Meiosis-Specific Stable Binding of Augmin to Acentrosomal Spindle Poles Promotes Biased Microtubule Assembly in Oocytes

In the oocytes of many animals including humans, the meiotic spindle assembles without centrosomes. It is still unclear how multiple pathways contribute to spindle microtubule assembly, and whether they are regulated differently in mitosis and meiosis. Augmin is a γ-tubulin recruiting complex which "amplifies" spindle microtubules by generating new microtubules along existing ones in mitosis. Here we show that in Drosophila melanogaster oocytes Augmin is dispensable for chromatin-driven assembly of bulk spindle microtubules, but is required for full microtubule assembly near the poles. The level of Augmin accumulated at spindle poles is well correlated with the degree of chromosome congression. Fluorescence recovery after photobleaching shows that Augmin stably associates with the polar regions of the spindle in oocytes, unlike in mitotic cells where it transiently and uniformly associates with the metaphase spindle. This stable association is enhanced by γ-tubulin and the kinesin-14 Ncd. Therefore, we suggest that meiosis-specific regulation of Augmin compensates for the lack of centrosomes in oocytes by actively biasing sites of microtubule generation within the spindle

The microtubule catastrophe promoter Sentin delays stable kinetochore-microtubule attachment in oocytes

The critical step in meiosis is to attach homologous chromosomes to the opposite poles. In mouse oocytes, stable microtubule end-on attachments to kinetochores are not established until hours after spindle assembly, and phosphorylation of kinetochore proteins by Aurora B/C is responsible for the delay. Here we demonstrated that microtubule ends are actively prevented from stable attachment to kinetochores until well after spindle formation in Drosophila melanogaster oocytes. We identified the microtubule catastrophe-promoting complex Sentin-EB1 as a major factor responsible for this delay. Without this activity, microtubule ends precociously form robust attachments to kinetochores in oocytes, leading to a high proportion of homologous kinetochores stably attached to the same pole. Therefore, regulation of microtubule ends provides an alternative novel mechanism to delay stable kinetochore–microtubule attachment in oocytes

Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes

In oocytes, where centrosomes are absent, the chromosomes direct the assembly of a bipolar spindle. Interactions between chromosomes and microtubules are essential for both spindle formation and chromosome segregation, but the nature and function of these interactions is not clear. We have examined oocytes lacking two kinetochore proteins, NDC80 and SPC105R, and a centromere-associated motor protein, CENP-E, to characterize the impact of kinetochore-microtubule attachments on spindle assembly and chromosome segregation in Drosophila oocytes. We found that the initiation of spindle assembly results from chromosome-microtubule interactions that are kinetochore-independent. Stabilization of the spindle, however, depends on both central spindle and kinetochore components. This stabilization coincides with changes in kinetochore-microtubule attachments and bi-orientation of homologs. We propose that the bi-orientation process begins with the kinetochores moving laterally along central spindle microtubules towards their minus ends. This movement depends on SPC105R, can occur in the absence of NDC80, and is antagonized by plus-end directed forces from the CENP-E motor. End-on kinetochore-microtubule attachments that depend on NDC80 are required to stabilize bi-orientation of homologs. A surprising finding was that SPC105R but not NDC80 is required for co-orientation of sister centromeres at meiosis I. Together, these results demonstrate that, in oocytes, kinetochore-dependent and -independent chromosome-microtubule attachments work together to promote the accurate segregation of chromosomes

Augmin is stably associated with spindle microtubules.

<p>(A–D) FRAP of spindle-associated GFP-Dgt2 in wild-type metaphase I oocytes (A), in wild-type prometaphase/metaphase syncytial embryos (B), in oocytes depleted of γ-tubulin37C by RNAi (C), and in <i>ncd^D</i> homozygous mutant oocytes (D). A typical meiotic figure used for FRAP is shown for each. Error bars are SEM. n≥15 in meiosis and n≥11 in mitosis. (E) Western blot of oocytes using an antibody which recognises all γ-tubulin in oocytes in wild type and after depletion of γ-tubulin37C by RNAi. (F) Two hypothetical models for stable association of Augmin with spindle microtubules. Our data are consistent with the “stabilise and then nucleate” model.</p

Augmin facilitates the generation of microtubules near spindle poles.

