Formation and Function of the Polar Body Contractile Ring in Spisula

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Developmental Biology 269 (2004) 421–432 www.elsevier.com/locate/ydbio

Formation and function of the polar body contractile ring in Spisula

Rafal M. Pielak, Valeriya A. Gaysinskaya, and William D. Cohen*

Department of Biological Sciences, Hunter College, New York, NY 10021, USA Marine Biological Laboratory, Woods Hole, MA 02543, USA Received for publication 2 November 2003, revised 24 December 2003, accepted 10 January 2004

Abstract

Initial studies suggested that spatial organization of the putative polar body contractile ring was determined by the peripheral aster in Spisula [Biol. Bull. 205 (2003) 192]. Here we report detailed supporting observations, including testing of aster and ring function with inhibitors. The metaphase peripheral aster was confirmed to spread cortically in an umbrella-like pattern, with microtubule-poor center. The aster disassembled during anaphase, leaving the spindle docked at the F-actin-poor center of a newly generated cortical F-actin ring that closely approximated the aster in location, measured diameter range, and pattern. Cytochalasin D and latrunculin-B permitted all events except ring and polar body formation. Nocodazole disassembly or taxol stabilization of the peripheral aster produced poorly defined rings or bulging anaphase asters within the ring center, respectively, inhibiting polar body formation. Polar body extrusion occurred at the ring center, the diameter of which diminished. Ring contractility—previously assumed—was verified using blebbistatin, a myosin-II ATPase inhibitor that permitted ring assembly but blocked polar body extrusion. The data support the hypothesis that peripheral aster spreading, perhaps dynein-driven, is causally related to polar body contractile ring formation, with anaphase entry and aster disassembly also required for polar body biogenesis. Previously reported astral spreading during embryonic micromere formation suggests that related mechanisms are involved in asymmetric somatic cytokinesis. D 2004 Elsevier Inc. All rights reserved.

Keywords: Polar body; Contractile ring; Aster; F-actin; Microtubules; Blebbistatin; Nocodazole; Latrunculin; Taxol; Spisula

Introduction differentiation (e.g., Knoblich, 2001; Schroeder, 1987; Yamashita et al., 2003). Cytokinesis is a field of active inquiry in cell and Cytokinetic inequality in terms of daughter cell size and developmental biology, with many studies analyzing the content typically involves furrow eccentricity, with polar mechanisms involved in signaling contractile ring assembly, body extrusion from meiotic eggs representing an extreme and in recruiting actin, myosin, and other proteins for ring example (Maro and Verlhac, 2002; Selman, 1966).Ina construction (e.g., Canman et al., 2003; Guertin et al., 2002; recent brief report on polar body formation in the surf clam, Rappaport, 1986; Sanger and Sanger, 2000). Recent work Spisula solidissima (Pielak et al., 2003), we noted possible also highlights furrow positioning and completion of cyto- involvement of the metaphase peripheral aster in determin- kinesis as being dependent upon the function of central ing the location, pattern, and dimensions of the thickened spindle proteins (Glotzer, 2003), and the discovery that ring of cortical F-actin that forms at anaphase. In addition, cytokinetic success requires extracellular mucopolysacchar- polar body formation appeared to require subsequent disas- ides (Shuster and Burgess, 2002; Skop et al., 2001; White sembly of the peripheral aster and passage of the associated and Bednarek, 2003). The function of specialized centro- centrosome and chromosomes through the center of the somes is of considerable interest as well, in relation to ring. Although direct evidence was lacking, our working asymmetric cell division in embryos and subsequent cellular assumption has been that this is a contractile ring similar to that of classic cytokinesis, though perhaps with some specialized modification. In the current studies, we sought to obtain insight into the * Corresponding author. Department of Biological Sciences, Hunter College, 695 Park Avenue, New York, NY 10021. Fax: +1-212-772-5227. mechanisms involved by asking the following questions: E-mail addresses: [email protected], [email protected] Does inhibition of F-actin ring assembly block polar body (W.D. Cohen). formation? Does interference with either assembly or disas-

