N Animal Cell Mitosis, The Cleavage Furrow Forms During Which Stage Of The Cell Cycle?

• Periodical List
• Mol Biol Jail cell
• v.x(ii); 1999 Feb
• PMC25170

Mol Biol Cell. 1999 Feb; 10(ii): 297–311.

Cleavage Furrows Formed between Centrosomes Lacking an Intervening Spindle and Chromosomes Contain Microtubule Bundles, INCENP, and CHO1 just Not CENP-E

Matthew S. Savoian

^*Department of Biomedical Sciences, State University of New York, Albany, New York 12222; ^†Sectionalisation of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509; and ^‡Institute of Cell and Molecular Biology, Academy at Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom

William C. Earnshaw

^*Department of Biomedical Sciences, Country Academy of New York, Albany, New York 12222; ^†Partition of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509; and ^‡Institute of Cell and Molecular Biology, University at Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom

Alexey Khodjakov

^*Department of Biomedical Sciences, Country University of New York, Albany, New York 12222; ^†Partitioning of Molecular Medicine, Wadsworth Center, New York Country Department of Health, Albany, New York 12201-0509; and ^‡Institute of Cell and Molecular Biology, University at Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom

Conly 50. Rieder

^*Department of Biomedical Sciences, State Academy of New York, Albany, New York 12222; ^†Partitioning of Molecular Medicine, Wadsworth Heart, New York State Department of Health, Albany, New York 12201-0509; and ^‡Institute of Jail cell and Molecular Biological science, University at Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom

Joseph Gall, Monitoring Editor

Received 1998 Sep 18; Accepted 1998 December 1.

Abstract

PtK_i cells containing two contained mitotic spindles tin can cleave between neighboring centrosomes, in the absence of an intervening spindle, as well as at the spindle equators. We used same-prison cell video, immunofluorescence, and electron microscopy to compare the structure and limerick of normal equatorial furrows with that of ectopic furrows formed between spindles. Every bit in controls, ectopic furrows contained midbodies composed of microtubule bundles and an electron-opaque matrix. Despite the absence of an intervening spindle and chromosomes, the midbodies associated with ectopic furrows also independent the microtubule-bundling protein CHO1 and the chromosomal passenger poly peptide INCENP. However, CENP-Due east, another passenger protein, was not plant in ectopic furrows but was ever present in controls. We too examined cells in which the ectopic furrow initiated but relaxed. Although relaxing furrows contained overlapping microtubules from opposing centrosomes, they lacked microtubule bundles as well as INCENP and CHO1. Together these data suggest that the mechanism defining the site of furrow germination during mitosis in vertebrates does not depend on the presence of underlying microtubule bundles and chromosomes or on the stable association of INCENP or CHO1. The information also suggest that the completion of cytokinesis requires the presence of microtubule bundles and specific proteins (e.g., INCENP, CHO1, etc.) that exercise not include CENP-E.

INTRODUCTION

Accurate cell division requires the coordinated segregation of the chromosomes (karyokinesis) too as sectionalization of the cytoplasm (cytokinesis). The latter is achieved in animal cells by the formation of an actin and/or myosin contractile ring (reviewed in Satterwhite and Pollard, 1992 ; Fishkind and Wang, 1995 ). Despite much report, the signals that decide the position of the contractile mechanism and the underlying molecular mechanism responsible for completing cytokinesis remain cryptic.

Usually cytokinesis is coordinated with the position of the mitotic spindle and so that it occurs along the spindle equator, between the groups of separated chromosomes. Currently there are two hypotheses for how this coordination is accomplished. In a now classic experiment, Rappaport (1961) found that sea urchin and starfish zygotes cleave between 2 contained spindles when they are manipulated so that the asters of opposing spindles can interact. The formation of a furrow between 2 radial or "astral" arrays of microtubules (Mts), in the absence of an intervening spindle and chromosomes, led to the idea that the asters release a factor responsible for determining the site of cleavage (reviewed in Oegema and Mitchison, 1997 ), either by causing the cortex to relax at the polar regions (White and Borisy, 1983 ) or by inducing contraction at the spindle equator (Devore et al., 1989 ). Alternatively, observations on cytokinesis in tissue culture cells have led to the hypothesis that the signal(south) determining furrow position come from the chromosomes and/or the spindle midzone (reviewed in Oegema and Mitchison, 1997 ; Glotzer, 1997 ). For example, tripolar spindles with a V-shaped metaphase plate undergo cytokinesis along each arm of the "V" but rarely between the arms (Wheatley and Wang, 1996 ; Eckley et al., 1997 ). This suggests that the chromosomes and/or something associated with the spindle midzone dictate the position of the cleavage furrow. In this regard it has been proposed that "chromosomal passenger proteins", including INCENP (Cooke et al., 1987 ), TD-lx (Andreassen et al., 1991 ), CENP-E (Yen et al., 1991 ), and others (reviewed in Earnshaw and Bernat, 1991 ; Rattner, 1992 ), are involved in defining the site of cytokinesis. These proteins are associated with the centromeric region of the chromosome during spindle formation and redistribute to the forming cleavage furrow during anaphase. Other spindle proteins that end upwards in the midbody during cytokinesis, including several kinesin-like proteins (CHO1/MKLP1/ZEN-four, KLP3A), have also been proposed to exist involved in furrow formation and function (Sellitto and Kuriyama, 1988 ; Nislow et al., 1992 ; Williams et al., 1995 ; Raich et al., 1998 ).

