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Department of Biology, Middlebury College, Bicentennial Way, Middlebury, VT 05753
Many students use "mitosis" and "cell division" interchangeably, apparently unaware that cell division includes mitosis as well as another, equally important process called cytokinesis. Mitosis separates duplicated chromosomes, whereas cytokinesis divides the parent cell (and duplicated chromosomes) into two daughter cells. Although these phenomena usually are linked temporally as well as spatially, mitosis can and does occur in the absence of cytokinesis (e.g., during the early development of Drosophila and other insects when many mitotic cycles occur before daughter nuclei become compartmentalized into separate cells; Alberts et al., 2002). Moreover, different mechanisms are responsible for each phenomenon. Thus, we give mitosis and cytokinesis different names, and it would be unfortunate (and especially confusing for students) if the definition of the former were expanded to include cytokinesis, as claimed in a recent review (Rieder and Khodjakov, 2003).
Thinking about these phenomena, I realized I was much more familiar with mitosis than I was with cytokinesis. Not only had I read contemporary reviews of the subject (Rieder and Khodjakov, 2003 [including 10 videos]; Mitchison and Salmon, 2001) and of mitosis Web sites (Blystone, 2003), I recently had reviewed videos and research articles dealing with the phenomenon in past Video Views and Reviews (Watters, 2003, 2002). My knowledge of cytokinesis, by contrast, was much less current and informed, and I was delighted when I had the opportunity in July 2004 to attend a conference on cytokinesis sponsored by the American Society for Cell Biology. This Feature arises from my experience at the conference and a subsequent search on High Wire Press (http://highwire.stanford.edu) for current research articles and videos on cytokinesis. Interested readers also may want to consult an older, comprehensive review written by a pioneer in the field (Rappaport, 1996) and the collaborative Web site maintained by the "Cytokinesis Mafia": http://www.bio.unc.edu/faculty/salmon/lab/mafia/index.html.
As most students know, mitosis entails the condensation of duplicated chromosomes during prophase; their alignment and separation along the mitotic apparatus (or spindle, as it is sometimes called) during, respectively, metaphase and anaphase; and the chromosomes' compartmentalization and relaxation during telophase. Once the chromosomes have been separated, cytokinesis begins, typically producing a cleavage furrow oriented at right angles to the axis of the spindle and passing through the plane of the metaphase plate. Anaphase, telophase, and furrow formation in a cultured animal cell are illustrated in Figure 1 (taken from Alsop and Zhang, 2003). Mechanistically, chromosomal movement occurs through the agency of the mitotic spindle, the kinetochores that attach chromosomes to various microtubular fibers of the spindle, the dynamic instability of microtubules themselves, and microtubular "motor" proteins (Rieder and Khodjakov, 2003). In contrast, cytokinesis and the formation of a cleavage furrow depend on the contraction of a cortical ring of actin and myosin filaments located just beneath the plasma membrane (Alberts et al., 2002). Ring constriction during cytokinesis is often referred to as purse-stringing, because the dividing daughter cells often resemble bags with gradually closing necks. The relative organization of the spindle and cortical ring is illustrated in Figure 2 (taken from Alsop and Zhang, 2004).
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Here I review recently published videos that depict the roles played by myosin II in contraction of the cortical ring during cellularization and cytokinesis in early development (Royou et al., 2004), by spindle and astral microtubules in regulating the formation of cleavage furrows during the cleavage of primary spermatocytes (Inoue et al., 2004), and by a novel kinase in the regulation of cortical ring formation in postembryonic development (D'Avino et al., 2004). All three sets of videos were obtained from Drosophila material and nicely illustrate the complexity of cytokinesis in a single organism. For the sake of simplicity, however, I have focused on those aspects of the articles that relate to phenomena depicted in the videos. The videos and articles are suitable for intermediate and advanced undergraduate students as well as graduate students, and their study could be extended to cytokinesis in other organisms through a High Wire search.
I appreciate hearing your reactions to these reviews and your suggestions of other peer-reviewed videos for possible review as educational material.
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MYOSIN II, CELLULATION, AND CYTOKINESIS IN DROSOPHILA EMBRYOS |
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The other four videos archived with Royou et al. (2004) concern the behavior of myosin II in cellularization. To understand this more complex phenomenon and the very striking videos, the viewer must first appreciate the idiosyncratic nature of early Drosophila development (Gilbert, 2003). Following fertilization, 13 rounds of mitotic division create a multinucleate (or syncytial) embryo in which nuclei first are evenly distributed throughout the very yolky, single-celled egg and then become localized around the periphery (Figure 4). At that time, cleavage furrows begin forming around the nuclei synchronously and perpendicular to the egg surface, to form the cellular blastoderm in a process called cellularization. Subsequent mitotic events during Drosophila development are accompanied by cytokinesis (as discussed in the previous paragraph).