0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.01.033 422 R.M. Pielak et al. / Developmental Biology 269 (2004) 421–432 sembly of the peripheral aster prevent ring and polar body in DF-NSW or in experimental media to a concentration of formation? Is a legitimate contractile process involved, approximately 1:100 (Veggs/VFSW), after which time-course requiring myosin function? Our experimental approach was samples were taken approximately every 2 min., and to combine carefully timed application of specific inhibitors prepared for fluorescence labeling and microscopy as with structural analysis by confocal fluorescence microscopy. described below. During experimental incubations, eggs Freshly obtained Spisula eggs, activated to undergo germinal were maintained in suspension by gentle rocking on a vesicle breakdown (GVBD), meiotic completion, and polar Nutator. body formation, by exposure to excess KCl, served as the In the course of the work, it was noted that the timing of experimental system. Cytochalasin D and latrunculin B were stages became somewhat extended with longer-term storage employed to block F-actin assembly, taxol to stabilize astral of the ripe animals. For example, GVBD occurred in microtubules, nocodazole to induce astral microtubule dis- approximately 7 min at 23jC for clams used initially, and assembly, and blebbistatin to inhibit nonmuscle myosin II in approximately 9–11 min at 23jC for those used about ATPase activity (Spector et al., 1989; Straight et al., 2003; 8 weeks later. This was adjusted for by monitoring a range Urbanik and Ware, 1989). Blebbistatin, a newly discovered of time point samples in each preparation. For this reason, inhibitor of cytokinesis in vertebrate cells, was reported to results with inhibitors are reported in terms of comparison have no effect on cells of D. melanogaster (Straight et al., with the stage reached by the specific control run within that 2003). Thus, it was of intrinsic interest to determine whether experiment, rather than by absolute sampling time post- blebbistatin would block cytokinetic events in a different cell GVBD. type and in an invertebrate. Near synchrony of KCl-activated Spisula eggs with Fluorescence labeling and microscopy respect to stages preceding polar body formation has permitted us to obtain answers to these questions with results Oocytes were simultaneously permeabilized and fixed in that are typical for most of the cells, characteristic for each a medium consisting of 0.6% Brij-58, 4% formaldehyde in type of inhibition, and consistent with previous work of PEM (100 mM PIPES, 5 mM EGTA, 1 mM MgCl2, pH 6.8, others. using NaOH). After 1 h of incubation, the samples were washed in PEM and then labeled. For F-actin, the eggs were stained with rhodamine or Alexa fluor 568-phalloidin. Materials and methods Microtubules were labeled with a 1:1 mass mixture of mouse monoclonal anti-a and anti-h tubulins (Sigma Ripe S. solidissima, maintained at 11–13jC to inhibit T9026, T-4026) pre-labeled with Alexa fluor 488-labeled shedding, were obtained from the Aquatic Resources Divi- anti-mouse Fab fragments (‘‘Zenonk’’; Molecular Probes), sion of the Marine Biological Laboratory (MBL). Gonadal and chromosomes and nuclei were labeled with DAPI. tissue was removed by dissection, suspended in approxi- These procedures followed protocols developed in previous mately 150 ml of filtered natural seawater (F-NSW; filtered work on rapid microtubule localization in a variety of cell through Whatman #2 paper), and the suspension filtered types with minimal morphological distortion (Lee et al., through a double layer of cheesecloth to remove the tissue. 2002). Labeled samples were viewed routinely by confocal The egg suspension that passed through was allowed to fluorescence microscopy with the Zeiss Laser Scanning settle (approximately 20–30 min), the supernate was re- System LSM 5 PASCAL, and image processing was done moved, and the eggs washed twice more by resuspension via Zeiss LSM Image Examiner and Adobe Photoshopn and settling, with final resuspension in 0.45 Am-filtered F- software. NSW (double-filtered or DF-NSW). Before use in experi- The methods employed for immunofluorescence labeling ments, aliquots were adjusted to a concentration of approx- of Spisula eggs in the present work (Lee et al., 2002; Pielak imately 1:10 (Veggs/VFSW) after determining egg volume by et al., 2003) were effective without removal of the vitelline gentle hand centrifugation. membrane, unlike those used in earlier studies (e.g., Kur- Activation of eggs was achieved by adding excess KCl iyama et al., 1986). This is probably attributable to our use to the egg suspension, according to standard methods for of detergent extraction at physiological temperature, rather this species (Allen, 1953; Costello and Henley, 1971; than ethanol extraction at low temperature. Since the vitel- Palazzo et al., 1999; Palazzo and Vogel, 1999). Eggs were line membrane consists of numerous plasma membrane- gently sedimented (hand centrifuge) and resuspended to enclosed egg microvilli enmeshed in a fibrous matrix 10% (Veggs/VFSW) in Tris-buffered artificial seawater (T- (Longo and Anderson, 1970), its removal is potentially ASW; MBL-formula ASW buffered with 10 mM Tris at pH injurious and is best avoided if possible. 8.0). One volume of the 10% egg suspension was gently mixed with 7.6 vol T-ASW, and 1.4 vol 0.5 M KCl. The Treatment with inhibitors time of KCl addition was recorded as t = 0, and eggs were then monitored in phase contrast for GVBD. Following As noted above, the times at which particular stages were GVBD, the eggs were sedimented gently and resuspended reached became somewhat extended during this study, R.M. Pielak et al. / Developmental Biology 269 (2004) 421–432 423

