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Department of Biology, Middlebury College, Bicentennial Way, Middlebury, VT 05753
Here I review videos depicting various aspects of microtubule dynamics, from papers that were published in Journal of Cell Biology (JCB). The papers describe, respectively, changes in the activity of the G protein Rho-A during cell division (Yoshizaki et al., 2003), the dynamic behavior of microtubules attached to kinetochores (Maddox et al., 2003), and the targeting of microtubules to sites of focal adhesion in migrating fibroblasts (Krylyshkina et al., 2003). In preparing this review, I used the extensive archive of video material at the JCB's Web site entitled "Annotated Video Collection" (AVC; (http://www.jcb.org/misc/annotatdvideo.shtml). I commend the editors of JCB for providing this valuable service, and I encourage editors of other journals that archive similar records to organize their collections in a similar fashion.
What makes the AVC especially valuable is evident in its title. AVC collates peer-reviewed research articles and their supplemental video clips conceptually by cellular topic and subtopic and provides a brief annotation for each video regarding content. Within each heading the articles are arranged in reverse chronological order, with the more recent articles appearing near the top of each listing. The annotations are brief summaries of research results and highlighted key words within each annotation provide hyperlinks to the complete article in JCB and to some of the supplemental videos. The annotations are terse and well written (apparently by a single individual), and occasionally the articles seem connected (if only by serial juxtaposition). Using AVC, Cell Biology Education readers and their students could locate videos of interest and, also, could organize journal club discussions or sections of advanced courses around the various topics or subtopics. Moreover, a link to the URL would be a useful addition to the Web page of any course interested in cell biology. AVC is current through the end of 2002 (Volume 159, Number 4, of JCB), which is probably the cutoff point for public access to the articles. I enthusiastically recommend AVC to readers of these reviews.
Again, I invite your comments on these reviews and your suggestions of other peer-reviewed videos for possible review as educational material.
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BEHAVIOR OF RHO-FAMILY GTPASES DURING CELL DIVISION |
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 TOP BEHAVIOR OF RHO-FAMILY GTPASES... MOVEMENT OF KINETOCHORES AND...TARGETING OF MICROTUBULES TO...REFERENCES |
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Following an introduction to FRET, students can also explore the cyclical inactivation and reactivation of Rho A as interphase alternates with mitosis and cytokinesis, and they can begin to see the cellular events in their biochemical context. Since the paper also presents similar data for other Rho-family G proteins and their regulators, study can be expanded to include other possible regulatory mechanisms on mitosis and possible interactions among the various factors. Finally, students with more of a molecular bent can use the paper and some of its citations to explore the creation and testing of genetically encoded Raichu probes of G proteins. This paper is a nice, varied teaching resource.
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MOVEMENT OF KINETOCHORES AND MICROTUBULES DURING MITOSIS |
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TOPBEHAVIOR OF RHO-FAMILY GTPASES...MOVEMENT OF KINETOCHORES AND...TARGETING OF MICROTUBULES TO...REFERENCES |
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Most students know the mitotic spindle forms during early prophase by microtubule growth outward from two poles (centrosomes). During spindle formation, duplicated chromosomes condense, remaining attached at a common centromere, and the nuclear envelope disappears. Those elongating microtubules that contact the centromere become attached at a specialized protein structure called a kinetochore and are called kinetochore tubules. Several tubules attached to the same kinetochore coalesce to form a kinetochore fiber. The kinetochores, in turn, are thought to "cap" the growing () ends of the anchored tubules. Paired chromosomes with+kinetochore fibers extending toward the opposing centrosomes are effectively "captured" within the spindle, which is formed by other, parallel (or polar) microtubules that have grown from pole to pole and are not attached to kinetochores (see, e.g., Lodish et al., 2003). Tubular bundles or fibers are the spindle units visible by light microscopy.
