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1
Bringmann, Henrik. "Mechanical and genetic separation of aster- and midzone-positioned cytokinesis." Biochemical Society Transactions 36, no. 3 (May 21, 2008): 381–83. http://dx.doi.org/10.1042/bst0360381.
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The mitotic spindle positions the cytokinesis furrow. The cytokinesis furrow then forms and ingresses at the site of the mitotic spindle, between the spindle poles. Two populations of spindle microtubules are implicated in cytokinesis furrow positioning: radial microtubule arrays called asters and bundled non-kinetochore microtubules called the spindle midzone. Here I will discuss our recent results that provided examples of how aster-positioned and midzone-positioned cytokinesis can be mechanically and genetically separated. These experiments illustrate how asters and midzone contribute to cytokinesis. ASS (asymmetric spindle severing) is a mechanical way to spatially separate the aster and midzone signals. In Caenorhabditis elegans embryos, asters and midzone provide two consecutive signals that position the cytokinesis furrow. The first signal is positioned midway between the microtubule asters; the second signal is positioned over the spindle midzone. Aster and midzone contribution can also be genetically separated. Mutants in spd-1 have no detectable midzone and are defective in midzone-positioned but not aster-positioned cytokinesis. Disruption of the function of LET-99 and the heterotrimeric G-proteins GOA-1/GPA-16 and their regulator GPR-1/2 causes defects in aster-positioned cytokinesis but not in midzone-positioned cytokinesis. In order to understand aster-positioned cytokinesis we have to understand how microtubule asters spatially control the activity of LET-99, GPR-1/2 and GOA-1/GPA-16 and how the activity of these G-protein pathway components control the assembly of a contractile ring.
2
Tse, Yu Chung, Alisa Piekny, and Michael Glotzer. "Anillin promotes astral microtubule-directed cortical myosin polarization." Molecular Biology of the Cell 22, no. 17 (September 2011): 3165–75. http://dx.doi.org/10.1091/mbc.e11-05-0399.
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Assembly of a cytokinetic contractile ring is a form of cell polarization in which the equatorial cell cortex becomes differentiated from the polar regions. Microtubules direct cytokinetic polarization via the central spindle and astral microtubules. The mechanism of central spindle–directed furrow formation is reasonably well understood, but the aster-directed pathway is not. In aster-directed furrowing, cytoskeletal factors accumulate to high levels at sites distal to the asters and at reduced levels at cortical sites near the asters. In this paper, we demonstrate that the cytoskeletal organizing protein anillin (ANI-1) promotes the formation of an aster-directed furrow in Caenorhabditis elegans embryos. Microtubule-directed nonmuscle myosin II polarization is aberrant in embryos depleted of ANI-1. In contrast, microtubule-directed polarized ANI-1 localization is largely unaffected by myosin II depletion. Consistent with a role in the induction of cortical asymmetry, ANI-1 also contributes to the polarization of arrested oocytes. Anillin has an evolutionarily conserved capacity to associate with microtubules, possibly providing an inhibitory mechanism to promote polarization of the cell cortex.
3
Sider, J. R., C. A. Mandato, K. L. Weber, A. J. Zandy, D. Beach, R. J. Finst, J. Skoble, and W. M. Bement. "Direct observation of microtubule-f-actin interaction in cell free lysates." Journal of Cell Science 112, no. 12 (June 15, 1999): 1947–56. http://dx.doi.org/10.1242/jcs.112.12.1947.
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Coordinated interplay of the microtubule and actin cytoskeletons has long been known to be crucial for many cellular processes including cell migration and cytokinesis. However, interactions between these two systems have been difficult to document by conventional approaches, for a variety of technical reasons. Here the distribution of f-actin and microtubules were analyzed in the absence of fixation using Xenopus egg extracts as an in vitro source of microtubules and f-actin, demembranated Xenopus sperm to nucleate microtubule asters, fluorescent phalloidin as a probe for f-actin, and fluorescent tubulin as a probe for microtubules. F-actin consistently colocalized in a lengthwise manner with microtubules of asters subjected to extensive washing in flow chambers. F-actin-microtubule association was heterogenous within a given aster, such that f-actin is most abundant toward the distal (plus) ends of microtubules, and microtubules heavily labeled with f-actin are found in close proximity to microtubules devoid of f-actin. However, this distribution changed over time, in that 5 minute asters had more f-actin in their interiors than did 15 minute asters. Microtubule association with f-actin was correlated with microtubule bending and kinking, while elimination of f-actin resulted in straighter microtubules, indicating that the in vitro interaction between f-actin and microtubules is functionally significant. F-actin was also found to associate in a lengthwise fashion with microtubules in asters centrifuged through 30% sucrose, and microtubules alone (i.e. microtubules not seeded from demembranated sperm) centrifuged through sucrose, indicating that the association cannot be explained by flow-induced trapping and alignment of f-actin by aster microtubules. Further, cosedimentation analysis revealed that microtubule-f-actin association could be reconstituted from microtubules assembled from purified brain tubulin and f-actin assembled from purified muscle actin in the presence, but not the absence, of Xenopus oocyte microtubule binding proteins. The results provide direct evidence for an association between microtubules and f-actin in vitro, indicate that this interaction is mediated by one or more microtubule binding proteins, and suggest that this interaction may be responsible for the mutual regulation of the microtubule and actomyosin cytoskeletons observed in vivo.
4
Nguyen, P. A., C. M. Field, and T. J. Mitchison. "Prc1E and Kif4A control microtubule organization within and between large Xenopus egg asters." Molecular Biology of the Cell 29, no. 3 (February 2018): 304–16. http://dx.doi.org/10.1091/mbc.e17-09-0540.
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Prc1E and Kif4A prune out anti-parallel microtubules in the huge asters that position cleavage furrows in Xenopus eggs. Within asters, this promotes radial order in the face of the randomizing effect of nucleation away from centrosomes. At boundaries between asters, it blocks growth of a microtubule from one aster into its neighbor.
5
Kotani, Tomoya, and Masakane Yamashita. "Overexpression of truncated γ-tubulins disrupts mitotic aster formation in Xenopus oocyte extracts." Biochemical Journal 389, no. 3 (July 26, 2005): 611–17. http://dx.doi.org/10.1042/bj20050243.
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Mechanisms of spindle pole formation rely on minus-end-directed motor proteins. γ-Tubulin is present at the centre of poles, but its function during pole formation is completely unknown. To address the role of γ-tubulin in spindle pole formation, we overexpressed GFP (green fluorescent protein)-fused γ-tubulin (γ-Tu-GFP) in Xenopus oocytes and produced self-assembled mitotic asters in the oocyte extracts. γ-Tu-GFP associated with endogenous α-, β- and γ-tubulin, suggesting that it acts in the same manner as that of endogenous γ-tubulin. During the process of aster formation, γ-Tu-GFP aggregated as dots on microtubules, and then the dots were translocated to the centre of the aster along microtubules in a manner dependent on cytoplasmic dynein activity. Inhibition of the function of γ-tubulin by an anti-γ-tubulin antibody resulted in failure of microtubule organization into asters. This defect was restored by overexpression of γ-Tu-GFP, confirming the necessity of γ-tubulin in microtubule recruitment for aster formation. We also examined the effects of truncated γ-tubulin mutants, which are difficult to solubly express in other systems, on aster formation. The middle part of γ-tubulin caused abnormal organization of microtubules in which minus ends of microtubules were not tethered, but dispersed. An N-terminus-deleted mutant prevented recruitment of microtubules into asters, similar to the effect of the anti-γ-tubulin antibody. The results indicate possible roles of γ-tubulin in spindle pole formation and show that the system developed in the present study could be useful for analysing roles of many proteins that are difficult to solubly express.
6
Dogterom, M., M. A. Félix, C. C. Guet, and S. Leibler. "Influence of M-phase chromatin on the anisotropy of microtubule asters." Journal of Cell Biology 133, no. 1 (April 1, 1996): 125–40. http://dx.doi.org/10.1083/jcb.133.1.125.
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In many eukaryotic cells going through M-phase, a bipolar spindle is formed by microtubules nucleated from centrosomes. These microtubules, in addition to being "captured" by kinetochores, may be stabilized by chromatin in two different ways: short-range stabilization effects may affect microtubules in close contact with the chromatin, while long-range stabilization effects may "guide" microtubule growth towards the chromatin (e.g., by introducing a diffusive gradient of an enzymatic activity that affects microtubule assembly). Here, we use both meiotic and mitotic extracts from Xenopus laevis eggs to study microtubule aster formation and microtubule dynamics in the presence of chromatin. In "low-speed" meiotic extracts, in the presence of salmon sperm chromatin, we find that short-range stabilization effects lead to a strong anisotropy of the microtubule asters. Analysis of the dynamic parameters of microtubule growth show that this anisotropy arises from a decrease in the catastrophe frequency, an increase in the rescue frequency and a decrease in the growth velocity. In this system we also find evidence for long-range "guidance" effects, which lead to a weak anisotropy of the asters. Statistically relevant results on these long-range effects are obtained in "high-speed" mitotic extracts in the presence of artificially constructed chromatin stripes. We find that aster anisotropy is biased in the direction of the chromatin and that the catastrophe frequency is reduced in its vicinity. In this system we also find a surprising dependence of the catastrophe and the rescue frequencies on the length of microtubules nucleated from centrosomes: the catastrophe frequency increase and the rescue frequency decreases with microtubule length.