Fig. 1. Behavior of peripheral aster preceding polar body formation in KCl-activated Spisula eggs; confocal optical sections (F-actin red, chromosomes blue, microtubules green throughout). (a) Metaphase, showing spread of astral microtubules outward along the cell cortex; diameter of spreading area is approximately 19 Am (arrowheads); central region (approximately 8 Am in diameter; arrow) contains relatively few microtubules. (b) Anaphase, with spindle closer to egg surface; aster is still spread over approximately 20 Am of cortex (arrowheads; note: F-actin signal omitted for clarity). Scale bar = 10 Am. presumably related to period of storage of ripe animals at muscle myosin function (Straight et al., 2003), using post- low temperature. Therefore, absolute times of inhibitor GVBD eggs resuspended in 100 AM blebbistatin, or in 1% addition are not as important as the stages reached when DMSO (solvent control). Nocodazole and taxol (Sigma) the inhibitor was added. Two basic times were employed: were employed to induce astral microtubule disassembly (1) ‘‘early’’, or ‘‘post-GVBD’’, with cells at metaphase and stabilization, respectively. Oocytes were transferred shortly after GVBD, but before movement of the spindle to10 Ag/ml nocodazole either early (post-GVBD) or late to the cortex (approximately 9–14 min at 23jC); (2) ‘‘late’’, (beginning of metaphase-to-anaphase transition), with a with metaphase spindles having moved to the cortex and 0.5% DMSO solvent control. Eggs were resuspended in some cells in the population beginning to enter anaphase 10 Ag/ml taxol either early or late, with a 0.4% DMSO (approximately 20–24 min at 23jC). solvent control. Nocodazole, taxol, and cytochalasin have For all inhibitors and their controls, the primary medium been used in earlier studies of meiotic Spisula eggs (Kur- was DF-NSW. Cytochalasin D or latrunculin B (Calbio- iyama, 1986; Kuriyama et al., 1986; Longo, 1972), but F- chem), used to inhibit actin polymerization, were added actin and microtubules were not colocalized. In addition, ‘‘early’’. Following GVBD, eggs were resuspended in 0.8 taxol was applied earlier than the time considered most Ag/ml of cytochalasin D, or 0.06 Ag/ml latrunculin B, or important to the present work, with different results. Taxol- 0.08% DMSO (solvent control), or DF-NSW alone (con- induced stabilization of early metaphase asters may interfere trol). Blebbistatin (Calbiochem.) was used to inhibit non- with normal spindle translocation, thus affecting metaphase-

Fig. 2. F-actin ring formation and apparent contraction; 3-D reconstructions from optical section stacks. (a) Late anaphase–telophase, showing thickened cortical ring approximately 20 Am in diameter, with F-actin-poor center approximately 7 Am in diameter; upper inset, the two chromosome sets; lower inset: interpolar spindle microtubules remaining after most astral microtubules have disassembled. (b) Composite of images in (a), showing spindle and chromosome location. Despite appearances, polar body extrusion has just begun, and there is only a slight bulge as determined from 3-D rotations. (c) Egg at later stage, with one pronucleus extruded (upper inset); F-actin ring diameter is now much reduced [compared with (a)], constricting the region between separating pronuclei. Some spindle microtubules remain (lower inset). Scale bar = 10 Am. 424 R.M. Pielak et al. / Developmental Biology 269 (2004) 421–432

Fig. 3. Details of F-actin ring and docked spindle; confocal optical sections. (a) F-actin-only survey view of two late anaphase eggs, each with thickened cortical F-actin ring and F-actin-poor center (arrows), and spindle location (inset). (b) Enlarged view, enhanced to show only thickest actin region; ring thickness appears to diminish gradually over approximately 10 Am distance on each side of the center. (c) Docking of spindles at center of the F-actin-poor regions shown in (b); most astral microtubules have disappeared, but interpolar spindle microtubule bundles remain. Scale bars = 10 Am. to-anaphase transition events only indirectly (Kuriyama, in a fountain-like pattern in which microtubules curve 1986). outward to become parallel with the surface (Fig. 1a). Counts were made to quantify the effect of inhibitors in The diameter of metaphase astral spread (Fig. 1a, arrow- experimental samples versus solvent-only controls. The heads) was in the range of 19–23 Am as measured in 11 percentages reported were obtained from counts of a min- different eggs. The center of this spread pattern, ranging in imum of 200 cells per sample. Measurements of aster and diameter from 7 to 13 Am, was typically nearly devoid of ring diameters were made only on images of optimum microtubules (Fig. 1a, arrow). clarity, using Zeiss Image Examiner software. Transition into anaphase occurred with the peripheral aster remaining in cortical contact (Fig. 1b; arrowheads). As anaphase proceeded, the aster disassembled and a ring of Results thickened F-actin appeared in the cortical region formerly occupied by the spreading aster (Fig. 2). This ring had an F- Normal sequence of cytoskeletal events leading to polar actin-poor circular center in the 6- to 9-Am-diameter range, body formation as measured in eight oocytes in optical sections or comput- er-generated 3-D images (Fig. 2a). Its outer diameter (ig- Activated Spisula eggs progress through GVBD, estab- noring edge irregularities) was in the range of 16–25 Am. lishment of an eccentrically located metaphase meiosis I Such rings were seen only in anaphase cells during the spindle, and migration of the metaphase spindle to the normal sequence, never in metaphase cells. Although astral cortex accompanied by unequal astral growth (Allen, microtubules were gone, the interpolar microtubule bundles 1953; Kuriyama et al., 1986; Longo and Anderson, of the spindle were still largely intact (Fig. 2a, lower inset), 1970). Our focus was on major cytoskeletal events imme- and the center of the ring was occupied by the peripheral diately preceding polar body formation. Critical stages in centrosome (Fig 2b). Subsequently, the peripheral chro- the normal sequence began with the spreading of meta- mosome set and associated centrosomal material passed phase peripheral astral microtubules along the cell cortex, through the ring center (Fig. 2c). Measurements showed

Fig. 4. Later stages in polar body formation; confocal optical sections. (a) Meiosis II anaphase spindle, with long axis perpendicular to cortex as in meiosis I; inset: three chromosome sets (outer one in polar body, two in anaphase II). (b) Egg with two extruded polar bodies. Scale bar = 10 Am. R.M. Pielak et al. / Developmental Biology 269 (2004) 421–432 425

spindle (Figs. 3a, b), and the spindle pole abutted against, but did not protrude through, the thinner central F-actin cortex (Fig. 3c). The meiosis II spindle assembled at the same site, oriented as in meiosis I (Fig. 4a), producing the second polar body adjacent to the first (Fig. 4b).