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At this point, most students may feel confused. On one hand, some of the very colorful aspects of cellularization and cytokinesis seem similar, especially the green honeycomb-like patterns of cortical myosin localization common to both. Yet, the two phenomena differ remarkably in their global aspects. Not only does cellularization seem longer and biphasic, but it occurs in the absence of mitosis, unlike cytokinesis. During cellularization, there are no spindles or metaphase plates to orient the cortical rings. (As suggested by classical histology, the rings seem to form in association with centrosomes and astral MT fibers that are located at the apical surfaces of blastoderm nuclei [see Figure 9.3 in Gilbert, 2003]) Indeed, these rings engirdle intact nuclei as furrowing proceeds, and they contract (as during cytokinesis) only during the final stages of cellularization. (The last part of authors' Movie 3 presents an excellent view of purse-stringing.) How does one make sense of these phenomena?
Heuristically, it may be worthwhile for students first to consider the possibility that cellularization and cytokinesis exhibit a similar sequence of steps or stages and then to hypothesize what these might be. One possible hypothesis contains the following sequence: 1) cortical recruitment of myosin and actin; 2) their assembly into a peripheral ring of fibers; 3) orientation or positioning of the ring; and, finally, 4) ring contraction. The two phenomena might then be understood to differ primarily in the length and prominence of the third step. During cytokinesis, cortical assembly and ring orientation around the metaphase plate seem to be rapid, nearly simultaneous events, whereas during cellularization, ring assembly occurs at the apical surface of a nucleus, and the ring then becomes oriented in a more protracted manner before contraction begins, by engirdling the nucleus and moving tens of microns in a basal direction in a process called ingression. Ring orientation and movement is also the prominent mechanistic feature of cellularization, because ingression of the cleavage furrow, which forms at the apical surface at the same time as the ring, seems tightly correlated with ring ingression. Following ingression, furrow closure during cellularization and both furrow formation and closure during cytokinesis require ring contraction, which is a relatively rapid event. Students are likely to raise many questions during their discussion. For example, are these four stages necessary and sufficient to describe both phenomena? Have some aspects of either phenomena been omitted? How might the existence of these stages be tested? Specifically, what causes and regulates step 3? The authors report the effects of cytochalasins and colchicine on cellularization, and examination of Movie 4 and authors' Figure 3 would prove instructive in answering this last question. So would the authors' data concerning the effect of the sqh1 mutant on cellularization (authors' Figure 5).
As it turns out, ring movement during cellularization requires the presence of MT, and although the ring must be present, actin and myosin do not seem to be actively involved in their own relocation. The MT requirement is consonant with older observations on fixed material showing the correlated elongation of blastoderm nuclei and the growth of MT and cleavage furrows (see Figure 9.3A in Gilbert, 2003). Students can conclude then, as do the authors, that furrow ingression and basal closure during cellularization are partially independent events. Reaching this conclusion then might lead to their wondering whether these two phenomena also might be partially independent, albeit concurrent, processes during cytokinesis.
Cellularization also provides a broad window for viewing other, more subtle aspects of cytokinesis, such as the increase in cell surface that accompanies cell division. More thoughtful students may wonder whether purse-stringing without the addition of new plasma membrane can separate daughter cells. In the case of cellularization, however, it should be obvious to most that furrow formation requires considerable expansion of the plasma membrane. The present study does not address how new membrane is added during cellularization, whether it is added during cytokinesis also, and in both instances, where the membrane might originate.
Students querying the more general importance of MT in organizing cytokinesis will find the next two articles especially interesting.
