ScienceWeek

CELL BIOLOGY: ON THE ORCHESTRATION OF THE MITOTIC SPINDLE

The following points are made by Paul R. Clarke (Science 2005 309:1334):

1) Eukaryotic cell division is entrancing when observed through a microscope. In a dramatic prelude, the internal structure of a cell is reorganized and pairs of duplicated chromosomes become arranged in the middle of the cell. Then each pair is separated into chromatids that are segregated one to each side, so that when the cell divides, each "daughter" cell receives an identical set of genes. This formidable feat is achieved by the mitotic spindle, a precision machine made from a bipolar array of microtubules that are focused at each end of the spindle by a centrosome or spindle pole body. Microtubules are themselves dynamic polymers that interact with chromosomes in the middle of the spindle and provide tracks to separate them toward the poles. Without the spindle, cell division would be impossible, and subtle defects in its function are likely to be involved with the genomic instability associated with cancer.

2) The mechanisms that orchestrate assembly of the mitotic spindle have been somewhat of an enigma. It has been proposed that signals emanating from chromosomes that promote microtubule growth play a key role[1]. New work[2] provides the clearest evidence yet that spindle assembly is coordinated by the generation, at chromosomes, of an intracellular gradient of the active guanosine triphosphate (GTP)-bound form of Ran, a small GTPase of the Ras super-family present in all eukaryotic cells.

3) The concept of signaling gradients is a familiar one in animal development[3]. Release of a diffusible and slowly degraded chemical, or morphogen, from a specific site can produce an extracellular concentration gradient that provides positional information to cells. The effect on a particular cell (for example, inducing differentiation) is determined by the cell's threshold in the response to the graded signal. If there are multiple thresholds, then the gradient can produce patterns of different cell responses. These may be limited to precise concentrations of the morphogen, and hence a precise position within a developing tissue. Intracellular gradients that provide positional cues can be generated through subcellular localization of mRNA, such as the localization of bicoid mRNA at the anterior pole of the Drosophila oocyte. Local translation subsequently produces a gradient of bicoid morphogen during early development[4].

4) Intracellular gradients can also be generated by enzyme activity. In this case, spatial information is provided by the concentration gradient of a diffusible substrate generated by a fixed enzyme. For instance, if phosphorylation of a diffusible protein is catalyzed by a localized protein kinase, and if the opposing protein phosphatase is dispersed, then a gradient in the phosphorylation status (and therefore in the activity of the substrate protein) can be generated[5]. Such a reaction-diffusion process creates a steadily changing gradient that is distinct from sharp concentration differences due to compartmentalization, such as the difference in ion concentration across an impermeable membrane. Previous work has shown, by mathematical simulation combined with molecular experiments using fluorescence reporters, that such a chemical gradient can be generated by phosphorylation of the microtubule-binding protein stathmin.

References (abridged):

1. E. Karsenti, J. Newport, M. Kirschner, J. Cell Biol. 99, 47s (1984)

2. M. Caudron, G. Bunt, P. Bastiaens, E. Karsenti, Science 309, 1373 (2005)

3. M. Osterfield, M. W. Kirschner, J. G. Flanagan, Cell 113, 425 (2003)

4. M. Kloc, N. R. Zearfoss, L. D. Etkin, Cell 108, 533 (2002)

5. G. C. Brown, B. N. Kholodenko, FEBS Lett. 457, 452 (1999)

Science http://www.sciencemag.org

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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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