Report |
Address correspondence to Dr. Dahong Zhang, Department of Zoology, Oregon State University, 3029 Cordley Hall, Corvallis, OR 97331. Tel.: (541) 737-6610. Fax: (541) 737-0501. email: zhangd{at}bcc.orst.edu
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Abstract |
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Key Words: cytokinesis; cleavage furrow; asters; microtubules; actin filaments
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Introduction |
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The evidence of asters in defining furrow position came largely from classical experiments in echinoderm embryos, cells with massive astral microtubule arrays as compared with their spindle (Larkin and Danilchik, 1999). When sand dollar eggs are experimentally distorted into a donut-like shape, subsequent cleavages occur not only at the spindle equators, but also between the asters of adjacent spindles (Rappaport, 1961). Furrow induction between the asters of two independent spindles has also been reported in fused somatic tissue culture cells (Eckley et al., 1997; Rieder et al., 1997). Further, in sea urchin eggs, mechanical aspiration of the central spindle does not impede furrow induction (Hiramoto, 1971), whereas hydrostatic pressureinduced disassembly of astral microtubule arrays prevents the induction (Salmon and Wolniak, 1990).
In contrast, the central spindle appears more important for furrow positioning in insect or mammalian cells whose asters are relatively small. In cultured rat kidney epithelial cells (NRK), placing a perforation between the spindle midzone and cell cortex before anaphase results in the formation of a furrow at the site of the perforation, but not the cortex where astral microtubules are localized (Cao and Wang, 1996). Spermatocytes of the Drosophila mutant asterless fail to form normal asters, but are fully capable of positioning a cleavage furrow with respect to the midzone of a normal-appearing central spindle (Bonaccorsi et al., 1998). The central spindle is comprised of antiparallel, overlapping microtubules at the midzone or the "midbody," which contains an amorphous deposit of electron-dense materials (McIntosh and Landis, 1971) that prevents binding of antitubulin antibodies. The midbody has been found to act as the site for accumulation of "chromosomal passenger proteins" (Earnshaw and Bernat, 1991) involved in furrow positioning (Martineau et al., 1995; Wheatley and Wang, 1996) and sustained cytokinesis (Wheatley and Wang, 1996; Savoian et al., 1999).
Chromosomal passenger proteins, such as INCENPs, TD-60, CHO-1, CENP-E, and CENP-F, are suggested to ride on chromosomes to the metaphase plate (Earnshaw and Mackay, 1994) where they redistribute at anaphase onset to the equatorial cortex, perhaps defining cleavage furrow position. However, removal of chromosomes from grasshopper spermatocytes during prometaphase does not affect cytokinesis (Zhang and Nicklas, 1996). Successful ectopic furrows in PtK1 cells appear to require the formation of a spindle midzone, which localizes INCENP and CHO-1, between disjoined asters that never possessed intervening chromosomes (Savoian et al., 1999). Such evidence suggests that spindle microtubules, but not chromosomes, are essential for proper distribution of chromosomal passenger proteins. Surprisingly, Caenorhabditis elegans embryos do not appear to require the spindle midzone for furrow initiation, though the midzone is necessary for the completion of cytokinesis (Jantsch-Plunger et al., 2000).
Even as different spindle constituents appear sufficient to initiate a cleavage furrow in different cell types, it is unclear how microtubules that are always present in these cells contribute to initiation. Microtubules comprise spindle constituents such as asters or the central spindle and act as the scaffolding on which the midzone assembles and localizes factors critical for cytokinesis. It remains to be tested, however, whether microtubules are sufficient to position the furrow in the absence of other structural constituents of the spindle apparatus. In this study, we mechanically altered grasshopper spermatocytes to test one structural constituent of the spindle apparatus at a time, so as to systematically narrow down the minimal requirement for induction of cell cleavage.