Despite the correlation between chromosome position and cytokinesis, the role of chromatin and its passenger proteins in this process remains unclear. The contempo observations that cytokinesis fails in cells overexpressing an INCENP:CENP-B chimera that is tethered at centromeres (Eckley et al., 1997 ), or a truncated nonmicrotubule binding grade of INCENP, provides experimental support for the idea that at to the lowest degree one passenger protein is involved in this process (Mackay et al., 1998 ). However, Zhang and Nicklas (1996) observed normal cytokinesis in grasshopper spermatocytes after all chromosomes were removed past micromanipulation. This implies that the signal(s) controlling furrow positioning is intrinsic to the spindle but not the chromosomes. That even the spindle itself is not needed for furrow positioning is evident from the study of Rieder et al. (1997) . These workers plant, as noted previously by Rappaport (1961) for echinoderm zygotes, that vertebrate (PtK₁) cells containing ii contained spindles tin can also form functional furrows between two centrosomes that lack an intervening spindle and chromosomes. This observation raises the question of whether chromosome and spindle-associated proteins are required for cytokinesis in vertebrates.

The observation that functional cleavage furrows tin form in vertebrate cells between two centrosomes, which are not connected by an intervening spindle or chromosomes, supports the hypothesis (discussed higher up) that the site of cytokinesis is dictated by where the radial "astral" arrays of Mts growing from each opposing centrosome overlap. Nonetheless the centrosomes contribute not merely the Mts that form the aster and spindle merely also those interpolar Mts that ultimately grade the interzone and midbody between the separating chromosomes during late anaphase and telophase (see Mastronarde et al., 1993 ). In this respect there is a growing torso of bear witness that bundles of interpolar Mts play an important role not just in establishing where cytokinesis will occur (e.g., Williams et al., 1995 ; Cao and Wang, 1996 ; Giansanti et al., 1998 ) but also in stabilizing the furrow after it forms (Wheatley and Wang, 1996 ; Wheatley et al., 1998 ; Raich et al., 1998 ). Although the mechanism by which these microtubule bundles form remains to exist resolved, information technology conspicuously involves one or more than kinesin-like proteins (due east.k., Nislow et al., 1992 ; Williams et al., 1995 ; Raich et al., 1998 ) and mayhap INCENP (Earnshaw and Cooke, 1991 ).

To evaluate the hypothesis that the positioning of a cytokinetic furrow involves Mt bundles, CHO1, CENP-Due east, and/or INCENP, we have thoroughly analyzed the structure and limerick of ectopic furrows that form during mitosis between 2 independent spindles in PtK_i cells. Because these furrows can produce 2 daughter cells (Rieder et al., 1997 ), they contain all of the machinery required for initiating and completing cytokinesis. The fact that they form between centrosomes that lack an intervening spindle and chromosomes provides a unique opportunity to identify spindle and chromosomal proteins that are normally found in the cleavage furrow but that are not required for cytokinesis in vertebrates.

MATERIALS AND METHODS

Cell Culture and Electrofusion

PtK₁ cells were grown on coverslips and electrofused with a ProGenetor 2 electroporator (Hoeffer, San Francisco, CA) every bit described previously (Rieder et al., 1997 ). Briefly, coverslip cultures that were 60–eighty% confluent were placed in fusion buffer (280 mM sucrose, 2 mM HEPES, 1 mM MgCl₂, pH vi.ix) between two platinum electrodes separated by ∼10 mm. They were then exposed to a 350 V pulse for 2 ms. Exposed cultures were then returned to conditioned medium for 1 h at 37°C. Coverslip cultures containing fused cells were then mounted in Rose chambers in fresh complete L-fifteen media and scanned 2 or more than hours later for cells containing two contained mitotic spindles. For cytochalasin D experiments, coverslip cultures were incubated overnight in conditioned media containing i μg/ml cytochalasin D. On the following day they were mounted in Rose chambers containing the same medium and followed by video stage-contrast microscopy.

Video Microscopy

Cells containing ii spatially separated mitotic spindles were followed by time-lapse video microscopy on an inverted Nikon Diaphot microscope, equipped with a Dage MTI VE-1000 video camera, using either a forty× (numerical aperture [N.A.] of 0.70) or a xx× (Due north.A. of 0.75) stage-contrast objective. The cells were maintained on the microscope stage at 37°C using a custom-built Rose chamber heater (Rieder and Cole, 1998 ) and were illuminated with estrus-filtered 546-nm (light-green) light that was shuttered between exposures. Recordings were made at iv–15 s intervals onto either super VHS tape or optical retentiveness disks, and background subtraction and averaging were conducted using either an ARGUS-10 or a Hamamatsu C2400 image processor. Selected frames were digitized using Scion Paradigm (Scion, Frederick, Medico) frame-grabbing software and were processed with Adobe Photoshop (Adobe Systems, Mountain View, CA).