Inhibition of F-actin assembly

Varying concentrations of both cytochalasin D and latrunculin B were tested to determine their useful range. At the higher cytochalasin and latrunculin concentrations (2–4 and 0.2–0.4 Ag/ml, respectively), the eggs rapidly lost their spheroidal shape and collapsed onto the substratum. Therefore, the concentrations of these drugs were reduced until eggs were found to maintain normal shape and progress through normal stages of meiosis I in terms of meiotic apparatus assembly and function. Cytochalasin D (0.8 Ag/ml), applied after GVBD (‘‘early’’), inhibited actin ring formation, cytokinesis, and conse- quently polar body release. Cortical F-actin was present in both experimental and control, and the first meiotic spindle formed at a time similar to that in the control, and migrated to the periphery normally. Microtubules of the peripheral aster curved and spread outward along the cell cortex at metaphase, as in the control (Fig. 5a). The spindle then moved closer to the cortex at anaphase, as normally (Fig. 5c), but the F-actin ring did not form and the first polar body was not extruded. An optically ‘‘clear region’’ appeared along the adjacent cortex as visualized in DIC (Fig. 5b). Subsequently, at the time when controls were in meiosis II, two meiotic apparatuses formed in most of the CD-treated cells, yielding four sets of chromosomes and four pronuclei (Fig. 5e) [note: we call these ‘‘pronuclei’’ here and below for want of a more accurate term; the true egg pronucleus has a nuclear envelope; Longo and Anderson, 1970]. In contrast, the controls at that time had produced two polar bodies and contained the normal single female pronucleus.

Fig. 5. Cytochalasin D, added ‘‘early’’; confocal optical sections, fluorescence and DIC. (a) Metaphase, with eccentric spindle and spreading peripheral aster, as in controls. (b) Anaphase egg in DIC; note clear area at upper right (arrowhead). (c) Same anaphase egg, with docked spindle containing interpolar microtubules but few astral microtubules; inset: anaphase chromosome sets; some F-actin appears localized at inner spindle pole (seen occasionally in other cells). (d) Same anaphase egg, F-actin only; cortical F-actin is present, but there is no ring. (e) Later CD stage, when controls had two polar bodies; CD-treated eggs produced no polar bodies, but most contained four pronuclei-produced in two meiosis-like divisions. Scale bar = 10 Am. the central ring opening to be less than half its original Fig. 6. Latrunculin B, added ‘‘early’’; confocal optical sections. (a) diameter during this stage, and the outer F-actin ring Metaphase, with peripheral aster spreading along cortex; note irregularity of diameter was also reduced (Figs. 2c vs. a). cortical F-actin, with concentrated spots throughout surface (arrowheads). (b) Docked late anaphase spindle, showing absence of contractile ring and The relationship of the late anaphase spindle to the F- bright discontinuities in cortical F-actin, particularly in the region of earlier actin ring is seen in greater detail in optical sections (Fig. 3). astral spreading (arrowheads). No polar bodies were produced, and eggs Ring thickness appeared to decrease with distance from the ultimately contained multiple pronuclei as in Fig. 5e. Scale bar = 10 Am. 426 R.M. Pielak et al. / Developmental Biology 269 (2004) 421–432

Fig. 7.

Fig. 8. R.M. Pielak et al. / Developmental Biology 269 (2004) 421–432 427

Latrunculin B (0.06 Ag/ml) had a similar effect on bulging protrusion, approximately 18–20 Am in diameter, contractile ring and polar body formation, ultimately yield- within a widened F-actin ring center (Figs. 8c–f). Astral ing four pronuclei. However, unlike cytochalasin D, it also microtubules were spread and curved in umbrella-like produced irregular discontinuities in the cortical F-actin. fashion against the thinner cortical layer of this bulge Foci of F-actin concentration were most frequent in the (Fig. 8d, arrow), and the astral center had a circular opening region in which the F-actin ring would normally have approximately 5 Am in diameter (Fig. 8f). Subsequently, the formed (Fig. 6, arrowheads). ring in such taxol-treated eggs did not close, and polar bodies were not produced. Induced microtubule disassembly and inhibition of microtubule disassembly Inhibition of nonmuscle myosin II ATPase activity