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Results and discussion |
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Cell cleavage can also be induced in the absence of asters
Despite the ability of asters to induce cell cleavage in grasshopper spermatocytes, we found that asters are not essential for cytokinesis in these cells. We produced cells lacking asters by cutting and removing entire spindle poles from late anaphase or early telophase cells (Fig. 2 A, ac). We then rotated the remaining central spindle fragment 90° from the equatorial cortex to avoid any predeposited furrow signals (Fig. 2 A, d). Despite being truncated (Fig. 2 B, 0 min onward; Fig. 2 C, 0 onward), lacking astral microtubules (MT and Overlay) and pronuclei (DAPI), the central spindle in all 21 cells produced remains well organized and initiates a cleavage furrow (Fig. 2 B, 10, arrows; Fig. 2 C, 8, arrows). The cytoskeleton in cells fixed shortly after furrowing shows the expected stage-specific structures: bundled microtubule arrays with a distinct midzone (Fig. 2 B, MT, arrows) surrounded by actin filaments (AF and Overlay, arrows). Furrow ingression (Fig. 2 C, 16) in such manipulated cells is normal, showing a well-organized central spindle and a contractile ring enriched in actin filaments at the midzone (Overlay).
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Cell cleavage can be induced by microtubules as the only spindle structural constituent
To produce cells containing microtubules as the only spindle structural constituent, we removed both asters and all chromosomes (Fig. 3 A, ac; Fig. 3 B, 08 min) from cells in metaphase (12 of 20 cells had furrow initiation and ingression). These manipulations not only remove other confounding spindle constituents, but also induce disassembly of the spindle and assembly of radiating microtubules bundled together with mitochondria (Fig. 3 A, d; Fig. 3 B, 37). Obviously, disassembly destroys bipolarity of the metaphase spindle and normal distribution of furrow signaling molecules, potentially from asters (Rappaport, 1996) or chromosomes (Earnshaw and Mackay, 1994). Radiating microtubules exhibit dynamics similar to that observed in metaphase (Cassimeris et al., 1988), repeatedly changing their length and distribution to transiently organize mono- or bipolar pseudospindles (Fig. 3 B, 70217; see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1), and ultimately give rise to relatively disorganized arrays of bundled microtubules (Fig. 3 B, 290296). Induction of cell cleavage occurs at midzones of bundled microtubule arrays (Fig. 3 B, 290296, arrows) organized by randomly formed pseudopoles (290296, *). Cells fixed at initiation (296) show no chromosomes (DAPI) and exhibit central spindlelike bundled microtubule arrays with lightly stained midzones (Fig. 3 B, MT, arrows), where actin filaments are localized (AF and Overlay). Often, furrow induction occurs at multiple locations (Fig. 3 C, 118128, arrows and arrowheads; see Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1) due to random distribution of bundled microtubule arrays (43 onward). Many of these furrows, however, are transient and regress as microtubules reorganize (Fig. 3 C, 118128, arrowheads). Successful furrows are usually induced independently at the midzones of persistent microtubule bundles (Fig. 3 C, 118137, arrows), supporting earlier discoveries that furrow ingression does not require a complete contractile ring (Rieder et al., 1997). During ingression, these independent furrows can change directions of inward movement and force bundled microtubule arrays together (Fig. 3 C, 128153, arrows). Cells fixed at completion of ingression (153) exhibit one central microtubule bundle with a distinct midzone (Fig. 3 C, MT, arrow) colocalized precisely with a contractile ring (AF and Overlay).
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Cleavage furrow abscission in micromanipulated cells
Certain spindle constituents, such as centrosomes (Piel et al., 2001), may play a critical role during furrow abscission, resolution of the midbody and separation of daughter cells (Ou and Rattner, 2002), which can be tested in micromanipulated cells containing defined spindle constituents.
