapid incorporation of G-actin into F-actin patches surrounding cortex towards the CF was detected (Fig. 4B). and F-actin bundles The mean speed of the movement during the stage 1 was We investigated how fast G-actin is incorporated into the 15.8±3.5 µm/min (n=11). We call this movement the ‘cortical contractile ring structure by injecting rhodamine-actin near the movement’. This movement actively occurred in the peri-CF contractile ring. The injections were made at sites 50 µm away zone. It started at the beginning of the stage 1, before the from the CF. Whole F-actin organization was visualized by formation of the short F-actin bundles or the long F-actin counterstaining with Bodipy-phallacidin after fixation of the bundles, indicating that the cortical movement was cell. Most of the injected eggs cleaved normally under the independent of the contraction of the contractile ring. Actin reorganization during cytokinesis 405

Fig. 1. F-actin-containing structures in the CFs of Xenopus eggs. F-actin structures in isolated CFs were stained with rhodamine-phalloidin. (B,D,E) Selected parts of A,C, at a higher magnification. (A,B) Stage 1 CF. Numerous F-actin patches and aggregates are seen. (C-E), Stage 2 CF. F-actin patches (0.5 ~ 1 µm in diameter) are emerging at the growing end (small arrows in E). Large F-actin aggregates (large arrow in E) are also seen. Short F-actin bundles, which are radially oriented are formed in addition to the F-actin patches at the growing end (E, arrowheads). Long F-actin bundles, which are aligned parallel to the axis of the CF, are formed in the central region (D, arrowheads). (F) Stage 3 CF. The furrow region becomes wider, and the long F-actin bundles (arrow) increase in number. (G) Stage 4 CF. The long F-actin bundles are suddenly packed to form the thinner contractile ring. (H) Stages of the 1st cleavage of Xenopus egg. Scale bar in A: 20 µm for A,C,F,G. Scale bar in B: 10 µm for B,D,E.

Accumulation of F-actin in the early CF and brightness, indicating that amount of F-actin in the patches The dominant F-actin structure in the CF in the stage 1 was the increased from the end to the middle region of the early CF, F-actin patch as described above (Fig. 1). The double staining being consistent with the result of the line profiling analysis. using rhodamine-phalloidin and FITC-WGA enabled us to compare the accumulation of F-actin and that of WGA-binding Effects of cytoskeleton inhibitors on the formation sites during CF formation (Fig. 5A,B). Line profiling analysis of WGA-blebs of the fluorescence intensity of both rhodamine-phalloidin and 5 mM 2, 3-butanedione monoxyme (BDM), which has been FITC-WGA along the furrow axis was carried out to quantify known to inhibit myosin II ATPase activity (Cramer and F-actin and the WGA-binding sites, respectively, of the early Mitchison, 1995), was microinjected into eggs near the furrow. It revealed that the amount of the WGA-binding sites growing end of the CF during stage 1 or stage 2. As judged by increased gradually from the end to the middle of the CF up to the FITC-WGA staining, the WGA-bleb formation at the 1.5±0.3-fold (n=9) (Fig. 5D). However, the amount of F-actin growing end was markedly inhibited by the injection (12/14 increased gradually at first and then steeply to reach a plateau CFs). During stage 2 when the contraction of the contractile near the middle. The total increase was 3.3±1.2-fold (2.3- to ring had started, the furrow partially regressed and the 5.2-fold, n=12). In other words, the slope of the F-actin contractile activity was significantly depressed by the BDM accumulation exceeded that of the accumulation of the cortex injection (7/16 CFs). The second and third cleavages occurred in the interior region of the CF. This suggests that a significant almost normally, suggesting that the inhibitory effect of BDM part of F-actin in the CF accumulates by a mechanism that is was reversed; the drug may have diffused out from the eggs. different from the cortical movement. In addition, it was When eggs were incubated either in 0.1 mM cytochalasin B estimated that the steeply increasing phase of F-actin for 40 minutes (n=25) or 2.3 µM latrunculin A for 20 minutes corresponded to about 1 minute. (n=10), before formation of the CF to disrupt cortical actin At a higher magnification, the FITC-WGA staining revealed filaments, the WGA-bleb formation was significantly disturbed numerous spherical structures on the surface of the CF (Fig. 5). in all the eggs examined. These results suggest that both These structures were extruded from the surface, which was myosin and cortical F-actin are required in the formation of the apparent by changing the focus of the microscope. We call this WGA-blebs. structure ‘WGA-binding bleb-like structure’ (WGA-bleb). The F-actin patch seemed to localize inside the WGA-bleb especially Immunofluorescent staining of myosin II in the CF near the neck region (Fig. 5E-J). At the end of the furrow marked In order to examine the distribution of myosin II and to compare by the FITC-WGA staining, the WGA-blebs colocalized with F- it with that of F-actin structures, antibodies were raised against actin patches containing a very low level of F-actin (Fig. 5K,M). myosin II heavy chains purified from Xenopus oocytes. A whole- However, in the middle region of the CF, they colocalized with cell lysate excluding yolk granules was subjected to immunoblot F-actin patches containing a high level of F-actin (Fig. 5L,N), analysis using the anti-myosin II serum to check the specificity although the blebs themselves did not seem to change both size of the antibody. An intense signal was detected with the 406 JOURNAL OF CELL SCIENCE 114 (2)

