Redistribution of fluorescently labeled tubulin in the mitotic apparatus of sand dollar eggs and the effects of taxol.

Hamaguchi, Yukihisa; Toriyama, Masaru; Sakai, Hikoichi; Hiramoto, Yukio

doi:10.1247/csf.12.43

Cited by 41 publications

(30 citation statements)

References 23 publications

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“…In many eukaryotes, the metaphase mitotic spindle maintains a constant length, and ϩTIPs that modulate MT dynamics are the major governors of spindle length dynamics (2). Previously, we have shown that mitotic spindle length and structure in Giardia is affected by both nocodazole and Taxol as in other eukaryotes (26,67; S. C. Dawson et al, unpublished data). Here we demonstrate a conserved mitotic localization of kinesin-13 at kinetochores of the dual giardial spindles (Fig.…”

Section: Identification Of Dynamic Microtubule Arrays In Giardiamentioning

confidence: 86%

Kinesin-13 Regulates Flagellar, Interphase, and Mitotic Microtubule Dynamics in Giardia intestinalis

et al. 2007

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Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle.Giardia intestinalis is the most frequently identified protozoan cause of intestinal morbidity worldwide (59) and has a two-stage life cycle: a "trophozoite," or flagellate, form that attaches to the intestinal microvilli and a cyst form that can persist in the environment (1, 21). As in other eukaryotes, the giardial microtubule cytoskeleton creates a stable scaffold for intracellular trafficking, for organelle attachment, and for cell polarization (21). However, other important functions of the microtubule cytoskeleton are dynamic and rely upon both intrinsic dynamic instability-stochastic switches between microtubule growth and shrinkage phases (43)-and active regulation of microtubule assembly and/or disassembly. Microtubule dynamics, for example, are critical during cell division in Giardia where the two nuclei (30) undergo mitosis with extranuclear spindles that penetrate at polar nuclear openings (58), followed by the duplication and repositioning of eight flagella into the daughter cells (75). Beyond descriptions of cytoskeletal movements, we currently have little understanding of the molecular mechanism of active regulation of interphase and mitotic microtubule dynamics in Giardia.One class of conserved regulators of microtubule dynamics that mediate interactions between microtubule plus ends and other organelles are t...

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Section: Identification Of Dynamic Microtubule Arrays In Giardiamentioning

confidence: 86%

Kinesin-13 Regulates Flagellar, Interphase, and Mitotic Microtubule Dynamics in Giardia intestinalis

et al. 2007

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“…4), a process termed microtubule poleward flux (Forer 1965;Hiramoto and Izutsu 1977;Bajer and Molè-Bajer 1972;Allen et al 1969;Hamaguchi et al 1987;Mitchison 1989). Flux, as an ordered translocation process, requires application of persistent forces.…”

Section: Spindle Microtubule Flux and Kinetochore/ Centromere Tensionmentioning

confidence: 99%

Maturation of the kinetochore-microtubule interface and the meaning of metaphase

Pereira

Maiato

2012

Chromosome Res

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Chromosome positioning at the equator of the mitotic spindle emerges out of a relatively entropic background. At this moment, termed metaphase, all kinetochores have typically captured microtubules leading to satisfaction of the spindle-assembly checkpoint, but the cell does not enter anaphase immediately. The waiting time in metaphase is related to the kinetics of securin and cyclin B1 degradation, which trigger sisterchromatid separation and promote anaphase processivity, respectively. Yet, as judged by metaphase duration, such kinetics vary widely between cell types and organisms, with no evident correlation to ploidy or cell size. During metaphase, many animal and plant spindles are also characterized by a conspicuous "flux" activity characterized by continuous poleward translocation of spindle microtubules, which maintain steady-state length and position. Whether spindle microtubule flux plays a specific role during metaphase remains arguable. Based on known experimental parameters, we have performed a comparative analysis amongst different cell types from different organisms and show that spindle length, metaphase duration and flux velocity combine within each system to obey a quasi-universal rule. As so, knowledge of two of these parameters is enough to estimate the third. This trend indicates that metaphase duration is tuned to allow approximately one kinetochore-to-pole round of microtubule flux. We propose that the time cells spend in metaphase evolved as a quality enhancement step that allows for the uniform stabilization/correction of kinetochore-microtubule attachments, thereby promoting mitotic fidelity.

