ScienceWeek

CELL BIOLOGY: SPINDLE ORGANIZATION AND FUNCTION

The following points are made by Eric Karsenti (Nature 2004 432:563):

1) Perhaps the most striking structure a cell can produce is the spindle -- a fine meshwork of filaments that carries out a fundamental task in cell division. Before one cell can make two, it must duplicate its DNA; the replicated chromosomes must then be separated and pulled to opposite poles of the cell, which splits into two. The mitotic spindle is the machine that segregates the duplicated chromosomes. It is built from an array of protein tubes -- the microtubules -- that assume a precise size and bipolar organization.

2) Several proteins, notably molecular motors and various signalling proteins[1,2], are essential for spindle organization and function, but no other types of molecule were thought to be involved. New work[3} has now changed that view, finding that a large, branched, polymeric molecule, known previously for its effects on chromosome structure, is also essential for microtubules to be organized into a functional spindle.

3) Research over the past 15 years has demonstrated that spindle assembly results from the self-organization of chromosomes, microtubules, and molecular motors[4]. While this is happening, some microtubules attach to specialized structures on chromosomes, the kinetochores; both kinetochores and microtubules are required to move chromosomes to the spindle poles[5]. This self-organization process involves the collective action of microtubule proteins, motor proteins, and signaling proteins[1,2]; Chang et al[3] have found that another type of chemical -- poly(ADP-ribose), PAR -- also has a key role. ADP (adenosine diphosphate) is a nucleotide, akin to the basic building blocks of DNA; ribose is a sugar molecule.

5) At first sight, all of this has no obvious connection with the mitotic spindle. What led Chang et al[3] to suspect that PAR might also be involved in spindle assembly was that all PARPs were known to be present in one way or another in mitotic spindles, and that the cellular concentration of PAR seems to increase significantly during mitosis. But that did not necessarily imply that this protein modification is involved in spindle assembly. So Chang et al[3] turned to the well-characterized in vitro system of frog egg extracts to address this question. These extracts are obtained by crushing frog eggs in a 10,000g gravitational field for 10 minutes. After sperm nuclei are added, functional spindles form. To test the role of PAR in spindle assembly, Chang et al[3] added an excess of the protein that antagonizes PAR addition (PARG) to such extracts, and examined the spindles formed. The effect was dramatic: instead of clear bipolar structures, they saw microtubules organized as two asters (star-shaped structures) on each side of the chromosomes. The microtubules remained dynamic, but could not interact properly at the equator.

References (abridged):

1. Wittmann, T., Hyman, A. & Desai, A. Nature Cell Biol. 3, E28-E34 (2001)

2. Hetzer, M., Gruss, O. J. & Mattaj, I. W. Nature Cell Biol. 4, E177-E184 (2002)

3. Chang, P., Jacobson, M. K. & Mitchison, T. J. Nature 432, 645-649 (2004)

4. Karsenti, E. & Vernos, I. Science 294, 543-547 (2001)

5. Scholey, J. M., Brust-Mascher, I. & Mogilner, A. Nature 422, 746-752 (2003)

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CELL BIOLOGY: MICROTUBULES, ACTIN, AND THE SPINDLE

The following points are made by Margaret A. Titus (Nature 2004 431:252):

1) The cytoskeleton is the complex of proteins responsible for cell shape and movement. One of the most important structures it forms is the spindle, which ensures the faithful delivery of replicated chromosomes to daughter cells following cell division. Spindle assembly was once believed to be the sole responsibility of the cytoskeletal components known as microtubules, and their associated motor proteins (the dyneins and kinesins). Recent research, however, demonstrates that the process depends -- in some circumstances at least -- on another cytoskeletal component, actin, and an associated motor protein, a member of the myosin family, which can bind directly to microtubules.

2) A spindle is necessary for both meiosis, the form of cell division that produces gametes for sexual reproduction, and mitosis, cell division for growth. Working with unfertilized eggs (oocytes), of the amphibian Xenopus, Weber et al(1) have demonstrated that one of the members of the highly diverse family of myosins, myosin-10 (Myo10), is involved in both spindle assembly and the subsequent positioning of the nuclei during meiosis.