Fig. 2. Reorganization of the F-actin patches into F-actin bundles at the growing end of the stage 2 CF in a living egg. Rhodamine-actin was injected to visualize the F-actin structures at the growing end of a CF in a living egg. (A-C) a CF-containing cortex of the rhodamine-actin-injected egg was isolated and examined for the rhodamine fluorescence in order to verify the incorporation of rhodamine- actin into F-actin structures in the CF (A). Whole F-actin in the cortex was counterstained with Bodipy-phallacidin (B). (C) Merged image of (A,B). (D,E) z-axis sections from outside, through surface, to inside of eggs (from the top to the bottom of the pictures). Twelve optical sections of 1 µm thick were piled up and the z-axis-sectioned images were reconstructed. In a rhodamine- actin-injected egg (D), a sharp peak of fluorescence (arrowhead) was recognized that we attributed to the signal from cortical F- actin. However, there was only diffused signal of fluorescence in cytoplasm without obvious peak in a rhodamine-BSA-injected egg (E). (F) The handmade chamber for scanning the cortex of a rhodamine-actin- injected egg. (G) A live image of a stage 2 CF of rhodamine-actin injected egg at low magnification. (H) The area of the scanning (blue rectangles) at the growing end of the CF shown in I. The dots, the broken lines and the black lines represent F-actin patches, the patches linked with each other and F-actin bundles, respectively. The blue arrow indicates the direction of the growth of the CF. (I) Sequential images of the F-actin structures at the growing end of the CF of a living egg at a higher magnification. The F-actin patches (small arrows) and the F-actin bundles (arrowheads) were clearly observed. In the left-hand picture (0 seconds), the dominant F-actin structures are the F-actin patches. As the furrow 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-actin patches move to line up and fuse with each other to form short F-actin bundles. Arrowheads indicate individual F-actin patches. Among them, some are newly developing (arrowheads 1 and 3). (K) Another magnified area of I. The short F-actin bundles further fuse with each other at their ends to form longer and thinner F-actin bundles. Arrowheads indicate the individual short F-actin bundles. The times (seconds) of the recordings antiserum as a single band at a position of 200 kDa, while no from 0 seconds are indicated in each image. Scale bar: 40 µm in signal was detected with pre-immune serum (Fig. 6A). A-C; 10 µm in D,E; 30 µm in G; 10 µm in I; 2 µm in J,K. Immunofluorescent staining with the anti-myosin II serum revealed that, at the growing end of the CF, myosin II assembly occurs to form dotty clusters (Fig. 6B,F). We call this structure seemed to form a fibrous structure along the long F-actin ‘myosin spot’. The size of the myosin spots throughout the bundles (Fig. 6H,I). In contrast, neither spot-like structure nor growing end region was quite homogeneous with a mean fibrous structure was observed when the furrow was stained diameter of 0.5±0.1 µm. At a higher magnification of the with the pre-immune serum as a control. growing end of the CF (Fig. 6F,G), the myosin spots colocalized with newly emerging F-actin patches. Some Appearance of myosin II, F-actin and WGA-binding myosin spots were arranged into tandem arrays along the short site at the growing end of CF F-actin bundles. Interestingly, at the periphery of the growing The relationship between the WGA-binding sites, the F-actin end of the CF, the myosin spots were not accompanied by the patches, and the myosin spots described above was investigated F-actin staining, suggesting that myosin spot formation by staining the CF at stage 1 with FITC-WGA, Bodipy- precedes the F-actin accumulation at F-actin patch. In an phallacidin and anti-myosin II antibodies (Fig. 7). interior region of the CF, the tandemly aligned myosin spots It seemed from the double-staining using anti-myosin Actin reorganization during cytokinesis 407