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“…Microtubule turnover is yet more rapid in the mitotic metaphase spindles of living mammalian cells and early echinoderm embryos, exhibiting half-times from 15 to 70 sec (1,2,9,10). This rapid assembly and disassembly of microtubules may play an important role in the proper positioning and segregation of the chromosomes.…”

mentioning

confidence: 99%

Microtubules in the metaphase-arrested mouse oocyte turn over rapidly.

Gorbsky

Simerly

Schatten

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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After ovulation mammalian oocytes arrest in second meiotic metaphase. We asked whether the microtubules that comprise the meiotic spindle of mouse oocytes were stable or were undergoing rapid cycles of assembly and disassembly. Porcine brain tubulin, derivatized with biotin or x-rhodamine [5-(and -6)-carboxy-x-rhodamine], was microhijected into living oocytes. Biotinylated tubulin incorporated into the meiotic spindle to apparent equilibrium within 15 min. To assess quantitatively the rates of disassembly and assembly of the microtubules, small domains within the spindles of oocytes injected with x-rhodamine-tubulin were photobleached and their recovery was analyzed by digital imaging microscopy. Fluorescence recovery in the spindles was rapid and extensive, plateauing to an average of 83% at 4 min. The calculated half-time for turnover of the spindle microtubules was 77 sec. In contrast, fluorescence recovery of the spindle midbodies in telophase oocytes was much more limited, averaging '=22% at 4 min. These data indicate that most microtubules within the arrested metaphase spindle of the mouse oocyte undergo rapid cycles of assembly and disassembly. Microtubules of the telophase midbody are more stable.Recent studies of living cells microinjected with fluorescent tubulin (1-4) have eroded the notion that microtubules are static structures. For the bulk of the microtubule array in cultured cells in interphase, estimates of the half-time for disassembly and reassembly range from 5 to 15 min (3-5). Rapid length changes at the ends of microtubules, growth at 3.5-7.2 pum/min and disassembly at 4.3-18.5 Aum/min, have been directly observed in the thin lamellae of well-spread cells (6-8). Microtubule turnover is yet more rapid in the mitotic metaphase spindles of living mammalian cells and early echinoderm embryos, exhibiting half-times from 15 to 70 sec (1, 2, 9, 10). This rapid assembly and disassembly of microtubules may play an important role in the proper positioning and segregation of the chromosomes.In contrast to the generally inexorable transit of cells through mitosis, oocytes are often arrested at particular stages of meiosis that are characteristic for each species (11). In mammals such as mice, mature oocytes within the ovary are arrested at meiotic prophase. The oocyte, under the influence of luteinizing hormone, is released from the ovary and undergoes the first meiotic division with the production of the first polar body. The egg then enters another period of stasis in development, arresting at second meiotic metaphase as it moves into the oviduct. The egg may remain arrested in this state for several hours awaiting fertilization and final maturation. Recently it was shown that expression of the c-mos protooncogene is the "cytostatic factor" that arrests Xenopus oocytes in metaphase of meiosis II (12). The mosencoded protein was found to be proteolyzed upon fertilization of Xenopus eggs (13). Presumably, destruction of the mos protein allows progression through meiosis and initiation of the mi...

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Redistribution of fluorescently labeled tubulin in the mitotic apparatus of sand dollar eggs and the effects of taxol.

Cited by 41 publications

References 23 publications

Kinesin-13 Regulates Flagellar, Interphase, and Mitotic Microtubule Dynamics in Giardia intestinalis

Kinesin-13 Regulates Flagellar, Interphase, and Mitotic Microtubule Dynamics in Giardia intestinalis

Maturation of the kinetochore-microtubule interface and the meaning of metaphase

Microtubules in the metaphase-arrested mouse oocyte turn over rapidly.

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