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ROLE OF MICROTUBULES IN CYTOKINESIS |
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Several features of cell division in Drosophila spermatocytes changed in response to reduced levels of the Orbit protein, and characterizing these changes could form the basis of a student discussion of the mechanics of furrow formation and cytokinesis. The changes evident in Video 5 (Figures 6E-G) include an irregularly shaped spindle, apparently fewer spindle MT, less regular arrays of both interior and peripheral MT, and a failure of microtubular detachment at the centrosome poles as anaphase proceeded. In spite of these changes, a cleavage furrow formed, albeit more slowly, taking 18 minutes to reach a stage of ingression comparable to the wild-type cell (compare Figures 6G and H with Figures 6C and D). Purse-stringing in these mutants never advanced to the same degree as in the wild-type cells, however. Control cells formed highly fluorescent midbodies, which consisted of thin necks of cytoplasm densely packed with MT and connecting daughter cells (see the 25-minute frame of Movie 1 and authors' Figure 1). Moreover, according to the authors, the furrows in mutant cells later regressed. They argue that cleavage furrow formation (and cytokinesis) was initiated by astral (peripheral) MT, but completed through the intercession of spindle (interior) MT. The germinal importance of astral MT is further strengthened by events portrayed in Video 4, where a furrow formed parallel to the spindle (rather than perpendicular to it) and passed more or less along a plane through both the centrosomes! Having considered the importance of centrosomes during the early stages of cellularization (discussed previously) some students will find it interesting that astral MT may be involved in spermatocyte cell division. Some students will also be frustrated that neither movie of an orbit mutant cell (Videos 4 and 5) shows furrow regression, precluding their tracking the terminal failure of cytokinesis with other changes in microtubular behavior.
Responding to these movies and to the authors' suggestion that peripheral and spindle MT play different roles in cytokinesis, students likely will pose numerous questions concerning, for example, the location of Orbit in wild-type spermatocytes and the consequence of its absence in mutants. These latter concerns are addressed in authors' Figures 3, 6, and 7, respectively, using immunofluorescence of fixed preparations. In similar preparations of mutant cells, actin and Anillin (a contractile ring protein) failed to become localized in a ring surrounding the midregion of the spindle, and this result will confuse many students who associate the formation of a cleavage furrow with the presence of a contractile ring of actin, myosin, and such ancillary proteins as Anillin. How could the furrows seen in the video have been generated by incomplete rings? Confused students also might want to know how GFP chimeras of these dislocated proteins behave in squashes of living, mutant cells and how that behavior correlates with furrows that form and later regress in the mutants. More advanced students, aware of the kinds of GFPs available for tracking protein movement, would likely also be interested in tracking the behavior of a red fluorescent protein chimera of Orbit (YFP-Orbit) and GFP-tubulin coexpressed in wild-type cells. Inquisitive students also will wonder how the absence of a critical protein at the kinetochore or (+) end of a spindle MT can affect the MT's attachment to the centrosome at its (-) end. All in all, students will find this paper and movies very provocative.
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KINASE REGULATION OF CYTOKINESIS: THE ROLE OF STICKY |
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Images of a control cell (transfected with dsRNA for GFP) undergoing cytokinesis are presented in Figure 7, showing the beginning of furrow formation (Figure 7A), the completion of the furrow (Figure 7B), and much later, after interphase nuclei have been reformed in the daughter cells (Figure 7C), which continue to be linked by a midbody. When viewing the control video, students will find it useful to review the final steps of mitosis, the stages of cytokinesis, and how the two are related temporally.
While watching the video of cells transfected with sti dsRNA (Figures 7D-F), I would subdivide the students into two groups, asking one group to list the similarities of events portrayed in each video and the other group to characterize the differences. As the students compare their lists, they will refine their own language in order to convey what they have seen, which seems more pedagogically useful than telling them what they are seeing. This procedure also would reinforce what they have learned in the previous two sets of videos. And examining both the videos and the figures in the article could generate more discussion.
In Video 3, the most obvious effect of STI depletion is an apparent increase in the number of furrows generated, which produce cytoplasmic blebs of variable size (Figures 7E and F), in addition to what appears to be a normal cleavage furrow formed at right angles to the mitotic spindle in the dividing experimental cell (Figure 7D). Most students who also examine Video 2, showing cytokinesis in another STI-depleted cell, might agree that although furrows formed in the inhibited cells, they were not as tightly regulated or spatially restricted in the experimental cells as in the control cell. To the extent that two daughter cells seemed to form, cytokinesis seemed temporally and spatially normal in both populations, at least based on what can be seen in the three archived videos (and authors' Figure 4 A). Unfortunately, students who also examine authors' Figure 3 and the related text discussion likely will become confused, because the authors conclude from their Figure 3 data that cytokinesis failed in the treated cells! Cytokinesis does indeed appear to have failed, because after 48 hours of RNAi treatment, 31% of the STI-depleted cells were binucleate, and after 72 hours of treatment, 60% of the cells were binucleate or multinucleate. Other students considering only the higher-magnification, fluorescent images in authors' Figures 4B and C and in the videos would not be persuaded easily that cytokinesis failed, because the control and experimental cells depicted all seemed to have completed the process, and treated cells differed from controls mainly in the larger size of their midbodies and in the presence of blebs. Critical students are unlikely to be convinced by the authors' reasons for choosing to ignore the data in their Figure 4 and in the videos in favor of data presented in Figure 3. The apparent disagreement at this point could provide a foundation for a good discussion on the reproduction of data, sampling errors in small data sets and the variability of RNAi treatment among different experiments.
