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

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 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

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