Immunofluorescence Microscopy

PtK₁ cells followed in vivo were fixed at the desired time in −20°C methanol for 10 min. They were then rinsed 3 times in phosphate-buffered saline containing 0.05% Tween 20 (PBST). The coverslips were then incubated for 1 h at 37°C in chief antibodies confronting either INCENP (pab1186 [meet Eckley et al., 1997 ]), CHO1 (Kuriyama et al., 1994 ) (courtesy of Dr. R. Kuriyama, Academy of Minnesota, Minneapolis, MN), or CENP-E (HX-1 [Schaar et al., 1997 ]) (courtesy of Dr. T. Yen, Fox Chase Cancer Center, Philadelphia, PA) diluted in PBST to 1:500, ane:500, and i:250, respectively. Subsequently primary antibody staining, coverslips were rinsed three times at five min each in PBST. They were then incubated at 37°C for 1 h in TRITC-labeled caprine animal anti-rabbit secondary antibodies diluted 1:50 in PBST.

Mts were labeled with a monoclonal α-tubulin antibody (T-5168, Sigma, St. Louis, MO) at i:300 dilution in PBST and were treated as described above with the exception that a FITC-labeled caprine animal anti-mouse secondary antibody was used. Cells treated with cytochalasin D were preextracted in i% Triton X-100 in PHEM (60 mM piperazine-N,Northward′-bis[2-ethanesulfonic acid], 25 mM HEPES, 10 mM EGTA, 2 mM MgCl₂, pH vi.9) for one min. They were then stock-still for x min in 1% glutaraldehyde in PHEM, reduced in NaBH_iv (0.i mg/ml in H_iiO), and labeled as described higher up.

After DNA labeling with Hoechst 33342, coverslips were mounted in glycerol/PBS (1:i) supplemented with 1 mg/ml p-phenylenediamine to foreclose photobleaching. Selected cells were viewed past epifluorescence using a Nikon Optiphot microscope equipped with lx× (N.A. of ane.4) and 100× (N.A. of one.four) objectives. Images were captured with a cooled charge-coupled device camera (KAF-1400, Photometrics, Tucson, AZ) and processed using Adobe Photoshop.

Electron Microscopy

Cells followed in vivo were stock-still for electron microscopy by perfusion with 2.5% glutaraldehyde in 0.ane M phosphate buffer (meet Khodjakov et al., 1997 ). They were and so post-stock-still in 2% OsO_iv for lx min at iv°C, washed in buffer, and treated with tannic acid (0.15% in phosphate buffer) for 1 min. Cultures were stained en bloc for 60 min at 4°C in 1% uranyl acetate, dehydrated in ethanol, and flat embedded in Epon (see Rieder and Cassels, 1999 ). Serial thin (xc-nm) sections were photographed on a Zeiss 910 TEM, and the negatives were subsequently digitized and processed in Adobe Photoshop.

RESULTS

Midbodies Are Associated with Ectopic Furrows

We followed 432 cells containing two independent mitotic spindles, separated on average by 35 μm (range of eighteen–75 μm), throughout mitosis past time-lapse light microscopy (LM). Ectopic furrows were initiated in 102 cells (24%). Of these 102 ectopic furrows, xxx (29%) relaxed before reaching the midbody stage (run into below). We found no correlation between the distances that separated the closest poles of neighboring spindles and the success of either ectopic furrow initiation and/or completion. We also found no correlation between the asynchrony betwixt the two spindles in anaphase onset (which could exist >xxx min) and the probability of forming an ectopic furrow. Ectopic furrows announced to course in a mode like to that of control furrows (Effigy ​ i).

During mitosis ectopic cleavage furrows and midbodies can form between spindles in a PtK₁ cell containing ii independent spindles. (A–F) Selected images from a time-lapse recording of a cell containing two independent mitotic spindles. During telophase (C and D), cytokinesis occurs as expected between the two groups of separating chromosomes (arrows in D) but likewise between the two spindles (arrowhead in D). This prison cell was fixed soon after the image in F when all three furrows contained phase-dense intercellular bridges. (G–I) Immunofluorescence images of tubulin distribution in the three intercellular bridges pictured in F. The control midbodies, formed between groups of separating chromosomes, are pictured in G (lower left-hand span in F) and I (upper right-hand bridge in F). The ectopic bridge (H and arrowhead in F) also contains microtubules. Bars: A–F, 10 μm; M–I, v μm.

An analysis of our video records revealed that ectopic furrows that did not relax always formed, within a 10–15 min period, a thin phase-dense intercellular bridge like to those formed past control furrows in the same cell (compare Figure ​ 1F, arrows and arrowhead). Anti-tubulin indirect immunofluoresence (Imf) assay revealed that, as in controls, these intercellular bridges always contained an Mt bundle that varied widely in its Mt content (n = 33; compare Effigy ​ i, M–I). The Mt bundles found in the intercellular bridge at the completion of furrowing in controls displayed a prominent nonstaining central gap (e.yard., Figure ​ 1, Chiliad and I) that has been shown by others to stand for at the electron microscopy (EM) level to the presence of an electron-opaque matrix material (Sellitto and Kuriyama, 1988 ). The Mt package within the intercellular span formed by an ectopic furrow also independent a like staining gap, simply in some cases it was barely discernable (eastward.thou., Figure ​ 1H; run across besides Figures ​ 7E and ​ 9E).

