Cell Biology International 1999, Vol. 23, No. 12, 805–812doi:10/1006/cbir.1999.0477, available online at http://www.idealibrary.com on

UNILATERAL AND WANDERING FURROWS DURING MITOSIS IN VERTEBRATES:IMPLICATIONS FOR THE MECHANISM OF CYTOKINESIS

M. S. SAVOIAN1,2, A. KHODJAKOV1,2 and C. L. RIEDER1,2,3*

1Division of Molecular Medicine, Wadsworth Center, P.O. Box 509, Albany, New York 12201-0509; 2Departmentof Biomedical Sciences, State University of New York, Albany, New York 1222; 3Marine Biology Laboratory,

Woods Hole, MA 02543, U.S.A.

Received 13 March 1999; accepted 20 April 1999

Vertebrate somatic cells sometimes form unilateral furrows during cytokinesis that ingress fromonly one edge of the cell. In some cases after a cell initiates a normal symmetrical circumferentialfurrow, one of its edges stops furrowing and regresses while the furrow associated with theopposing edge continues across the cell. In cells containing two independent spindles unilateralfurrows are sometimes formed that do not follow a linear path but instead sharply change theirdirection and wander for >40 �m through the cell. These observations reveal that the‘contractile ring’ normally seen during cytokinesis is composed of multiple independent‘furrowing units’ that are normally coordinated to form a symmetrical furrow around the cell,and that once formed this so-called contractile band does not function as a ‘purse string’ ascommonly envisioned. Individual furrowing units can work independently of one another, andcytokinesis in vertebrates can be consummated by the formation of a single functional furrowingunit in a localized region of the cell cortex that is then propagated across the cell. How thispropagation occurs remains an important question for the future. � 1999 Academic Press

K: cytokinesis; vertebrates; fused-cells; furrowing unit; unilateral furrow; mitosis.

*To whom correspondence should be addressed: Dr Conly L. Rieder,Division of Molecular Medicine, Wadsworth Center, P.O. Box 509,Albany, New York, 12201-0509, U.S.A. Tel.: +1 518-474-6774; fax:+1 518-486-4901; e-mail: <Rieder@Wadsworth.org>.

INTRODUCTION

Cytokinesis in higher eukaryotes is thought to beeffected by an actin–myosin-based ‘contractile ring’that functions much like a purse string to progres-sively constrict the cell in a plane around itsequator (reviewed in Satterwhite and Pollard, 1992;Fishkind and Wang, 1993, 1995). Because this ringis positioned in the cell cortex and anchored to theinside of the plasma membrane, it pulls the mem-brane towards the center of the cell as it constricts.Over time this circumferential constriction leadsto the formation of a thin microtubule (Mt)-containing intercellular bridge known as a mid-body (Mullins and Bisele, 1977). Once formed, themid-body is then discarded after the daughter cellsbegin to migrate away from one another.

1065–6995/99/120805+08 $35.00/0

The process of cytokinesis can be temporallysubdivided into three stages. During the first stage,the site of where a furrow is to form is established,and an underlying mechanochemical apparatuscontaining actin, myosin, and other proteins isformed. In echinoderm zygotes furrow positioningappears to be established just after the chromatidsseparate to initiate anaphase, and multiple furrowscan be initiated after this time simply by movingthe spindle within the cell (e.g. Rappaport andEbstein, 1965). Although the site of cytokinesisalways corresponds to where two opposing anti-parallel arrays of Mts overlap and bundle, howthese arrays define the position of the furrowremains to be resolved. It appears to involve,however, phosphorylation of the kinesin-like Mtbundling protein MKLP-1 (e.g. Adams et al.,1998), perhaps by polokinase (Carmena et al.,1998). Through its role in positioning the spindle,the Mt minus-end-directed motor cytoplasmicdynein has also been implicated in specifying the

� 1999 Academic Press

806 Cell Biology International, Vol. 23, No. 12, 1999

plane of cytokinesis in C. elegans (Skop and White,1998). Recent work suggests that the small GTPaseRho (Drechsel et al., 1996; Madaule et al., 1998;O’Connell et al., 1999) and the actin-bindingprotein anillin (Field and Alberts, 1995) are alsoinvolved in positioning and forming the contractileapparatus.

