Model for acentrosomal spindle assembly and chromosome orientation.

<p>The spindle is composed of microtubules that make end-on attachments to kinetochores (dark green) and those that do not, such as those with plus ends that overlap in the central spindle (light green). (A) Meiotic spindle assembly begins with the accumulation of microtubules around the DNA and the centromeres clustered. These centromeres (shown in black) are not attached to microtubules. (B) Prometaphase is characterized by the presence of kinetochores (red) interacting laterally with microtubules. The process of building a bipolar spindle between panels A and B involves organizing a central spindle and pole-focusing and has been described in detail elsewhere [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005605#pgen.1005605.ref082" target="_blank">82</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005605#pgen.1005605.ref083" target="_blank">83</a>]. (C) At metaphase, all kinetochores have end-on attachments (white) and little interaction with the central spindle. Metaphase is also characterized by less reliance on the central spindle for stability. (D-F) A model for bi-orientation showing an enlargement of the spindle around the chromosomes. Kinetochores can move laterally along microtubules in a CENP-E-dependent plus-end direction or a minus-end direction by an unknown force. (D) In this example, two homologous centromeres are moving laterally in the minus-end direction along central spindle microtubules of the same polarity; therefore, they move towards the same pole. (E) This error is detected by an unknown mechanism, leading at least one of the kinetochores to fail to make an end-on attachment and move towards the other pole in a CENP-E dependent manner. (F’) If the kinetochore makes an end-on attachment following this movement, the homologs are bi-oriented. The kinetochores maintain their position by balancing the minus-end force with either the chiasmata or CENP-E. In the absence of CENP-E, errors cannot be corrected, leading to mono-orientation as in (E). (F”) Alternatively, the minus-end force can continue to move centromeres towards the poles, either through a lateral or end-on interaction, resulting in a splitting of the karyosome.</p

Loss of SPC105R or the CPC disrupts kinetochore assembly in oocytes.

<p>Confocal images of localization of (A) NDC80, (B) SPC105R, and (C) NSL1 (Mis12 complex) in wild-type oocytes and after knockdown of <i>Ndc80</i>, <i>Spc105R</i>, or <i>aurB</i>. DNA is shown in blue and tubulin is shown in green in merged images. Kinetochore components are shown in red in merged images and white in single channel images. Scale bars represent 10 μm.</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

Loss of CENP-E disrupts chromosome alignment in oocytes.

<p>Confocal images of karyosome and spindle organization in oocytes from wild type, <i>cana/Df</i>, after knockdown of <i>cmet</i>, <i>Cenp-E</i> germline clones, and after knockdown of <i>Ndc80</i>, <i>Spc105R</i> (HMS01548), <i>Ndc80</i> and <i>cmet</i>, or <i>Spc105R</i> (HMS01548) and <i>cmet</i>. DNA is in blue and tubulin is in green in merged images. Single channel images show DNA in white. 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

Centromere bi-orientation in the absence of CANA and CMET.

<p>^a Fisher’s exact test comparing bi/mono-orientation to wild type</p><p>^b Oocytes in which orientation could not be determined because spindle poles were poorly defined, centromeres were not pulled towards a pole, and/or the chromosomes were dispersed.</p><p>Centromere bi-orientation in the absence of CANA and CMET.</p

Prometaphase spindle stability depends on both kinetochore and central spindle components in oocytes.

<p>In all images, DNA is in blue, tubulin is in green, and the scale bars represent 10 μm. (A,B) Confocal images of wild-type oocytes and after <i>Spc105R</i> knockdown from prometaphase-enriched (A) and metaphase-enriched (B) collections. The CPC component INCENP is in red in merged images, white in single channel images. (C) Confocal images of <i>sub</i>-depleted and <i>sub Spc105R</i> double-depleted oocytes. For <i>sub</i>-depleted oocytes, a tripolar (left) and bipolar (right) spindle are shown. Monopolar spindles were also observed [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005605#pgen.1005605.ref009" target="_blank">9</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005605#pgen.1005605.ref047" target="_blank">47</a>]. In all <i>sub</i>-depleted oocytes, the prominent central spindle is missing. For <i>sub Spc105R</i> double-depleted oocytes, the absence of a spindle (left) and a spindle with thin and disorganized microtubules (right) are shown. (D) Graph showing the percentage of weak/absent spindles during prometaphase in wild-type oocytes (n = 171) and after <i>Spc105R</i> (n = 86), <i>sub</i> (n = 23), or <i>sub Spc105R</i> (n = 24) depletion, and metaphase in wild-type oocytes (n = 110) and after <i>Spc105R</i> depletion (n = 42). The frequency of weak/absent spindles at metaphase in <i>sub</i>- and <i>sub Spc105R</i>-depleted oocytes was not determined. Error bars show 95% confidence intervals.</p

Homologous chromosome bi-orientation depends on kinetochores and CENP-E in oocytes.

<p>Confocal images of FISH probes marking the 2^nd (red) and 3^rd (white) chromosome centromeres. DNA is in blue and tubulin is in green in merged images. Only FISH probes are shown in the panel below each merged image. Scale bars represent 10 μm. (A) Oocytes from wild type and after knockdown of <i>Ndc80</i> or <i>Spc105R</i>. (B) Dot plot of the distance between pairs of FISH foci in wild-type oocytes and after knockdown of <i>Ndc80</i> or <i>Spc105R</i>. Horizontal dotted lines show the mean, error bars show 95% confidence intervals. (C) Oocytes from <i>cana/Df</i>, after knockdown of <i>cmet</i>, and <i>Cenp-E</i> germline clones. The data is summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005605#pgen.1005605.t002" target="_blank">Table 2</a>.</p

Chromosome orientation depends on the kinetochore in oocytes.

<p>(A) Confocal images of the centromere protein CENP-C (red in merged images, white in single channel) in wild-type oocytes, after knockdown of <i>Ndc80</i> or <i>Spc105R</i>, and in <i>ord</i> mutants. Single channel images are zoomed in relative to merged to highlight CENP-C foci. In <i>ord</i> mutants, due to defects in cohesion or crossing over, precocious anaphase is observed [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005605#pgen.1005605.ref080" target="_blank">80</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005605#pgen.1005605.ref081" target="_blank">81</a>]. DNA is in blue and tubulin is in green in merged images. Scale bars represent 10 μm in merged, 5 μm in CENP-C single channel images. (B) Bar graph showing the ratio of CENP-C foci closer to (dark gray) and further from (light gray) the spindle than 0.2 μM. Inset shows a centromere in red, a microtubule in green, and the distance measurement denoted by “x”. (C) Dot plot of the number of CENP-C foci per oocyte in wild type, after <i>Ndc80</i> or <i>Spc105R</i> knockdown, in <i>ord</i> mutants, and in <i>mei-S332</i> mutants (encoding the <i>Drosophila</i> homolog of Shugoshin). Horizontal dotted lines show the mean, error bars show 95% confidence intervals. (D) Dot plot of the angle of CENP-C foci with respect to the spindle axis in wild-type oocytes and after knockdown of <i>Ndc80</i> or <i>Spc105R</i>. Horizontal dotted lines show the median. The red bar in the inset shows a line perpendicular to the spindle axis: centromeres located on this line would result in 90 degree angle measurements, while centromeres in line with the spindle axis measure 0 degrees. Differences in the width of the karyosome, in wild-type oocytes (3.13 μm), and after knockdown of <i>Ndc80</i> (3.43 μm) or <i>Spc105R</i> (3.32 μm), are not great enough to explain the differences in angles of CENP-C foci.</p