7
Tanimoto, Hirokazu, Akatsuki Kimura, and Nicolas Minc. "Shape–motion relationships of centering microtubule asters." Journal of Cell Biology 212, no. 7 (March 28, 2016): 777–87. http://dx.doi.org/10.1083/jcb.201510064.
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Although mechanisms that contribute to microtubule (MT) aster positioning have been extensively studied, still little is known on how asters move inside cells to faithfully target a cellular location. Here, we study sperm aster centration in sea urchin eggs, as a stereotypical large-scale aster movement with extreme constraints on centering speed and precision. By tracking three-dimensional aster centration dynamics in eggs with manipulated shapes, we show that aster geometry resulting from MT growth and interaction with cell boundaries dictates aster instantaneous directionality, yielding cell shape–dependent centering trajectories. Aster laser surgery and modeling suggest that dynein-dependent MT cytoplasmic pulling forces that scale to MT length function to convert aster geometry into directionality. In contrast, aster speed remains largely independent of aster size, shape, or absolute dynein activity, which suggests it may be predominantly determined by aster growth rate rather than MT force amplitude. These studies begin to define the geometrical principles that control aster movements.
8
Zhang, C., M. Hughes, and P. R. Clarke. "Ran-GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts." Journal of Cell Science 112, no. 14 (July 15, 1999): 2453–61. http://dx.doi.org/10.1242/jcs.112.14.2453.
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Ran is an abundant GTPase of the Ras superfamily that is highly conserved in eukaryotes. In interphase cells, Ran is mainly nuclear and thought to be predominantly GTP-bound, but it is also present in the cytoplasm, probably GDP-bound. This asymmetric distribution plays an important role in directing nucleocytoplasmic transport. Ran has also been implicated in cell cycle control, including the transition from mitosis to interphase when the compartmentalisation of the nucleus is established. Here, we have examined the role of Ran in this transition using a cell-free system of Xenopus egg extracts supplemented with sperm heads that provides a model for microtubule aster formation and post-M phase nuclear assembly. Ran-GTP, added as wild-type protein, a mutant defective in GTPase activity (Q69L), or generated by addition of the specific nucleotide exchange factor RCC1, stabilises large microtubule asters nucleated at the sperm centrosome, prevents the redistribution of NuMA from the aster to the nucleus and blocks chromatin decondensation. In contrast, Ran GDP does not stabilise microtubules or inhibit nuclear assembly. RanT24N and RanBP1, which oppose the generation of Ran-GTP by RCC1, arrest nuclear growth after disappearance of the aster. Ran associates with microtubule asters in egg extracts and with mitotic spindles in somatic Xenopus cells, suggesting that it may affect microtubule stability directly. These results show that Ran has a novel function in the control of microtubule stability that is clearly distinct from nucleocytoplasmic transport. The Ran GDP/GTP switch may play a role in co-ordinating changes in the structure of microtubules and the assembly of the nucleus associated with the transition from mitosis to interphase.
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Schroeder, M. M., and D. L. Gard. "Organization and regulation of cortical microtubules during the first cell cycle of Xenopus eggs." Development 114, no. 3 (March 1, 1992): 699–709. http://dx.doi.org/10.1242/dev.114.3.699.
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Anti-tubulin antibodies and confocal immunofluorescence microscopy were used to examine the organization and regulation of cytoplasmic and cortical microtubules during the first cell cycle of fertilized Xenopus eggs. Appearance of microtubules in the egg cortex temporally coincided with the outgrowth of the sperm aster. Microtubules of the sperm aster first reached the animal cortex at 0.25, (times normalized to first cleavage), forming a radially organized array of cortical microtubules. A disordered network of microtubules was apparent in the vegetal cortex as early as 0.35. Cortical microtubule networks of both animal and vegetal hemispheres were reorganized at times corresponding to the cortical rotation responsible for specification of the dorsal-ventral (D-V) axis. Optical sections suggest that the cortical microtubules are continuous with the microtubules of the sperm aster in fertilized eggs, or an extensive activation aster in activated eggs. Neither assembly and organization, nor disassembly of the cortical microtubules coincided with MPF activation during mitosis. However, cycloheximide or 6-dimethylaminopurine, which arrest fertilized eggs at interphase, blocked cortical microtubule disassembly. Injection of p13, a protein that specifically inhibits MPF activation, delayed or inhibited cortical microtubule breakdown. In contrast, eggs injected with cyc delta 90, a truncated cyclin that arrest eggs in M-phase, showed normal microtubule disassembly. Finally, injection of partially purified MPF into cycloheximide-arrested eggs induced cortical microtubule breakdown. These results suggest that, despite a lack of temporal coincidence, breakdown of the cortical microtubules is dependent on the activation of MPF.
10
Schroeder, T. E., and D. E. Battaglia. ""Spiral asters" and cytoplasmic rotation in sea urchin eggs: induction in Strongylocentrotus purpuratus eggs by elevated temperature." Journal of Cell Biology 100, no. 4 (April 1, 1985): 1056–62. http://dx.doi.org/10.1083/jcb.100.4.1056.
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"Spiral asters" composed of swirls of subcortical microtubules were recently described in fertilized eggs of the sea urchin Strongylocentrotus purpuratus. In our study, these structures did not occur at culture temperatures below 16 degrees C. When the culture temperature was elevated, however, "spiral asters" routinely appeared during a susceptible period before mitotic prophase when the sperm aster-diaster normally exists. A massive and protracted rotation of the cytoplasm (excluding an immobile cortex and perinuclear region) began within 1 min of exposure to elevated temperature. Fibrils of the "spiral aster" could be seen within this rotating mass even by bright-field microscopy. The identity of microtubules in these structures was confirmed by indirect immunofluorescence microscopy. A mechanistic association between "spiral aster" formation and cytoplasmic rotation was indicated by the simultaneous inhibitory effects of microtubule and dynein poisons. Inhibitors of microfilaments, however, had no effect. We infer that elevated temperature induces unique changes in the microtubules of the pre-prophase sperm aster-diaster, resulting in cytoplasmic rotation and the spiral configuration of microtubules. Comparative cytological evidence supports the idea that "spiral asters" do not normally occur in fertilized sea urchin eggs. Biogeographic evidence for S. purpuratus indicates that fertilization and development naturally occur below 15 degrees C, hence "spiral asters" in eggs of this species should be regarded as abnormalities induced in the laboratory by unnaturally elevated temperatures.
11
Field, Christine M., Aaron C. Groen, Phuong A. Nguyen, and Timothy J. Mitchison. "Spindle-to-cortex communication in cleaving, polyspermic Xenopus eggs." Molecular Biology of the Cell 26, no. 20 (October 15, 2015): 3628–40. http://dx.doi.org/10.1091/mbc.e15-04-0233.
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Mitotic spindles specify cleavage planes in early embryos by communicating their position and orientation to the cell cortex using microtubule asters that grow out from the spindle poles during anaphase. Chromatin also plays a poorly understood role. Polyspermic fertilization provides a natural experiment in which aster pairs from the same spindle (sister asters) have chromatin between them, whereas asters pairs from different spindles (nonsisters) do not. In frogs, only sister aster pairs induce furrows. We found that only sister asters recruited two conserved furrow-inducing signaling complexes, chromosome passenger complex (CPC) and Centralspindlin, to a plane between them. This explains why only sister pairs induce furrows. We then investigated factors that influenced CPC recruitment to microtubule bundles in intact eggs and a cytokinesis extract system. We found that microtubule stabilization, optimal starting distance between asters, and proximity to chromatin all favored CPC recruitment. We propose a model in which proximity to chromatin biases initial CPC recruitment to microtubule bundles between asters from the same spindle. Next a positive feedback between CPC recruitment and microtubule stabilization promotes lateral growth of a plane of CPC-positive microtubule bundles out to the cortex to position the furrow.
12
Mountain, Vicki, Calvin Simerly, Louisa Howard, Asako Ando, Gerald Schatten, and Duane A. Compton. "The Kinesin-Related Protein, Hset, Opposes the Activity of Eg5 and Cross-Links Microtubules in the Mammalian Mitotic Spindle." Journal of Cell Biology 147, no. 2 (October 18, 1999): 351–66. http://dx.doi.org/10.1083/jcb.147.2.351.
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We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.
