The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts.

Chromosome condensation is required for the physical resolution and segregation of sister chromatids during cell division, but the precise role of higher order chromatin structure in mitotic chromosome functions is unclear. Here, we address the role of the major condensation machinery, the condensin complex, in spindle assembly and function in Xenopus laevis egg extracts. Immunodepletion of condensin inhibited microtubule growth and organization around chromosomes, reducing the percentage of sperm nuclei capable of forming spindles, and causing dramatic defects in anaphase chromosome segregation. Although the motor CENP-E was recruited to kinetochores pulled poleward during anaphase, the disorganized chromosome mass was not resolved. Inhibition of condensin function during anaphase also inhibited chromosome segregation, indicating its continuous requirement. Spindle assembly around DNA-coated beads in the absence of kinetochores was also impaired upon condensin inhibition. These results support an important role for condensin in establishing chromosomal architecture necessary for proper spindle assembly and chromosome segregation

New ⁴⁰Ar/³⁹Ar dating of the Antrim Plateau Volcanics, Australia: clarifying an age for the eruptive phase of the Kalkarindji continental flood basalt province

The Kalkarindji flood basalt province of northern Australia erupted in the mid-Cambrian. Today the province consists of scattered volcanic and intrusive suites, the largest being the Antrim Plateau Volcanics (APV) in Northern Territory. Accurate dating of Kalkarindji has proved challenging with previous studies focused on minor volcanics and intrusive dykes in Northern Territory and Western Australia. These previously published data, corrected to the same decay constants, range from 512.8 to 509.6 ± 2.5 Ma [2σ], placing Kalkarindji in apparent synchronicity with the Cambrian Stage 4–5 biotic crisis at 510 ± 1 Ma. This study utilises 40Ar/39Ar dating of basalts from the APV to accurately date the major volcanic eruptions in this province. Results yield an age of 508.0–498.3 ± 5.5 Ma [2σ], indicating the APV is younger than the intrusives. These dates allude to a relative timing discrepancy, where intrusive activity in the North Australian Craton preceded the eruption of the APV as the last magmatic activity in the region. The determination of these largest eruptions to be later than 510 Ma, effectively disassociates Kalkarindji lavas from being a major cause of the 510 Ma biotic crisis, but cannot definitively discount any deleterious effects on the fragile Cambrian ecosystem

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

Interplay between microtubule bundling and sorting factors ensures acentriolar spindle stability during <i>C</i>. <i>elegans</i> oocyte meiosis

<div><p>In many species, oocyte meiosis is carried out in the absence of centrioles. As a result, microtubule organization, spindle assembly, and chromosome segregation proceed by unique mechanisms. Here, we report insights into the principles underlying this specialized form of cell division, through studies of <i>C</i>. <i>elegans</i> KLP-15 and KLP-16, two highly homologous members of the kinesin-14 family of minus-end-directed kinesins. These proteins localize to the acentriolar oocyte spindle and promote microtubule bundling during spindle assembly; following KLP-15/16 depletion, microtubule bundles form but then collapse into a disorganized array. Surprisingly, despite this defect we found that during anaphase, microtubules are able to reorganize into a bundled array that facilitates chromosome segregation. This phenotype therefore enabled us to identify factors promoting microtubule organization during anaphase, whose contributions are normally undetectable in wild-type worms; we found that SPD-1 (PRC1) bundles microtubules and KLP-18 (kinesin-12) likely sorts those bundles into a functional orientation capable of mediating chromosome segregation. Therefore, our studies have revealed an interplay between distinct mechanisms that together promote spindle formation and chromosome segregation in the absence of structural cues such as centrioles.</p></div

Coordinating cohesion, co-orientation, and congression during meiosis: lessons from holocentric chromosomes

Organisms that reproduce sexually must reduce their chromosome number by half during meiosis to generate haploid gametes. To achieve this reduction in ploidy, organisms must devise strategies to couple sister chromatids so that they stay together during the first meiotic division (when homologous chromosomes separate) and then segregate away from one another during the second division. Here we review recent findings that shed light on how Caenorhabditis elegans, an organism with holocentric chromosomes, deals with these challenges of meiosis by differentiating distinct chromosomal subdomains and remodeling chromosome structure during prophase. Furthermore, we discuss how features of chromosome organization established during prophase affect later chromosome behavior during the meiotic divisions. Finally, we illustrate how analysis of holocentric meiosis can inform our thinking about mechanisms that operate on monocentric chromosomes

SPD-1 and KLP-18 are required for KLP-15/16-independent spindle reorganization during anaphase.