The second stage of cytokinesis involves acti-vation of the contractile apparatus to produce aprogressive dimpling or furrowing around theplasma membrane in a plane perpendicular to theinterpolar axis of the mitotic spindle. In verte-brates this requires the degradation of CDK1 (e.g.Wheatley et al., 1997), which also leads to chroma-tid separation and anaphase onset (e.g. Clute andPines, 1999). Under normal conditions, once fur-rowing is activated it proceeds until the mid-body isformed, after which the completed furrow is stab-ilized during the third stage of cytokinesis. Anumber of proteins have recently been identified inC. elegans and other organisms that are requiredfor furrow propagation and/or stabilization. Al-though mutations in these proteins, which include,e.g. the Aurora/Ip11-related protein kinase AIR-2(Schumacher et al., 1998), the FH protein cyk-1(Swan et al., 1998), and INCENP (e.g. Eckleyet al., 1997; Mackay et al., 1998) do not inhibitfurrow formation, the furrows are not stable andultimately regress or relax. Anti-parallel bundles ofMts below the furrow, which are formed fromthe activity of a kinesin-like Mt bundling protein(human MKLP, Nislow et al., 1992; C. elegansZEN 4, Raich et al., 1998; Powers et al., 1998;Drosophila KLP3A/pavarotti, Williams et al., 1995;Adams et al., 1998), are also required for furrowpropagation and/or stabilization (Cao and Wang,1996; Wheatley and Wang, 1996; Giansanti et al.,1998).

We have recently developed a model system thatenables us to study the formation of furrows be-tween two centrosomes in vertebrate somatic cellsthat lack an intervening spindle and chromosomes.This system is based on following mitosis in fusedPtK1 cells that form two independent spindles. Inthese cells cytokinesis can occur, as in echinodermzygotes (Rappaport, 1961), not only at the spindleequator but also between the centrosomes of neigh-boring spindles (Rieder et al., 1997). An immuno-cytochemical analysis of these ‘ectopic’ furrowsreveals that they lack CENP-E, which is inevitablyfound in control furrows, but that they alwayscontain Mt bundles, INCENP, and the CHO1protein (Savoian et al., 1999), as well as anillin.During the course of these investigations we madeseveral observations relevant to the mechanism of

cytokinesis that have not been previously reported.Specifically we have documented a number ofinstances in which a furrow forms only on one sideof the cell, and then wanders randomly and exten-sively throughout the cell (sometimes over 40 �m)often making sharp turns. Similar unilateral fur-rowing has been reported during mitosis in multi-nucleated Dictyostelium cells lacking myosin II(Neujahr et al., 1998), however it has not beendescribed for vertebrate cells. Here we documentthese unusual furrows, and discuss their impli-cations for the mechanism of cytokinesis.

MATERIALS AND METHODS

Cell culture

Stock cultures of PtK1 cells were maintained in 5%CO2 in antibiotic-free Ham’s F12 supplementedwith 10% fetal calf serum (FBS). The PtK1 line wasinitially purchased from ATCC (Rockville, MD,U.S.A.) at passage number 66, and only cells atpassage numbers 70–100 were used for this study.For experiments, cells were trypsinized from stockflasks seeded into plastic petri dishes contain-ing 25�25 mm glass coverslips, and incubatedat 37�C.

Cell fusion

We electrofused PtK1 cells as previously described(Rieder et al., 1997; Savoian et al., 1999). For thisprocess subconfluent mitotically-active PtK1coverslip cultures were placed between two elec-trodes separated by �10 mm and bathed in fusionbuffer (280 m sucrose, 2 m Hepes, 1 m MgCl2,pH 6.9). A single 2 ms pulse of 350 V was appliedby a Progenitor II electroporation device (HoeferScientific Instruments, CA, U.S.A.). The cultureswere then quickly returned to conditioned Ham’sF12 media containing 10% FBS for 2 h or moreat 37�C.