Fig. 3. Fluorescent images of CFs isolated from eggs into which rhodamine-actin had been microinjected. (A,C,F,H) Rhodamine fluorescence images. (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 of microinjection are indicated by large arrows. (A,B) Rhodamine-actin was microinjected near the growing end of a stage 2 CF and the egg was fixed 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 the egg was fixed within 90 seconds. (H,I) Magnified micrographs of F,G, respectively. Rhodamine-actin was incorporated homogeneously into long 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. antibodies and Bodipy-phallacidin that accumulation of the contractile ring of Xenopus eggs. The Methods and Results myosin II precedes that of F-actin at the growing end of the presented in this report contribute to determining the steps at CF in all CFs examined (n=16, Fig. 7A,B). Line profiling which various factors are involved in cytokinesis function. analysis revealed that myosin II accumulation occurs gradually from the end to the middle of the CF (Fig. 7D, average extent F-actin reorganization and relationship between of increased myosin; 1.7±0.4 fold, n=13). This contrasted with each F-actin structures the accumulation of F-actin, which was first gradual and then Our observations demonstrate that there are three distinct F-actin steep as described above. This suggests that myosin spot structures in the growing end of the CF of Xenopus eggs during formation precedes the accumulation of F-actin at the F-actin early stages of cytokinesis. The first sign of reorganization of the patches in the CF, and the F-actin accumulation continues after actin cytoskeleton is the F-actin patch formation which initiates the completion of the myosin spot formation. at stage 1. During stage 2, some of the F-actin patches are fused The double staining of stage 1 CF using anti-myosin II with each other and obviously organized into a radial array of antibodies and FITC-WGA showed that assembly of the short F-actin bundles. Finally, in the central region of the furrow, WGA-blebs and that of the myosin spots occurred in a similar the short F-actin bundles seem to become reorganized into the manner (Fig. 7E,F). Both extents and slopes of increase of long F-actin bundles. The long bundles may correspond to the myosin and WGA-binding sites from the end to the middle of F-actin bundles in the CF of newt eggs, previously demonstrated the early furrow were comparable (not shown for Fig. 7E,F; by EM to compose the contractile ring (Perry et al., 1971; see Figs 5D, 7D). The myosin spot and the WGA-bleb Mabuchi et al., 1988). This sequence was confirmed by the live colocalized with each other at the end of CF (Fig. 7G,H). In recordings of rhodamine-actin incorporated in the F-actin the bleb, myosin was concentrated at the neck region. structures in the CF. The long F-actin bundles are finally tightly packed into the thin contractile ring at stage 4. In case of the sea urchin egg, F-actin becomes accumulated DISCUSSION at the equator at the anaphase-telophase transition. The F-actin then forms bundles, and the bundles are arranged to form the Xenopus egg has been a good system for studying factors tightly packed contractile ring (Mabuchi, 1994; Yonemura and involved in cytokinesis, because of the ease with which it can Kinoshita, 1986). The transient region at the growing end of be micromanipulated and microinjected. However, little is the CF of Xenopus eggs, where the F-actin patches and the known about the organization of F-actin structures in the CF short F-actin bundles are formed, may correspond to the F- of this cell both during and after its formation. Here, we actin accumulation in the equatorial cortex of the sea urchin demonstrated process of organization of actin and myosin into egg. The long F-actin bundle formation and its packing at stage 408 JOURNAL OF CELL SCIENCE 114 (2)

