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UNILATERAL AND WANDERING FURROWS DURING MITOSIS IN VERTEBRATES: IMPLICATIONS FOR THE MECHANISM OF CYTOKINESIS
Cell Biology International 1999, Vol. 23, No. 12, 805–812 doi: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* 1
Division of Molecular Medicine, Wadsworth Center, P.O. Box 509, Albany, New York 12201-0509; 2Department of 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 from only one edge of the cell. In some cases after a cell initiates a normal symmetrical circumferential furrow, one of its edges stops furrowing and regresses while the furrow associated with the opposing edge continues across the cell. In cells containing two independent spindles unilateral furrows are sometimes formed that do not follow a linear path but instead sharply change their direction 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’ as commonly envisioned. Individual furrowing units can work independently of one another, and cytokinesis in vertebrates can be consummated by the formation of a single functional furrowing unit in a localized region of the cell cortex that is then propagated across the cell. How this 1999 Academic Press propagation occurs remains an important question for the future. K: cytokinesis; vertebrates; fused-cells; furrowing unit; unilateral furrow; mitosis.
INTRODUCTION Cytokinesis in higher eukaryotes is thought to be effected by an actin–myosin-based ‘contractile ring’ that functions much like a purse string to progressively constrict the cell in a plane around its equator (reviewed in Satterwhite and Pollard, 1992; Fishkind and Wang, 1993, 1995). Because this ring is positioned in the cell cortex and anchored to the inside of the plasma membrane, it pulls the membrane towards the center of the cell as it constricts. Over time this circumferential constriction leads to the formation of a thin microtubule (Mt)containing intercellular bridge known as a midbody (Mullins and Bisele, 1977). Once formed, the mid-body is then discarded after the daughter cells begin to migrate away from one another. *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:. 1065–6995/99/120805+08 $35.00/0
The process of cytokinesis can be temporally subdivided into three stages. During the first stage, the site of where a furrow is to form is established, and an underlying mechanochemical apparatus containing actin, myosin, and other proteins is formed. In echinoderm zygotes furrow positioning appears to be established just after the chromatids separate to initiate anaphase, and multiple furrows can be initiated after this time simply by moving the spindle within the cell (e.g. Rappaport and Ebstein, 1965). Although the site of cytokinesis always corresponds to where two opposing antiparallel arrays of Mts overlap and bundle, how these arrays define the position of the furrow remains to be resolved. It appears to involve, however, phosphorylation of the kinesin-like Mt bundling 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 cytoplasmic dynein has also been implicated in specifying the 1999 Academic Press
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plane of cytokinesis in C. elegans (Skop and White, 1998). Recent work suggests that the small GTPase Rho (Drechsel et al., 1996; Madaule et al., 1998; O’Connell et al., 1999) and the actin-binding protein anillin (Field and Alberts, 1995) are also involved in positioning and forming the contractile apparatus. The second stage of cytokinesis involves activation of the contractile apparatus to produce a progressive dimpling or furrowing around the plasma membrane in a plane perpendicular to the interpolar axis of the mitotic spindle. In vertebrates this requires the degradation of CDK1 (e.g. Wheatley et al., 1997), which also leads to chromatid separation and anaphase onset (e.g. Clute and Pines, 1999). Under normal conditions, once furrowing is activated it proceeds until the mid-body is formed, after which the completed furrow is stabilized during the third stage of cytokinesis. A number of proteins have recently been identified in C. elegans and other organisms that are required for furrow propagation and/or stabilization. Although 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. Eckley et al., 1997; Mackay et al., 1998) do not inhibit furrow formation, the furrows are not stable and ultimately regress or relax. Anti-parallel bundles of Mts below the furrow, which are formed from the activity of a kinesin-like Mt bundling protein (human MKLP, Nislow et al., 1992; C. elegans ZEN 4, Raich et al., 1998; Powers et al., 1998; Drosophila KLP3A/pavarotti, Williams et al., 1995; Adams et al., 1998), are also required for furrow propagation and/or stabilization (Cao and Wang, 1996; Wheatley and Wang, 1996; Giansanti et al., 1998). We have recently developed a model system that enables us to study the formation of furrows between two centrosomes in vertebrate somatic cells that lack an intervening spindle and chromosomes. This system is based on following mitosis in fused PtK1 cells that form two independent spindles. In these cells cytokinesis can occur, as in echinoderm zygotes (Rappaport, 1961), not only at the spindle equator but also between the centrosomes of neighboring spindles (Rieder et al., 1997). An immunocytochemical analysis of these ‘ectopic’ furrows reveals that they lack CENP-E, which is inevitably found in control furrows, but that they always contain Mt bundles, INCENP, and the CHO1 protein (Savoian et al., 1999), as well as anillin. During the course of these investigations we made several observations relevant to the mechanism of