Light microscopy

Coverslip cultures containing fused PtK1 cells weremounted in Rose chambers (see Rieder and Hard,1990) containing L-15 media supplemented with10% FBS, 10 m HEPES and antibiotics (100 u/mlpenicillin and 100 �g/ml streptomycin). Thesechambers were then placed on the stage of a Nikon(Melville, NY, U.S.A.) Diaphot inverted lightmicroscope (LM) and maintained at 35–37�C witha custom-built Rose chamber heater (described in

Cell Biology International, Vol. 23, No. 12, 1999 807

Rieder and Cole, 1998). Single cells containing twoseparate and independent spindles were locatedand followed from prometaphase through ana-phase by time-lapse video LM using a framing rateof 15 frames/min. Cells were illuminated with shut-tered, monochromatic (546 nm) light obtainedfrom a 50 W tungsten filament. They were viewedand followed with a 40� phase contrast (NA=0.7)objective and a 0.7 NA condenser. Images werecaptured by a DAGE MTI VE-1000 video camera,and real-time background subtraction and frameaveraging was conducted using either anARGUS-10 or a Hamamatsu C2400 image proces-sor. Processed images were then recorded on toSVHS tape using a Panasonic AG 6740 time-lapserecorder or on to optical memory disks using aPanasonic TQ 2028F recorder. Selected framesfrom the time-lapse recordings were digitized into aPC with the Scion Image (Scion Corporation, MD,U.S.A.) frame-grabbing package and processedwith Adobe Photoshop (Adobe Systems Inc., CA,U.S.A.).

Measurements

Sequential images (recorded at 4 s intervals) weredigitized into a PC with the Image 1 frame-grabbing system (Universal Imaging, Westchester,PA, U.S.A.). A stationary point in the field of viewwas used as the origin for all subsequent measure-ments. Following calibration of Image 1’s onboardmeasuring function with a slide micrometer, thepositions of two points were measured for eachfurrow; one marking the side closest to the station-ary point and the other labeling the position of theopposing side of the furrow. Over 300 measure-ments were made at each of the four points corre-sponding to the most actively moving part of thatportion of the furrow. Data points were thenentered into Microsoft Excel (Microsoft Corp.,WA, U.S.A.) and plotted.

RESULTS

PtK1 cells that contain two independent spindlesusually form two cleavage furrows, one in associ-ation with each of the two spindles, and eachfurrow usually forms and functions in a mannerconsistent with the action of a contractile ring, i.e.the cytoplasm becomes constricted progressivelyand uniformly around the circumference of the cellat the spindle equator, and in a plane perpendicularto the spindle long-axis (e.g. arrow in Fig. 1).During the furrowing process a linear phase-lucent

band is usually seen to form across the cell, in thefurrow plane, as the dorsal and ventral membranesurfaces become constricted (Fig. 1B, arrow).

In five of the 432 fused cells that entered andcompleted mitosis in the presence of two indepen-dent spindles, one of the furrows was initiated andproceeded from a single side of the cell (e.g. arrow-head in Fig. 1). Several features distinguish these‘unilateral’ furrows from the more symmetricalones that are usually formed. First, they tend to beformed in association with spindles that are posi-tioned many microns from the lateral edges of thecell. In addition, during the furrowing process thereis no evidence that the dorsal and ventral cellsurface are involved. Instead, the furrow appearson one side of the cell, and then progresses to theother side in a manner similar to the way a knifeedge under pressure cuts (e.g. arrowhead Fig. 1C–D). In some cells these unilateral furrows moved asmuch as 40 �m before contacting the other side ofthe cell.

To visualize better the differences between uni-lateral furrows and their symmetrical counterpartswe plotted, in the same cell, the progression of eachfurrow type as described in the Materials andMethods. The graph shown in Fig. 2 was generatedfrom the cell shown in Fig. 1. The two thin blacklines represent the motion of the lateral cell sur-faces towards one another in the symmetrical fur-row noted by the arrow in Fig. 1. It is evident fromthese plots that both sides of the furrow approacheach other at approximately the same rate(�1.2 �m/min), and that it took approximately10 min for the furrow to progress 20 �m (to themid-body stage). The two thick black lines simi-larly represent the motion of the unilateral furrow.From these lines it is evident that one edge of thecell remained relatively stationary (top line) whilethe other furrowing edge (bottom line) movedtowards it. When compared to the symmetricalfurrow, this unilateral furrow moved �27 �m in�20 min. This graph also reveals that the uni-lateral furrow moves at about the same rate as thesymmetrical furrow (1.9 �m/min versus �1.2 �m/min), and also that (in this case) the whole cellbegan to crawl near the 10 min mark towards thestationary reference point prior to completion ofthe unilateral cytokinesis.