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Sallé, Jérémy, Jing Xie, Dmitry Ershov, Milan Lacassin, Serge Dmitrieff, and Nicolas Minc. "Asymmetric division through a reduction of microtubule centering forces." Journal of Cell Biology 218, no. 3 (December 18, 2018): 771–82. http://dx.doi.org/10.1083/jcb.201807102.
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Asymmetric divisions are essential for the generation of cell fate and size diversity. They implicate cortical domains where minus end–directed motors, such as dynein, are activated to pull on microtubules to decenter asters attached to centrosomes, nuclei, or spindles. In asymmetrically dividing cells, aster decentration typically follows a centering phase, suggesting a time-dependent regulation in the competition between microtubule centering and decentering forces. Using symmetrically dividing sea urchin zygotes, we generated cortical domains of magnetic particles that spontaneously cluster endogenous dynein activity. These domains efficiently attract asters and nuclei, yielding marked asymmetric divisions. Remarkably, aster decentration only occurred after asters had first reached the cell center. Using intracellular force measurement and models, we demonstrate that this time-regulated imbalance results from a global reduction of centering forces rather than a local maturation of dynein activity at the domain. Those findings define a novel paradigm for the regulation of division asymmetry.
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Verde, F., J. M. Berrez, C. Antony, and E. Karsenti. "Taxol-induced microtubule asters in mitotic extracts of Xenopus eggs: requirement for phosphorylated factors and cytoplasmic dynein." Journal of Cell Biology 112, no. 6 (March 15, 1991): 1177–87. http://dx.doi.org/10.1083/jcb.112.6.1177.
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Taxol, a microtubule stabilizing drug, induces the formation of numerous microtubule asters in the cytoplasm of mitotic cells (De Brabander, M., G. Geuens, R. Nuydens, R. Willebrords, J. DeMey. 1981. Proc. Natl. Acad. Sci. USA. 78:5608-5612). The center of these asters share with spindle poles some characteristics such as the presence of centrosomal material and calmodulin. We have recently reproduced the assembly of taxol asters in a cell-free system (Buendia, B., C. Antony, F. Verde, M. Bornens, and E. Karsenti. 1990. J. Cell Sci. 97:259-271) using extracts of Xenopus eggs. In this paper, we show that taxol aster assembly requires phosphorylation, and that they do not grow from preformed centers, but rather by a reorganization of microtubules first crosslinked into bundles. This process seems to involve sliding of microtubules along each other and we show that cytoplasmic dynein is required for taxol aster assembly. This result provides a possible functional basis to the recent findings, that dynein is present in the spindle and enriched near spindle poles (Pfarr, C. M., M. Cove, P. M. Grissom, T. S. Hays, M. E. Porter, and J. R. McIntosh. 1990. Nature (Lond.). 345:263-265; Steuer, E. R., L. Wordeman, T. A. Schroer, and M. P. Sheetz. 1990. Nature (Lond.). 345:266-268).
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Félix, MA, C. Antony, M. Wright, and B. Maro. "Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation." Journal of Cell Biology 124, no. 1 (January 1, 1994): 19–31. http://dx.doi.org/10.1083/jcb.124.1.19.
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Centrioles organize microtubules in two ways: either microtubules elongate from the centriole cylinder itself, forming a flagellum or a cilium ("template elongation"), or pericentriolar material assembles and nucleates a microtubule aster ("astral nucleation"). During spermatogenesis in most species, a motile flagellum elongates from one of the sperm centrioles, whereas after fertilization a large aster of microtubules forms around the sperm centrioles in the egg cytoplasm. Using Xenopus egg extracts we have developed an in vitro system to study this change in microtubule-organizing activity. An aster of microtubules forms around the centrioles of permeabilized frog sperm in egg extracts, but not in pure tubulin. However, when the sperm heads are incubated in the egg extract in the presence of nocodazole, they are able to nucleate a microtubule aster after isolation and incubation with pure calf brain tubulin. This provides a two-step assay that distinguishes between centrosome assembly and subsequent microtubule nucleation. We have studied several centrosomal antigens during centrosome assembly. The CTR2611 antigen is present in the sperm head in the peri-centriolar region. gamma-tubulin and certain phosphorylated epitopes appear in the centrosome only after incubation in the egg extract. gamma-tubulin is recruited from the egg extract and associated with electron-dense patches dispersed in a wide area around the centrioles. Immunodepletion of gamma-tubulin and associated molecules from the egg extract before sperm head incubation prevents the change in microtubule-organizing activity of the sperm heads. This suggests that gamma-tubulin and/or associated molecules play a key role in centrosome formation and activity.
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Luetjens, Craig Marc, and Adriaan W. C. Dorresteijn. "Dynamic changes of the microtubule system corresponding to the unequal and spiral cleavage modes in the embryo of the zebra mussel, Dreissena polymorpha (Mollusca, Bivalvia)." Zygote 6, no. 3 (August 1998): 239–48. http://dx.doi.org/10.1017/s0967199498000185.
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Unequal cleavage requires a highly organised cytoskeleton. We investigated the localisation of both tubulins and microtubular arrays in Dreissena eggs during and after fertilisation using confocal laser scanning microscopy. Freshly spawned eggs are arrested in metaphase I. A maternal pool of γ-tubulin is found mainly in the centre of the asters of the meiotic spindle. The paternal pool of γ-tubulin, present in the fertilising sperm, could not be traced within the egg, but a microtubule-organising centre forms near the male pronucleus at anaphase II. Male and female pronuclei grow as they migrate in the wake of their aster and rendezvous. First cleavage is unequal and starts without pronuclear fusion. At metaphase the two equal-sized asters span the entire egg in a symmetrical arrangement. At late metaphase the spindle shifts along its longitudinal axis into an eccentric position and the peripheral aster takes on an umbrella-like appearance, whereas the central aster remains spherical. The cleavage furrow becomes determined in the circumferential overlap of the asters. The inequality at second cleavage, however, is due to the unequal size of the asters. The third cleavage spindle also has asymmetrical asters and spindle shift was only observed in the D-cell. The spiral character is a result of an asymmetrical organisation of the larger, vegetal aster. Our results show that the arrangement of the γ-tubulin clusters and of microtubules changes and develops during early development of Dreissena in a way that can explain the axis-generating asymmetries in cell pattern and the spiral sense of cleavage. The major cytological characters expected to direct pattern formation in this phase of development are: size, position, and symmetry or asymmetry of both spindle and asters.
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Chakravarty, Arijit, Louisa Howard, and Duane A. Compton. "A Mechanistic Model for the Organization of Microtubule Asters by Motor and Non-Motor Proteins in a Mammalian Mitotic Extract." Molecular Biology of the Cell 15, no. 5 (May 2004): 2116–32. http://dx.doi.org/10.1091/mbc.e03-08-0579.
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We used computer simulation to understand the functional relationships between motor (dynein, HSET, and Eg5) and non-motor (NuMA) proteins involved in microtubule aster organization. The simulation accurately predicted microtubule organization under all combinations of motor and non-motor proteins, provided that microtubule cross-links at minus-ends were dynamic, and dynein and HSET were restricted to cross-linking microtubules in parallel orientation only. A mechanistic model was derived from these data in which a combination of two aggregate properties, Net Minus-end–directed Force and microtubule Cross-linking Orientation Bias, determine microtubule organization. This model uses motor and non-motor proteins, accounts for motor antagonism, and predicts that alterations in microtubule Cross-linking Orientation Bias should compensate for imbalances in motor force during microtubule aster formation. We tested this prediction in the mammalian mitotic extract and, consistent with the model, found that increasing the contribution of microtubule cross-linking by NuMA compensated for the loss of Eg5 motor activity. Thus, this model proposes a precise mechanism of action of each noncentrosomal protein during microtubule aster organization and suggests that microtubule organization in spindles involves both motile forces from motors and static forces from non-motor cross-linking proteins.
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Meaders, Johnathan L., and David R. Burgess. "Microtubule-Based Mechanisms of Pronuclear Positioning." Cells 9, no. 2 (February 23, 2020): 505. http://dx.doi.org/10.3390/cells9020505.
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The zygote is defined as a diploid cell resulting from the fusion of two haploid gametes. Union of haploid male and female pronuclei in many animals occurs through rearrangements of the microtubule cytoskeleton into a radial array of microtubules known as the sperm aster. The sperm aster nucleates from paternally-derived centrioles attached to the male pronucleus after fertilization. Nematode, echinoderm, and amphibian eggs have proven as invaluable models to investigate the biophysical principles for how the sperm aster unites male and female pronuclei with precise spatial and temporal regulation. In this review, we compare these model organisms, discussing the dynamics of sperm aster formation and the different force generating mechanism for sperm aster and pronuclear migration. Finally, we provide new mechanistic insights for how sperm aster growth may influence sperm aster positioning.
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Steffen, W., H. Fuge, R. Dietz, M. Bastmeyer, and G. Müller. "Aster-free spindle poles in insect spermatocytes: evidence for chromosome-induced spindle formation?" Journal of Cell Biology 102, no. 5 (May 1, 1986): 1679–87. http://dx.doi.org/10.1083/jcb.102.5.1679.
