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Kinetochore-independent chromosome poleward movement during anaphase of meiosis II in mouse eggs.

Deng M, Gao J, Suraneni P, Li R - PLoS ONE (2009)

Bottom Line: Disruption of dynein function abolished the poleward movements of DNA beads but not of the meiotic chromosomes, suggesting the existence of different dynein-dependent and dynein-independent force generation mechanisms for the chromosome poleward movement, and the latter may be dependent on the presence of kinetochores.Consistent with the observed DNA bead poleward movement, sperm haploid chromatin (which also induced bipolar spindle formation after injection to a metaphase egg without forming detectable kinetochore structures) also underwent similar poleward movement at anaphase as DNA beads.The results suggest that in the chromatin-induced meiotic spindles, kinetochore attachments to spindle microtubules are not absolutely required for chromatin poleward movements at anaphase.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America. MQD@stowers-institute.org

ABSTRACT
Kinetochores are considered to be the key structures that physically connect spindle microtubules to the chromosomes and play an important role in chromosome segregation during mitosis. Due to different mechanisms of spindle assembly between centrosome-containing mitotic cells and acentrosomal meiotic oocytes, it is unclear how a meiotic spindle generates the poleward forces to drive two rounds of meiotic chromosome segregation to achieve genome haploidization. We took advantage of the fact that DNA beads are able to induce bipolar spindle formation without kinetochores and studied the behavior of DNA beads in the induced spindle in mouse eggs during meiosis II. Interestingly, DNA beads underwent poleward movements that were similar in timing and speed to the meiotic chromosomes, although all the beads moved together to the same spindle pole. Disruption of dynein function abolished the poleward movements of DNA beads but not of the meiotic chromosomes, suggesting the existence of different dynein-dependent and dynein-independent force generation mechanisms for the chromosome poleward movement, and the latter may be dependent on the presence of kinetochores. Consistent with the observed DNA bead poleward movement, sperm haploid chromatin (which also induced bipolar spindle formation after injection to a metaphase egg without forming detectable kinetochore structures) also underwent similar poleward movement at anaphase as DNA beads. The results suggest that in the chromatin-induced meiotic spindles, kinetochore attachments to spindle microtubules are not absolutely required for chromatin poleward movements at anaphase.

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Lack of CENP-A in DNA bead- and sperm chromatin-assembled spindles in MII eggs.(A) CENP-A in a DNA bead-injected egg. The arrow indicates the location of MII chromosomes and the arrowhead indicates the position of DNA beads. (B) DIC image of (A) showing the position of the injected DNA beads (arrowhead). (C) The same egg of (A) showing spindle (red) and CENP-A (green) and DNA (blue). Note that the two spindle poles induced by the DNA beads are not in the same focal plane. (D–F) A sperm chromatin-injected egg showing CENP-A (green), DNA (blue) and spindle (red). The arrow indicates the location of MII chromosomes and the arrowhead indicates the location of the injected sperm chromatin. MII represents the meiotic chromosomes and Sp indicates sperm chromatin.
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pone-0005249-g003: Lack of CENP-A in DNA bead- and sperm chromatin-assembled spindles in MII eggs.(A) CENP-A in a DNA bead-injected egg. The arrow indicates the location of MII chromosomes and the arrowhead indicates the position of DNA beads. (B) DIC image of (A) showing the position of the injected DNA beads (arrowhead). (C) The same egg of (A) showing spindle (red) and CENP-A (green) and DNA (blue). Note that the two spindle poles induced by the DNA beads are not in the same focal plane. (D–F) A sperm chromatin-injected egg showing CENP-A (green), DNA (blue) and spindle (red). The arrow indicates the location of MII chromosomes and the arrowhead indicates the location of the injected sperm chromatin. MII represents the meiotic chromosomes and Sp indicates sperm chromatin.

Mentions: To test if the observed DNA bead poleward movement is a general phenomenon of kinetochore-free chromosomes during meiosis, we injected mouse sperm chromatin into eggs to induce spindle formation and followed their behavior during meiosis II. It was previously observed that Xenopus sperm chromatin is able to induce spindle formation in egg extracts without forming kinetochores or kinetochore microtubules [35]. This was confirmed by negative immunostaining of centromere protein A (CENP-A), a kinetochore marker, in both DNA bead- and sperm-assembled spindles (Figure 3A, 3D, arrowheads). Consistent with that observed in DNA bead-spindles, the condensed sperm chromatin moved poleward without disjunction during anaphase of meiosis II (Figure 1F, arrow). The monopolar movements of DNA beads and sperm chromatin might be due to physical stickiness of the beads or the highly condensed sperm chromatin. To test this possibility, DNA beads were co-injected with sperm chromatin into an egg to induce formation of a single spindle containing both DNA beads (Figure 1G, arrowhead) and sperm chromatin (Figure 1G, arrow). After inducing anaphase onset, the co-injected DNA beads and sperm chromatin either segregated from each other and moved toward opposite poles (Figure 1H, arrow and arrowhead, n = 7/20), or moved together to the same pole (Figure 1I, arrow and arrowhead, n = 13/20). This suggests that in the absence of kinetochore, the chromosome-induced spindle has the ability to move chromosomes toward opposite poles but loses the assurance of equal segregation.