So far our data suggest that unilateral furrowsare not formed from the action of contractile rings,but instead from dynamic contractile elements thatare positioned and maintained only on one side ofthe cell. That this is the case is clearly illustrated inFig. 3, which documents the formation and behav-ior of an ectopic unilateral furrow that wanders

808 Cell Biology International, Vol. 23, No. 12, 1999

many microns through the cell. Initially this furrowappeared to start on both sides of the cell betweenthe two spindles (white arrowheads in B). However,as cytokinesis progressed, one side relaxed whilethe other continued across the cell (white arrow-heads in Fig. 3B–D). Just prior to reaching theother relaxing furrow, the active furrow made a 45�turn and proceeded to cut through the cytoplasmfor another 20 �m (white arrowhead in Fig. 3D–F).

Fig. 1. (A–F) Selected frames from a time-lapse video sequence of cytokinesis in a PtK1 cell containing two independent spindles.Two independent furrows formed during late anaphase (B–C) in association with each of the two spindle midzones. One of these(black arrow) was symmetrical and effected cytokinesis by ingression from both sides of the midzone. During a symmetricalcytokinesis a phase-lucent band is usually formed in the constriction plane as the dorsal and ventral membranes approach oneanother (arrow in B). By contrast, the other furrow (black arrowhead) was asymmetric in that its activity was only evident onone side of the cell. Time in h:min:s at bottom right corner of each frame. Bar in F=10 �m.

DISCUSSION

As emphasized by Satterwhite and Pollard (1992)‘the actomyosin contractile-ring mechanism re-mains the paradigm for cytokinesis after 20 yearsof experimental testing’. In this mechanism a po-larized array of actin filaments, anchored in andpositioned around a narrow equatorial band, inter-act with myosin II filaments and other proteins toapply constant tension on the overlying membrane.This tension then progressively constricts the cell,like a purse string, to ultimately pinch it in two.

Evidence that the contractile elements respon-sible for furrow formation can be organized into aring comes from those systems in which the furrowingresses simultaneously around the entire cellperiphery. This occurs, for example, during thecleavage divisions in many types of fertilized eggsand also during cytokinesis in most animal somaticcells. As a result, with few exceptions, mostworkers model cytokinesis around the premise thatthe process is initiated through and mediated by theformation of a contractile ring (e.g. see Fishkindand Wang, 1995; Glotzer, 1998). However, thereare a number of examples in which cleavage inembryos, and even cytokinesis in somatic cells, isclearly not mediated by contractile elements organ-ized into a ring. For example, the zygotes ofcephalopods, elasmobranchs, teleosts, birds, andreptiles exhibit a partial, or meroblastic, type ofcleavage during early development in which thelower hemisphere of the egg does not divide(Wilson, 1925). In these organisms a unilateralfurrow is formed that spreads laterally across onesurface of the egg while it simultaneously ingresses

Cell Biology International, Vol. 23, No. 12, 1999 809

Fig. 2. Plot depicting progression of the two furrows shown in Fig. 1 relative to a stationary point positioned between the twofurrows and external to the cell. The thin black lines represent changes in the distance between the stationary point and both sidesof the symmetrical furrow noted by the arrow in Fig. 1. The thick black lines depict changes in distance from the same point andboth sides of the asymmetric furrow noted by the arrowhead in Fig. 1. Note that once formed, the mid-body (parallel lines near10 min time point) in the symmetric furrow slowly moved towards the stationary point. A similar motion is also seen in thestationary edge of the asymmetric furrow (top thick black line). See text for details.

to a set depth (usually to the yolk layer; e.g. seeJesuthasan, 1998). Unilateral furrows, which ulti-mately regress, are also formed during the syncytialmitoses in Drosophila zygotes (e.g. see Sullivanet al., 1990), and when cellularization finally occursit is effected by multiple unilateral furrows. Mostrecently Neujahr et al. (1998) have shown thatunilateral furrows similar to those we describe herefor PtK1 cells are routinely formed during mitosisin multinucleated myosin II null Dictyosteliumcells. As noted by these authors such furrows arenot consistent with a contractile ring model forcytokinesis.