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Tipulid spermatocytes form normally functioning bipolar spindles after one of the centrosomes is experimentally dislocated from the nucleus in late diakinesis (Dietz, R., 1959, Z. Naturforsch., 14b:749-752; Dietz, R., 1963, Zool. Anz. Suppl., 23:131-138; Dietz, R., 1966, Heredity, 19:161-166). The possibility that dissociated pericentriolar material (PCM) is nevertheless responsible for the formation of the spindle in these cells cannot be ruled out based on live observation. In studying serial sections of complete cells and of lysed cells, it was found that centrosome-free spindle poles in the crane fly show neither pericentriolar-like material nor aster microtubules, whereas the displaced centrosomes appear complete, i.e., consist of a centriole pair, aster microtubules, and PCM. Exposure to a lysis buffer containing tubulin resulted in an increase of centrosomal asters due to aster microtubule polymerization. Aster-free spindle poles did not show any reaction, also indicating the absence of PCM at these poles. The results favor the hypothesis of chromosome-induced spindle pole formation at the onset of prometaphase and the dispensability of PCM in Pales.
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Maiato, Helder, Paula Sampaio, Catarina L. Lemos, John Findlay, Mar Carmena, William C. Earnshaw, and Claudio E. Sunkel. "MAST/Orbit has a role in microtubule–kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity." Journal of Cell Biology 157, no. 5 (May 28, 2002): 749–60. http://dx.doi.org/10.1083/jcb.200201101.
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Multiple asters (MAST)/Orbit is a member of a new family of nonmotor microtubule-associated proteins that has been previously shown to be required for the organization of the mitotic spindle. Here we provide evidence that MAST/Orbit is required for functional kinetochore attachment, chromosome congression, and the maintenance of spindle bipolarity. In vivo analysis of Drosophila mast mutant embryos undergoing early mitotic divisions revealed that chromosomes are unable to reach a stable metaphase alignment and that bipolar spindles collapse as centrosomes move progressively closer toward the cell center and eventually organize into a monopolar configuration. Similarly, soon after depletion of MAST/Orbit in Drosophila S2 cells by double-stranded RNA interference, cells are unable to form a metaphase plate and instead assemble monopolar spindles with chromosomes localized close to the center of the aster. In these cells, kinetochores either fail to achieve end-on attachment or are associated with short microtubules. Remarkably, when microtubule dynamics is suppressed in MAST-depleted cells, chromosomes localize at the periphery of the monopolar aster associated with the plus ends of well-defined microtubule bundles. Furthermore, in these cells, dynein and ZW10 accumulate at kinetochores and fail to transfer to microtubules. However, loss of MAST/Orbit does not affect the kinetochore localization of D-CLIP-190. Together, these results strongly support the conclusion that MAST/Orbit is required for microtubules to form functional attachments to kinetochores and to maintain spindle bipolarity.
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Sulerud, Taylor, Abdullah Bashar Sami, Guihe Li, April Kloxin, John Oakey, and Jesse Gatlin. "Microtubule-dependent pushing forces contribute to long-distance aster movement and centration in Xenopus laevis egg extracts." Molecular Biology of the Cell 31, no. 25 (December 1, 2020): 2791–802. http://dx.doi.org/10.1091/mbc.e20-01-0088.
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In this work, we demonstrate that microtubule asters are able to center in a variety of cell geometries and can do so over long distances, even when the activity of cytoplasmic dynein is inhibited. This observation, along with additional characterizations of aster movements, is consistent with a microtubule-based pushing mechanism.
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Horne, Melinda M., and Thomas M. Guadagno. "A requirement for MAP kinase in the assembly and maintenance of the mitotic spindle." Journal of Cell Biology 161, no. 6 (June 23, 2003): 1021–28. http://dx.doi.org/10.1083/jcb.200304144.
Full textAbstract:
Circumstantial evidence has suggested the possibility of microtubule-associated protein (MAP) kinase's involvement in spindle regulation. To test this directly, we asked whether MAP kinase was required for spindle assembly in Xenopus egg extracts. Either the inhibition or the depletion of endogenous p42 MAP kinase resulted in defective spindle structures resembling asters or half-spindles. Likewise, an increase in the length and polymerization of microtubules was measured in aster assays suggesting a role for MAP kinase in regulating microtubule dynamics. Consistent with this, treatment of extracts with either a specific MAP kinase kinase inhibitor or a MAP kinase phosphatase resulted in the rapid disassembly of bipolar spindles into large asters. Finally, we report that mitotic progression in the absence of MAP kinase signaling led to multiple spindle abnormalities in NIH 3T3 cells. We therefore propose that MAP kinase is a key regulator of the mitotic spindle.
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Ault, J. G., A. J. DeMarco, E. D. Salmon, and C. L. Rieder. "Studies on the ejection properties of asters: astral microtubule turnover influences the oscillatory behavior and positioning of mono-oriented chromosomes." Journal of Cell Science 99, no. 4 (August 1, 1991): 701–10. http://dx.doi.org/10.1242/jcs.99.4.701.
Full textAbstract:
The position of a mono-oriented chromosome changes as it oscillates to and from the pole to which it is attached. Such oscillatory behavior reveals that the net force on a mono-oriented chromosome is constantly changing. Fluctuations may occur in both the polewardly directed force acting at the kinetochore and the opposing outwardly directed force associated with the aster. We have examined the ejection properties of the aster—as well as the oscillatory behavior and positioning of mono-oriented chromosomes—in relation to astral microtubule turnover. We treated cells containing monopolar spindles with drugs that affect microtubule turnover, either by promoting the depletion of dynamically unstable astral microtubules (nocodazole and colcemid) or by augmenting their numbers and stability (taxol). Both types of drugs stopped the oscillatory behavior of mono-oriented chromosomes within seconds. The final position of the chromosomes depended on how microtubule turnover was affected. In the case of nocodazole and colcemid, non-kinetochore astral microtubules were depleted first and the kinetochore-to-pole distance shortened. In these cells chromosome fragments generated by laser microsurgery were no longer expelled from the center of the aster. By contrast, with taxol the number of non-kinetochore microtubules increased and the astral ejection force became stronger as shown by the finding that the chromosomes moved away from the pole to the periphery of the monaster. Moreover, arms severed from chromosomes at the periphery of the taxol monaster failed to move further away from the aster's center. From these observations we conclude that the oscillatory movements and changing position of a mono-oriented chromosome relative to the pole are mediated by changes in the number of astral microtubules. The dynamic instability of astral microtubules that leads to a rapid turnover may contribute to the astral ejection force by allowing the continual growth of microtubules out from the aster. Growing astral microtubules may exert a pushing force that their rigidity maintains until their depolymerization.
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Waterman-Storer, Clare, Devin Y. Duey, Kari L. Weber, John Keech, Richard E. Cheney, E. D. Salmon, and William M. Bement. "Microtubules Remodel Actomyosin Networks in Xenopus Egg Extracts via Two Mechanisms of F-Actin Transport." Journal of Cell Biology 150, no. 2 (July 24, 2000): 361–76. http://dx.doi.org/10.1083/jcb.150.2.361.
Full textAbstract:
Interactions between microtubules and filamentous actin (F-actin) are crucial for many cellular processes, including cell locomotion and cytokinesis, but are poorly understood. To define the basic principles governing microtubule/F-actin interactions, we used dual-wavelength digital fluorescence and fluorescent speckle microscopy to analyze microtubules and F-actin labeled with spectrally distinct fluorophores in interphase Xenopus egg extracts. In the absence of microtubules, networks of F-actin bundles zippered together or exhibited serpentine gliding along the coverslip. When microtubules were nucleated from Xenopus sperm centrosomes, they were released and translocated away from the aster center. In the presence of microtubules, F-actin exhibited two distinct, microtubule-dependent motilities: rapid (∼250–300 nm/s) jerking and slow (∼50 nm/s), straight gliding. Microtubules remodeled the F-actin network, as F-actin jerking caused centrifugal clearing of F-actin from around aster centers. F-actin jerking occurred when F-actin bound to motile microtubules powered by cytoplasmic dynein. F-actin straight gliding occurred when F-actin bundles translocated along the microtubule lattice. These interactions required Xenopus cytosolic factors. Localization of myosin-II to F-actin suggested it may power F-actin zippering, while localization of myosin-V on microtubules suggested it could mediate interactions between microtubules and F-actin. We examine current models for cytokinesis and cell motility in light of these findings.
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Houliston, E., and R. P. Elinson. "Patterns of microtubule polymerization relating to cortical rotation in Xenopus laevis eggs." Development 112, no. 1 (May 1, 1991): 107–17. http://dx.doi.org/10.1242/dev.112.1.107.