In any event, most observers would likely agree the cortical rings appear to have contracted normally in both control and STI-depleted cells, and it would be useful to postulate how cytokinesis could have been successful in the former instance, but could have failed in the latter. To formulate hypotheses, students likely will want to know where STI is located normally and whether its expression is restricted and/or its location affected in RNAi-treated cells. Authors' Figure 5 provides useful data in this regard, but students also may wonder about the variability of STI in RNAi-treated cells. Some students also may want to know what happens to midbodies at the end of cytokinesis and whether midbody scission occurs in control cells, but presumably fails in treated cells. This is a crucial point, because one resolution of the paradox raised by the apparently contradictory data in authors' Figures 3 and 4 (and the videos) entails the dissolution of contractile rings in both populations. Such disassembly would be inconsequential (and normal) in control cells already completely separated by scission, but the constricted midbodies of incompletely separated, treated cells could secondarily expand following ring disassembly, resulting in the formation of large, binucleate cells as seen in Figure 3. Unfortunately, midbody scission is not addressed in this paper and little seems to be known about the process. Perceptive students who have read the article closely, however, may also note that Anillin, an actin-binding protein thought to link the contractile ring to the plasma membrane (Somma et al., 2002), is localized in the scission zone of control cell midbodies, but more broadly dispersed in sti RNAi-treated cells. Such dislocation could account for the failure of membrane fusion that must accompany the final stages of scission, and it would be interesting to learn whether STI and Anillin interact, and, given the kinase function of STI orthologs in other cells, whether Anillin is phosphorylated in control cells and dephosphorylated in sti RNAi-treated cells. Finally, based on genetic data (authors' Figure 6H), the authors infer that one of the kinase targets of STI was the myosin RLC coded by the spaghetti squash gene. Thoughtful consideration of this effect could produce an alternative hypothesis: Phosphorylated RLC produces a more stable contractile ring than the dephosphorylated form thought to result from sti iRNA-treatment. If true, multi-nucleation of treated cells could have resulted from premature contractile ring disassembly and not from the absence of scission.
Testing these alternative hypotheses—indeed, understanding how contraction of an actomyosin ring actually separates one cell into two—requires more visual and molecular information about the final steps of cytokinesis than has been provided by any of these videos or papers. Final discussion of these papers, therefore, might well involve hypothesis-building and literature searches concerning other instances of membrane expansion and fusion.
Address correspondence to: Christopher Watters (watters{at}middlebury.edu).
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REFERENCES |
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TOPMYOSIN II, CELLULATION, AND...ROLE OF MICROTUBULES IN...KINASE REGULATION OF...REFERENCES |
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Alsop, G.B., and Zhang, D. (2003). Microtubules continuously dictate distribution of actin filaments and positioning of cell cleavage in grasshopper spermatocytes. J. Cell Sci.117 , 1591-1602.
Alsop, G.B., and Zhang, D. (2004). Microtubules are the only structural constituent of the spindle apparatus required for induction of cell cleavage. J. Cell Biol.162 , 383-390.
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Mitchison, T.J., and Salmon, E.D. (2001). Mitosis: a history of division. Nat. Cell Biol.3 (1), E17-21.[CrossRef][Medline]
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Rappaport, R. (1996). Cytokinesis in Animal Cells. Developmental and Cell Biology Series, ed. P.W. Barlow, J.B.L. Bard, P.B. Green, and D.L. Kirk. Cambridge, UK: Cambridge University Press.
Rieder, C.L., and Khodjakov, A. (2003). Mitosis
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Royou, A., Field, C., Sisson, J.C., Sullivan, W., and Karess, R.
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Schneider, I. (1969). Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morphol. 27,353 -365.
Somma, M.P., Fasulo, B., Cenci, G., Cundari, E., and Gatti, M.
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Watters, C. (2003). Video Views and Reviews.Cell Biol. Ed. 2(1),9 -13.[CrossRef]
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