<p>(A) Timing of the first microtubule assembly from nuclear envelope breakdown in wild-type and <i>wacΔ</i> mutant oocytes. The error bars are SEM. n≥11, p = 0.06. (B) Normalised tubulin intensity plots along the long axis of the wild-type and <i>wacΔ</i> mutant spindles. Pixel intensity was measured along a line from one pole to the other as in the diagrams below. Box plots show the central 50% of the data (box), the median (central bisecting line), and 1.5X the interquartile range (whiskers). The tubulin intensity of the sub-polar spindle regions (the regions 3,8) relative to that of the equator region (5,6) is significantly lower in the <i>wacΔ</i> mutant than wild type (p<0.01). (C) Spindle poles are often missing or weak in <i>wacΔ</i> oocytes expressing GFP-tubulin, while they are robust in wild-type oocytes expressing GFP-tubulin. Scale bar = 10 µm. (D) The frequencies of various spindle morphologies in wild-type and <i>wacΔ</i> oocytes expressing GFP-tubulin. The spindles with at least one weak or missing pole were significantly more frequent in the <i>wac</i> mutant (p<0.01, n≥48).</p

Chromosomes fail to congress in <i>wac</i> mutant oocytes.

<p>(A) Chromosome movement in wild-type and <i>wacΔ</i> oocytes expressing Rcc1-mCherry. Scale bar = 10 µm. Time = min:sec. (B) The degree of chromosome congression in wild-type and <i>wacΔ</i> oocytes. The spread of the chromosome mass along the spindle axis, excluding the 4^th chromosome (the double arrow in the diagram), in six oocytes each plotted from nuclear envelope breakdown (time 0) over time.</p

Stable association of Augmin with spindle poles compensate for the lack of centrosomes in oocytes.

<p>Stable association of Augmin with spindle poles compensate for the lack of centrosomes in oocytes.</p

Augmin accumulation at spindle poles is correlated with chromosome congression.

<p>(A, B) GFP-Dgt2 and Wac-GFP localise to wild-type acentrosomal spindle poles. (C) Dgt6 localises to spindle poles in wild-type oocytes by immunostaining. (D) The Augmin level in spindle pole regions is well correlated with the level of chromosome congression. Live oocytes expressing GFP-Dgt2 and Rcc1-mCherry were used to measure two parameters: the spread of the chromosome mass (including the 4th chromosomes) along the spindle axis (as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003562#pgen-1003562-g001" target="_blank">Figure 1B</a>), and the intensity of GFP-Dgt2 signal above the background (as in Methods & Materials) for each spindle. Correlation between the chromosome spread and the log of GFP-Dgt2 intensity is significant (r = −0.772, p<0.01, n = 26).</p

Loss of NDC80 or SPC105R disrupts interactions between kinetochores and microtubules in oocytes.

<p>Confocal images of wild-type oocytes (A,B) and after knockdown of <i>Ndc80</i> (C,D) or <i>Spc105R</i> (E,F). Oocytes were treated with either ethanol (EtOH) (A,C,E) or colchicine (B,D,F). DNA is shown in blue, tubulin is shown in green, and CENP-C is in red. Scale bars represent 10 μm</p

Prometaphase karyosome configurations in the absence of kinetochore components.

<p>^a Prometaphase defined as karyosome in a figure eight shape and/or 4th chromosomes separated from main karyosome mass. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005605#pgen.1005605.s003" target="_blank">S3 Fig</a>.</p><p>^b Karyosome is separated into two or more masses of chromosomes</p><p>^c Fisher's exact test comparing prometaphase/non-prometaphase to wild type</p><p>^d Fisher’s exact test comparing split/non-split to wild type</p><p>Prometaphase karyosome configurations in the absence of kinetochore components.</p