Midbodies formed in association with ectopic furrows contain the CHO1 protein. (A–D) Images from a time-lapse recording of a jail cell that formed an ectopic furrow between a tripolar spindle and a normal, bipolar spindle. Arrows in C and D indicate control furrows. The arrowhead in C indicates the forming ectopic furrow, and that in D indicates the ectopic midbody. (E–K) Immunofluorescence images of the aforementioned cell shown in A–D, fixed shortly after the image in D. (Due east) Comparison of tubulin distribution between the control midbodies (arrows) and the ectopic midbody (arrowhead). This ectopic midbody exhibits the nonstaining central region found at the controls. (F) Distribution of the CHO1 protein. Arrowhead indicates the ectopic furrow. Although weaker in intensity, the location and localization patterns of the signal are identical to those of the controls. Note that the tripolar spindle region in the upper portion of the panel just shows a signal at one site, suggesting that two and not three daughter cells would have resulted upon completion of cleavage. (G) Dna distribution. Bars: A–D and E–Yard, ten μm.

CENP-Eastward is not found in midbodies formed from ectopic furrows. (A–D) Images from a time-lapse sequence from a cell that formed an ectopic furrow (arrowhead in C and D). (Due east and F) Immunofluorescence localization of microtubules (E) and CENP-Due east (F) in the same cell fixed soon later on the image in D. At this time the ectopic furrow (arrowhead) had progressed to a point between the ii control furrows (arrows), one of which (upper) had not notwithstanding formed a midbody (run into text for details). All three furrow sites contain microtubule bundles and a nonstaining central band (E). Although the two control furrows vary in their state of progression, both incorporate CENP-E (arrows in F), but this poly peptide is not constitute in the ectopic furrow (arrowhead in F). Bars: A–D and Eastward and F, ten μm.

We used serial-section EM to examine the ultrastructure of the intercellular span produced past ectopic furrows (Effigy ​ twoA–D). In all cells (north = nine) this span contained a variable number of tightly packed Mts as well as an electron-opaque matrix material (Effigy ​ 2D). Because the construction of this span was indistinguishable from the packet of "midbody" Mts (east.grand., come across Buck and Tisdale, 1962 ) associated with the command intercellular bridges in the same cell (Effigy ​ two, B and C), we conclude that midbodies are e'er present in the thin intercellular bridges formed from the activeness of an ectopic furrow. Our EM assay likewise revealed that the midbodies associated with ectopic furrows sometimes contained a large number of 10-nm filaments (our unpublished observations) that are likely keratin filaments that are known to surround the spindle during mitosis in epithelia (e.g., Mandeville and Rieder, 1990 ).

Midbodies formed in association with ectopic cleavage furrows are structurally similar to control midbodies. (A) Stage-dissimilarity image of a cell containing two control midbodies (arrows) and one ectopic midbody (arrowhead) only earlier fixation for series-department EM. (B–D) Selected electron micrographs through the midbodies pictured in A. The letter by the arrow or arrowhead in A indicates the respective micrograph depicting its ultrastructure. The midbody formed in association with the ectopic furrow (D, also arrowhead in A) is structurally similar to those of the controls (B and C). All comprise bundles of microtubules that are embedded in an electron-opaque matrix. Confined: A, 10 μm; B–D, 0.75 μm.

Of the 432 cells followed for this study, 330 (76%) failed to initiate furrows betwixt the independent spindles. We fixed and stained 15 of these cells, after the control furrows had formed midbodies, for an International monetary fund analysis of Mt distribution (e.m., Figure ​ 3). In all cases the region between the two spindles where an ectopic furrow would be predicted lacked Mt bundles. Nevertheless, information technology e'er contained large numbers of Mts derived from the two opposing late telophase centrosomes (Figure ​ 3D, short arrows).

Cells that neglect to initiate an ectopic furrow between the spindles incorporate numerous microtubules, but not microtubule bundles, betwixt the opposing spindles. (A–C) Selected images from a time-lapse recording of a cell that did non initiate cleavage betwixt separate mitotic spindles. Arrows in B indicate furrow initiation at the location of the metaphase plates, whereas those in C betoken the resultant midbodies. (D) Immunofluorescence image of microtubule distribution in this same cell stock-still soon after the image in C. The control midbodies (long arrows) contain characteristic microtubule bundles bisected by a nonstaining region. Past contrast, the region between the two spindles (arrowhead) contains numerous microtubules derived from the opposing centrosomes (short arrows) but no microtubule bundles. Bars: A–C and D, 10 μm.

With few exceptions (<one%) later on a control furrow became detectable by video LM, it proceeded without intermission to grade, over a 10–15 min period, a midbody. In contrast, we found that 29% of the ectopic furrows that started between two spindles ultimately relaxed before midbody formation. As a rule in our experimental cells, the ectopic furrow began to class at approximately the same time as the command furrows. Still, at variable times thereafter (and earlier midbody formation), ectopic furrows that were destined to relax began to increase steadily in bore, and 5–10 min later they were no longer detectable (e.chiliad., Figures ​ iv, A–D, and ​ v, A–D). To determine how Mts are distributed in the region of relaxing ectopic furrows, we followed ten cells containing two spindles through anaphase and fixed them for International monetary fund five min after their associated ectopic furrows had begun to relax. An analysis of these cells revealed that the region of the relaxing ectopic furrow ever contained numerous Mts but lacked the distinct Mt bundles characteristic of progressing control and ectopic furrows (see and compare Figures ​ 4F and ​ fiveF with ​ 1H and ​ 9E; see also below).