To disassemble microtubules at a critical point, nocoda- Whereas counts showed that polar body formation oc- zole (10 Ag/ml) was added ‘‘late’’ when most cells contained curred in 95% of solvent-only control eggs, blebbistatin metaphase spindles with spreading peripheral asters, but (100 AM), introduced after GVBD (‘‘early’’), inhibited some had already entered anaphase. Metaphase cells now cytokinesis and polar body extrusion in >90% of the eggs. remained at metaphase even after the second polar body However, it did not visibly alter earlier events. The meta- formed in controls. However, cells in anaphase had abnor- phase spindle migrated to the egg periphery, microtubules of mal, poorly defined F-actin rings (Fig. 7). The polar body the peripheral aster spread outward on the actin cortex, and did not form in 98% of these oocytes (vs. 94% formed in the chromosome sets separated at anaphase as in the normal controls), and subsequently, decondensation of the two sets sequence (Fig. 9). The F-actin ring appeared to form of anaphase chromosomes occurred, producing two pronu- normally, accompanied by peripheral aster disassembly clei. In contrast, anaphase cells in the solvent-only control (Figs. 9b–d), but the polar body was not produced. Subse- preparation formed normal F-actin rings, and later, 94% quently, when solvent-only control eggs that had produced produced polar bodies. the first polar body were in meiosis II, blebbistatin-treated When nocodazole was added ‘‘early’’, that is, with all eggs assembled a meiotic apparatus for both the interior and eggs in metaphase shortly after GVBD, microtubule disas- peripheral nucleus. The reappearing peripheral aster was sembly resulted in long-term retention of metaphase chro- again spread against the cortex, and spindles appeared to be mosome sets. Counts showed that when approximately 50% interlinked by microtubules (Fig. 9e). The long axis of the of the controls had entered anaphase, 98% of the experi- second meiotic spindle was now typically oriented perpen- mental eggs were still at metaphase. When last observed at dicular to that of the first, with three to five asters observed the time of polar body formation in controls, their chromo- in different eggs. In most cases, the chromosomes later somes had remained in metaphase configuration without decondensed to four pronuclei. forming pronuclei. To block disassembly and enhance assembly of astral microtubules, taxol (paclitaxel; 10 Ag/ml) was also added Discussion ‘‘late’’, when most cells contained metaphase spindles with cortex-spreading peripheral asters, but some had entered The normal sequence of cytoskeletal events before polar anaphase. In metaphase eggs, both asters were exaggerated body formation in size and microtubule content, but astral asymmetry was still evident and the peripheral aster followed the egg The pattern, size, and location of the polar body con- contour (Fig. 8a). The anaphase peripheral aster did not tractile ring appear to be determined by the peripheral aster. disassemble to any great extent, producing a slight bulge in Peripheral aster spreading along the cortex at metaphase was early anaphase (Fig. 8b). In samples taken from the same followed by F-actin ring formation at anaphase (Figs. 10a, preparation several minutes later, anaphase eggs had a b), and the dimensions of aster and ring essentially matched.

Fig. 7. Nocodazole, added ‘‘late’’; confocal optical sections and 3-D reconstruction from section stacks. (a) Optical section, anaphase; two pronuclei present. Microtubules are absent, and some F-actin is concentrated at cortical region adjacent to outer pronucleus (arrow). (b) Another anaphase example, with F-actin thickened at cortical region near outer pronucleus (arrow), and no microtubules. (c) Rotated view of (b) from 3-D stack, showing poorly defined F-actin patch rather than a typical ring. No polar bodies were produced in nocodazole-treated oocytes; ultimately, when controls had two polar bodies, experimental metaphase eggs retained their metaphase chromosome configuration, but anaphase eggs contained two pronuclei. Scale bar = 10 Am.

Fig. 8. Taxol, added ‘‘late’’; 3-D reconstructions from confocal optical section stacks. (a) Metaphase, showing enlargement of both asters but retention of their distinctly different form and localization; peripheral aster is spread, without a surface bulge. (b) Early anaphase, with continued spreading and slight surface bulge evident. (c) Later anaphase, with bulging aster in abnormally wide center of F-actin ring; microtubules are spread umbrella-like against the thinner cortical layer of the bulge. (d) Same cell as (c), with microtubules not shown, to increase visibility of thin cortex at center (arrow). (e) Another anaphase egg, triple-labeled, showing enlarged center of F-actin ring; Microtubules and chromosomes are shown separately in lower and upper insets, respectively. (f) Taxol- induced aster of another cell, enlarged to illustrate dimensions and umbrella-like organization. Scale bar = 10 Am in panels (a) to (e). 428 R.M. Pielak et al. / Developmental Biology 269 (2004) 421–432

Fig. 9.