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cytokinesis that have not been previously reported. Specifically we have documented a number of instances in which a furrow forms only on one side of the cell, and then wanders randomly and extensively throughout the cell (sometimes over 40 m) often making sharp turns. Similar unilateral furrowing has been reported during mitosis in multinucleated Dictyostelium cells lacking myosin II (Neujahr et al., 1998), however it has not been described for vertebrate cells. Here we document these unusual furrows, and discuss their implications 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 supplemented with 10% fetal calf serum (FBS). The PtK1 line was initially purchased from ATCC (Rockville, MD, U.S.A.) at passage number 66, and only cells at passage numbers 70–100 were used for this study. For experiments, cells were trypsinized from stock flasks seeded into plastic petri dishes containing 2525 mm glass coverslips, and incubated at 37C. Cell fusion We electrofused PtK1 cells as previously described (Rieder et al., 1997; Savoian et al., 1999). For this process subconfluent mitotically-active PtK1 coverslip cultures were placed between two electrodes separated by 10 mm and bathed in fusion buffer (280 m sucrose, 2 m Hepes, 1 m MgCl2, pH 6.9). A single 2 ms pulse of 350 V was applied by a Progenitor II electroporation device (Hoefer Scientific Instruments, CA, U.S.A.). The cultures were then quickly returned to conditioned Ham’s F12 media containing 10% FBS for 2 h or more at 37C. Light microscopy Coverslip cultures containing fused PtK1 cells were mounted in Rose chambers (see Rieder and Hard, 1990) containing L-15 media supplemented with 10% FBS, 10 m HEPES and antibiotics (100 u/ml penicillin and 100 g/ml streptomycin). These chambers were then placed on the stage of a Nikon (Melville, NY, U.S.A.) Diaphot inverted light microscope (LM) and maintained at 35–37C with a custom-built Rose chamber heater (described in
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Rieder and Cole, 1998). Single cells containing two separate and independent spindles were located and followed from prometaphase through anaphase by time-lapse video LM using a framing rate of 15 frames/min. Cells were illuminated with shuttered, monochromatic (546 nm) light obtained from a 50 W tungsten filament. They were viewed and followed with a 40 phase contrast (NA=0.7) objective and a 0.7 NA condenser. Images were captured by a DAGE MTI VE-1000 video camera, and real-time background subtraction and frame averaging was conducted using either an ARGUS-10 or a Hamamatsu C2400 image processor. Processed images were then recorded on to SVHS tape using a Panasonic AG 6740 time-lapse recorder or on to optical memory disks using a Panasonic TQ 2028F recorder. Selected frames from the time-lapse recordings were digitized into a PC with the Scion Image (Scion Corporation, MD, U.S.A.) frame-grabbing package and processed with Adobe Photoshop (Adobe Systems Inc., CA, U.S.A.). Measurements Sequential images (recorded at 4 s intervals) were digitized into a PC with the Image 1 framegrabbing system (Universal Imaging, Westchester, PA, U.S.A.). A stationary point in the field of view was used as the origin for all subsequent measurements. Following calibration of Image 1’s onboard measuring function with a slide micrometer, the positions of two points were measured for each furrow; one marking the side closest to the stationary point and the other labeling the position of the opposing side of the furrow. Over 300 measurements were made at each of the four points corresponding to the most actively moving part of that portion of the furrow. Data points were then entered into Microsoft Excel (Microsoft Corp., WA, U.S.A.) and plotted. RESULTS PtK1 cells that contain two independent spindles usually form two cleavage furrows, one in association with each of the two spindles, and each furrow usually forms and functions in a manner consistent with the action of a contractile ring, i.e. the cytoplasm becomes constricted progressively and uniformly around the circumference of the cell at the spindle equator, and in a plane perpendicular to the spindle long-axis (e.g. arrow in Fig. 1). During the furrowing process a linear phase-lucent
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band is usually seen to form across the cell, in the furrow plane, as the dorsal and ventral membrane surfaces become constricted (Fig. 1B, arrow). In five of the 432 fused cells that entered and completed mitosis in the presence of two independent spindles, one of the furrows was initiated and proceeded from a single side of the cell (e.g. arrowhead in Fig. 1). Several features distinguish these ‘unilateral’ furrows from the more symmetrical ones that are usually formed. First, they tend to be formed in association with spindles that are positioned many microns from the lateral edges of the cell. In addition, during the furrowing process there is no evidence that the dorsal and ventral cell surface are involved. Instead, the furrow appears on one side of the cell, and then progresses to the other side in a manner similar to the way a knife edge under pressure cuts (e.g. arrowhead Fig. 1C– D). In some cells these unilateral furrows moved as much as 40 m before contacting the other side of the cell. To visualize better the differences between unilateral furrows and their symmetrical counterparts we plotted, in the same cell, the progression of each furrow type as described in the Materials and Methods. The graph shown in Fig. 2 was generated from the cell shown in Fig. 1. The two thin black lines represent the motion of the lateral cell surfaces towards one another in the symmetrical furrow noted by the arrow in Fig. 1. It is evident from these plots that both sides of the furrow approach each other at approximately the same rate (1.2 m/min), and that it took approximately 10 min for the furrow to progress 20 m (to the mid-body stage). The two thick black lines similarly represent the motion of the unilateral furrow. From these lines it is evident that one edge of the cell remained relatively stationary (top line) while the other furrowing edge (bottom line) moved towards it. When compared to the symmetrical furrow, this unilateral furrow moved 27 m in 20 min. This graph also reveals that the unilateral furrow moves at about the same rate as the symmetrical furrow (1.9 m/min versus 1.2 m/ min), and also that (in this case) the whole cell began to crawl near the 10 min mark towards the stationary reference point prior to completion of the unilateral cytokinesis. So far our data suggest that unilateral furrows are not formed from the action of contractile rings, but instead from dynamic contractile elements that are positioned and maintained only on one side of the cell. That this is the case is clearly illustrated in Fig. 3, which documents the formation and behavior of an ectopic unilateral furrow that wanders
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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 symmetrical cytokinesis a phase-lucent band is usually formed in the constriction plane as the dorsal and ventral membranes approach one another (arrow in B). By contrast, the other furrow (black arrowhead) was asymmetric in that its activity was only evident on one side of the cell. Time in h:min:s at bottom right corner of each frame. Bar in F=10 m.
