Microtubule pushing on an organelle
Pushing forces on a non-dividing nucleus
Aster. During interphase in the budding yeast Saccharomyces cerevisiae, microtubules are organized in an aster or a conical array radiating from the spindle pole body (a yeast centrosome). The microtubules of the aster exhibit dynamic instability and push against the cell cortex, thus propelling the nucleus in the opposite direction (Shaw et al. 1997).
Parallel array. In cylindrically shaped cells of the fission yeast Schizosaccharomyces pombe, interphase microtubules lie parallel with the cell axis, surrounding the nucleus like a barrel. They are attached to the nuclear envelope at several points, including the spindle pole body and interphase microtubule-organizing centers. These microtubules grow from the nuclear region towards the cell tips, and exert a transient pushing force on the nucleus upon reaching the cell tips (Tran et al. 2001). Microtubule pushing keeps the nucleus at the cell center and can even re-center a displaced nucleus. This was shown previously by experiments where the nucleus was moved away from the cell center using optical tweezers (Tolić-Nørrelykke et al. 2005) or cell centrifugation (Daga et al. 2006). Interestingly, when the microtubule organization was changed from a parallel array into an aster by ectopically expressing the meiosis-specific spindle pole body component Hrs1p/Mcp6p, pushing forces by astrally arranged microtubules were not able to center the nucleus, but generated large nuclear excursions instead (Tanaka et al. 2005). Thus, the ability of microtubule pushing forces to center the nucleus depends on the arrangement of microtubules, besides its more obvious dependence on microtubule dynamics and cell geometry.
Pushing forces on a dividing nucleus
Pushing on the spindle. In S. pombe, spindle movements involve microtubule-pushing forces. At the transition from interphase to mitosis, interphase microtubules are attached to the duplicated spindle pole body. Pushing forces exerted by these microtubules against the cell tips position the duplicated spindle pole body in the cell center, thereby setting the central location and the alignment of the future spindle (Vogel et al. 2007). Later in mitosis, during anaphase B, astral microtubules grow from the spindle pole bodies in a direction nearly perpendicular to the central spindle. Without contributing to spindle elongation, these astral microtubules help to align the spindle with the cell axis by pushing against the cell sides (Tolić-Nørrelykke et al. 2004).
Pushing on chromosomes. Experiments, where a prometaphase chromosome was cut by a laser microbeam, demonstrated that microtubules can push on the chromosomes in the spindle. The fragment without the kinetochore moved rapidly away from the spindle pole, suggesting that “polar ejection forces” push chromosome arms away from the proximal spindle pole (Rieder et al. 1986). Recent studies have revealed the molecular entity generating ejection forces to be the chromokinesin Kid (Antonio et al. 2000; Brouhard and Hunt 2005; Funabiki and Murray 2000). This motor is bound to chromosome arms and “walks” towards the microtubule plus-end, which points away from the spindle pole.
An additional motor protein contributes to chromosome movements away from the spindle pole. The plus-end directed motor CENP-E binds the chromosome at the kinetochore and slides it along another microtubule bound to the kinetochore of another chromosome (Kapoor et al. 2006). This movement is not only microtubule-based “pushing” of an organelle away from the spindle pole, but it also involves the idea of “sliding” of an organelle along a microtubule (see below).
Microtubule pulling on an organelle
Pulling forces on a non-dividing nucleus
In meiotic prophase of S. pombe, the nucleus shows a peculiar movement: it oscillates from one end of the cell to the other with a period of 5–10 min repeated over 2–3 h. The oscillatory movement of the nucleus is lead by the spindle pole body and depends on microtubules growing from the spindle pole body, and on the motor protein cytoplasmic dynein (Yamamoto et al. 1999). Observation of the spindle pole body movements in relation to the microtubule position (Yamamoto et al. 2001), as well as laser-cutting of microtubules (Vogel et al. 2008), indicated that the driving force for nuclear oscillations is pulling by astral microtubules. This pulling force is generated by cortically anchored dynein motors that walk along the microtubules towards microtubule minus-ends, which are focused at the spindle pole body.
After fertilization in multicellular organisms, the male pronucleus migrates towards the center of the egg to reach the female pronucleus. This movement depends on the aster of microtubules that grow from two centrosomes attached to the male pronucleus. Minus-end directed motors, anchored at organelles in the cytoplasm, move the aster and the associated male pronucleus by walking along the astral microtubules (Hamaguchi and Hiramoto 1986; Kimura and Onami 2005). Simultaneously, the female pronucleus does not passively wait, but moves towards the male pronucleus by sliding along the same astral microtubules (see below).
Pulling forces on a dividing nucleus
Pulling on the spindle. Since the pioneering experiments of Aist and Berns (1981), forces exerted by astral microtubules on the spindle poles have been explored by laser-cutting of the spindle in a variety of cell types. Cutting of the spindle in the fungus Fusarium solani showed that extranuclear forces, presumably involving astral microtubules, pull on the spindle poles and that the central spindle limits the separation rate (Aist and Berns 1981). Similar conclusions were obtained by laser ablation of centrosomes in rat kangaroo kidney epithelium (PtK2) cells (Aist et al. 1993). In the single-cell stage C. elegans embryo, cutting of the central spindle revealed that pulling forces external to the spindle act on the two spindle poles, a stronger net force acting on the posterior pole. The asymmetry in the pulling force results in an asymmetric spindle position, which translates into an asymmetric cell division (Grill et al. 2001; Grill and Hyman 2005). Laser-cutting has identified pulling forces by microtubules and dynein to be responsible for rapid elongation and positioning of the central spindle in the fungus Ustilago maydis (Fink et al. 2006). Observation of astral microtubules and spindle pole body movements have suggested that astral microtubules pull one spindle pole into the bud during budding yeast mitosis (Adames and Cooper 2000; Yeh et al. 2000). In fibroblasts, astral microtubules transiently anchored at the bottom of the cell exert pulling forces to position the spindle (Schultz and Onfelt 2001). These mechanisms of pulling the organelles via cortically anchored molecular motors that move along astral microtubules can be described as “pulling” from the organelle viewpoint, and as “sliding” from the cortex perspective.