Full textAbstract:
Following fertilization, the Xenopus egg cortex rotates relative to the cytoplasm by 30 degrees about a horizontal axis. The direction of rotation, and as a result the orientation of the embryonic body axes, is normally specified by the position of sperm entry. The mechanism of rotation appears to involve an array of aligned microtubules in the vegetal cortex (Elinson and Rowning, 1988, Devl Biol. 128, 185–197). We performed anti-tubulin immunofluorescence on sections to follow the formation of this array. Microtubules disappear rapidly from the egg following fertilization, and reappear first in the sperm aster. Surprisingly, astral microtubules then extend radially through both the animal and vegetal cytoplasm. The cortical array arises as they reach the vegetal cell surface. The eccentric position of the sperm aster gives asymmetry to the formation of the array and may explain its alignment since microtubules reaching the cortex tend to bend away from the sperm entry side. The radial polymerization of cytoplasmic microtubules is not dependent on the sperm aster or on the female pronucleus: similar but more symmetric patterns arise in artificially activated and enucleate eggs, slightly later than in fertilized eggs. These observations suggest that the cortical microtubule array forms as a result of asymmetric microtubule growth outward from cytoplasm to cortex and, since cortical and cytoplasmic microtubules remain connected throughout the period of the rotation, that the microtubules of the array rotate with the cytoplasm.
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Dikovskaya, Dina, Ian P. Newton, and Inke S. Näthke. "The Adenomatous Polyposis Coli Protein Is Required for the Formation of Robust Spindles Formed in CSF Xenopus Extracts." Molecular Biology of the Cell 15, no. 6 (June 2004): 2978–91. http://dx.doi.org/10.1091/mbc.e03-08-0613.
Full textAbstract:
Mutations in the adenomatous polyposis coli (APC) protein occur early in colon cancer and correlate with chromosomal instability. Here, we show that depletion of APC from cystostatic factor (CSF) Xenopus extracts leads to a decrease in microtubule density and changes in tubulin distribution in spindles and asters formed in such extracts. Addition of full-length APC protein or a large, N-terminally truncated APC fragment to APC-depleted extracts restored normal spindle morphology and the intact microtubule-binding site of APC was necessary for this rescue. These data indicate that the APC protein plays a role in the formation of spindles that is dependent on its effect on microtubules. Spindles formed in cycled extracts were not sensitive to APC depletion. In CSF extracts, spindles predominantly formed from aster-like intermediates, whereas in cycled extracts chromatin was the major site of initial microtubule polymerization. These data suggest that APC is important for centrosomally driven spindle formation, which was confirmed by our finding that APC depletion reduced the size of asters nucleated from isolated centrosomes. We propose that lack of microtubule binding in cancer-associated mutations of APC may contribute to defects in the assembly of mitotic spindles and lead to missegregation of chromosomes.
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Zimmerman, Wendy C., James Sillibourne, Jack Rosa, and Stephen J. Doxsey. "Mitosis-specific Anchoring of γ Tubulin Complexes by Pericentrin Controls Spindle Organization and Mitotic Entry." Molecular Biology of the Cell 15, no. 8 (August 2004): 3642–57. http://dx.doi.org/10.1091/mbc.e03-11-0796.
Full textAbstract:
Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by γ tubulin ring complexes (γ TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors γ TuRCs at spindle poles through an interaction with γ tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted γ tubulin localization and spindle organization in mitosis but had no effect on γ tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin–γ TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled γ TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of γ tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.
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Cao, Tracy T., Wakam Chang, Sarah E. Masters, and Mark S. Mooseker. "Myosin-Va Binds to and Mechanochemically Couples Microtubules to Actin Filaments." Molecular Biology of the Cell 15, no. 1 (January 2004): 151–61. http://dx.doi.org/10.1091/mbc.e03-07-0504.
Full textAbstract:
Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (∼1:24 Myosin-Va:tubulin; Kd = 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca2+, ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 μM Ca2+, ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments.
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Mangal, Sriyash, Jennifer Sacher, Taekyung Kim, Daniel Sampaio Osório, Fumio Motegi, Ana Xavier Carvalho, Karen Oegema, and Esther Zanin. "TPXL-1 activates Aurora A to clear contractile ring components from the polar cortex during cytokinesis." Journal of Cell Biology 217, no. 3 (January 8, 2018): 837–48. http://dx.doi.org/10.1083/jcb.201706021.
Full textAbstract:
During cytokinesis, a signal from the central spindle that forms between the separating anaphase chromosomes promotes the accumulation of contractile ring components at the cell equator, while a signal from the centrosomal microtubule asters inhibits accumulation of contractile ring components at the cell poles. However, the molecular identity of the inhibitory signal has remained unknown. To identify molecular components of the aster-based inhibitory signal, we developed a means to monitor the removal of contractile ring proteins from the polar cortex after anaphase onset. Using this assay, we show that polar clearing is an active process that requires activation of Aurora A kinase by TPXL-1. TPXL-1 concentrates on astral microtubules coincident with polar clearing in anaphase, and its ability to recruit Aurora A and activate its kinase activity are essential for clearing. In summary, our data identify Aurora A kinase as an aster-based inhibitory signal that restricts contractile ring components to the cell equator during cytokinesis.
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Palazzo, R. E., E. A. Vaisberg, D. G. Weiss, S. A. Kuznetsov, and W. Steffen. "Dynein is required for spindle assembly in cytoplasmic extracts of Spisula solidissima oocytes." Journal of Cell Science 112, no. 9 (May 1, 1999): 1291–302. http://dx.doi.org/10.1242/jcs.112.9.1291.
Full textAbstract:
Meiosis I spindle assembly is induced in lysate-extract mixtures prepared from clam (Spisula solidissima) oocytes. Unactivated lysate prepared from unactivated oocytes contain nuclei (germinal vesicles, GVs) which house condensed chromosomes. Treatment of unactivated lysate with clarified activated extract prepared from oocytes induced to complete meiosis by treatment with KCl induces GV breakdown (GVBD) and assembly of monopolar, bipolar, and multipolar aster-chromosome complexes. The process of in vitro meiosis I spindle assembly involves the assembly of microtubule asters and the association of these asters with the surfaces of the GVs, followed by GVBD and spindle assembly. Monoclonal antibody m74-1, known to react specifically with the N terminus of the intermediate chain of cytoplasmic dynein, recognizes Spisula oocyte dynein and inhibits in vitro meiosis I spindle assembly. Control antibody has no affect on spindle assembly. A similar inhibitory effect on spindle assembly was observed in the presence of orthovanadate, a known inhibitor of dynein ATPase activity. Neither m74-1 nor orthovanadate has any obvious affect on GVBD or aster formation. We propose that dynein function is required for the association of chromosomes with astral microtubules during in vitro meiosis I spindle assembly in these lysate-extract mixtures. However, we conclude that dynein function is not required for centrosome assembly and maturation or for centrosome-dependent aster formation.
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Gaglio, Tirso, Mary A. Dionne, and Duane A. Compton. "Mitotic Spindle Poles are Organized by Structural and Motor Proteins in Addition to Centrosomes." Journal of Cell Biology 138, no. 5 (September 8, 1997): 1055–66. http://dx.doi.org/10.1083/jcb.138.5.1055.
Full textAbstract:
The focusing of microtubules into mitotic spindle poles in vertebrate somatic cells has been assumed to be the consequence of their nucleation from centrosomes. Contrary to this simple view, in this article we show that an antibody recognizing the light intermediate chain of cytoplasmic dynein (70.1) disrupts both the focused organization of microtubule minus ends and the localization of the nuclear mitotic apparatus protein at spindle poles when injected into cultured cells during metaphase, despite the presence of centrosomes. Examination of the effects of this dynein-specific antibody both in vitro using a cell-free system for mitotic aster assembly and in vivo after injection into cultured cells reveals that in addition to its direct effect on cytoplasmic dynein this antibody reduces the efficiency with which dynactin associates with microtubules, indicating that the antibody perturbs the cooperative binding of dynein and dynactin to microtubules during spindle/aster assembly. These results indicate that microtubule minus ends are focused into spindle poles in vertebrate somatic cells through a mechanism that involves contributions from both centrosomes and structural and microtubule motor proteins. Furthermore, these findings, together with the recent observation that cytoplasmic dynein is required for the formation and maintenance of acentrosomal spindle poles in extracts prepared from Xenopus eggs (Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman, and E. Karsenti. 1996. Nature (Lond.). 382: 420–425) demonstrate that there is a common mechanism for focusing free microtubule minus ends in both centrosomal and acentrosomal spindles. We discuss these observations in the context of a search-capture-focus model for spindle assembly.
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Fujinami, N., Y. Hosoi, H. Kato, T. Mitani, K. Matsumoto, K. Saeki, and A. Iritani. "313 EFFECTS OF ETHANOL TREATMENT AFTER INTRACYTOPLASMIC SPERM INJECTION (ICSI) ON SPERM AFTER FORMATION AND THE MICROTUBULE ORGANIZATION OF BOVINE OOCYTES." Reproduction, Fertility and Development 17, no. 2 (2005): 307. http://dx.doi.org/10.1071/rdv17n2ab313.