Relaxing ectopic furrows lack prominent microtubule bundles and the CHO1 protein. (A–D) Images from a time-lapse recording of a prison cell that initiated an ectopic furrow between the independent spindles (arrowhead in C). This furrow subsequently and rapidly relaxed (arrowhead in D). The control furrows (arrows) progressed to completion without incident and formed midbodies (D). (E–Chiliad) This cell fixed soon subsequently the epitome in D for an immunofluorescence assay of microtubules (Eastward and F) and CHO1 (Chiliad). Note that the region of the relaxing furrow contains a loftier density of microtubules (arrowhead in E) that at a higher magnification (F) practise not announced to exist organized into conspicuous bundles. Although each control furrow (arrows) also contains CHO1, this protein is not institute in the region of the relaxing furrow (arrowhead in G). Bars: A–D, Due east and Yard, and F, 10 μm.

Relaxing ectopic furrows lack bundled microtubules and INCENP. (A–D) Selected images from a time-lapse recording of an ectopic furrow (arrowhead in C) that goes almost to completion (i.e., the midbody phase) earlier rapidly relaxing (D). (E–G) This cell fixed shortly after the epitome in D for an immunofluorescence analysis of microtubules (East and F) and INCENP (G). Unlike the control midbodies (arrows), each of which contains a prominent bundle of Mts (Eastward), the region of the relaxing ectopic furrow (arrowhead in E and shown at a college magnification in F) is rich in microtubules that are non bundled. Note also that the INCENP antigen is present in both control furrows (arrows) just absent from the relaxing ectopic furrow (arrowhead in G). Bars: A–D, East and G, and F, 10 μm.

Inhibiting Furrow Formation with Cytochalasin D Does Non Inhibit the Bundling of Interzonal Microtubules

To investigate whether the absence of Mt bundles in relaxing ectopic furrows is caused by the regression of the furrow or by the absenteeism of some internal activeness required for bundling, nosotros treated PtK₁ cells with cytochalasin D. This drug specifically inhibits actin polymerization and thus cytokinesis just not progression through mitosis (east.1000., Aubin et al., 1981 ). As expected, PtK_ane cells treated with cytochalasin D at 1 μg/ml showed no evidence of furrow germination (Effigy ​ 6, A–D). An International monetary fund assay of the interzonal Mts in these cells (north = 7), which were fixed when untreated cells comprise midbodies (30–35 min after anaphase onset), revealed that fifty-fifty in the absence of a constricting action many of the interpolar Mts were bundled (Effigy ​ vi, E and F). As in the midbodies of untreated cells, these bundles also independent a cardinal equatorial region that failed to stain with anti-tubulin antibody. Thus the formation of interzone Mt bundles during telophase does non depend on furrowing activity. Because these bundles have been shown to exist extremely stable after they are formed (e.chiliad., Salmon et al., 1976 ; Mullins and Biesele, 1977 ), the lack of Mt bundles in relaxing ectopic furrows is caused by the absenteeism of an internal component needed for bundling and not past the relaxation of the furrow.

Interpolar microtubules between the separating groups of anaphase chromosomes form bundles even when furrow formation is inhibited with cytochalasin D. (A–D) Images from a time-lapse recording of a cytochalasin-treated prison cell equally it progressed from metaphase through telophase of mitosis. (E) This cell stock-still soon afterward the image in D, and ∼thirty min afterward anaphase onset, for an immunofluorescence analysis of microtubule distribution. Note that even when cytokinesis is inhibited, the interzonal microtubules positioned between the two separated nuclei form prominent bundles and comprise a nonstaining equatorial band. (F) Microtubule distribution in another cytochalasin-treated cell that was followed past video LM until it was fixed during telophase. Over again, even though this prison cell did not initiate furrowing, information technology contains prominent bundles of interzonal microtubules, many of which are bisected by a nonstaining region. Bars: A–D and Eastward and F, x μm.

Ectopic Furrows Incorporate CHO1 and INCENP just Not CENP-Due east

The structural data presented above demonstrate that the midbodies associated with ectopic furrows are similar to those of controls. Nosotros adjacent determined whether proteins known to be associated with control furrows and midbodies were likewise present in the ectopic furrows that formed in the absenteeism of an intervening spindle and chromosomes. For these analyses we chose to assay for the presence of the kinesin-like CHO1 motor protein (e.k., Sellitto and Kuriyama, 1988 ; Raich et al., 1998 ) and 2 chromosomal passenger proteins, INCENP (Cooke et al., 1987 ) and CENP-E (e.g., Brown et al., 1996 ).

The distribution of CHO1 was examined in cells fixed 5 or more minutes after both the control and ectopic furrows had formed midbodies (northward = 12; e.g., Figure ​ 7). In all cases we found a variable, but clearly detectable, level of CHO1 in the ectopic midbody (Figure ​ seven, D–F, arrowhead). As in control midbodies, CHO1 was restricted to the fundamental region of the midbody that does not stain for tubulin by International monetary fund (compare Figure ​ seven, E and F, arrowheads). This is the region that corresponds to the location of the electron-dense matrix material (Sellitto and Kuriyama, 1988 ).

Ectopic midbodies formed in cells containing two spindles were also examined for the presence of the chromosomal passenger protein INCENP (north = 12). Because no chromosomes were present in this region, we were surprised to observe INCENP consistently in the ectopic midbodies (e.chiliad., Effigy ​ 8). In controls INCENP was found, as reported by others (reviewed in Rattner, 1992 ), on opposite sides of the CHO1-containing matrix fabric that excludes Mt staining (Figure ​ viii, B, B′, C, and C′). Although in virtually cells this was also the case for the ectopic midbodies, in some INCENP appeared to be distributed in an uninterrupted linear pattern (Figure ​ 8, D and D′).