Unilateral furrows are not an artifact of ourfused cell system—they are also seen at approxi-mately the same frequency (1%) in control PtK1cells containing a single mitotic spindle (Fig. 4).The initiation of a furrow on just one side of thecell during cytokinesis has also been documentedbut not discussed in other studies of untreated (e.g.Fig. 3A–D in Wheatley et al., 1997) or drug-treated(e.g. Fig. 7D–G in Wheatley et al., 1998; Fig. 8F–Jin O’Connell et al., 1999) tissue culture cells, andthey also form in association with the telophasedisk when HeLa cells are released from a cyto-chalasin B mediated block of cytokinesis (Fig. 10Bin Martineau et al., 1995). The formation andingression of the cleavage furrow from just the

dorsal cell surface has also been detailed in NRKcells by Fishkind and Wang (1993). In these cellsactin filaments are found in a band around theentire circumference of the cell, but more areconcentrated on the dorsal cell surface where fur-rowing does not occur. This was interpreted toindicate that unilateral furrows are formed whenthe cell cannot overcome strong cell-to-cell or cell-to-substrate adhesions at other points around itscircumference. However, the actin filaments in uni-lateral (single sided) furrows formed in HeLa cellsafter release from cytochalasin B appear to beconcentrated primarily at the unilateral furrowingsite (Martineau et al., 1995).

As noted by Fishkind and Wang (1993) theunilateral furrows seen in untreated PtK1 cellscould result from a contractile ring that is somehowinhibited from working in those regions of thecortex that do not manifest a furrow. Alternatively,as suggested by the HeLa data of Martineau et al.(1995) a unilateral furrow could also be the productof an incomplete ring (e.g. a contractile crescent).Our observation that during a symmetrical cyto-kinesis one side of the furrow can suddenly relaxand regress while the other side continues to ingressdemonstrates that there need not be a direct con-nection between two furrows working from oppos-ing sides of the cell. Furthermore, our observation

810 Cell Biology International, Vol. 23, No. 12, 1999

that a unilateral furrow can sharply change itsdirection, as in Fig. 3, clearly demonstrates that acontractile ring is not required for furrow ingres-sion. Instead, our data support the hypothesis thatcytokinesis is normally effected by the coordinationof multiple furrows that form independently in thecleavage plane, i.e. that the contractile ring seenduring cytokinesis in most organisms is composedof multiple furrowing units that are normallylinked and coordinated, but which can also workindependently of each other.

An important unresolved question is whether theunilateral furrows we observed that wander exten-sively are following a pre-determined ‘track’ orpath within the cell as they ingress, or if theypropagate through the cell by simply forming theirown track immediately in front of them. Based onthe fact that unilateral furrows in Dictyostelium canexceed the length of normal furrows, and that thecontinued presence of centrosomes (i.e. Mts) is notrequired for continued furrowing, Neujahr et al.(1998) concluded that progression of unilateralfurrows in Dictyostelium is a self-sustaining pro-cess. In their view the signals that initiate furrow

formation are not required for subsequently main-taining the furrow, which propagates itself by con-tinuously recruiting contractile elements to itsleading edge. However, these conclusions may notbe valid for other types of cells including verte-brates: numerous experimental conditions havebeen defined in worms, flies and vertebrates (seeIntroduction) in which furrows suddenly stop ad-vancing and then regress. Although the initialsignals for furrow formation may not be requiredfor furrow progression in these systems, some othercomponent(s) clearly are. Whether these compo-nents are constantly recruited to the leading edge ofthe furrow, or if they already exist in a preformedtrack that the furrow must follow, remains to beresolved.