Full textAbstract:
The cleavage rate of bovine embryos is very low without activation of oocytes after intracytoplasmic sperm injection (ICSI), although both male and female pronuclei are formed. We previously reported that the stimulus due to the injected sperm alone was sufficient to lower the MPF activity of bovine oocytes after ICSI, and the activation treatment of oocytes with ethanol at 4 h after ICSI served to maintain the low levels of MPF activity until the next cell cycle started (Fujinami et al. 2004 J. Reprod. Dev. 50, 171–178). These results suggested that activation treatment is necessary to improve the embryonic development after bovine ICSI. In bovine fertilization, the sperm introduces the centrosome into the oocyte. The centrosome acts as the microtubule-organizing center and microtubules are organized within the oocyte. It is reported that the sperm aster is important for the normal fertilization process. Therefore, failure of sperm aster formation possibly causes the failure of cleavage following fertilization. To investigate the reason of the low cleavage rate after bovine ICSI without artificial activation treatment, we examined sperm aster formation and the microtubule organization in bovine oocytes with or without activation treatment after ICSI. Bull spermatozoa immobilized by piezopulse was injected into bovine oocytes matured in vitro. At 4 h after ICSI, oocytes were treated with 7% ethanol in TCM199 for 5 min for activation. Oocytes were fixed at 6 and 12 h after ICSI, and the microtubule organization was examined by using specific antibodies and immunofluorescence microscopy. The cleavage rate (51% vs. 15%) and the developmental rate to the blastocyst stage (13% vs. 3%) were increased by ethanol treatment after ICSI (with or without ethanol treatment, respectively, P < 0.05). In oocytes activated with ethanol after ICSI, both the sperm aster formation rate at 6 h and the microtubule organization rate at 12 h after ICSI were significantly higher than in oocytes without activation treatment (58%, 80% vs. 12%, 26%, P < 0.05). It was reported that the sperm aster has an important role for the pronuclear movement to make the male and female pronuclei come into close apposition. From these results, it was concluded that oocyte activation after bovine ICSI promoted sperm aster formation and microtubule organization, and was effective to improve embryonic development. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.
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Harris, P. J., and B. P. Rubin. "Transition from mitosis to interphase in sea urchin first division: immunofluorescence studies of tubulin distribution in methacrylate sections." Journal of Histochemistry & Cytochemistry 35, no. 3 (March 1987): 343–49. http://dx.doi.org/10.1177/35.3.3546483.
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Previous immunofluorescence studies of microtubule distribution in fertilized sea urchin eggs have suffered from poor resolution caused by cell thickness, unavoidable artifacts resulting from excessive flattening, or extraction by detergents of membranes and other lipid-containing structures that may be of interest in relation to the microtubules. To avoid these difficulties, we have developed a fixation and embedding protocol based on buffered paraformaldehyde fixation and butyl-methyl methacrylate embedment, which allows immunofluorescence staining of 0.5-1 micron sections. Polymerization artifacts are reduced by polymerizing the methacrylate at a relatively low temperature (40-45 degrees C) and by flat embedding for more uniform polymerization. Using this method, we have examined mitotic stages in the first cleavage cycle of the sea urchin Strongylocentrotus purpuratus. We provide evidence that the interphase microtubules that appear after first division are not derived from the mitotic asters but are new structures growing from organizing centers within the degenerating mitotic asters. During the transition from mitosis to interphase, there is a temporary overlap of old and new microtubules to form a very large composite aster at telophase before the old structure finally disappears.
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Nédélec, François. "Computer simulations reveal motor properties generating stable antiparallel microtubule interactions." Journal of Cell Biology 158, no. 6 (September 16, 2002): 1005–15. http://dx.doi.org/10.1083/jcb.200202051.
Full textAbstract:
An aster of microtubules is a set of flexible polar filaments with dynamic plus ends that irradiate from a common location at which the minus ends of the filaments are found. Processive soluble oligomeric motor complexes can bind simultaneously to two microtubules, and thus exert forces between two asters. Using computer simulations, I have explored systematically the possible steady-state regimes reached by two asters under the action of various kinds of oligomeric motors. As expected, motor complexes can induce the asters to fuse, for example when the complexes consist only of minus end–directed motors, or to fully separate, when the motors are plus end directed. More surprisingly, complexes made of two motors of opposite directionalities can also lead to antiparallel interactions between overlapping microtubules that are stable and sustained, like those seen in mitotic spindle structures. This suggests that such heterocomplexes could have a significant biological role, if they exist in the cell.
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Kobayashi, Toshihiro, Kazue Amemiya, Kana Takeuchi, Tomomi Tsujioka, Keiichiro Tominaga, Masumi Hirabayashi, Hajime Ishikawa, Yutaka Fukui, and Shinichi Hochi. "Contribution of spermatozoal centrosomes to the microtubule-organizing centre in Antarctic minke whale (Balaenoptera bonaerensis)." Zygote 14, no. 1 (February 2006): 45–51. http://dx.doi.org/10.1017/s0967199406003522.
Full textAbstract:
Using an interspecies microinsemination assay with bovine oocytes, it was examined whether centrosomes of Antarctic minke whale spermatozoa function as the microtubule-organizing centre (MTOC). Bull and rat spermatozoa were used as positive and negative controls, respectively. Vitrified-warmed bovine mature oocytes were subjected to immunostaining against α-tubulin 4–6 h after intracytoplasmic injection (ICSI) of 5 mM dithiothreitol-treated spermatozoa. Aster formation occurred from whale spermatozoa (33%) and bull spermatozoa (33%), but very little from rat spermatozoa (3%). Activation treatment for the microinseminated oocytes with 7% ethanol + 2 mM 6-dimethylaminopurine resulted in a similar proportion of oocytes forming a whale sperm aster (35% vs 27% in the non-treated group; 4 h after ICSI) but a significantly larger aster (ratio of aster diameter to oocyte diameter, 0.57 vs 0.30 in the non-treated group). These results indicate that the centrosome introduced into bovine oocytes by whale spermatozoa contributes to the MTOC and that assembly of the microtubule network is promoted by oocyte activation.
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Kim, Bong-Ki, Youn-Jeong Lee, Xiang-Shun Cui, and Nam-Hyung Kim. "Chromatin and microtubule organisation in maturing and pre-activated porcine oocytes following intracytoplasmic sperm injection." Zygote 10, no. 2 (May 2002): 123–29. http://dx.doi.org/10.1017/s0967199402002174.
Full textAbstract:
Chromatin and microtubule organisation was determined in maturing and activated porcine oocytes following intracytoplasmic sperm injection in order to obtain insights into the nature of sperm chromatin decondensation and microtubule nucleation activity. Sperm chromatin was slightly decondensed at 8 h following injection into germinal vesicle stage oocytes. Sperm-derived microtubules were not seen in these oocytes. Following injection into metaphase I (MI)-stage oocytes, sperm chromatin went to metaphase in most cases. A meiotic-like spindle was seen in the sperm metaphase chromatin. In a few MI-stage oocytes, sperm chromatin decondensed at 8 h after injection, and a small sperm aster was seen. Sperm injection into oocytes at 5 h following activation failed to yield pronuclear formation. Maternally derived microtubules were organised near the female chromatin in these oocytes, and seemed to move condensed male chromatin closer to the female pronucleus. At 18 h after sperm injection into pre-activated oocytes, a condensed sperm nucleus was located in close proximity to the female pronucleus. These results suggest that the sperm nuclear decondensing activity and microtubule nucleation abilities of the male centrosome are cell cycle dependent. In the absence of a functional male centrosome, microtubules of female origin take over the role of microtubule nucleation for nuclear movement.
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Zhu, Jie, Anton Burakov, Vladimir Rodionov, and Alex Mogilner. "Finding the Cell Center by a Balance of Dynein and Myosin Pulling and Microtubule Pushing: A Computational Study." Molecular Biology of the Cell 21, no. 24 (December 15, 2010): 4418–27. http://dx.doi.org/10.1091/mbc.e10-07-0627.
Full textAbstract:
The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces—dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles—is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility.
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Ems-McClung, Stephanie C., Kathleen M. Hertzer, Xin Zhang, Mill W. Miller, and Claire E. Walczak. "The Interplay of the N- and C-Terminal Domains of MCAK Control Microtubule Depolymerization Activity and Spindle Assembly." Molecular Biology of the Cell 18, no. 1 (January 2007): 282–94. http://dx.doi.org/10.1091/mbc.e06-08-0724.