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 growing end surface of a stage 1-2 CF double-stained with rhodamine-WGA (parts 1 and 3) and WGA-fluorescent beads (parts 2 and 4). Yellow lines indicate the division plane. An arrow in part 3 indicates the position of the CF. Red spots with numbers in parts 2 and 4 indicate some individual WGA-beads. Numbers in the bottom right-hand corner indicate the time (seconds) of the recording. At 0 seconds, the growing end has not appeared in the microscopic area, while it reaches the upper right-hand corner by 30 seconds. The WGA-beads gradually move towards the cleavage plane as the growing end advances. (C) The cortical movement at the growing end. The cortical movement towards the division plane initiates at the beginning of stage 1. Scale bars: 100 µm in A; 10 µm in B.

4 in the Xenopus egg CF may correspond to the formation of wide parallel arrays of F-actin bundles at the equator and their packing at later stages in the sea urchin egg, respectively (see Fig. 8). New actin polymerization at the growing end The steep increase of F-actin occurred during the early furrowing in the CF region between the growing end and the middle. The slope of the increase exceeded that of the cortex, which was monitored by the WGA-binding site. This result indicates that a significant portion of F-actin accumulated as the patches cannot be accounted for by transportation of cortical F-actin by the cortical movement. A high-resolution analysis revealed that the accumulation occurred underneath the WGA-bleb. Moreover, we directly visualized a rapid incorporation of microinjected rhodamine-actin into the F- actin patches and the short F-actin bundles at the growing end.

Fig. 5. Distributions of F-actin patches and WGA-binding sites in stage 1 CFs. (A-D) Comparison of distribution of WGA- binding sites on the surface as revealed by FITC-WGA staining (A) and F-actin distribution as revealed by rhodamine- phalloidin staining of a stage 1 CF (B). (C) Merged image of A,B. White rectangle indicates the area where the line profiling analysis was performed. (D) Fluorescence intensities of rhodamine-phalloidin (red line) and FITC-WGA (green line). Right-hand arrows indicate the tip of the FITC signal, while left-hand arrows indicate the middle of the CF. (E-J) z-axis sectioned micrographs of WGA-blebs and F-actin patches taken at different focal planes by the Delta Vision system. The optical sections were 0.2 µm thick. At the middle level of the WGA blebs (E), weak signal of rhodamine-phalloidin was observed inside of the bleb (F), while at the bottom level of the blebs (H), strong signal of rhodamine-phalloidin was observed 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. At the tip of the furrow (K,M), WGA-blebs colocalized with F-actin patches, which were only dimly stained. In an interior region of the CF (L,N), WGA-blebs colocalized 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 bar in N: 2.5 µm for K-N. Actin reorganization during cytokinesis 409