Fig. 3. (A–F) Same conditions as in Fig. 1. In this cell a symmetrical ectopic furrow (B–C, arrowheads) formed between the topmultipolar spindle and the bottom bipolar spindle. As this furrow progressed, the cell became progressively pinched from bothsides (B–C; arrowheads). However, instead of forming a midbody the furrow on the right side of the cell relaxed (D), and theother active furrow (D–F; white arrowhead) turned �45� and began to wander over the next 14 min in an unpredictable fashionthroughout the cell. Bar in F=10 �m.

ACKNOWLEDGEMENTS

The authors thank Ms Cindy Hughes for tissueculture assistance, Mr Richard Cole for assist-ance with the microscopy, and Drs R. Sloboda(Dartmouth College), R. Palazzo (University of

Cell Biology International, Vol. 23, No. 12, 1999 811

Kansas, Lawrence) and M. Koonce (WadsworthCenter) for stimulating discussions related to cyto-kinesis. This work was supported, in part, by NIHGMS R01 40198 (to C.L.R.), and was conducted inconjunction with the Wadsworth Center’s VideoLight Microscopy Core Facility.

Fig. 4. (A–F) Cytokinesis in untreated PtK1 cells can also occur primarily through the activity of a unilateral furrow. In thisexample the plasma membrane on one side of the spindle midzone remained relatively stationary (black arrowhead in B–E) whilethe furrow approached from the other side. Bar in F=10 �m.

REFERENCES

A RR, T AAM, S A, B HJ, GDM, 1998. Pavarotti encodes a kinesin-like protein requiredto organize the central spindle and contractile ring forcytokinesis. Genes Dev 12: 1483–1494.

C L, W Y, 1996. Signals from the spindle midzone arerequired for the stimulation of cytokinesis in cultured epi-thelial cells. Mol Biol Cell 7: 225–232.

C M, R MG, M G, T AM,A R, C G, G DM, 1998. Drosophila polokinase is required for cytokinesis. J Cell Biol 143: 659–671.

C P, P J, 1999. Temporal and spatial regulation ofcyclin B1 destruction in metaphase. Nature Cell Biol 1:82–87.

D DN, H AA, H A, G M, 1996. Arequirement for Rho and cdc42 during cytokinesis in Xeno-pus embryos. Curr Biol 7: 12–23.

E DM, A AM, M A, G IG,E WC, 1997. Chromosomal proteins and cyto-kinesis: patterns of cleavage furrow formation and the

inner-centromere protein position in mitotic heterokaryonsand midanaphase cells. J Cell Biol 136: 1169–1183.

F CM, A BM, 1995. Anillin, a contractile ringprotein that cycles from the nucleus to the cell cortex. J CellBiol 131: 165–178.

F DJ, W Y, 1993. Orientation and three-dimensional organization of actin filaments in dividingcultured cells. J Cell Biol 123: 837–848.

F DJ, W Y, 1995. New horizons for cytokinesis.1995. Curr Opin Cell Biol 7: 23–31.

G MG, B S, W B, W EV,S C, G ML, G M, 1998. Co-operative interactions between the central spindle and thecontractile ring during Drosophila cytokinesis. Genes Dev 12:396–410.

G M, 1998. The mechanism and control of cytokinesis.Curr Opin Cell Biol 9: 815–823.

J S, 1998. Furrow-associated microtubule arrays arerequired for the cohesion of zebrafish blastomeres followingcytokinesis. J Cell Sci 111: 3695–3703.

M AM, A AM, E DM, E WC,1998. A dominant mutant of Inner Centromere Protein(INCENP), a chromosomal protein, disrupts prometaphasecongression and cytokinesis. J Cell Biol 140: 991–1002.

M P, E M, W N, F K, MT, B H, I T, N S, 1998. Role of citronkinase as a target of the small GTPase Rho in cytokinesis.Nature 394: 491–494.

M S, A PR, M RL, 1995. Delay ofHeLa cell cleavage into interphase using dihydrocyto-chalasin B: retention of a postmitotic spindle and telophase

812 Cell Biology International, Vol. 23, No. 12, 1999

disc correlates with synchronous cleavage recovery. J CellBiol 131: 191–205.