Fig. 10. R.M. Pielak et al. / Developmental Biology 269 (2004) 421–432 429

Average diameter for normal spreading metaphase periph- cortex as to the size, pattern, and location of the F-actin eral asters was 21 Am (Fig. 10a; y), and average initial ring, perhaps stabilizing the ring in the process. Does diameter of the F-actin ring was 22 Am (Figs. 2a, 3, and disassembly of the peripheral aster with nocodazole affect 10b;). The F-actin-poor central circular area of the ring also F-actin ring and polar body formation? correlated with the consistently observed ‘‘microtubule- Yes. With nocodazole added ‘‘late’’, microtubules were poor’’ central region of the earlier aster (Figs. 10a, b; x). absent from both metaphase and anaphase cells in samples Lack of microtubules in this region was convincing; for taken 5 min after drug exposure, that is, there were none in example, it could not be attributed to artifactual blockage of spindles or asters. Metaphase cells did not contain F-actin antibody penetration by densely packed microtubules be- rings, but the anaphase cells had ill-defined rings lacking a cause they were well-labeled in taxol-treated eggs (Fig. 8). distinct F-actin-poor central region (Fig. 7). Since, in the Thus, the present experiments confirm and extend our normal sequence, the ring assembles less than 3 min after earlier observations on the spatial and temporal astral the metaphase-to-anaphase transition, we conclude that exit microtubule-F-actin ring relationship in prepolar body from metaphase is necessary but not sufficient to generate stages in Spisula (Pielak et al., 2003), and are in accord normal rings. Something additional is required, and our with previous work on Spisula oocyte microtubule cycles observations indicate that it is the maintenance of patterned (Kuriyama et al., 1986). physical contact between peripheral aster and cortex. How- Our work implies that if an oocyte were to produce two ever, in interpreting the correlation between peripheral aster polar bodies simultaneously, two spreading peripheral asters disassembly and absence of normal rings, there is a proviso, would be required. Fortunately, nature provides such an because both astral and spindle microtubules are disas- experiment: the metaphase-I spindle in oocytes of the sembled by nocodazole. Thus, further experiments are androgenetic clam Corbicula leana aligns parallel to the desirable in which the peripheral aster is disrupted more surface, and both asters flatten against the cortex before specifically. simultaneous extrusion of two polar bodies (Komaru et al., If nocodazole was added ‘‘early’’, nearly all eggs 2000). Although not yet demonstrated, our working hypoth- remained at metaphase when solvent controls entered ana- esis predicts that the two asters will match two F-actin rings phase. Observed again later, when controls produced polar in terms of size, location, and pattern. bodies, the chromosomes of nocodazole-treated eggs remained at metaphase, without having formed pronuclei. Function of prepolar body structures and events, as probed Thus, ‘‘early’’ addition of nocodazole was not very infor- with inhibitors mative with respect to critical events of polar body formation. However, these results were consistent with a require- Use of specific inhibitors at selected stages provided ment for anaphase entry to produce pronuclei, as discussed answers to several questions about the normal prepolar body further below. cytoskeletal sequence (Fig. 10). To simplify discussion, the terms ‘‘early’’ and ‘‘late’’ are used again to indicate, Requirement for peripheral aster disassembly respectively, inhibitor addition at post-GVBD metaphase, In the normal sequence, peripheral aster microtubules or addition just before metaphase–anaphase transition in the disassemble before polar body extrusion. Does taxol inhi- population. In the latter case, inhibitors elicited their effect bition of microtubule disassembly prevent polar body during this transition. formation? Yes. ‘‘Late’’ taxol exposure produced a bulging mass with Function of the peripheral aster in ring formation a large peripheral aster dominating the ring center (Fig. 8), The normal cytoskeletal sequence (Fig. 10) suggests that and neither ring closure nor polar body extrusion occurred. It the peripheral aster signals or otherwise influences the remains to be determined whether this was due to a non-

Fig. 9. Blebbistatin, added ‘‘early’’; confocal optical sections and 3-D stack reconstructions. (a) Anaphase egg, DIC; note slightly bulging clear area (arrowhead). (b) Same egg, triple-labeled, showing docked spindle and absence of astral microtubules; upper inset: anaphase chromosome sets; lower inset, spindle interpolar microtubules. (c) Same egg, F-actin only; F-actin ring is visible in cortex (arrowheads). (d) 3-D rotation view of F-actin ring in another blebbistatin-treated anaphase egg. (e) Later stage, when controls were in meiosis II. No polar bodied formed; instead, nuclei underwent a second meiotic division in most blebbistatin-treated cells. The peripheral aster reappeared essentially in its original location, again spread against the cortex, with inner spindle parallel to the cortex instead of perpendicular to it (arrow; compare with Fig. 4c). Microtubules appeared to interlink all asters. Scale bars = 10 Am.