many microns through the cell. Initially this furrow appeared to start on both sides of the cell between the two spindles (white arrowheads in B). However, as cytokinesis progressed, one side relaxed while the other continued across the cell (white arrowheads in Fig. 3B–D). Just prior to reaching the other relaxing furrow, the active furrow made a 45 turn and proceeded to cut through the cytoplasm for another 20 m (white arrowhead in Fig. 3D–F). DISCUSSION As emphasized by Satterwhite and Pollard (1992) ‘the actomyosin contractile-ring mechanism remains the paradigm for cytokinesis after 20 years of experimental testing’. In this mechanism a polarized array of actin filaments, anchored in and positioned around a narrow equatorial band, interact with myosin II filaments and other proteins to apply 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 responsible for furrow formation can be organized into a ring comes from those systems in which the furrow ingresses simultaneously around the entire cell periphery. This occurs, for example, during the cleavage divisions in many types of fertilized eggs and also during cytokinesis in most animal somatic cells. As a result, with few exceptions, most workers model cytokinesis around the premise that the process is initiated through and mediated by the formation of a contractile ring (e.g. see Fishkind and Wang, 1995; Glotzer, 1998). However, there are a number of examples in which cleavage in embryos, and even cytokinesis in somatic cells, is clearly not mediated by contractile elements organized into a ring. For example, the zygotes of cephalopods, elasmobranchs, teleosts, birds, and reptiles exhibit a partial, or meroblastic, type of cleavage during early development in which the lower hemisphere of the egg does not divide (Wilson, 1925). In these organisms a unilateral furrow is formed that spreads laterally across one surface of the egg while it simultaneously ingresses
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Fig. 2. Plot depicting progression of the two furrows shown in Fig. 1 relative to a stationary point positioned between the two furrows and external to the cell. The thin black lines represent changes in the distance between the stationary point and both sides of the symmetrical furrow noted by the arrow in Fig. 1. The thick black lines depict changes in distance from the same point and both sides of the asymmetric furrow noted by the arrowhead in Fig. 1. Note that once formed, the mid-body (parallel lines near 10 min time point) in the symmetric furrow slowly moved towards the stationary point. A similar motion is also seen in the stationary edge of the asymmetric furrow (top thick black line). See text for details.
to a set depth (usually to the yolk layer; e.g. see Jesuthasan, 1998). Unilateral furrows, which ultimately regress, are also formed during the syncytial mitoses in Drosophila zygotes (e.g. see Sullivan et al., 1990), and when cellularization finally occurs it is effected by multiple unilateral furrows. Most recently Neujahr et al. (1998) have shown that unilateral furrows similar to those we describe here for PtK1 cells are routinely formed during mitosis in multinucleated myosin II null Dictyostelium cells. As noted by these authors such furrows are not consistent with a contractile ring model for cytokinesis. Unilateral furrows are not an artifact of our fused cell system—they are also seen at approximately the same frequency (1%) in control PtK1 cells containing a single mitotic spindle (Fig. 4). The initiation of a furrow on just one side of the cell during cytokinesis has also been documented but 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–J in O’Connell et al., 1999) tissue culture cells, and they also form in association with the telophase disk when HeLa cells are released from a cytochalasin B mediated block of cytokinesis (Fig. 10B in Martineau et al., 1995). The formation and ingression of the cleavage furrow from just the
dorsal cell surface has also been detailed in NRK cells by Fishkind and Wang (1993). In these cells actin filaments are found in a band around the entire circumference of the cell, but more are concentrated on the dorsal cell surface where furrowing does not occur. This was interpreted to indicate that unilateral furrows are formed when the cell cannot overcome strong cell-to-cell or cellto-substrate adhesions at other points around its circumference. However, the actin filaments in unilateral (single sided) furrows formed in HeLa cells after release from cytochalasin B appear to be concentrated primarily at the unilateral furrowing site (Martineau et al., 1995). As noted by Fishkind and Wang (1993) the unilateral furrows seen in untreated PtK1 cells could result from a contractile ring that is somehow inhibited from working in those regions of the cortex 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 product of an incomplete ring (e.g. a contractile crescent). Our observation that during a symmetrical cytokinesis one side of the furrow can suddenly relax and regress while the other side continues to ingress demonstrates that there need not be a direct connection between two furrows working from opposing sides of the cell. Furthermore, our observation
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Fig. 3. (A–F) Same conditions as in Fig. 1. In this cell a symmetrical ectopic furrow (B–C, arrowheads) formed between the top multipolar spindle and the bottom bipolar spindle. As this furrow progressed, the cell became progressively pinched from both sides (B–C; arrowheads). However, instead of forming a midbody the furrow on the right side of the cell relaxed (D), and the other active furrow (D–F; white arrowhead) turned 45 and began to wander over the next 14 min in an unpredictable fashion throughout the cell. Bar in F=10 m.