In cells containing asters (Fig. 5 A, 0, *) alone, furrow ingression (68, arrows) tightly constricts the bundled microtubule array (88). Surprisingly, despite the presence of centrosomes, such furrows (n = 5) eventually regress (Fig. 5 A, 16 h). In contrast, furrow ingression (Fig. 5 B, 1272, arrows; see Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1) in cells (n = 11) lacking asters and pronuclei sustains and leads to midbody abscission, yielding two daughter cells (17.1 h). Owing to observations that grasshopper spermatocytes remain connected after cytokinesis (Carlson and Handel, 1988) and cell cleavage regresses in C. elegans Zen-4 mutants even after daughter cells enter G1 phase (Severson et al., 2000), we compared visually abscised experimental cells (n = 5) with nonmanipulated controls (n = 5). In both cases, injection of rhodamine dextran (Bukauskas et al., 1992) into one daughter cell results in accumulation of fluorescence in the other (Fig. 5 B'), showing that micromanipulation does not alter the final stage of cell cleavage. When microtubules are the only remaining spindle constituent (n = 6; Fig. 5 C), furrow ingression (155243, arrows) is usually followed by regression (Fig. 5 C, 315390, arrow; see Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1), except in one cell where successful abscission is observed (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1; Video 5).
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In conclusion, our results demonstrate that microtubules, whether from asters, the central spindle, or even a collapsed spindle lacking both asters and chromosomes, are sufficient to induce cell cleavage and maintain furrow ingression. We do not imply that microtubules act as an independent source of the furrowing signal, as a plethora of motor proteins and regulatory factors are critical for proper cytokinesis (for reviews see Earnshaw and Mackay, 1994; Larochelle et al., 2000; Robinson and Spudich, 2000; Glotzer, 2001; Guertin et al., 2002). However, without asters and chromosomes, microtubules appear sufficient to mediate the distribution of associated cytokinetic factors, as judged by their ability to form midzone-bearing microtubule bundles and initiate cell cleavage. We therefore propose that microtubules, regardless of source, are the only structural constituent of the spindle apparatus required for induction of cell cleavage.
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Materials and methods |
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Micromanipulation with digital-enhanced polarization microscopy
Micromanipulations, such as chromosome/aster removal and cell cutting (Zhang and Nicklas, 1999), were performed using a fine glass needle with a tip diameter of 0.1 µm, maneuvered using a Burleigh MIS-5000 series piezoelectric micromanipulator. Cells were observed with a high extinction/high resolution polarization microscope modified according to Inoué and Spring (1997), except from an Axiovert 100 microscope equipped with a 1.4 NA achromatic-aplanatic condenser, an infinity-corrected 1.4 NA/63x Plan-Apochromat objective lens (Carl Zeiss MicroImaging, Inc.), and a cooled CCD digital camera (ORCA-100; model C474295; Hamamatsu). Images were acquired and processed using Image Pro Plus software (Media Cybernetics).
Immunofluorescence microscopy
Cells were microfixed (Nicklas et al., 1979) at the moment of interest and stained for microtubules, actin filaments, and chromosomes. In brief, target cells on the coverslip were fixed by micropipetting microfixative (2% glutaraldehyde, 1% CHAPS, 0.33 µM rhodamine-phalloidin [Molecular Probes] in Pipes buffer) in cells' vicinity. The coverslip was then transferred into macrofixative (0.1% glutaraldehyde, 0.5% NP-40 in Pipes buffer). Microtubules were stained with antiß tubulin (clone KMX-1; Chemicon) primary antibody and Alexa® Fluor 488conjugated secondary antibody (Molecular Probes). Actin filaments were stained with 0.165 µM rhodamine-phalloidin (Molecular Probes). Coverslips were mounted in Vectashield (Vector Laboratories) containing DAPI to stain chromosomes. Image stacks were acquired using a confocal microscope (Leica TCS), processed in Adobe Photoshop 5.0®, and reconstructed using Simple PCI software (C-imaging Systems).
Microinjection
We have succeeded in microinjection of grasshopper spermatocytes. In brief, micropipettes were pulled with a Flaming/Brown puller (model P-87; Sutter Instrument Co.), loaded with 2 mg/ml rhodamine-labeled dextran in a Pipes buffer, and maneuvered with a piezoelectric micromanipulator. A home-made high pressure (up to 60 psi) pneumatic system was used to simultaneously drive needle penetration and delivery of the fluorescent dye.