3) The view that the spindle depends solely on the microtubule cytoskeleton arose from experiments showing that up until its final act -- the creation of daughter cells via a process called cytokinesis -- cell division was a myosin-free event. Early studies(2,3) looked at only one form of myosin, Myo2. But subsequent work(4) demonstrated that mutants lacking other myosins (such as types 1, 5, 6, 7 and 15) can undergo mitosis perfectly well.

4) The first hint of direct interaction between a myosin and microtubules came from an initially puzzling observation(5) --that in a certain cell type, Myo5, known as a motor that powers organelle transport, localizes to microtubules as well as to the centrosome (a structure from which the spindle develops), in addition to being present in regions where actin resides. Consistent with these observations, it later emerged that Myo5 binds directly to microtubules in vitro with high affinity via its tail region.

References (abridged):

1. Weber, K. L., Sokac, A. M., Berg, J. S., Cheney, R. E. & Bement, W. M. Nature 431, 325-329 (2004)

2. Kiehart, D. P. et al. J. Cell Biol. 94, 165-178 (1982)

3. Neujahr, R., Heizer, C. & Gerisch, G. J. Cell Sci. 110, 123-137 (1997)

4. Kieke, M. C. & Titus, M. A. in Molecular Motors (ed. Schliwa, M.) 3-44 (Wiley-VCH, Weinheim, 2003)

5. Espreafico, E. M. et al. Proc. Natl Acad. Sci. USA 95, 8636-8641 (1998)

Nature http://www.nature.com/nature

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CELL BIOLOGY: ON MITOTIC SPINDLE DYNAMICS

The following points are made by Rebecca W. Heald (Nature 2004 427:300):

1) A dramatic event in the life of a cell is its transformation into two genetically identical progeny. This is achieved during mitosis, when an exact complement of chromosomes is partitioned to each half of the cell, just before it pinches into two(1). Errors in this process can result in cell death or contribute to cancer. The molecular properties of the mitotic spindle -- the apparatus that distributes the chromosomes -- have been studied for decades, but the molecular mechanisms underlying chromosome transport have remained elusive.

2) The events of cell division require dynamic elements of the cell's internal skeleton to assemble into macromolecular structures capable of performing work. The spindle is one such structure: this highly dynamic yet ordered assemblage is composed of cytoskeletal elements known as microtubule filaments, along with many associated proteins which form a bipolar array(3).

3) Microtubules are polymers made up of tubulin proteins, and they grow or shrink when tubulin is added or lost from their ends. These ends are termed "plus" and "minus" to distinguish their behaviors. Minus ends are focused at each pole of the spindle, emanating from a nucleating structure called the centrosome. Plus ends grow outwards from the poles, capturing and moving chromosomes or overlapping at the center of the spindle. Several classes of microtubule-based motor proteins are also required for chromosome segregation. Each type of motor moves in a set direction along microtubules. Together they cross-link and sort the microtubules according to their structural polarity, and mediate chromosome interactions with the spindle(1).

4) Before embarking on mitosis, a cell duplicates its chromosomes, producing pairs of "sister chromatids". The members of each pair are identical. In what is known as "prometaphase" of mitosis, these chromatids become tethered to the spindle, such that one member of each pair is attached to a bundle of microtubules emanating from one pole, and the other member is connected to the other pole. "Kinetochores" are the microtubule landing pads on chromosomes(3). A tug-of-war ensues, driven by the addition or loss of tubulin (that is, microtubule polymerization or depolymerization) at the kinetochores. This tug-of-war results in chromosomes becoming aligned during metaphase of mitosis, such that each sister faces its final destination. Finally, "anaphase" begins when the glue holding sisters together is dissolved, and they take off towards opposite spindle poles.(2)

References (abridged):

1. Scholey, J. M. et al. Nature 422, 746-752 (2003)

2. Rogers, G. et al. Nature 427, 364 370 (2004)

3. Rieder, C. L. & Salmon, E. D. Trends Cell Biol. 8, 310 318 (1998)

4. Mitchison, T. J. J. Cell Biol. 109, 637 652 (1989)

5. Brust-Mascher, I. & Scholey, J. M. Mol. Cell. Biol. 13, 3967 3975 (2002)

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