that a unilateral furrow can sharply change its direction, as in Fig. 3, clearly demonstrates that a contractile ring is not required for furrow ingression. Instead, our data support the hypothesis that cytokinesis is normally effected by the coordination of multiple furrows that form independently in the cleavage plane, i.e. that the contractile ring seen during cytokinesis in most organisms is composed of multiple furrowing units that are normally linked and coordinated, but which can also work independently of each other. An important unresolved question is whether the unilateral furrows we observed that wander extensively are following a pre-determined ‘track’ or path within the cell as they ingress, or if they propagate through the cell by simply forming their own track immediately in front of them. Based on the fact that unilateral furrows in Dictyostelium can exceed the length of normal furrows, and that the continued presence of centrosomes (i.e. Mts) is not required for continued furrowing, Neujahr et al. (1998) concluded that progression of unilateral furrows in Dictyostelium is a self-sustaining process. In their view the signals that initiate furrow
formation are not required for subsequently maintaining the furrow, which propagates itself by continuously recruiting contractile elements to its leading edge. However, these conclusions may not be valid for other types of cells including vertebrates: numerous experimental conditions have been defined in worms, flies and vertebrates (see Introduction) in which furrows suddenly stop advancing and then regress. Although the initial signals for furrow formation may not be required for furrow progression in these systems, some other component(s) clearly are. Whether these components are constantly recruited to the leading edge of the furrow, or if they already exist in a preformed track that the furrow must follow, remains to be resolved. ACKNOWLEDGEMENTS The authors thank Ms Cindy Hughes for tissue culture assistance, Mr Richard Cole for assistance with the microscopy, and Drs R. Sloboda (Dartmouth College), R. Palazzo (University of
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Fig. 4. (A–F) Cytokinesis in untreated PtK1 cells can also occur primarily through the activity of a unilateral furrow. In this example the plasma membrane on one side of the spindle midzone remained relatively stationary (black arrowhead in B–E) while the furrow approached from the other side. Bar in F=10 m.
Kansas, Lawrence) and M. Koonce (Wadsworth Center) for stimulating discussions related to cytokinesis. This work was supported, in part, by NIH GMS R01 40198 (to C.L.R.), and was conducted in conjunction with the Wadsworth Center’s Video Light Microscopy Core Facility. REFERENCES A RR, T AAM, S A, B HJ, G DM, 1998. Pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes Dev 12: 1483–1494. C L, W Y, 1996. Signals from the spindle midzone are required for the stimulation of cytokinesis in cultured epithelial cells. Mol Biol Cell 7: 225–232. C M, R MG, M G, T AM, A R, C G, G DM, 1998. Drosophila polo kinase is required for cytokinesis. J Cell Biol 143: 659–671. C P, P J, 1999. Temporal and spatial regulation of cyclin B1 destruction in metaphase. Nature Cell Biol 1: 82–87. D DN, H AA, H A, G M, 1996. A requirement for Rho and cdc42 during cytokinesis in Xenopus embryos. Curr Biol 7: 12–23. E DM, A AM, M A, G IG, E WC, 1997. Chromosomal proteins and cytokinesis: patterns of cleavage furrow formation and the
inner-centromere protein position in mitotic heterokaryons and midanaphase cells. J Cell Biol 136: 1169–1183. F CM, A BM, 1995. Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex. J Cell Biol 131: 165–178. F DJ, W Y, 1993. Orientation and threedimensional organization of actin filaments in dividing cultured 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. Cooperative interactions between the central spindle and the contractile 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 are required for the cohesion of zebrafish blastomeres following cytokinesis. 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 prometaphase congression and cytokinesis. J Cell Biol 140: 991–1002. M P, E M, W N, F K, M T, B H, I T, N S, 1998. Role of citron kinase as a target of the small GTPase Rho in cytokinesis. Nature 394: 491–494. M S, A PR, M RL, 1995. Delay of HeLa cell cleavage into interphase using dihydrocytochalasin B: retention of a postmitotic spindle and telophase
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disc correlates with synchronous cleavage recovery. J Cell Biol 131: 191–205. M JM, B JJ, 1977. Terminal phase of cytokinesis in D-98S cells. J Cell Biol 73: 672–684. N R, A R, K J, M M, S JM, W M, G G, 1998. Microtubule-mediated centrosome motility and the positioning of cleavage furrows in 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 antiparallel microtubules in vitro localizes to the interzone of mitotic spindles. Nature 359: 543–547. O’C CB, W SP, A S, W YL, 1999. The small GTP-binding protein Rho regulates cortical activities 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 advancement. Curr Biol 8: 1133–1136. R WB, M AN, R JH, H J, 1998. Cytokinesis and midzone microtubule organization in Caenorhabditis elegans require the kinesin-like protein ZEN-4. Mol Biol Cell 9: 2037–2049. R R, 1961. Experiments concerning the cleavage stimulus in sand dollar eggs. J Exp Zool 148: 81–89. R R, E RP, 1965. Duration of stimulus and latent periods preceding furrow formation in sand dollar eggs. J Exp Zool 158: 373–382. R CL, H R, 1990. Newt lung epithelial cells: cultivation, use and advantages for biomedical research. Int Rev Cytol 122: 153–220. R CL, K A, P LV, C RW, S G, 1997. Mitosis in vertebrate somatic cells with two spindles: Implications for the metaphase/anaphase transition checkpoint and cleavage. Proc Natl Acad Sci USA 94: 5107–5112. R CL, C R, 1998. Perfusion chambers for highresolution video-light microscopic studies of vertebrate cell monolayers: some considerations and a design. Meth Cell Biol 56: 253–275.