Fig. 6. Distribution of myosin II in the CFs of Xenopus eggs. (A) Specificity of anti-Xenopus oocyte myosin II antibodies. Immunoblot analysis against a total lysate of Xenopus oocytes excluding yolk granules with pre-immune serum (lane 1; diluted to 1/10,000) and with anti-myosin II serum (lane 2; diluted to 1/10,000), respectively. (B-I) Immunofluorescence microscopy of myosin II in the stage 2 CFs. (B,F,H) Staining with anti- Xenopus myosin II serum. (D) Staining with pre- immune serum. (C,E,G,I) Staining with Bodipy- phallacidin. (F-I) Micrographs at higher magnifications. (B) Numerous myosin spots are formed and aligned into radial arrays (small arrows in B) at the growing end. (D) These spots are not observed in a CF stained with pre-immune serum. (F,G) A number of the myosin spots colocalize with emerging F-actin patches (small arrows) near the growing end of the CF, while those in periphery (arrowheads) have no corresponding F-actin patches. Some myosin spots are arranged into tandem arrays along the short F-actin bundles (F,G, large arrows). (H,I) An interior region of the CF. The myosin spots (small arrowheads) are aligned on the long F-actin bundles (arrows) showing a fibrous appearance. Scale bar in D: 20 µm for B-E. Scale bar in G: 10 µm 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 myosin spot formation apparently precedes the F-actin accumulation at F-actin patches. (C) Merged image of A,B. White rectangle indicates the area where the line profiling analysis was performed. (D) Fluorescence intensities of myosin II (red line) and Bodipy-phallacidin (green line). Right-hand arrows indicate the tip of the myosin staining, while left-hand arrows indicate the middle of the CF. (E,F) Comparison of myosin II distribution (E) with distribution of WGA-binding sites (F) in a CF. The myosin spot formation and the WGA-bleb formation start in the same region. Arrows on the right indicate the tip of the staining. (G,H) Myosin spots and the WGA-blebs, respectively, at the very tip of the CF taken at a higher magnification at a same focal plane. At the tip of the CF, myosin spots colocalize 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 JOURNAL OF CELL SCIENCE 114 (2)

Fig. 8. Schematic representation of sequential reorganization of actin-myosin cytoskeleton at Cortical movement the growing end of the CF in the Xenopus egg. WGA-bleb Myosin spot formation The incorporation took place within 30 seconds of the injection, at a position 50 µm from furrow region. We estimated the F-actin patch formation time required for rhodamine-actin to travel (Actin polymerization?) this distance in the Xenopus egg cytoplasm by observing the injected protein to be less Reorganization of the F-actin patches than 20 seconds (not shown). This is in into F-actin bundles good agreement with an estimate of 22 seconds (Berg, 1983), using the diffusion (Elongation and fusion of F-actin patches) coefficient of G-actin in cytoplasm of sea Side view urchin eggs (Wang et al., 1982; Salmon et al., 1984; Hiramoto and Kaneda, 1988). Top view However, it would take more than 3 minutes by cortical movement. Thus, the speed of the several minutes after microinjection to label the incorporation incorporation is explained by diffusion followed by site with fluorescently labeled G-actin, and the incorporation polymerization, but not by transportation by cortical sites are distributed in a punctuate manner along the stress movement. We also observed emergence and growth of the fiber. The whole stress fiber is labeled at about 40 minutes after actin patches by live recording of the rhodamine-actin-injected the injection, while the whole contractile ring was labeled in egg in a CF region where no cortical movement occurs. The less than two minutes. Therefore, the contractile ring is the timing of the emergence was different between patches. This structure much more dynamic than the stress fiber. It has been may be explained by spontaneous polymerization occurring in shown that actin-depolymerizing factor/cofilin, which can individual patch, but may not be explained by accumulation of accelerate the turnover rate of actin (Carlier et al., 1997; F-actin by cortical movement, which would cause a Rosenblatt et al., 1997; Theriot, 1997), is present in the CF of synchronized formation of the patches. By the line profiling Xenopus eggs (Abe et al., 1996). This protein may facilitate the analysis, the extent of the F-actin increase in the middle CF actin turnover in the contractile ring. was double that of the increase of the WGA-binding sites. These results suggest that a half amount of F-actin in the CF The sequential relationship of the myosin spot may be derived from the new actin polymerization. The rest of formation and F-actin patch formation F-actin in the CF might be transported from the surrounding Myosin II first formed spots at the growing end. The size of cortex through the cortical movement. F-actin in the dimly the myosin spots was close to that of the cytoplasmic ‘myosin stained patches at the tip of the CF could represent this spots’, seen in the active lamella undergoing protrusion, population of F-actin. previously reported for fibroblastic cells (Verkhovsky and Borisy, 1993; Verkhovsky et al., 1995). These spots have been Rhodamine-actin incorporation into the contractile found to be clusters of short myosin filaments called ring ‘minifilaments’. Fishkind et al. have recently reported that the We found that rhodamine-actin is evenly incorporated into the minifilaments are localized to the CF of tissue cultured cells long actin bundles at the middle region of the furrow at a (Fishkind et al., 1996). The myosin spots in the Xenopus egg considerable speed, although it was a little slower than the one CF may also consist of the minifilaments. It is of interest that at the growing end. This result suggests that the turnover of myosin II in fission yeast also forms spots around the division actin in the contractile ring actively occurs in vivo, and the site, and then the spots are interconnected and packed into the actin incorporation sites distribute homogeneously along the contractile ring structure (Motegi et al., 2000). The assembly contractile ring. These features are distinct from those of stress of myosin II as spots prior to the formation of the contractile fibers in cultured cells (Turnacioglu et al., 1998): it takes ring may be common in cytokinesis in animal and fungal cells.

