It has been shown by Nicklas that stable connections between kinetochores and spindle microtubules are formed only when this junction is under tension. What properties could to attribute to the kinetochore-MT junction so it would become stable only when under tension?
One approach to this question is to give the molecules that connect a kinetochore to a microtubule the properties you want. For example, a motor enzyme that is stably attached to the kinetochore might have motile (and therefore labile) connections with the wall of the microtubule, particularly if it is not a “processive” motor (i.e., one that stays on the MT for long periods). If the motor is trying to walk along the microtubule, but it is prevented from doing so by tension acting in the opposite direction, the motor will stall. If the motor has to change its shape to go through a full cycle of force-producing motor activity, the tension would prevent this shape change and hold it in some intermediate stage. If the release of the motor from the microtubule (to continue with its motility cycle) requires that it finish its shape changes, then tension would lock the motor in this intermediate state, and it would not be able to release, providing a bond with the kinetochore that is stabilized by tension.
The spindle generates forces at multiple places (kinetochores, poles, zones where MTs from the two ends of the spindle interdigitate, etc.), and the structure of MTs allows them to resist tension, compression, and bending stresses. Draw your own diagram of the spindle and put into it all of the forces you can think of that are relevant to understanding the mechanics of spindle-mediated chromosome motion.
The start for an answer to this question is found in the slide from Pollard et al. presented around the middle of this lecture in which they provide their own answer to this question. Beyond that, one can add the forces that are generated by MT polymerization and depolymerization, forces added by motors that influence MT polymerization, bending forces that throw the MTs of many spindles into arc-shaped fibers, forces that result from the viscous drag on moving chromosomes, and forces that may be generated by the interaction of any spindle component (fibers, motors, chromosomes) with a “matrix”, i.e. a not yet understood structural material that pervades at least some spindles.
Recent evidence shows that chromosomes can initiate MT polymerization in association with either their arms or their kinetochores. What impact do you expect these features of at least some spindles to have on spindle structure and/or function?
This situation poses many questions: What is the polarity of the MTs that form on chromosome arms and/or kinetochores? What kind of mechanical attachment is there between the chromosomes and these MTs? Do the MTs exert forces on the chromosomes or simply swim or diffuse away? Are there factors that impose order on these MTs, so they take on a global pattern, or are they simply random? Little is actually known about the answers to these structural and mechanical questions. Information about such MTs can be found in Heald, R., R. Tournebize, et al. (1996). "Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts." Nature 382: 420-425. Hyman, A. A. and E. Karsenti (1996). "Morphogenetic properties of microtubules and mitotic spindle assembly." Cell 84(3): 401-10. Caudron, et al. (2005). "Spatial coordination of spindle assembly by chromosome-mediated signaling gradients." Science 309(5739): 1373-6. Yokoyama, et al. (2008). "Cdk11 is a RanGTP-dependent microtubule stabilization factor that regulates spindle assembly rate." J Cell Biol 180(5): 867-75. Khodjakov, A. et al. (2003). "Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis." J Cell Biol 160(5): 671-83.
The flux of spindle MTs suggests that these polymers are polymerizing and depolymerizing at different places. Where are these events occurring, and how would you test your answer experimentally?
The continued flux of spindle MTs towards the two poles suggests that each spindle MT is elongating by the addition of tubulin subunits at its plus (pole-distal) end, sliding under the action of motors, and depolymerizing at the pole. The site of tubulin addition could be identified by injecting a small amount of brightly labeled tubulin into a mitotic cell and seeing where it added to the spindle. It could also be found by photobleaching various part of the spindle and asking where the unbleached fluorescence returned into the now-dim region of the spindle. Finding sites of depolymerization is more difficult. It could be done by using a labeled tubulin whose fluorescence can be activated by irradiation with near UV light. (Thereafter, its fluorescence is followed with blue light.) Such reagents are available either as alleles of the green fluorescent protein or as synthesized chemical reagents.
What spindle components do you think are responsible for MT flux? How would you test your ideas?
Flux almost certainly requires motors to push the sliding of MTs away from the spindle midplane, factors (probably motors) that induce MT depolymerization at spindle poles, factors that keep these moving MTs associated with one another where they interdigitate near the midplane and associated with the poles, where they are depolymerizing. Tests of these ideas would involve identifying the relevant motors and cross-bridging protein and modifying their function by genetic, immunological, or pharmacological methods.
What do you suppose would happen if a single kinetochore became attached to two poles? How might the cell deal with such a problem?
This condition does arise in nature, and it is one of the sources of errors in chromosome segregation. It is called a “merotelic attachment”, and it is particularly common in cells whose normal mitosis has been bothered by genetic or other experimental treatment. Cimini, D., B. Howell, et al. (2001). "Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells." J Cell Biol 153(3): 517-27. Cells can deal with it by having a mechanism for loosening the strength of the attachment between a kinetochore and the spindle MTs. Such a mechanism is found in the action of the protein kinase called Aurora-B, which is localized to the kinetochores. Cimini, D., X. Wan, et al. (2006). "Aurora kinase promotes turnover of kinetochore microtubules to reduce chromosome segregation errors." Curr Biol 16(17): 1711-8. Just how it accomplishes this loosening job is not yet known. Interesting field for future work.
Diagram a spindle in late prometaphase in which one chromosome has not yet arrived at the spindle midplane. Consider what must go on at that chromosome for it to move to join the other chromosomes. Add to your diagram all the polymerization and depolymerization events that must occur and what forces must act on the chromosome to get it where it belongs. Identify all the ways you can think of to develop the necessary forces.
The diagram is straightforward. The point to recognize is that the MTs associated with one kinetochore must elongate while the ones associated with the other kinetochore must shorten for the chromosome to move. The forces that move the chromosome could be the pushing forces from the poles, identified in the Rieder experiment described in the lecture. They could also come from a chromosome knowing by some mechanism that it is not at the spindle midplane and manipulating its kinetochores to get it to the right place. This hypothesis has been called the “Smart kinetochore” model.
The speed of chromosome motion is generally quite slow (micrometers per minute). Calculate the amount of force necessary to overcome the viscous drag on a chromosome at such speeds. Each motor enzyme can generate around 5 x 10-12 N, so how many motors of this kind would be required to move a chromosome? Does the answer surprise you in any way? What do you think your result suggests about force generation in mitosis?
The force necessary to move a chromosome against viscous drag can be estimated very roughly with the Stokes equation, F = 6p?rv, in which F is the viscous drag force measured in Newtons, ? is the viscosity of the medium (the solution all around the chromosome) measured in Pascal-seconds, r is the radius of the sphere we will use to represent the chromosome measured in meters, and v is the speed at which the chromosome is moving, measured in meters/sec. The viscosity of water is about 1x10-3 Pascal-sec, and the medium of the spindle is unlikely to be more than 100 times this viscous. r is about 0.5x10-6 M, i.e., half a micrometer, and a high value for v is 1x10-7 M/sec. This works out to about 10-13 N, about one 50th of the force developed by a single motor enzyme. Thus, the amount of force necessary to move chromosomes against the cell’s viscous drag is VERY small.
This result poses several questions to which the answers now available are only speculation, but they are intriguing: why does a cell move its chromosomes so slowly? How is chromosome speed regulated so the motors that are present do not cause the chromosomes to speed up? Are the forces generated by the spindle being used for a more subtle purpose, such as tearing the spindle apart as the chromosomes move?