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S LL, P TD, 1992. Cytokinesis. Curr Opin Cell Biol 4: 43–52. S M, E WC, K A, R CL, 1999. Cleavage furrows formed between centrosomes lacking an intervening spindle and chromosomes contain microtubule bundles, INCENP, and CHO1 but not CENP-E. Mol Biol Cell 10: 297–311. S JM, G A, D PJ, 1998. AIR-2: an aurora/Ip11-related protein kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caernorhabditis elegans embryos. J Cell Biol 143: 1635–1646. S AR, W JG, 1998. The dynactin complex is required for cleavage plane specification in early Caenoirhabditis elegans embryos. Curr Biol 8: 1110–1116. S W, M JS, A BM, 1990. Daughterlessabo-like, a Drosophila maternal-effect mutation that exhibits abnormal centrosome separation during the late blastoderm divisions. Development 110: 311–323. S KA, S AF, C JC, M PR, S H, S R, B B, 1998. Cyk-1: a C. elegans FH gene required for a late step in embryonic cytokinesis. J Cell Sci 111: 2017–2027. W SP, W Y, 1996. Midzone microtubules are continuously required for cytokinesis in cultured epithelial cells. J Cell Biol 135: 981–989. W SP, H EH, G M, H AA, S S, W Y, 1997. CDK1 inactivation regulates anaphase spindle dynamics and cytokinesis in vivo. J Cell Biol 138: 385–393. W SP, O’C CB, W Y, 1998. Inhibition of chromosomal separation provides insights into cleavage furrow stimulation in cultured epithelial cells. Mol Biol Cell 9: 2173–2184. W BC, R MF, W EV, G M, G ML, 1995. The Drosophila kinesin-like protein KLP3A is a midbody component required for central spindle assembly and initiation of cytokinesis. J Cell Biol 129: 709–723. W EB, 1925. The Cell in Development and Heredity. 3rd Edition. New York, NH, MacMillan Co.
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* 1
Division of Molecular Medicine, Wadsworth Center, P.O. Box 509, Albany, New York 12201-0509; 2Department of 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 from only one edge of the cell. In some cases after a cell initiates a normal symmetrical circumferential furrow, one of its edges stops furrowing and regresses while the furrow associated with the opposing edge continues across the cell. In cells containing two independent spindles unilateral furrows are sometimes formed that do not follow a linear path but instead sharply change their direction 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’ as commonly envisioned. Individual furrowing units can work independently of one another, and cytokinesis in vertebrates can be consummated by the formation of a single functional furrowing unit in a localized region of the cell cortex that is then propagated across the cell. How this 1999 Academic Press propagation occurs remains an important question for the future. K: cytokinesis; vertebrates; fused-cells; furrowing unit; unilateral furrow; mitosis.
INTRODUCTION Cytokinesis in higher eukaryotes is thought to be effected by an actin–myosin-based ‘contractile ring’ that functions much like a purse string to progressively constrict the cell in a plane around its equator (reviewed in Satterwhite and Pollard, 1992; Fishkind and Wang, 1993, 1995). Because this ring is positioned in the cell cortex and anchored to the inside of the plasma membrane, it pulls the membrane towards the center of the cell as it constricts. Over time this circumferential constriction leads to the formation of a thin microtubule (Mt)containing intercellular bridge known as a midbody (Mullins and Bisele, 1977). Once formed, the mid-body is then discarded after the daughter cells begin to migrate away from one another. *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:
The process of cytokinesis can be temporally subdivided into three stages. During the first stage, the site of where a furrow is to form is established, and an underlying mechanochemical apparatus containing actin, myosin, and other proteins is formed. In echinoderm zygotes furrow positioning appears to be established just after the chromatids separate to initiate anaphase, and multiple furrows can be initiated after this time simply by moving the spindle within the cell (e.g. Rappaport and Ebstein, 1965). Although the site of cytokinesis always corresponds to where two opposing antiparallel arrays of Mts overlap and bundle, how these arrays define the position of the furrow remains to be resolved. It appears to involve, however, phosphorylation of the kinesin-like Mt bundling 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 cytoplasmic dynein has also been implicated in specifying the 1999 Academic Press
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plane of cytokinesis in C. elegans (Skop and White, 1998). Recent work suggests that the small GTPase Rho (Drechsel et al., 1996; Madaule et al., 1998; O’Connell et al., 1999) and the actin-binding protein anillin (Field and Alberts, 1995) are also involved in positioning and forming the contractile apparatus. The second stage of cytokinesis involves activation of the contractile apparatus to produce a progressive dimpling or furrowing around the plasma membrane in a plane perpendicular to the interpolar axis of the mitotic spindle. In vertebrates this requires the degradation of CDK1 (e.g. Wheatley et al., 1997), which also leads to chromatid separation and anaphase onset (e.g. Clute and Pines, 1999). Under normal conditions, once furrowing is activated it proceeds until the mid-body is formed, after which the completed furrow is stabilized during the third stage of cytokinesis. A number of proteins have recently been identified in C. elegans and other organisms that are required for furrow propagation and/or stabilization. Although 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. Eckley et al., 1997; Mackay et al., 