Table 1. Incorporation of rhodamine-actin into the F-actin patches, the short actin bundles and the long actin bundles in the cleavage furrow Incorporation into CF F-actin (number incorporated/ Injection site Injectant structure stained total number examined) Growing end 4 mg/ml rhodamine-actin F-actin patches 26/28 Short actin bundles 23/28 4 mg/ml rhodamine-BSA None 0/11 Central 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. Actin reorganization during cytokinesis 411 The double staining experiments lead us to two important peri-CF zone could be the cortical region that is responding to features of the myosin assembly. First, myosin colocalizes with the signal of the CF formation. It is tempting to speculate that the WGA-bleb at the tip of the CF. The WGA-blebs were a role of the cleavage signal is to stimulate the equatorial area formed by the accumulation of WGA-binding proteins of the cortex to induce the cortical movement in order to transported by the cortical movement. These observations accumulate further signaling molecules and materials suggest that myosin is also transported from outside of the CF necessary to construct the contractile ring. Similar movements by the cortical movement. Second, the formation of the myosin of the cortex around the CF have been observed in sea urchin spots precedes the F-actin accumulation at F-actin patches. eggs by means of attachment of carbon particles on the surface This suggests that the actin polymerization takes place after the (Dan, 1943), and in cultured mammalian cells by attachment assembly of myosin at the same site. In budding yeast and of concanavalin A-beads (Wang et al., 1994). A traction force fission yeast, myosin II accumulates in the division site earlier in the equatorial cortex of dividing cultured cells demonstrated than does F-actin (Lippincott and Li, 1998; Motegi et al., by a silicone-rubber method (Burton and Taylor, 1997) may 2000). In addition, myosin assembles faster than F-actin in an also reflect the cortical movement. artificial wound in Xenopus oocyte (Bement et al., 1999). The WGA-bleb formation was inhibited by application of However, in the sea urchin egg, we have reported that the BDM, cytochalasin B or latrunculin A. This result suggests that assembly of myosin and F-actin occurs at the same time in the both a myosin family motor protein and F-actin are involved process of the formation of the contractile ring (Mabuchi, in the cortical movement. It is tempting to speculate that 1994). This difference could be due to the difference of the myosin II binds to the membrane, generates the force for the species, or to resolution of the microscopic systems used. cortical movement, and translocates itself to the CF along the Although the myosin spot formation preceded the growth of actin cytoskeleton. However, other types of myosin cannot be the F-actin patch, it is still not clear whether the myosin spot excluded from the candidates of the motor protein for the formation is independent of F-actin or not. As discussed above, cortical movement. The mechanism by which the cortical a part of F-actin could be transported to the CF through the movement and the myosin II assembly are induced needs cortical movement, and there were actually dimly stained F- clarification, in order to understand how the CF is induced actin patches before the accumulation of F-actin at the growing during cytokinesis. end of the CF (Fig. 5). Thus, it is possible that a ‘precursor’ of the F-actin patch may simultaneously be formed on the myosin We thank Dr Hiroshi Kubota of the Kyoto University for help with spot through the cortical movement. Structural investigation by keeping frogs in the laboratory; the late Mr Masao Shinoda for advice EM is required to understand how myosin and F-actin interact and help in constructing the frog aquarium; and Dr Richard Elinson to construct the substructure of the contractile ring at the of the University of Toronto for suggesting the staining of F-actin in the cortex of Xenopus eggs. growing end.

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