Nocodazole treatment
1 mg/ml nocodazole in Belars ringers was prepared from 10 mg/ml nocodazole in DMSO stock (Sigma-Aldrich) and micropipetted to the cell culture to a final concentration 20 µg/ml.
Online supplemental material
The videos of polarization microscope sequences corresponding to Fig. 1 E (Video 1), Fig. 3 B (Video 2), Fig. 3 C (Video 3), Fig. 5 B (Video 4), and Fig. 5 C (Video 5) and Figs. S1 and S2 are available at http://www.jcb.org/cgi/content/full/jcb.200301073/DC1.
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Acknowledgments |
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This work was supported in part by a National Science Foundation (NSF) GK-12 Program fellowship to G.B. Alsop and an NSF Cellular Organization grant to D. Zhang.
Submitted: 21 January 2003
Accepted: 16 June 2003
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References |
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Bonaccorsi, S., M.G. Giansanti, and M. Gatti. 1998. Spindle self-organization and cytokinesis during male meiosis in asterless mutants of Drosophila melanogaster. J. Cell Biol. 142:751761.
Bucciarelli, E., M.G. Giansanti, S. Bonaccorsi, and M. Gatti. 2003. Spindle assembly and cytokinesis in the absence of chromosomes during Drosophila male meiosis. J. Cell Biol. 160:993999.
Bukauskas, F.F., C. Kempf, and R. Weingart. 1992. Cytoplasmic bridges and gap junctions in an insect cell line (Aedes albopictus). Exp. Physiol. 77:903911.[Abstract]
Canman, J.C., D.B. Hoffman, and E.D. Salmon. 2000. The role of pre- and post-anaphase microtubules in the cytokinesis phase of the cell cycle. Curr. Biol. 10:611614.[CrossRef][Medline]
Cao, L., and Y.L. Wang. 1996. Signals from the spindle midzone are required for the stimulation of cytokinesis in cultured epithelial cells. Mol. Biol. Cell. 7:225232.[Abstract]
Carlson, J.G., and M.A. Handel. 1988. Intercellular bridges and factors determining their patterns in the grasshopper testis. J. Morphol. 196:173185.[Medline]
Cassimeris, L., S. Inoué, and E.D. Salmon. 1988. Microtubule dynamics in the chromosomal spindle fiber: analysis by fluorescence and high-resolution polarization microscopy. Cell Motil. Cytoskeleton. 10:185196.[Medline]
Earnshaw, W.C., and R.L. Bernat. 1991. Chromosomal passengers: towards an integrated view of mitosis. Chromosoma. 100:139146.[Medline]
Earnshaw, W.C., and A.M. Mackay. 1994. Role of nonhistone proteins in the chromosomal events of mitosis. FASEB J. 8:947956.
Eckley, D.M., A.M. Ainsztein, A.M. Mackay, I.G. Goldberg, and W.C. Earnshaw. 1997. Chromosomal proteins and cytokinesis: patterns of cleavage furrow formation and inner centromere protein positioning in mitotic heterokaryons and mid-anaphase cells. J. Cell Biol. 136:11691183.
Glotzer, M. 2001. Animal cell cytokinesis. Annu. Rev. Cell Dev. Biol. 17:351386.[CrossRef][Medline]
Guertin, D.A., S. Trautmann, and D. McCollum. 2002. Cytokinesis in eukaryotes. Microbiol. Mol. Biol. Rev. 66:155178.
Hiramoto, Y. 1971. Analysis of cleavage stimulus by means of micromanipulation of sea urchin eggs. Exp. Cell Res. 68:291298.[Medline]
Inoué, S., and K.R. Spring. 1997. Video Microscopy: the Fundamentals. Plenum Publishing Corp., New York/London. 709 pp.