1998) do not inhibit furrow formation, the furrows are not stable and ultimately regress or relax. Anti-parallel bundles of Mts below the furrow, which are formed from the activity of a kinesin-like Mt bundling protein (human MKLP, Nislow et al., 1992; C. elegans ZEN 4, Raich et al., 1998; Powers et al., 1998; Drosophila KLP3A/pavarotti, Williams et al., 1995; Adams et al., 1998), are also required for furrow propagation and/or stabilization (Cao and Wang, 1996; Wheatley and Wang, 1996; Giansanti et al., 1998). We have recently developed a model system that enables us to study the formation of furrows between two centrosomes in vertebrate somatic cells that lack an intervening spindle and chromosomes. This system is based on following mitosis in fused PtK1 cells that form two independent spindles. In these cells cytokinesis can occur, as in echinoderm zygotes (Rappaport, 1961), not only at the spindle equator but also between the centrosomes of neighboring spindles (Rieder et al., 1997). An immunocytochemical analysis of these ‘ectopic’ furrows reveals that they lack CENP-E, which is inevitably found in control furrows, but that they always contain Mt bundles, INCENP, and the CHO1 protein (Savoian et al., 1999), as well as anillin. During the course of these investigations we made several observations relevant to the mechanism of
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cytokinesis that have not been previously reported. Specifically we have documented a number of instances in which a furrow forms only on one side of the cell, and then wanders randomly and extensively throughout the cell (sometimes over 40 m) often making sharp turns. Similar unilateral furrowing has been reported during mitosis in multinucleated Dictyostelium cells lacking myosin II (Neujahr et al., 1998), however it has not been described for vertebrate cells. Here we document these unusual furrows, and discuss their implications 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 supplemented with 10% fetal calf serum (FBS). The PtK1 line was initially purchased from ATCC (Rockville, MD, U.S.A.) at passage number 66, and only cells at passage numbers 70–100 were used for this study. For experiments, cells were trypsinized from stock flasks seeded into plastic petri dishes containing 2525 mm glass coverslips, and incubated at 37C. Cell fusion We electrofused PtK1 cells as previously described (Rieder et al., 1997; Savoian et al., 1999). For this process subconfluent mitotically-active PtK1 coverslip cultures were placed between two electrodes separated by 10 mm and bathed in fusion buffer (280 m sucrose, 2 m Hepes, 1 m MgCl2, pH 6.9). A single 2 ms pulse of 350 V was applied by a Progenitor II electroporation device (Hoefer Scientific Instruments, CA, U.S.A.). The cultures were then quickly returned to conditioned Ham’s F12 media containing 10% FBS for 2 h or more at 37C. Light microscopy Coverslip cultures containing fused PtK1 cells were mounted in Rose chambers (see Rieder and Hard, 1990) containing L-15 media supplemented with 10% FBS, 10 m HEPES and antibiotics (100 u/ml penicillin and 100 g/ml streptomycin). These chambers were then placed on the stage of a Nikon (Melville, NY, U.S.A.) Diaphot inverted light microscope (LM) and maintained at 35–37C with a custom-built Rose chamber heater (described in
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Rieder and Cole, 1998). Single cells containing two separate and independent spindles were located and followed from prometaphase through anaphase by time-lapse video LM using a framing rate of 15 frames/min. Cells were illuminated with shuttered, monochromatic (546 nm) light obtained from a 50 W tungsten filament. They were viewed and followed with a 40 phase contrast (NA=0.7) objective and a 0.7 NA condenser. Images were captured by a DAGE MTI VE-1000 video camera, and real-time background subtraction and frame averaging was conducted using either an ARGUS-10 or a Hamamatsu C2400 image processor. Processed images were then recorded on to SVHS tape using a Panasonic AG 6740 time-lapse recorder or on to optical memory disks using a Panasonic TQ 2028F recorder. Selected frames from the time-lapse recordings were digitized into a PC with the Scion Image (Scion Corporation, MD, U.S.A.) frame-grabbing package and processed with Adobe Photoshop (Adobe Systems Inc., CA, U.S.A.). Measurements Sequential images (recorded at 4 s intervals) were digitized into a PC with the Image 1 framegrabbing system (Universal Imaging, Westchester, PA, U.S.A.). A stationary point in the field of view was used as the origin for all subsequent measurements. Following calibration of Image 1’s onboard measuring function with a slide micrometer, the positions of two points were measured for each furrow; one marking the side closest to the stationary point and the other labeling the position of the opposing side of the furrow. Over 300 measurements were made at each of the four points corresponding to the most actively moving part of that portion of the furrow. Data points were then entered into Microsoft Excel (Microsoft Corp., WA, U.S.A.) and plotted. RESULTS PtK1 cells that contain two independent spindles usually form two cleavage furrows, one in association with each of the two spindles, and each furrow usually forms and functions in a manner consistent with the action of a contractile ring, i.e. the cytoplasm becomes constricted progressively and uniformly around the circumference of the cell at the spindle equator, and in a plane perpendicular to the spindle long-axis (e.g. arrow in Fig. 1). During the furrowing process a linear phase-lucent