Kinetochore-independent chromosome poleward movement during anaphase of meiosis II in mouse eggs.

Deng M, Gao J, Suraneni P, Li R - PLoS ONE (2009)

Lack of CENP-A in DNA bead- and sperm chromatin-assembled spindles in MII eggs.(A) CENP-A in a DNA bead-injected egg. The arrow indicates the location of MII chromosomes and the arrowhead indicates the position of DNA beads. (B) DIC image of (A) showing the position of the injected DNA beads (arrowhead). (C) The same egg of (A) showing spindle (red) and CENP-A (green) and DNA (blue). Note that the two spindle poles induced by the DNA beads are not in the same focal plane. (D–F) A sperm chromatin-injected egg showing CENP-A (green), DNA (blue) and spindle (red). The arrow indicates the location of MII chromosomes and the arrowhead indicates the location of the injected sperm chromatin. MII represents the meiotic chromosomes and Sp indicates sperm chromatin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664963&req=5

pone-0005249-g003: Lack of CENP-A in DNA bead- and sperm chromatin-assembled spindles in MII eggs.(A) CENP-A in a DNA bead-injected egg. The arrow indicates the location of MII chromosomes and the arrowhead indicates the position of DNA beads. (B) DIC image of (A) showing the position of the injected DNA beads (arrowhead). (C) The same egg of (A) showing spindle (red) and CENP-A (green) and DNA (blue). Note that the two spindle poles induced by the DNA beads are not in the same focal plane. (D–F) A sperm chromatin-injected egg showing CENP-A (green), DNA (blue) and spindle (red). The arrow indicates the location of MII chromosomes and the arrowhead indicates the location of the injected sperm chromatin. MII represents the meiotic chromosomes and Sp indicates sperm chromatin.
Mentions: To test if the observed DNA bead poleward movement is a general phenomenon of kinetochore-free chromosomes during meiosis, we injected mouse sperm chromatin into eggs to induce spindle formation and followed their behavior during meiosis II. It was previously observed that Xenopus sperm chromatin is able to induce spindle formation in egg extracts without forming kinetochores or kinetochore microtubules [35]. This was confirmed by negative immunostaining of centromere protein A (CENP-A), a kinetochore marker, in both DNA bead- and sperm-assembled spindles (Figure 3A, 3D, arrowheads). Consistent with that observed in DNA bead-spindles, the condensed sperm chromatin moved poleward without disjunction during anaphase of meiosis II (Figure 1F, arrow). The monopolar movements of DNA beads and sperm chromatin might be due to physical stickiness of the beads or the highly condensed sperm chromatin. To test this possibility, DNA beads were co-injected with sperm chromatin into an egg to induce formation of a single spindle containing both DNA beads (Figure 1G, arrowhead) and sperm chromatin (Figure 1G, arrow). After inducing anaphase onset, the co-injected DNA beads and sperm chromatin either segregated from each other and moved toward opposite poles (Figure 1H, arrow and arrowhead, n = 7/20), or moved together to the same pole (Figure 1I, arrow and arrowhead, n = 13/20). This suggests that in the absence of kinetochore, the chromosome-induced spindle has the ability to move chromosomes toward opposite poles but loses the assurance of equal segregation.

Bottom Line: Disruption of dynein function abolished the poleward movements of DNA beads but not of the meiotic chromosomes, suggesting the existence of different dynein-dependent and dynein-independent force generation mechanisms for the chromosome poleward movement, and the latter may be dependent on the presence of kinetochores.Consistent with the observed DNA bead poleward movement, sperm haploid chromatin (which also induced bipolar spindle formation after injection to a metaphase egg without forming detectable kinetochore structures) also underwent similar poleward movement at anaphase as DNA beads.The results suggest that in the chromatin-induced meiotic spindles, kinetochore attachments to spindle microtubules are not absolutely required for chromatin poleward movements at anaphase.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America. MQD@stowers-institute.org

ABSTRACT
Kinetochores are considered to be the key structures that physically connect spindle microtubules to the chromosomes and play an important role in chromosome segregation during mitosis. Due to different mechanisms of spindle assembly between centrosome-containing mitotic cells and acentrosomal meiotic oocytes, it is unclear how a meiotic spindle generates the poleward forces to drive two rounds of meiotic chromosome segregation to achieve genome haploidization. We took advantage of the fact that DNA beads are able to induce bipolar spindle formation without kinetochores and studied the behavior of DNA beads in the induced spindle in mouse eggs during meiosis II. Interestingly, DNA beads underwent poleward movements that were similar in timing and speed to the meiotic chromosomes, although all the beads moved together to the same spindle pole. Disruption of dynein function abolished the poleward movements of DNA beads but not of the meiotic chromosomes, suggesting the existence of different dynein-dependent and dynein-independent force generation mechanisms for the chromosome poleward movement, and the latter may be dependent on the presence of kinetochores. Consistent with the observed DNA bead poleward movement, sperm haploid chromatin (which also induced bipolar spindle formation after injection to a metaphase egg without forming detectable kinetochore structures) also underwent similar poleward movement at anaphase as DNA beads. The results suggest that in the chromatin-induced meiotic spindles, kinetochore attachments to spindle microtubules are not absolutely required for chromatin poleward movements at anaphase.

Show MeSH