While many students may appreciate the structure and the origin of the spindle, as presented above, some, especially introductory students, may also equate the "finished" product with the static images they have seen in texts or histological material. They may only vaguely be aware that the spindle, including both kinetochore and polar tubules and fibers, is a dynamic structure. Microtubules, in fact, are steady-state organelles: that is, at times they may appear to be constant in length because assembly at the (-) ends is balanced more or less by disassembly at the+ () ends located in the centrosome. Such steady-state behavior was deduced from different experiments and observations, most recently from viewing the poleward movement of spots or "speckles" of labeled subunits (tubulin) in metaphase tubules that for the most part were unlabeled (Mitchison and Salmon, 2001).
A spindle consisting of speckled microtubules looks as if it has measles, and when examined during metaphase in living material, individual measles spots (speckles) were not static. They moved poleward at similar velocities, although the spindle fibers themselves appeared to be quite stable and constant in length (see also Lodish et al., 2003). Most students will appreciate the simplest explanation of such movement, which entails tubulin subunits "treadmilling" from their point of assembly at the (+) ends of microtubules to the (-) ends where disassembly occurs. In kinetochore tubules, treadmilling occurs from the kinetochore towards the centrosome.
In the present report, Maddox et al. (2003) extend the collaborative effort that produced the 2001 study by Mitchison and Salmon to include measurements of the anaphase movements toward the poles of both labeled kinetochores and tubule speckles. In isolated egg spindle preparations, kinetochores were labeled with red fluorescent antibodies prepared against the kinetochore protein CENPH-A and green-speckled microtubules were created by introducing a substoichometric "pulse" of X-rhodamine-labeled tubulin.
Figure 2 summarizes the behavior of kinetochores and spindle microtubules at roughly the beginning (Figure 2A), the middle (Figure 2B), and the end of anaphase (Figure 2C) of anaphase. While colorful, they do not do adequate justice to the vivid impressions conveyed by the time-lapse video (Maddox et al., 2003, Video 3), which should be examined closely and viewed repetitively. In the video, students will note that speckles, as well as kinetochores, move poleward as chromosomes separate during anaphase, and perhaps they can also detect differences in the respective rates of movement. Data from this video (summarized in Maddox et al., 2003, Fig. 3) indicate that speckles usually moved at different rates than did their respective kinetochores—sometimes more slowly, sometimes more rapidly—but only rarely did a kinetochore and the speckles of its attached tubules move at the same velocity. This is an unexpected result and one likely to puzzle most students (and many of their teachers), and the quantitative reduction of these data presented by Maddox et al. 2003, (Fig. 3) is not easy to follow. Their discussion and explanatory models (Fig. 1) are quite helpful, however. When dissecting this paper with intermediate or advanced students, I suggest it would be appropriate to view the video several times and then to try tracking the behavior of specific kinetochores and their respective tubule speckles. Then it would be useful to explain how the velocities of single kinetochores and their respective tubule speckles were obtained from kymograph traces along single fibers, using the Mitchison and Salmon (2001) paper and Figure 20-38 of Lodish et al. (2003) as aids. Following these steps, students should be in a stronger position to appreciate the authors' three models for the various movements (Maddox et al., 2003, Fig. 1).
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TARGETING OF MICROTUBULES TO FOCAL ADHESIONS |
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TOPBEHAVIOR OF RHO-FAMILY GTPASES...MOVEMENT OF KINETOCHORES AND...TARGETING OF MICROTUBULES TO...REFERENCES |
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In their brief report, Krylyshkina et al. (2003) describe the dynamic behavior of microtubules polymerizing near focal adhesions in goldfish fin fibroblasts doubly transfected with genes for two chimeric proteins, GFP-CLIP-170 and DsRed-zyxin. The polymerizing (+) tips of microtubules were marked by the presence of CLIP-170, a microtubule cross-linking protein with a Mr of 170 kDa, joined with GFP (cf. Schroer, 2001). Focal adhesions were identified through the presence of zyxin, an ancillary adhesion protein, labeled with DsRed. The movement or putative growth of green microtubule tips into red adhesion sites was then documented by video fluorescence microscopy. Several confocal images obtained over a 6 s interval and abstracted from a longer dual-color sequence (Supplemental Video 5) are presented in Figure 3.