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band is usually seen to form across the cell, in the furrow plane, as the dorsal and ventral membrane surfaces become constricted (Fig. 1B, arrow). In five of the 432 fused cells that entered and completed mitosis in the presence of two independent spindles, one of the furrows was initiated and proceeded from a single side of the cell (e.g. arrowhead in Fig. 1). Several features distinguish these ‘unilateral’ furrows from the more symmetrical ones that are usually formed. First, they tend to be formed in association with spindles that are positioned many microns from the lateral edges of the cell. In addition, during the furrowing process there is no evidence that the dorsal and ventral cell surface are involved. Instead, the furrow appears on one side of the cell, and then progresses to the other side in a manner similar to the way a knife edge under pressure cuts (e.g. arrowhead Fig. 1C– D). In some cells these unilateral furrows moved as much as 40 m before contacting the other side of the cell. To visualize better the differences between unilateral furrows and their symmetrical counterparts we plotted, in the same cell, the progression of each furrow type as described in the Materials and Methods. The graph shown in Fig. 2 was generated from the cell shown in Fig. 1. The two thin black lines represent the motion of the lateral cell surfaces towards one another in the symmetrical furrow noted by the arrow in Fig. 1. It is evident from these plots that both sides of the furrow approach each other at approximately the same rate (1.2 m/min), and that it took approximately 10 min for the furrow to progress 20 m (to the mid-body stage). The two thick black lines similarly represent the motion of the unilateral furrow. From these lines it is evident that one edge of the cell remained relatively stationary (top line) while the other furrowing edge (bottom line) moved towards it. When compared to the symmetrical furrow, this unilateral furrow moved 27 m in 20 min. This graph also reveals that the unilateral furrow moves at about the same rate as the symmetrical furrow (1.9 m/min versus 1.2 m/ min), and also that (in this case) the whole cell began to crawl near the 10 min mark towards the stationary reference point prior to completion of the unilateral cytokinesis. So far our data suggest that unilateral furrows are not formed from the action of contractile rings, but instead from dynamic contractile elements that are positioned and maintained only on one side of the cell. That this is the case is clearly illustrated in Fig. 3, which documents the formation and behavior of an ectopic unilateral furrow that wanders
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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 symmetrical cytokinesis a phase-lucent band is usually formed in the constriction plane as the dorsal and ventral membranes approach one another (arrow in B). By contrast, the other furrow (black arrowhead) was asymmetric in that its activity was only evident on one side of the cell. Time in h:min:s at bottom right corner of each frame. Bar in F=10 m.
many microns through the cell. Initially this furrow appeared to start on both sides of the cell between the two spindles (white arrowheads in B). However, as cytokinesis progressed, one side relaxed while the other continued across the cell (white arrowheads in Fig. 3B–D). Just prior to reaching the other relaxing furrow, the active furrow made a 45 turn and proceeded to cut through the cytoplasm for another 20 m (white arrowhead in Fig. 3D–F). DISCUSSION As emphasized by Satterwhite and Pollard (1992) ‘the actomyosin contractile-ring mechanism remains the paradigm for cytokinesis after 20 years of experimental testing’. In this mechanism a polarized array of actin filaments, anchored in and positioned around a narrow equatorial band, interact with myosin II filaments and other proteins to apply 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 responsible for furrow formation can be organized into a ring comes from those systems in which the furrow ingresses simultaneously around the entire cell periphery. This occurs, for example, during the cleavage divisions in many types of fertilized eggs and also during cytokinesis in most animal somatic cells. As a result, with few exceptions, most workers model cytokinesis around the premise that the process is initiated through and mediated by the formation of a contractile ring (e.g. see Fishkind and Wang, 1995; Glotzer, 1998). However, there are a number of examples in which cleavage in embryos, and even cytokinesis in somatic cells, is clearly not mediated by contractile elements organized into a ring. For example, the zygotes of cephalopods, elasmobranchs, teleosts, birds, and reptiles exhibit a partial, or meroblastic, type of cleavage during early development in which the lower hemisphere of the egg does not divide (Wilson, 1925). In these organisms a unilateral furrow is formed that spreads laterally across one surface of the egg while it simultaneously ingresses
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Fig. 2. Plot depicting progression of the two furrows shown in Fig. 1 relative to a stationary point positioned between the two furrows and external to the cell. The thin black lines represent changes in the distance between the stationary point and both sides of the symmetrical furrow noted by the arrow in Fig. 1. The thick black lines depict changes in distance from the same point and both sides of the asymmetric furrow noted by the arrowhead in Fig. 1. Note that once formed, the mid-body (parallel lines near 10 min time point) in the symmetric furrow slowly moved towards the stationary point. A similar motion is also seen in the stationary edge of the asymmetric furrow (top thick black line). See text for details.