M JM, B JJ, 1977. Terminal phase of cytokinesisin D-98S cells. J Cell Biol 73: 672–684.

N R, A R, K J, M M, SJM, W M, G G, 1998. Microtubule-mediatedcentrosome motility and the positioning of cleavage furrowsin multinucleate myosin II-null cells. J Cell Sci 111: 1227–1240.

N C, L VA, K R, MI J, 1992.A plus-end-directed motor enzyme that moves antiparallelmicrotubules in vitro localizes to the interzone of mitoticspindles. Nature 359: 543–547.

O’C CB, W SP, A S, W YL, 1999.The small GTP-binding protein Rho regulates cortical ac-tivities in culture cells during division. J Cell Biol 144:305–313.

P J, B O, R D, S S, S W, 1998.A nematode kinesin required for cleavage furrow advance-ment. Curr Biol 8: 1133–1136.

R WB, M AN, R JH, H J, 1998.Cytokinesis and midzone microtubule organization inCaenorhabditis elegans require the kinesin-like proteinZEN-4. Mol Biol Cell 9: 2037–2049.

R R, 1961. Experiments concerning the cleavagestimulus in sand dollar eggs. J Exp Zool 148: 81–89.

R R, E RP, 1965. Duration of stimulus andlatent periods preceding furrow formation in sand dollareggs. J Exp Zool 158: 373–382.

R CL, H R, 1990. Newt lung epithelial cells: culti-vation, use and advantages for biomedical research. Int RevCytol 122: 153–220.

R CL, K A, P LV, C RW, SG, 1997. Mitosis in vertebrate somatic cells with two spin-dles: Implications for the metaphase/anaphase transitioncheckpoint and cleavage. Proc Natl Acad Sci USA 94:5107–5112.

R CL, C R, 1998. Perfusion chambers for high-resolution video-light microscopic studies of vertebrate cellmonolayers: some considerations and a design. Meth CellBiol 56: 253–275.

S LL, P TD, 1992. Cytokinesis. Curr OpinCell Biol 4: 43–52.

S M, E WC, K A, R CL,1999. Cleavage furrows formed between centrosomes lack-ing an intervening spindle and chromosomes contain micro-tubule bundles, INCENP, and CHO1 but not CENP-E. MolBiol Cell 10: 297–311.

S JM, G A, D PJ, 1998. AIR-2: anaurora/Ip11-related protein kinase associated with chromo-somes and midbody microtubules is required for polarbody extrusion and cytokinesis in Caernorhabditis elegansembryos. J Cell Biol 143: 1635–1646.

S AR, W JG, 1998. The dynactin complex is requiredfor cleavage plane specification in early Caenoirhabditiselegans embryos. Curr Biol 8: 1110–1116.

S W, M JS, A BM, 1990. Daughterless-abo-like, a Drosophila maternal-effect mutation that exhibitsabnormal centrosome separation during the late blastodermdivisions. Development 110: 311–323.

S KA, S AF, C JC, M PR, SH, S R, B B, 1998. Cyk-1: a C. elegansFH gene required for a late step in embryonic cytokinesis.J Cell Sci 111: 2017–2027.

W SP, W Y, 1996. Midzone microtubules arecontinuously required for cytokinesis in cultured epithelialcells. J Cell Biol 135: 981–989.

W SP, H EH, G M, H AA,S S, W Y, 1997. CDK1 inactivation regulatesanaphase spindle dynamics and cytokinesis in vivo. J CellBiol 138: 385–393.

W SP, O’C CB, W Y, 1998. Inhibition ofchromosomal separation provides insights into cleavagefurrow stimulation in cultured epithelial cells. Mol Biol Cell9: 2173–2184.

W BC, R MF, W EV, G M, GML, 1995. The Drosophila kinesin-like protein KLP3A is amidbody component required for central spindle assemblyand initiation of cytokinesis. J Cell Biol 129: 709–723.

W EB, 1925. The Cell in Development and Heredity. 3rdEdition. New York, NH, MacMillan Co.