Pulling on chromosomes. Experiments with laser-cutting of prometaphase chromosomes, mentioned above in the context of pushing forces, have also revealed a pulling force on chromosomes. Whereas the chromosome fragment without the kinetochore moved away from the proximal spindle pole, the fragment with the kinetochore moved towards the pole, suggesting that kinetochore microtubules pull on the kinetochore (Rieder et al. 1986). Recent work has shown that the Dam1 ring complex sliding along the microtubule translates the force generated by microtubule depolymerization into a poleward kinetochore movement along the microtubule lattice to drive chromosome segregation (Westermann et al. 2006).
In this class of intracellular movements the organelle is loosely bound to, and moves with respect to, the microtubule, which serves as a track for the movement. The movement is driven by molecular motors. Plus-end directed motors, such as kinesins, distribute the endoplasmic reticulum and the Golgi complex along microtubules (Lippincott-Schwartz et al. 1995). Kinesins and other proteins from the kinesin-superfamily transport mitochondria along microtubules (Fujita et al. 2007), as well as precursors of synaptic vesicles and axonal membranes in neurons (Kondo et al. 1994). The minus-end directed motor dynein moves the endoplasmic reticulum in U. maydis (Wedlich-Soldner et al. 2002). After fertilization in multicellular organisms, the female pronucleus moves towards the male pronucleus along the astral microtubules extending from the centrosome associated with the male pronucleus (Hamaguchi and Hiramoto 1986; Schatten 1981).
Similar movement of membrane-bound organelles along microtubules contributes to skin pigmentation. In keratinocytes, a “microparasol” made of phagocytosed melanosomes filled with the dark pigment melanin protects the nucleus from UV-induced DNA damage. Cytoplasmic dynein is the motor driving the perinuclear-directed aggregation of melanosomes along microtubules (Byers et al. 2003).
Microtubules growing from the two spindle poles meet in an anti-parallel arrangement in the central part of the spindle. The spindle length is nearly constant during metaphase while the kinetochore microtubules are capturing the chromosomes. In anaphase B, on the other hand, the spindle elongates in order to separate the two sets of chromosomes. The spindle length is controlled by a “push-me-pull-you” mechanism: motors of opposite polarity cross-link the overlapping microtubules in the central spindle. Their opposing forces slide the spindle microtubules with respect to one another. The plus-end directed motors (BimC kinesins, e.g., KLP61F in Drosophila) push the spindle poles apart, whereas minus-end directed motors (kinesin-14 family, e.g., Ncd in Drosophila) pull them together (Sharp et al. 1999). The balance of forces exerted by these two classes of motors helps to set the constant spindle length in metaphase and the elongation onset at the transition to anaphase.
In S. pombe, spindle elongation in anaphase B is driven by pushing forces in the central spindle, as opposed to pulling-apart of the spindle poles by astral microtubules as described above. The role of pushing forces was revealed by laser-cutting experiments where the poles of severed spindles did not continue to move apart, suggesting that the forces external to the spindle do not have a significant impact on spindle elongation. Thus, the elongation forces are instead generated in the central spindle (Khodjakov et al. 2004; Tolić-Nørrelykke et al. 2004).
Spindle pole focusing
Minus-end directed motor proteins, such as dynein, can cross-link and slide single free microtubules with respect to each other. If several motor molecules are connected, their activity can rearrange the microtubules into an aster with a focus of minus-ends (Verde et al. 1991). This mechanism of motor-based microtubule sliding and convergence, together with the nucleation of microtubules from centrosomes, is required for focusing of minus-ends into spindle poles in vertebrate somatic cells (Gaglio et al. 1997).
Cilia and flagella
Another specialized force-generating structure based on microtubules and dynein is the axoneme, the core of motile cilia and flagella. A key structural feature of the axoneme, compared to the central spindle and asters, is that the parallel microtubules in the axoneme are not free to move, but are connected to their neighbors by protein links. This prevents sliding of the microtubules with respect to one another. The motor activity of the axonemal dynein is, therefore, converted into bending of the microtubules, resulting in the beating of cilia and the wave motion of flagella (Lindemann 2003).
Microtubule-actin push–pull mechanism
Besides pushing or pulling on the cell cortex, nucleus, spindle, chromosomes, and various membrane-bound organelles, microtubules exert forces on other cytoskeleton systems such as the actin network. A model of cell mechanics called “tensegrity,” which is a contraction of “tensional integrity,” describes the cell as a structure consisting of a continuous, tensed network of structural elements together with other isolated support elements that resist compression (Ingber 1993). According to this model, which is used to describe cell shape changes, cell deformability depends on the level of tension in the cell. In the simplest embodiment of the cellular tensegrity model, the actin–myosin contractile system is under tension, which is balanced by the compression borne by microtubules and the attachment points to the external substrate. The observed correlation between the extent of microtubule buckling and the level of tension in the actin cytoskeleton supports the idea that microtubules bear compression as they balance a substantial portion of the contractile stress (Wang et al. 2001). Another prediction of the model is that, upon microtubule disruption, the portion of stress balanced by microtubules would shift to the substrate, thereby causing an increase in the forces exerted by the cell at the attachment points to the substrate. This was observed in adherent smooth muscle cells (Stamenovic et al. 2002), providing further evidence in favor of the model of the push–pull relationship between microtubules and actin.