to a set depth (usually to the yolk layer; e.g. see Jesuthasan, 1998). Unilateral furrows, which ultimately regress, are also formed during the syncytial mitoses in Drosophila zygotes (e.g. see Sullivan et al., 1990), and when cellularization finally occurs it is effected by multiple unilateral furrows. Most recently Neujahr et al. (1998) have shown that unilateral furrows similar to those we describe here for PtK1 cells are routinely formed during mitosis in multinucleated myosin II null Dictyostelium cells. As noted by these authors such furrows are not consistent with a contractile ring model for cytokinesis. Unilateral furrows are not an artifact of our fused cell system—they are also seen at approximately the same frequency (1%) in control PtK1 cells containing a single mitotic spindle (Fig. 4). The initiation of a furrow on just one side of the cell during cytokinesis has also been documented but 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–J in O’Connell et al., 1999) tissue culture cells, and they also form in association with the telophase disk when HeLa cells are released from a cytochalasin B mediated block of cytokinesis (Fig. 10B in Martineau et al., 1995). The formation and ingression of the cleavage furrow from just the
dorsal cell surface has also been detailed in NRK cells by Fishkind and Wang (1993). In these cells actin filaments are found in a band around the entire circumference of the cell, but more are concentrated on the dorsal cell surface where furrowing does not occur. This was interpreted to indicate that unilateral furrows are formed when the cell cannot overcome strong cell-to-cell or cellto-substrate adhesions at other points around its circumference. However, the actin filaments in unilateral (single sided) furrows formed in HeLa cells after release from cytochalasin B appear to be concentrated primarily at the unilateral furrowing site (Martineau et al., 1995). As noted by Fishkind and Wang (1993) the unilateral furrows seen in untreated PtK1 cells could result from a contractile ring that is somehow inhibited from working in those regions of the cortex 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 product of an incomplete ring (e.g. a contractile crescent). Our observation that during a symmetrical cytokinesis one side of the furrow can suddenly relax and regress while the other side continues to ingress demonstrates that there need not be a direct connection between two furrows working from opposing sides of the cell. Furthermore, our observation
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Fig. 3. (A–F) Same conditions as in Fig. 1. In this cell a symmetrical ectopic furrow (B–C, arrowheads) formed between the top multipolar spindle and the bottom bipolar spindle. As this furrow progressed, the cell became progressively pinched from both sides (B–C; arrowheads). However, instead of forming a midbody the furrow on the right side of the cell relaxed (D), and the other active furrow (D–F; white arrowhead) turned 45 and began to wander over the next 14 min in an unpredictable fashion throughout the cell. Bar in F=10 m.
that a unilateral furrow can sharply change its direction, as in Fig. 3, clearly demonstrates that a contractile ring is not required for furrow ingression. Instead, our data support the hypothesis that cytokinesis is normally effected by the coordination of multiple furrows that form independently in the cleavage plane, i.e. that the contractile ring seen during cytokinesis in most organisms is composed of multiple furrowing units that are normally linked and coordinated, but which can also work independently of each other. An important unresolved question is whether the unilateral furrows we observed that wander extensively are following a pre-determined ‘track’ or path within the cell as they ingress, or if they propagate through the cell by simply forming their own track immediately in front of them. Based on the fact that unilateral furrows in Dictyostelium can exceed the length of normal furrows, and that the continued presence of centrosomes (i.e. Mts) is not required for continued furrowing, Neujahr et al. (1998) concluded that progression of unilateral furrows in Dictyostelium is a self-sustaining process. In their view the signals that initiate furrow
formation are not required for subsequently maintaining the furrow, which propagates itself by continuously recruiting contractile elements to its leading edge. However, these conclusions may not be valid for other types of cells including vertebrates: numerous experimental conditions have been defined in worms, flies and vertebrates (see Introduction) in which furrows suddenly stop advancing and then regress. Although the initial signals for furrow formation may not be required for furrow progression in these systems, some other component(s) clearly are. Whether these components are constantly recruited to the leading edge of the furrow, or if they already exist in a preformed track that the furrow must follow, remains to be resolved. ACKNOWLEDGEMENTS The authors thank Ms Cindy Hughes for tissue culture assistance, Mr Richard Cole for assistance with the microscopy, and Drs R. Sloboda (Dartmouth College), R. Palazzo (University of
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Fig. 4. (A–F) Cytokinesis in untreated PtK1 cells can also occur primarily through the activity of a unilateral furrow. In this example the plasma membrane on one side of the spindle midzone remained relatively stationary (black arrowhead in B–E) while the furrow approached from the other side. Bar in F=10 m.
Kansas, Lawrence) and M. Koonce (Wadsworth Center) for stimulating discussions related to cytokinesis. This work was supported, in part, by NIH GMS R01 40198 (to C.L.R.), and was conducted in conjunction with the Wadsworth Center’s Video Light Microscopy Core Facility. REFERENCES A RR, T AAM, S A, B HJ, G DM, 1998. Pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes Dev 12: 1483–1494. C L, W Y, 1996. Signals from the spindle midzone are required for the stimulation of cytokinesis in cultured epithelial cells. Mol Biol Cell 7: 225–232. C M, R MG, M G, T AM, A R, C G, G DM, 1998. Drosophila polo kinase is required for cytokinesis. J Cell Biol 143: 659–671. C P, P J, 1999. Temporal and spatial regulation of cyclin B1 destruction in metaphase. Nature Cell Biol 1: 82–87. D DN, H AA, H A, G M, 1996. A requirement for Rho and cdc42 during cytokinesis in Xenopus embryos. Curr Biol 7: 12–23. E DM, A AM, M A, G IG, E WC, 1997. Chromosomal proteins and cytokinesis: patterns of cleavage furrow formation and the
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