The apparent targeting of growing microtubules toward a single focal adhesion site is evident in this very striking video sequence, and the close proximity of the green organelles and the red adhesion site is suggested by the yellow spot within the adhesion site and by the very thin focal plane obtained with confocal imaging. Observant students will note that while numerous microtubule tips sequentially enter a single adhesion site and the center of the adhesion site "flickers" over time, the yellow spot does not seem to increase in size or intensity. The more curious and vocal will want to know why not—that is, how the center maintains a steady-state association of adhesive integral membrane proteins and tubular tips—and a good discussion of microtubule dynamics and the optics of confocal imaging can ensue. To address this question further, more advanced students may wish to examine the videos produced by the other imaging technique employed by the authors—total internal reflection fluorescence microscopy, or TIRFM. These data support the confocal observations summarized in Figure 1, but the optics of TIRFM seem too technical for most undergraduate audiences.
Other students may question whether the tips are moving only by microtubular growth—that is, through assembly at their (+) ends—or whether entire organelles might be moving as the result of CLIP-coated tips sliding forward. Possibly, both modes of motility are involved. Addressing this concern will require close reading of Krylyshkina et al. (2003) and further work in the literature (e.g., Schuyler and Pellman, 2001), and an interesting journal club discussion could be developed around this point. It is also interesting to ask, as have Krylyshkina et al., how growing microtubules could be directed or targeted to focal adhesions. The mechanistic model presented in their Figure 5 is especially clear and thoughtful in addressing this matter. Discussion could also be generated toward how the model might be tested experimentally (and the short report expanded into a full length research article). Finally, some students may want to know how such dynamic intracellular behavior relates to cell locomotion, since none of the cells imaged in the eight video sequences archived with this paper seems to be moving across the substratum. In extending their investigation of microtubule targeting and fibroblast motility, students may find an earlier review of microfilament dynamics in lamellipodia helpful (Small et al., 2002). All in all, the paper by Krylyshkina et al. (2003) presents a striking set of video records and a provocative study.
E-mail address: watters{at}middlebury.edu.
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REFERENCES |
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TOPBEHAVIOR OF RHO-FAMILY GTPASES...MOVEMENT OF KINETOCHORES AND...TARGETING OF MICROTUBULES TO...REFERENCES |
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Lodish, H., Berk, A., Matsudaira, P., Kaiser, C.A., Krieger, M., Scott, M.P., Zipursky, S.L., and Darnell, J. (2003).Molecular Cell Biology, 5th ed. , New York: W.H. Freeman.
Maddox, P., Straight, A., Coughlin, P., Mitchison, T.J., and
Salmon, E.D. (2003). Direct observations of microtubule dynamics
at kinetochores in Xenopus extract spindles: Implications for spindle
mechanics. J. Cell Biol.
162(3),377
-382.
Mitchison, T.J., and Salmon, E.D. (2001). Mitosis: A history of division. Nat. Cell Biol. 3,E17 -E21.[CrossRef][Medline]
Schroer, T.A. (2001). Microtubules don and off their caps: Dynamic attachments at plus and minus ends. Curr. Opin. Cell Biol. 13,92 -96.[CrossRef][Medline]
Small, J.V., Stradal, T., Vignal, E., and Rottner, K. (2002). The lamellipodium: Where motility begins. Trends Cell Biol. 12(3),112 -120.[CrossRef][Medline]
Yoshizaki, H., Ohba, Y., Kurokawa, K., Itoh, R.E., Nakamura, T.,
Mochizuki, N. Nagashima, K., and Matsuda, M. (2003). Activity of
Rho-family GTPases during cell division as visualized with FRET-based probes.J. Cell Biol.
162(2),223
-232.
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