Midbodies formed between spindles contain INCENP. (A) The distribution of microtubules in the jail cell shown in Figure ​ ane. The arrows betoken control midbodies, whereas the arrowhead indicates the midbody formed in clan with the ectopic furrow. (B–D) The microtubule patterns in these midbodies at a higher magnification. B is from the upper command midbody shown in A, C is from the lower control, and D is from the ectopic midbody. (B′–D′) The corresponding images depicting the distribution of INCENP. Note that all of the midbodies, including the ectopic 1, incorporate this protein. Bars: A, 10 μm; B–D and B′–D′, 5 μm.

We too attempted to look at TD-60 (reviewed in Margolis and Andreassen, 1993 ) simply were thwarted because this antibiotic does not recognize TD-60 in PtK₁ cells, and our attempts to create cells containing two independent spindles that stain with TD-sixty (e.grand., CV-1) were not successful. Nosotros discovered that the independent spindles in prison cell types that normally round during mitosis, which include all fibroblasts and near epithelial cells, rapidly associated and fused to class a big common multipolar spindle. However, with regards to INCENP, a contempo study found that the distribution of this protein and TD-60 was indistinguishable under a wide variety of weather condition (Martineau-Thuillier et al., 1999 ).

Finally, we examined ectopic furrows for the presence of CENP-E. CENP-E is another chromosomal passenger protein that relocates during anaphase to the interzone Mts, and its location ultimately becomes restricted to the midbody. Nevertheless, because it is degraded one-time after midbody germination but before the final separation of girl cells (Dark-brown et al., 1994 ), it is present in all young but not older midbodies. We therefore stock-still our experimental cells (due north = 9) after furrowing at the ectopic site was well underway but before midbody germination (e.thou., Figure ​ 9). Although this poly peptide was present in all forming control furrows and midbodies (east.g., Figure ​ nine, E and F, arrows), we never found it in ectopic furrows or forming ectopic midbodies (Figure ​ 9, E and F, arrowhead).

Relaxing Ectopic Furrows Lack INCENP and CHO1

Equally noted previously, 76% of the cells followed past video LM failed to initiate cytokinesis between the two spindles after anaphase. When we examined those regions where an ectopic furrow should have formed, nosotros always found numerous Mts that were derived from opposing centrosomes (see Figure ​ 3D). Withal, in these cells nosotros never saw INCENP or CHO1 protein at locations other than the control midbodies (our unpublished observations).

We speculate that ectopic furrows that relax are probable to be scarce in some component(south) required for the last stages of cytokinesis. 1 prediction is that the CHO1 poly peptide is non present in relaxing ectopic furrows because these furrows lack Mt bundles (our unpublished observation) that require CHO1 (east.g., Kuriyama et al., 1994 ; Raich et al., 1998 ). Some other prediction is that they too lack INCENP that has recently been implicated in the latter but non in early stages of cytokinesis (Mackay et al., 1998 ). To evaluate these predictions we examined relaxing furrows for the presence of these proteins. In every example we found that, as predicted, these regions lacked detectable CHO1 (due north = v; Figure ​ 41000, arrowhead) and INCENP (n = v; Figure ​ vThou, arrowhead).

DISCUSSION

PtK₁ cells that contain two independent mitotic spindles tin can be produced by electrofusion. Every bit described by Rieder et al. (1997) , these spindles remain independent throughout mitosis unless they wander close enough (within ∼twenty μm) for their Mt arrays to overlap, at which time they and so speedily fuse to grade a unmarried large multipolar spindle. In their original report, Rieder et al. (1997) noted that cells containing 2 spindles sometimes undergo cytokinesis between the spindles in a region that lacked an intervening spindle and chromosomes. Although it was noted that many of these "ectopic" furrows were fully functional in that they led to the production of two independent daughter cells, no attempt was fabricated at the time to study their structure or composition.

The demonstration that cytokinesis can occur in vertebrate somatic cells betwixt two centrosomes, in the absence of an intervening spindle or chromosomes, is like to that reported earlier for echinoderm zygotes (reviewed in Rappaport, 1991 ). The fact that both vertebrate somatic cells and large invertebrate zygotes play past the same rules implies that cytokinesis occurs by similar mechanisms in diverse systems. Because cytokinesis can occur in the absence of a spindle and chromosomes, the role these structures play in this procedure remains unclear.

As emphasized by Oegema and Mitchison (1997) , data concerning the construction and composition of ectopic furrows are potentially useful for evaluating models of cytokinesis. We therefore used same-prison cell correlative LM, Imf, and EM to narrate the structure of ectopic furrows and to determine whether they comprise some of the chromosomal rider (INCENP and CENP-Eastward) and spindle (CHO1) proteins thought to be involved in cytokinesis.