Full textAbstract:
Spindle assembly and accurate chromosome segregation require the proper regulation of microtubule dynamics. MCAK, a Kinesin-13, catalytically depolymerizes microtubules, regulates physiological microtubule dynamics, and is the major catastrophe factor in egg extracts. Purified GFP-tagged MCAK domain mutants were assayed to address how the different MCAK domains contribute to in vitro microtubule depolymerization activity and physiological spindle assembly activity in egg extracts. Our biochemical results demonstrate that both the neck and the C-terminal domain are necessary for robust in vitro microtubule depolymerization activity. In particular, the neck is essential for microtubule end binding, and the C-terminal domain is essential for tight microtubule binding in the presence of excess tubulin heterodimer. Our physiological results illustrate that the N-terminal domain is essential for regulating microtubule dynamics, stimulating spindle bipolarity, and kinetochore targeting; whereas the C-terminal domain is necessary for robust microtubule depolymerization activity, limiting spindle bipolarity, and enhancing kinetochore targeting. Unexpectedly, robust MCAK microtubule (MT) depolymerization activity is not needed for sperm-induced spindle assembly. However, high activity is necessary for proper physiological MT dynamics as assayed by Ran-induced aster assembly. We propose that MCAK activity is spatially controlled by an interplay between the N- and C-terminal domains during spindle assembly.
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SAKAUSHI, Shinji, Kaori SENDA-MURATA, Shigenori OKA, and Kenji SUGIMOTO. "Visualization of Aberrant Perinuclear Microtubule Aster Organization by Microtubule-Destabilizing Agents." Bioscience, Biotechnology, and Biochemistry 73, no. 5 (May 23, 2009): 1192–96. http://dx.doi.org/10.1271/bbb.80754.
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Hamaguchi, Y., M. Toriyama, H. Sakai, and Y. Hiramoto. "Distribution of fluorescently labeled tubulin injected into sand dollar eggs from fertilization through cleavage." Journal of Cell Biology 100, no. 4 (April 1, 1985): 1262–72. http://dx.doi.org/10.1083/jcb.100.4.1262.
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Porcine brain tubulin labeled with fluorescein isothiocyanate (FITC) was able to polymerize by itself and co-polymerize with tubulin purified from starfish sperm flagella. When we injected the FITC-labeled tubulin into unfertilized eggs of the sand dollar, Clypeaster japonicus, and the eggs were then fertilized, the labeled tubulin was incorporated into the sperm aster. When injected into fertilized eggs at streak stage, the tubulin was quickly incorporated into each central region of growing asters. It was clearly visualized that the labeled tubulin, upon reaching metaphase, accumulated in the mitotic apparatus and later disappeared over the cytoplasm during interphase. The accumulation of the fluorescence in the mitotic apparatus was observed repeatedly at successive cleavage. After lysis of the fertilized eggs with a microtubule-stabilizing solution, fluorescent fibrous structures around the nucleus and those of the sperm aster and the mitotic apparatus were preserved and coincided with the fibrous structures observed by polarization and differential interference microscopy. We found the FITC-labeled tubulin to be incorporated into the entire mitotic apparatus within 20-30 s when injected into the eggs at metaphase or anaphase. This rapid incorporation of the labeled tubulin into the mitotic apparatus suggests that the equilibrium between mitotic microtubules and tubulin is attained very rapidly in the living eggs. Axonemal tubulin purified from starfish sperm flagella and labeled with FITC was also incorporated into microtubular structures in the same fashion as the FITC-labeled brain tubulin. These results suggest that even FITC-labeled heterogeneous tubulins undergo spatial and stage-specific regulation of assembly-disassembly in the same manner as does sand dollar egg tubulin.
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Stefanovic, Sandra, Miriam Windsor, Koh-ici Nagata, Masaki Inagaki, and Thomas Wileman. "Vimentin Rearrangement during African Swine Fever Virus Infection Involves Retrograde Transport along Microtubules and Phosphorylation of Vimentin by Calcium Calmodulin Kinase II." Journal of Virology 79, no. 18 (September 15, 2005): 11766–75. http://dx.doi.org/10.1128/jvi.79.18.11766-11775.2005.
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ABSTRACT African swine fever virus (ASFV) infection leads to rearrangement of vimentin into a cage surrounding virus factories. Vimentin rearrangement in cells generally involves phosphorylation of N-terminal domains of vimentin by cellular kinases to facilitate disassembly and transport of vimentin filaments on microtubules. Here, we demonstrate that the first stage in vimentin rearrangement during ASFV infection involves a microtubule-dependent concentration of vimentin into an “aster” within virus assembly sites located close to the microtubule organizing center. The aster may play a structural role early during the formation of the factory. Conversion of the aster into a cage required ASFV DNA replication. Interestingly, viral DNA replication also resulted in the activation of calcium calmodulin-dependent protein kinase II (CaM kinase II) and phosphorylation of the N-terminal domain of vimentin on serine 82. Immunostaining showed that vimentin within the cage was phosphorylated on serine 82. Significantly, both viral DNA replication and Ser 82 phosphorylation were blocked by KN93, an inhibitor of CaM kinase II, suggesting a link between CaM kinase II activation, DNA replication, and late gene expression. Phosphorylation of vimentin on serine 82 may be necessary for cage formation or may simply be a consequence of activation of CaM kinase II by ASFV. The vimentin cage may serve a cytoprotective function and prevent movement of viral components into the cytoplasm and at the same time concentrate late structural proteins at sites of virus assembly.
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Gundersen, G. G., S. Khawaja, and J. C. Bulinski. "Postpolymerization detyrosination of alpha-tubulin: a mechanism for subcellular differentiation of microtubules." Journal of Cell Biology 105, no. 1 (July 1, 1987): 251–64. http://dx.doi.org/10.1083/jcb.105.1.251.
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Tyrosinated (Tyr) and detyrosinated (Glu) alpha-tubulin, species interconverted by posttranslational modification, are largely segregated in separate populations of microtubules in interphase cultured cells. We sought to understand how distinct Tyr and Glu microtubules are generated in vivo, by examining time-dependent alterations in Tyr and Glu tubulin levels (by immunoblots probed with antibodies specific for each species) and distributions (by immunofluorescence) after microtubule regrowth and stabilization. When microtubules were allowed to regrow after complete depolymerization by microtubule antagonists, Glu microtubules reappeared with a delay of approximately 25 min after the complete array of Tyr microtubules had regrown. In these experiments, Tyr tubulin immunofluorescence first appeared as an aster of distinct microtubules, while Glu tubulin staining first appeared as a grainy pattern that was not altered by detergent extraction, suggesting that Glu microtubules were created by detyrosination of Tyr microtubules. Treatments with taxol, azide, or vinblastine, to stabilize polymeric tubulin, all resulted in time-dependent increases in polymeric Glu tubulin levels, further supporting the hypothesis of postpolymerization detyrosination. Analysis of monomer and polymer fractions during microtubule regrowth and in microtubule stabilization experiments were also consistent with postpolymerization detyrosination; in each case, Glu polymer levels increased in the absence of detectable Glu monomer. The low level of Glu monomer in untreated or nocodazole-treated cells (we estimate that Glu tubulin comprises less than 2% of the monomer pool) also suggested that Glu tubulin entering the monomer pool is efficiently retyrosinated. Taken together these results demonstrate that microtubules are polymerized from Tyr tubulin and are then rapidly converted to Glu microtubules. When Glu microtubules depolymerize, the resulting Glu monomer is retyrosinated. This cycle generates structurally, and perhaps functionally, distinct microtubules.
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Kim, N. H., M. R. Shin, and S. H. Park. "47BOVINE OOCYTE CYTOPLASM SUPPORTS NUCLEAR REMODELING BUT NOT REPROGRAMMING OF MURINE FIBROBLASTS." Reproduction, Fertility and Development 16, no. 2 (2004): 145. http://dx.doi.org/10.1071/rdv16n1ab47.
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Nuclear transfer (NT) is used to elucidate fundamental biological functions such as cell differentiation reversibility and interactions between the cytoplasm and nucleus. In the present study, we compared nuclear and microtubule dynamics in bovine oocytes following NT of bovine and murine fibroblast cells. To clarify the nuclear reprogramming procedures, we additionally examined the expression of development-related genes (Octamer-binding transcription factors, Oct-4; E-cadherin, E-cad) and housekeeping genes (Heat shock protein 70.1, Hsp; Bos taurus apoptosis regulator box-a, Bax; Glucose transporter 1, Glut-1) in bovine embryos that had received nuclei from bovine and murine fibroblast cells. Bovine oocytes were matured in vitro and enucleated after 22h. The oocytes reconstructed with mouse embryonic fibroblast cells or bovine somatic fibroblast cells were cultured in CR1aa media. While the embryos that received nuclei from bovine fibroblast cells developed into blastocysts, those that received nuclei from murine fibroblasts did not develop beyond the 8-cell stage. Similar nuclear and microtubule dynamics were observed in oocytes reconstructed with murine and bovine fibroblast cells. A small microtubule aster-containing γ-tubulin spot was observed in association with decondensed chromatin following NT of mouse fibroblasts. Within 1h of fusion of enucleated, non-activated cytoplasm, most mouse fibroblast nuclei were transformed to premature chromosome condensation (PCC). Randomly arrayed microtubules were tightly associated with PCC and formed meiotic-like microtubular spindles in all cases. Condensed chromosomes were divided into two or three chromatin masses and developed into multiple pronuclear-like structures. Microtubule asters were observed near the pronuclear-like structures during apposition in the cytoplasm. Two poles of the γ-tubulin spot evident at the mitotic metaphase stage are involved in the formation of the astral microtubule spindle for initial mitosis. A number of housekeeping mouse genes (hsp70, bax and glt-1) were abnormally expressed in embryos that had received nuclei from mouse fibroblast cells. However, development-related genes, such as Oct-4 and E-cad, were not expressed. The results collectively suggest that the bovine oocyte cytoplasm supports nuclear remodeling, but not reprogramming of mouse fibroblast cells. Table 1 Relative abundance of mRNA expression (mean±SEM) in mouse and xenonuclear-transferred (X-NT) embryos
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Houliston, E., D. Carre, J. A. Johnston, and C. Sardet. "Axis establishment and microtubule-mediated waves prior to first cleavage in Beroe ovata." Development 117, no. 1 (January 1, 1993): 75–87. http://dx.doi.org/10.1242/dev.117.1.75.