<p>(A and D) DNA (blue), tubulin (green), AIR-2 (not in merge) and SEP-1 (red in merge); all spindles shown are mid/late anaphase, with SEP-1 gone and AIR-2 relocalized to the microtubules. (A) Singly depleting/inhibiting <i>klp-15/16</i>, <i>spd-1</i>, or <i>zen-4</i> or doubly depleting/inhibiting <i>klp-15/16;zen-4</i> and <i>spd-1;zen-4</i> all resulted in anaphase spindles that were able to bundle microtubules and segregate chromosomes, while double depletion of <i>klp-15/16;spd-1</i> abolished microtubule bundling and chromosome segregation. (B) Quantification of the experiment shown in (A). The simple matching coefficient for microtubule bundling and chromosome segregation = 0.82 (n = 251); in other words, 82% of the spindles either showed chromosome segregation when microtubules were bundled or did not show chromosome segregation when microtubules were not bundled. (C) Box plots showing anaphase microtubule length measurements; for a given image, the most prominent and longest microtubule bundle in the spindle was measured. Box represents first quartile, median, and third quartile. Lines extend to data points within 1.5 interquartile range. Asterisks (***) represent significant difference (p < 0.001, two tailed t-test) compared to the other three conditions; (*) represents significant difference (p < 0.05, two tailed t-test) compared to control conditions. (D) Anaphase spindle reorganization and chromosome segregation are not observed in <i>klp-18(tm2841)</i> following either control or <i>klp-15/16</i> RNAi; in both conditions microtubules are disorganized and segregation fails, suggesting that KLP-18 could potentially mediate the anaphase spindle reorganization observed in KLP-15/16-depleted oocytes. n represents the number of spindles observed for each condition. (E) DNA (blue), tubulin (green), AIR-2 (not in merge) and SPD-1 (red in merge); SPD-1 localizes to spindle microtubule bundles in <i>klp-18(tm2841)</i> and <i>klp-18(tm2841);klp-15/16 (RNAi)</i> oocytes. n represents the number of spindles observed for each condition. Bars = 2.5 μm.</p

Consequences of the anaphase defects observed in <i>klp-15/16(RNAi)</i> oocytes.

<p>(A and B) Analysis of polar bodies and maternal pronuclei was done using live worms expressing GFP::tubulin, GFP::histone. (A) Quantification of the number of polar bodies per embryo for each condition listed. (B) Quantification of the number of maternal pronuclei per embryo for each condition listed. For A and B, embryos were only scored if the paternal pronucleus was decondensed to ensure that the meiotic divisions were complete. (C) Example mitotic embryos showing DNA (blue) and tubulin (green) to show some of the phenotypes observed in the quantification displayed in A and B. Asterisks denote polar bodies and arrowheads denote maternal pronuclei. We observe extra polar bodies and pronuclei following <i>klp-15/16(RNAi)</i>, indicative of meiotic defects. Moreover, co-depletion of KLP-15/16 and SPD-1 often results in ejection of all maternal chromosomes into a single polar body, resulting in no maternal pronucleus. Bars = 10 μm.</p

SPD-1 and centralspindlin localize to anaphase spindle microtubules.

<p>(A and B) DNA (blue), tubulin (green) and ZEN-4 (top) or SPD-1 (bottom) (red) in control (A) or <i>klp-15/16(RNAi)</i> (B) oocytes. ZEN-4 does not localize to metaphase spindles in either case. In anaphase, ZEN-4 localizes to a distinct region in the midzone of control spindles (A, top zoom), but is sometimes not seen on spindles with a short chromosome segregation distance. Following <i>klp-15/16(RNAi)</i>, ZEN-4 localizes to the microtubule bundles but is not concentrated to as distinct a band as in the control (B, top zoom). In both control and <i>klp-15/16(RNAi)</i> oocytes, SPD-1 does not localize to metaphase spindles. In control oocytes, SPD-1 localizes to the microtubules between the chromosomes in early anaphase and then becomes concentrated in a band on the microtubule bundles in the center of the spindle (A, bottom zoom). Following <i>klp-15/16(RNAi)</i>, SPD-1 localizes to microtubule bundles between segregating chromosomes, even when bundles are not all oriented along the same axis (B, bottom zoom) (quantification in Materials and Methods). (C) DNA (blue), tubulin (green), ZEN-4 (red) and SPD-1 (green in final merge). In both control and <i>klp-15/16(RNAi)</i> oocytes, ZEN-4 and SPD-1 have similar but distinct localization patterns. (D) Movie stills of oocytes expressing mCherry::histone and SPD-1::GFP in both control and <i>klp-15/16(RNAi)</i>. In control spindles, SPD-1 localizes between chromosomes as they begin to segregate and continues to accumulate as anaphase progresses, consistent with previous studies [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006986#pgen.1006986.ref019" target="_blank">19</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006986#pgen.1006986.ref020" target="_blank">20</a>]. Following <i>klp-15/16(RNAi)</i>, SPD-1 begins to load all over the microtubules, and as SPD-1 accumulates, long bundles begin to form that then orient along the same axis and the chromosomes segregate (4/4 movies analyzed). White dashed lines = cell cortex. Bars = 2.5 μm.</p

KLP-15/16 localize to spindle microtubules during oocyte meiosis.

<p>(A) Western blot from control, <i>klp-15 or klp-16(RNAi)</i> worms probed with the KLP-16 antibody or tubulin as a loading control. (B and C) DNA (blue), tubulin (green) and KLP-15/16 (red). (B) Wild-type meiotic spindles at all stages of spindle assembly; KLP-15/16 begin to accumulate on microtubules at the multipolar stage and remain associated through anaphase (quantification in Materials and Methods). (C) The KLP-15/16 signal is lost following <i>klp-15/16(RNAi)</i>, though staining of <i>klp-15(ok1958)</i> and <i>klp-16(wig1)</i> is not different from wild-type spindles (quantification in Materials and Methods). (D and E) Live imaging of worms expressing KLP-16::GFP and mCherry::histone shows that KLP-16 localizes to microtubule bundles at the cage stage (which was not apparent in the KLP-16 antibody staining) and remains associated with the spindle (D). The multipolar image is a partial projection of the entire structure. Stages of spindle assembly were discerned by spindle morphology, chromosome organization, and position in the germline. (E) KLP-16 is also present on mitotic spindle microtubules and centrosomes at the one-cell stage (in 5/5 embryos analyzed). Bars = (B, C, and D) 2.5 μm; (E) 10 μm.</p