Fig. 10. Diagrammatic interpretation of the normal cytoskeletal sequence (metaphase to polar body extrusion; plasma membrane, black; F-actin, red; microtubules, green; chromosomes blue). (a) Metaphase spindle with peripheral aster spreading. x = diameter of central microtubule-poor area; y = diameter of astral spread. The F-actin-containing cortex consists of vitelline membrane (matrix-embedded egg microvilli) plus underlying cortical layer. (b) Late anaphase, after F-actin ring formation and astral disassembly. Here, the diameter of central F-actin-poor area (x) and outer diameter of F-actin ring (y) approximate the x and y astral dimensions in (a). The anaphase spindle retains its interpolar microtubules, and its peripheral centrosome abuts against the original cortex at the ring center. The F-actin ring is artificially illustrated as separated from the original cortex to indicate its interior addition to the latter. (c) Hypothetical: polar body nucleus and associated centrosome begin to push outward by an unknown mechanism, possibly spindle elongation (see Discussion). (d) Ring contraction posterior to nucleus separates polar body; the original cortex, including vitelline membrane, covers its outer surface (Longo and Anderson, 1970). 430 R.M. Pielak et al. / Developmental Biology 269 (2004) 421–432 functional ring or to physical presence of the stabilized aster. were produced in 95% of solvent-only controls. This prob- This ‘‘taxol aster’’ appeared to be derived from the earlier ably reflects the near-synchronous progression of the KCl- normal peripheral aster, not one newly assembled at a late activated egg population through prepolar body stages, as anaphase centrosome, as it had a microtubule-poor center compared with use of nonsynchronized cell cultures in some and similar inner and outer diameters (approximately 5 and of the previous experiments (Straight et al., 2003). In stage- 20 Am, respectively). Moreover, such bulging asters were not specific in vitro analysis of molecular mechanisms underly- present in the same egg population a few minutes earlier, ing polar body contractile ring formation and function, when asters were already taxol-stabilized. However, in myosin-II-based actin filament motility has been reported interpreting results, the same proviso applies as for nocoda- in M-phase extracts of Spisula oocytes, in which F-actin zole: the primary taxol effect appeared to be stabilization and networks assemble spontaneously (Wo¨llert et al., 2001). enlargement of the peripheral aster, but it probably affected spindle microtubules as well. Properties of pronuclei and centrosomes Although peripheral aster disassembly appears to be necessary for polar body formation, the egg waits until late Our results with CD, LB, and blebbistatin support anaphase to bring it about. This is another indicator that the previous work in showing that the prepolar body centro- earlier astral spreading onto the cortex is functionally some and chromosomes are not irreversibly marked for significant. discard or dysfunction. Inhibition of polar body formation with all three agents frequently yielded four pronuclei (Fig. Formation and function of the F-actin ring 5e), and thus the meiosis II egg accommodates both meiosis Based on earlier electron microscopic observations I daughter pronuclei. Multiple pronuclei were similarly (Longo and Anderson, 1970), we interpret ring formation noted in earlier work on cytochalasin B-treated Spisula as occurring by assembly of F-actin and associated proteins and starfish eggs (Longo, 1972; Tsukahara and Ishikawa, onto the inner cortical surface (Fig. 10b). Thus, CD inhibition 1980), and starfish polar bodies underwent a second divi- of F-actin assembly blocked ring formation, with the cortical sion when provided more cytoplasm by hexylene glycol actin layer still visible (Fig. 5). However, LB both inhibited enlargement (Saiki and Hamaguchi, 1997). Polar body ring assembly and caused cortical actin disruption (Fig. 6), nuclei are also genetically competent to produce embryos as expected for its mode of action (Spector et al., 1989). in both invertebrates and vertebrates (e.g., Tsukahara and Does inhibiting ring assembly block polar body forma- Ishikawa, 1980; Wakayama and Yanagimachi, 1998). Thus, tion? Yes. Polar bodies did not form in the presence of either physical isolation of cytoplasm-depleted polar body con- CD or LB (Figs. 5 and 6). Similar inhibition by cytochalasin tents serves to suppress its division. B was demonstrated previously in Spisula (Longo, 1972) Nocodazole-induced microtubule disassembly during and in other species (e.g., Tsukahara and Ishikawa, 1980; metaphase caused chromosomes to remain in metaphase starfish). However, the present work has enabled us to show configuration, whereas at anaphase, the chromosomes that essentially normal docking of the metaphase spindle and formed two pronuclei well before decondensation occurred cortical spreading of its peripheral aster occur in the presence in controls. With nocodazole present, these remained as two of such actin assembly inhibitors. pronuclei at least until most solvent-only control eggs With respect to ring function, the observed reduction in produced polar bodies. In contrast, cells treated with inhib- central ring diameter provides evidence of ring closure itors that did not disassemble microtubules consistently during polar body extrusion, as expected for a contractile formed pronuclei following naturally occurring microtubule ring (Figs. 2 and 10c, d). Is the ring truly contractile as disassembly, as in controls. This suggests that oocyte previously assumed, requiring myosin motor activity? Yes. chromosomal decondensation is linked to microtubule dis- Blebbistatin, an inhibitor of nonmuscle myosin II ATPase assembly during anaphase. activity (Straight et al., 2003), did not block Spisula meiotic spindle formation, chromosome movement, spindle docking, Polar body cytokinesis versus somatic cytokinesis astral spreading, aster disassembly, or F-actin ring formation (Figs. 9a–d). However, the polar body did not form, in Are polar body cytokinetic mechanisms unique, or are agreement with the highly specific effects of the drug on they similar to those of somatic cells and thus relevant to vertebrate cells (Straightetal.,2003). Extra asters and cytokinesis in general? Certainly, one critical feature differs: multiple (usually four) pronuclei ultimately appeared in most the polar body F-actin ring assembles initially at the cortex of these eggs, as with actin assembly inhibitors (Fig. 9e). on one side of the egg, not between two potential daughter One feature, not observed with the other inhibitors, was the cell masses (Fig. 3). In other words, the ring forms before deviation of some late anaphase eggs from circularity, with a there is visual evidence of what it will separate. slight bulge near the outer spindle pole (Figs. 9b, c). Its However, when comparison is made with highly asym- possible significance remains to be determined. metric somatic cytokinesis, certain similarities become ev- Counts showed that blebbistatin blocked polar body ident. Sea urchin micromeres, produced at the fourth round formation in at least 91% of the eggs, while polar bodies of embryonic cleavage, are larger than Spisula polar bodies R.M. Pielak et al. / Developmental Biology 269 (2004) 421–432 431