Jantsch-Plunger, V., P. Gönczy, A. Romano, H. Schnabel, D. Hamill, R. Schnabel, A.A. Hyman, and M. Glotzer. 2000. CYK-4: a Rho family GTPase activating protein (GAP) required for central spindle formation and cytokinesis. J. Cell Biol. 149:13911404.
Larkin, K., and M.V. Danilchik. 1999. Microtubules are required for completion of cytokinesis in sea urchin eggs. Dev. Biol. 214:215226.[CrossRef][Medline]
Larochelle, D.A., N. Gerald, and A. De Lozanne. 2000. Molecular analysis of racE function in Dictyostelium. Microsc. Res. Tech. 49:145151.[CrossRef][Medline]
Martineau, S.N., P.R. Andreassen, and R.L. Margolis. 1995. Delay of HeLa cell cleavage into interphase using dihydrocytochalasin B: retention of a postmitotic spindle and telophase disc correlates with synchronous cleavage recovery. J. Cell Biol. 131:191205.[Abstract]
McIntosh, J.R., and S.C. Landis. 1971. The distribution of spindle microtubules during mitosis in cultured human cells. J. Cell Biol. 49:468497.
Mishima, M., S. Kaitna, and M. Glotzer. 2002. Central spindle assembly and cytokinesis require a kinesin-like protein/RhoGAP complex with microtubule bundling activity. Dev. Cell. 2:4154.[Medline]
Nicklas, R.B., B.R. Brinkley, D.A. Pepper, D.F. Kubai, and G.K. Rickards. 1979. Electron microscopy of spermatocytes previously studied in life: methods and some observations on micromanipulated chromosomes. J. Cell Sci. 35:87104.[Abstract]
Ou, Y., and J.B. Rattner. 2002. Post-karyokinesis centrosome movement leaves a trail of unanswered questions. Cell Motil. Cytoskeleton. 51:123132.[CrossRef][Medline]
Piel, M., J. Nordberg, U. Euteneur, and M. Bornens. 2001. Centrosome-dependent exit of cytokinesis in animal cells. Science. 291:15501553.
Rappaport, R. 1961. Experiments concerning the cleavage stimulus in sand dollar eggs. J. Exp. Zool. 234:167171.
Rappaport, R. 1996. Cytokinesis in Animal Cells. Cambridge University Press, Cambridge, UK. 400 pp.
Rieder, C.L., A. Khodjakov, L.V. Paliulis, T.M. Fortier, R.W. Cole, and G. Sluder. 1997. Mitosis in vertebrate somatic cells with two spindles: implications for the metaphase/anaphase transition checkpoint and cleavage. Proc. Natl. Acad. Sci. USA. 94:51075112.
Robinson, D.N., and J.A. Spudich. 2000. Towards a molecular understanding of cytokinesis. Trends Cell Biol. 10:228237.[CrossRef][Medline]
Salmon, E.D., and S.M. Wolniak. 1990. Role of microtubules in stimulating cytokinesis in animal cells. Ann. NY Acad. Sci. 582:8898.[Abstract]
Savoian, M.S., W.C. Earnshaw, A. Khodjakov, and C.L. Rieder. 1999. Cleavage furrows formed between centrosomes lacking an intervening spindle and chromosomes contain microtubule bundles, INCENP, and CHO-1 but not CENP-E. Mol. Biol. Cell. 10:297311.
Severson, A.F., D.R. Hamill, J.C. Carter, J. Schumacher, and B. Bowerman. 2000. The aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis. Curr. Biol. 10:11621171.[CrossRef][Medline]
Wheatley, S., and Y.L. Wang. 1996. Midzone microtubule bundles are continuously required for cytokinesis in cultured epithelial cells. J. Cell Biol. 135:981989.[Abstract]
Zhang, D., and R.B. Nicklas. 1996. "Anaphase" and cytokinesis in the absence of chromosomes. Nature. 382:466468.[CrossRef][Medline]
Zhang, D., and R.B. Nicklas. 1999. Micromanipulation of chromosomes and spindles in insect spermatocytes. Methods Cell Biol. 61:209218.[Medline]
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