Ectopic Furrows Lead to the Germination of Midbodies

During anaphase the interzonal region betwixt the groups of separating chromosomes contains 2 overlapping interpolar Mt arrays, of opposite polarity, that are derived from the opposing centrosomes (e.g., Mastronarde et al., 1993 ). During belatedly anaphase and near the initiation of cytokinesis, adjacent Mts within this interpolar array brainstorm to coalesce into multiple bundles, each of which contains a detached patch of an electron-opaque matrix textile (Buck and Tisdale, 1962 ; reviewed in Rattner, 1992 ; Kuriyama et al., 1994 ). As furrowing is initiated, these multiple Mt bundles and their associated matrix coalesce into a single Mt parcel referred to equally a midbody (Buck and Tisdale, 1962 ; reviewed in Rattner, 1992 ) (Effigy ​ two, B–D). The midbody so bridges the ii girl cells until information technology is broken and discarded (encounter Mullins and Biesele, 1977 ).

The preliminary results of Rieder et al. (1997) suggested that ectopic furrows in PtK_i cells course in a region between the spindles devoid of Mts and lack a midbody. However, our extensive analyses of cells containing 2 contained spindles reveal that by telophase the region between the two spindles always contains numerous Mts (Figures ​ 3D, ​ fourEastward, and ​ fiveF). Because these Mts are not nowadays between the spindles at the fourth dimension of anaphase onset (Rieder et al., 1997 ), they likely ascend during telophase equally each of the centrosomes nucleate a new array of cytoplasmic Mts (e.yard., see Vandre et al., 1984 ).

We plant that the intercellular bridge formed past an ectopic furrow contained a midbody that was duplicate from those formed past control furrows in the aforementioned cell (compare Figure ​ 2, B–D). In some cases the ectopic midbody contained just a few bundled Mts (e.1000., Figures ​ oneH and ​ viiDue east) and little matrix material, but in others it was as robust as those of control midbodies formed between separating groups of chromosomes (e.g., Figure ​ 2D). By contrast, although overlapping arrays of interpolar Mts were always found in the region betwixt 2 independent telophase spindles, nosotros never saw bundles of Mts or midbodies in this region in cells that failed to initiate an ectopic furrow (Figure ​ 3D) or in those cells in which the ectopic furrow relaxed (Figures ​ 4F and ​ 5F). Because the fact that after being formed, interzonal Mt bundles are extremely stable (Salmon et al., 1976 ; Mullins and Biesele, 1977 ; Mullins and McIntosh, 1982 ), nosotros conclude that ectopic furrows tin be initiated in the absence of Mt bundling. Nevertheless, Mt bundling seems to exist required for the furrow to propagate to the midbody phase because nosotros always establish bundles in the midbodies of ectopic furrows and, as evident from our cytochalasin D-treated cells, bundling is non a product of furrow constriction but an intrinsic property of the spindle interzone (e.g., Figure ​ 6). In this context it is noteworthy that we e'er found the Mt-bundling protein CHO1 in both command and ectopic midbodies merely never in relaxing ectopic furrows. Our decision that Mt bundling is required for furrow propagation but non for initiation is consistent with the recent report that furrows are initiated, but then ultimately relax, in ZEN-iv null Caenorhabditis elegans embryonic cells that also lack the chapters to bundle interzone Mts (Raich et al., 1998 ).

CHO1 and INCENP Are Nowadays in Ectopic Midbodies and Are Therefore Likely Required for Cytokinesis, whereas CENP-Eastward Is Not

The formation of midbodies between next spindles occurred < twenty% of the time (i.east., in 72 of 432 or 17.six% of the cells), just when these midbodies did course, they typically appeared to exist both functionally and structurally normal. Their formation therefore provides a model arrangement for analyzing the office of specific proteins during cytokinesis. In particular, proteins that are required for midbody formation must be present in both normal (control) and ectopic furrows.

Our written report reveals that the kinesin-like Mt motor protein CENP-E, which becomes full-bodied in command cleavage furrows and midbodies, is not essential for cytokinesis. Its location within the furrow and its association with midbody Mts have led to the suggestion that this chromosomal passenger protein is involved in this process (e.g., encounter Yen et al., 1991 ). Yet, this hypothesis has not been directly tested considering the inhibition of CENP-E role, by antibody injection (Yen et al., 1991 ) or overexpressing a mutant (Schaar et al., 1997 ), inhibits the anaphase onset that is a prerequisite for cytokinesis. Our data reveal that although CENP-East is always present in command furrows and midbodies, it is never seen in ectopic furrows or ectopic midbodies (Effigy ​ 6). Thus we conclude that CENP-E is not required for cytokinesis.

The converse of this logic is that proteins that are always associated with both control and ectopic furrows and midbodies are excellent candidates for components that have an obligatory role in cytokinesis. Although localization data by itself cannot demonstrate a functional involvement, it is striking that two of the proteins nosotros institute to be always associated with normal and ectopic furrows have been independently suggested to be involved in cytokinesis.

The start of these is the CHO1 poly peptide that bundles Mts in vitro (Nislow et al., 1992 ) and in vivo (Kuriyama et al., 1994 ) and is known to accept human (MKLP1 [Nislow et al., 1992 ]) and C. elegans (ZEN-four [Raich et al., 1998 ]) homologues. During spindle germination, this poly peptide is distributed diffusely along the spindle fibers, but during anaphase, information technology redistributes into prominent streaks inside the interzone that correspond to MT bundles. Finally during telophase, it becomes restricted to the very center of the midbody where the electron-opaque matrix associated with overlapping Mt ends is located (Sellitto and Kuriyama, 1988 ). Similar CENP-East, CHO1 is a member of the kinesin superfamily, but unlike CENP-E, our data reveal that CHO1 is present in every ectopic midbody, suggesting that information technology is required for cytokinesis in vertebrates equally its homologue (ZEN-four) is required in C. elegans (Raich et al., 1998 ). How CHO1 becomes associated with ectopic furrows, which can form many micrometers from a spindle and chromosomes, remains to be determined. Because it is a plus-end Mt motor, present in the centrosome and nucleus before spindle assembly, it is possible that it moves into the region where the ectopic furrow will form during telophase as the centrosomes begin to renucleate extensive arrays of cytoplasmic Mts.