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The single axis (oral-aboral) and two planes of symmetry of the ctenophore Beroe ovata become established with respect to the position of zygote nucleus formation and the orientation of first cleavage. Bisection of Beroe eggs at different times revealed that differences in egg organisation are established in relation to the presumptive oral-aboral axis before first cleavage. Lateral fragments produced after but not before the time of first mitosis developed into larvae lacking comb-plates on one side. Time-lapse video demonstrated that waves of cytoplasmic reorganisation spread through the layer of peripheral cytoplasm (ectoplasm) of the egg during the 80 minute period between pronuclear fusion and first cleavage, along the future oral-aboral axis. These waves are manifest as the progressive displacement and dispersal of plaques of accumulated organelles around supernumerary sperm nuclei, and a series of surface movements. Their timing and direction of propagation suggest they may be involved in establishing cytoplasmic differences with respect to the embryonic axis. Inhibitor experiments suggested that the observed cytoplasmic reorganisation involves microtubules. Nocodazole and taxol, which prevent microtubule turnover, blocked plaque dispersal and reduced surface movements. The microfilament-disrupting drug cytochalasin B did not prevent plaque dispersal but induced abnormal surface contractions. We examined changes in microtubule organisation using immunofluorescence on eggs fixed at different times and in live eggs following injection of rhodamine-tubulin. Giant microtubule asters become associated with each male pronucleus after the end of meiosis. Following pronuclear fusion they disappear successively, those nearest the zygote nucleus shrinking first, to establish gradients of aster size within single eggs. Regional differences in microtubule behaviour around the time of mitosis were revealed by brief taxol treatment, which induced the formation of small microtubule asters in the region of the nucleus or spindle during both first and second cell cycles. The observed wave of change may thus reflect the local appearance and spreading of mitotic activity as the zygote nucleus approaches mitosis.
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Żyłkiewicz, Eliza, Monika Kijańska, Won-Chan Choi, Urszula Derewenda, Zygmunt S. Derewenda, and P. Todd Stukenberg. "The N-terminal coiled-coil of Ndel1 is a regulated scaffold that recruits LIS1 to dynein." Journal of Cell Biology 192, no. 3 (January 31, 2011): 433–45. http://dx.doi.org/10.1083/jcb.201011142.
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Ndel1 has been implicated in a variety of dynein-related processes, but its specific function is unclear. Here we describe an experimental approach to evaluate a role of Ndel1 in dynein-dependent microtubule self-organization using Ran-mediated asters in meiotic Xenopus egg extracts. We demonstrate that extracts depleted of Ndel1 are unable to form asters and that this defect can be rescued by the addition of recombinant N-terminal coiled-coil domain of Ndel1. Ndel1-dependent microtubule self-organization requires an interaction between Ndel1 and dynein, which is mediated by the dimerization fragment of the coiled-coil. Full rescue by the coiled-coil domain requires LIS1 binding, and increasing LIS1 concentration partly rescues aster formation, suggesting that Ndel1 is a recruitment factor for LIS1. The interactions between Ndel1 and its binding partners are positively regulated by phosphorylation of the unstructured C terminus. Together, our results provide important insights into how Ndel1 acts as a regulated scaffold to temporally and spatially regulate dynein.
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Huang, Shu-Ching, Eva S. Liu, Siu-Hong Chan, Indira D. Munagala, Heidi T. Cho, Ramasamy Jagadeeswaran, and Edward J. Benz. "Mitotic Regulation of Protein 4.1R Involves Phosphorylation by cdc2 Kinase." Molecular Biology of the Cell 16, no. 1 (January 2005): 117–27. http://dx.doi.org/10.1091/mbc.e04-05-0426.
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The nonerythrocyte isoform of the cytoskeletal protein 4.1R (4.1R) is associated with morphologically dynamic structures during cell division and has been implicated in mitotic spindle function. In this study, we define important 4.1R isoforms expressed in interphase and mitotic cells by RT-PCR and mini-cDNA library construction. Moreover, we show that 4.1R is phosphorylated by p34cdc2kinase on residues Thr60 and Ser679 in a mitosis-specific manner. Phosphorylated 4.1R135isoform(s) associate with tubulin and Nuclear Mitotic Apparatus protein (NuMA) in intact HeLa cells in vivo as well as with the microtubule-associated proteins in mitotic asters assembled in vitro. Recombinant 4.1R135is readily phosphorylated in mitotic extracts and reconstitutes mitotic aster assemblies in 4.1R-immunodepleted extracts in vitro. Furthermore, phosphorylation of these residues appears to be essential for the targeting of 4.1R to the spindle poles and for mitotic microtubule aster assembly in vitro. Phosphorylation of 4.1R also enhances its association with NuMA and tubulin. Finally, we used siRNA inhibition to deplete 4.1R from HeLa cells and provide the first direct genetic evidence that 4.1R is required to efficiently focus mitotic spindle poles. Thus, we suggest that 4.1R is a member of the suite of direct cdc2 substrates that are required for the establishment of a bipolar spindle.
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Maryshev, Ivan, Alexander Morozov, Andrew B. Goryachev, and Davide Marenduzzo. "Pattern formation in active model C with anchoring: bands, aster networks, and foams." Soft Matter 16, no. 38 (2020): 8775–81. http://dx.doi.org/10.1039/d0sm00927j.
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Ookata, K., S. Hisanaga, J. C. Bulinski, H. Murofushi, H. Aizawa, T. J. Itoh, H. Hotani, E. Okumura, K. Tachibana, and T. Kishimoto. "Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics." Journal of Cell Biology 128, no. 5 (March 1, 1995): 849–62. http://dx.doi.org/10.1083/jcb.128.5.849.
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We previously demonstrated (Ookata et al., 1992, 1993) that the p34cdc2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule-associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34cdc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34cdc2/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34cdc2/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34cdc2, cyclin B, and the COOH-terminal domain of MAP4, PA4, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34cdc2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.
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Jin, Yong-Xun, Xiang-Shun Cui, Xian-Feng Yu, Sung-Hyun Lee, Qing-Ling Wang, Wei-Wei Gao, Yong-Nan Xu, Shao-Chen Sun, IL-Keun Kong, and Nam-Hyung Kim. "Cat fertilization by mouse sperm injection." Zygote 20, no. 4 (July 27, 2011): 371–78. http://dx.doi.org/10.1017/s0967199411000451.
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SummaryInterspecies intracytoplasmic sperm injection has been carried out to understand species-specific differences in oocyte environments and sperm components during fertilization. While sperm aster organization during cat fertilization requires a paternally derived centriole, mouse and hamster fertilization occur within the maternal centrosomal components. To address the questions of where sperm aster assembly occurs and whether complete fertilization is achieved in cat oocytes by interspecies sperm, we studied the fertilization processes of cat oocytes following the injection of cat, mouse, or hamster sperm. Male and female pronuclear formations were not different in the cat oocytes at 6 h following cat, mouse or hamster sperm injection. Microtubule asters were seen in all oocytes following intracytoplasmic injection of cat, mouse or hamster sperm. Immunocytochemical staining with a histone H3-m2K9 antibody revealed that mouse sperm chromatin is incorporated normally with cat egg chromatin, and that the cat eggs fertilized with mouse sperm enter metaphase and become normal 2-cell stage embryos. These results suggest that sperm aster formation is maternally dependent, and that fertilization processes and cleavage occur in a non-species specific manner in cat oocytes.
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Nédélec, François, and Thomas Surrey. "Dynamics of microtubule aster formation by motor complexes." Comptes Rendus de l'Académie des Sciences - Series IV - Physics-Astrophysics 2, no. 6 (August 2001): 841–47. http://dx.doi.org/10.1016/s1296-2147(01)01227-6.
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