(15–20 Am vs. approximately 5–10 Am in diameter), but (Busson et al., 1998), with outward microtubule sliding in less than half the macromere diameter. The two centrosomes their plus-end direction causing the peripheral aster to of micromere-producing cells are structurally and function- flatten against the surface, pulling its centrosome and ally distinct (Holy and Schatten, 1991), and both aster size chromosomes close to the cortex for subsequent expulsion. inequality and metaphase spindle cortical docking have been documented in two urchin species (Holy and Schatten, Polar body extrusion 1991; Schroeder, 1987). In both cases, the peripheral aster appears to spread upon, and flatten against, the cell cortex. Results with blebbistatin indicate that myosin-based ring In addition, peripheral astral deformation against the cell contraction is required for polar body formation. This might cortex was observed in unequal cleavage in Spisula embryos help generate an expulsive force, but what mechanism (Dan and Inoue, 1987). This indicates that rather than being produces an initial bulge at the site of polar body formation unique, cytokinetic mechanisms related to those of polar (Fig. 10c)? Ring contraction appears to occur behind this bodies are utilized during embryonic development. A closer initial bulge (Figs. 2 and 4b), but such contraction can be comparative look at micromere formation or other types of effective only if occurring between two predefined masses unequal cleavage, using multiple labeling and confocal to be separated. One possibility is that the docked anaphase reconstruction, is warranted. spindle, in which interpolar microtubules are retained (Figs. 2 and 3), provides the required initial push by active Aster involvement in polar body formation elongation via microtubule motors. These microtubules correspond to those of the central spindle, numerous pro- Cytokinetic eccentricity varies from slight to extreme in teins of which have been shown to be critical for cytokinesis different animal cell types, and is typically preceded by in other model systems (Glotzer, 2003). This could account varying degrees of astral asymmetry. In nonmammalian for the slight bulge observed in blebbistatin-treated eggs, eggs and some other cell types, unequal astral growth alone since such motors would not be inhibited by the drug. appears to determine furrow position by spindle transloca- Exaggerated central spindle elongation might also be re- tion, and astral microtubules may be involved in delivery of sponsible for the taxol-induced bulge (Fig. 8). However, any signals and/or materials to establish the contractile ring and such elongation-driven mechanism would require that the furrow (Rappaport, 1986, 1996; Sanger and Sanger, 2000). spindle be held in position by physical binding to the cortex However, asters are not universal; centrioles and typical after astral microtubules have disassembled. asters are absent in mammalian eggs (Brunet et al., 1999; Mechanical anchorage of the meiotic spindle to a spe- Szollosi et al., 1972), in which peripheral spindle movement cialized cortical site has been demonstrated in Chaetopterus appears to involve actin-based motility via formin attach- oocytes (Lutz et al., 1988). There is no similar evidence in ments, not microtubules (Leader et al., 2002; Maro and Spisula, but the region of astral spreading sometimes Verlhac, 2002). Further enriching things mechanistically, retained enhanced optical clarity at later stages (e.g., DIC; spindle midzone microtubules and associated proteins have Figs. 5b and 9a, arrowheads). Organelles and inclusions are been shown to be active in cytokinetic furrow positioning, excluded from that region (Longo and Anderson, 1970), and maintenance, and function in at least some cell types in the normal sequence, the retained meiosis I centrosome (Glotzer, 2003; Wheatley and Wang, 1996). appeared to remain there for meiosis II spindle generation With respect to polar body formation and the peripheral (Fig. 4a). In addition, the peripheral aster in blebbistatin- aster, there are profound consequences even in a simple treated eggs reappeared essentially in its original location scenario in which the two meiosis I centrosomes nucleate upon meiosis II-induced reassembly (Fig. 9e), as if the non- the same number of microtubules but differ in rate of astral expelled polar body centrosome had been held there in a microtubule growth. Leading the resulting spindle translo- nonmicrotubule, non-F-actin matrix. Recent work points to cation, the peripheral aster meets the surface closer to its the existence of specialized spindle matrix proteins (e.g., centrosome, producing greater microtubule density per unit Johansen and Johansen, 2002; Scholey et al., 2001; Walker cortical area than at the internal aster periphery. Such et al., 2000) and a mitotic apparatus matrix was suggested in cortical microtubule density is a critical factor in several older work (Cohen, 1968). The hypothetical roles of spindle proposed cytokinetic mechanisms (Rappaport, 1996), per- elongation and a spindle-anchoring matrix in polar body haps involving microtubule end capture for surface delivery initiation remain to be tested. of key molecular components (Shuster and Burgess, 2002; Skop et al., 2001; Straight et al., 2003). Although astral delivery of signals and/or materials may Acknowledgments be involved in Spisula polar body formation as well, the process is not simply one of contact between microtubule We thank Drs. David Burgess, Kyeng-Gea Lee, and ends and the cortex. Rather, microtubule bending and Robert Palazzo for assistance and helpful discussion, and we spreading positions much of their length parallel to the are indebted to Louie Kerr of the MBL and Rudi Rotten- cortical surface. This suggests a dynein-based mechanism fusser of Carl Zeiss Inc. for providing the excellent confocal 432 R.M. 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