The chromosomal passenger protein INCENP was originally suggested to play a role in cytokinesis (Cooke et al., 1987 ) and becomes concentrated at the site of furrow germination before myosin (Eckley et al., 1997 ). Recent studies reveal that cytokinesis ultimately fails in cells overexpressing unlike dominant-negative mutant forms of INCENP (Eckley et al., 1997 ; Mackay et al., 1998 ). Yet, a function for this protein in cytokinesis has been questioned because it is difficult to envision how a chromosomal protein tin go positioned in ectopic furrows that form many micrometers from the closest chromosome (Oegema and Mitchison, 1997 ). Here we show that INCENP is present in all ectopic furrows. This upshot reveals that this poly peptide does not accept to exist delivered past a chromosome simply tin can movement freely within the prison cell. Because we found INCENP in all ectopic furrows that formed midbodies, the data also strongly support the hypothesis that it plays an obligatory function in cytokinesis.

I advantage of our system is that information technology allows i to compare the structure and composition of completed furrows with those that began to form merely then ultimately relaxed. As in echinoderm zygotes (Rappaport, 1961 ), some (∼thirty%) of the ectopic furrows formed in PtK₁ cells relaxed before reaching the midbody phase (Rieder et al., 1997 ) (our unpublished observations). Because cytokinesis has long been known to be a multistep procedure (reviewed in Rappaport, 1991 ; Adachi et al., 1997 ), this result is non unexpected. It means that the conditions for forming and initiating the activity of a furrow are not the same as those for maintaining and propagating the furrow. Therefore, an analysis of regressing ectopic furrows may be useful for defining which components are needed for maintaining and stabilizing a furrow afterwards information technology is formed. Our findings that CHO1 and INCENP are always present in ectopic midbodies, but never in regressing ectopic furrows, advise that they are not involved in furrow initiation just instead stabilize the activity of the furrow until the midbody is formed. This hypothesis is consequent with the recent observation that furrowing is initiated in C. elegans zygotes lacking the CHO1 homologue (ZEN-4) merely that these furrows ultimately backslide (Raich et al., 1998 ). It is too consistent with the demonstration of Eckley et al. (1997) (meet also Mackay et al., 1998 ) that overexpressing a mutant course of INCENP disrupts cytokinesis only after the furrow has already formed.

Although it is not known which molecules signal the location of either normal or ectopic furrows, our data allow united states of america to conclude that the initiation of furrow formation requires more than just overlapping antiparallel arrays of Mts in a telophase cytoplasm; seventy% of our cells met this condition without exhibiting whatsoever detectable signs of furrow formation. The bundles of interpolar Mts that form within the interzone during anaphase and telophase are extremely stable (meet above), and their formation requires CHO1 only not the presence of a contractile furrow (eastward.g., see Figure ​ six). Contempo observations of mutant Drosophila spermatocytes that fail to undergo cytokinesis propose that the bundles of interpolar Mts formed in the interzone during anaphase are involved in positioning and initiating furrow germination (due east.yard., Williams et al., 1995 ; Giansanti et al., 1998 ). However, we practise not notice bundles of interzonal Mts in regressing ectopic furrows where, because of their farthermost stability, they would be expected if they were required for furrow germination. Indeed, when the germination of interzonal Mt bundles is inhibited in C. elegans past knocking out the CHO1 homologue ZEN-4, cytokinesis is still initiated but ultimately fails (Raich et al., 1998 ).

In determination, the ability to examine the structure and composition of ectopic furrows that complete furrowing to the midbody stage and of regressing ectopic furrows provides a novel and powerful system for exploring the mechanism of cytokinesis. We take used this system to evaluate the role of several proteins thought to be involved in this procedure and have identified one (CENP-E) that is not. Furthermore, our data implicate the Mt-bundling CHO1 protein and INCENP in the propagation and/or stabilization of furrows merely not in their germination. The assay described hither should exist useful in the future for determining whether proteins suspected of being involved in cytokinesis are and if so whether they are involved in the initial or latter stages of this process.

ACKNOWLEDGMENTS

We gratefully admit Ms. Cindy Hughes for her tireless assistance with prison cell culture, Ms. Grisel Cassels for help with the EM aspects of this study, and Mr. Richard Cole for thoughtful discussions and technical input. We as well give thanks Drs. T. Yen (Fox Chase Cancer Center, Philadelphia, PA) and R. Kuriyama (Department of Cell Biology and Neuroanatomy, Academy of Minnesota, Minneapolis, MN) for their generous gifts of the CENP-E and CHO1 antibodies, respectively. This work was supported by National Institutes of Health/GMS grant 40198 (to C.L.R.) and a Chief Research Fellowship from the Welcome Trust (to West.C.Due east.) and made utilise of the Wadsworth Center'southward video LM core facility.

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