Cytoskeletal forces span the nuclear envelope to coordinate meiotic chromosome pairing and synapsis.

During meiosis, each chromosome must pair with its unique homologous partner, a process that usually culminates with the formation of the synaptonemal complex (SC). In the nematode Caenorhabditis elegans, special regions on each chromosome known as pairing centers are essential for both homologous pairing and synapsis. We report that during early meiosis, pairing centers establish transient connections to the cytoplasmic microtubule network. These connections through the intact nuclear envelope require the SUN/KASH domain protein pair SUN-1 and ZYG-12. Disruption of microtubules inhibits chromosome pairing, indicating that these connections promote interhomolog interactions. Dynein activity is essential to license formation of the SC once pairing has been accomplished, most likely by overcoming a barrier imposed by the chromosome-nuclear envelope connection. Our findings thus provide insight into how homolog pairing is accomplished in meiosis and into the mechanisms regulating synapsis so that it occurs selectively between homologs. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

Making an effective switch at the kinetochore by phosphorylation and dephosphorylation.

The kinetochore, the proteinaceous structure on the mitotic centromere, functions as a mechanical latch that hooks onto microtubules to support directional movement of chromosomes. The structure also brings in a number of signaling molecules, such as kinases and phosphatases, which regulate microtubule dynamics and cell cycle progression. Erroneous microtubule attachment is destabilized by Aurora B-mediated phosphorylation of multiple microtubule-binding protein complexes at the kinetochore, such as the KMN network proteins and the Ska/Dam1 complex, while Plk-dependent phosphorylation of BubR1 stabilizes kinetochore-microtubule attachment by recruiting PP2A-B56. Spindle assembly checkpoint (SAC) signaling, which is activated by unattached kinetochores and inhibits the metaphase-to-anaphase transition, depends on kinetochore recruitment of the kinase Bub1 through Mps1-mediated phosphorylation of the kinetochore protein KNL1 (also known as Blinkin in mammals, Spc105 in budding yeast, and Spc7 in fission yeast). Recruitment of protein phosphatase 1 to KNL1 is necessary to silence the SAC upon bioriented microtubule attachment. One of the key unsolved questions in the mitosis field is how a mechanical change at the kinetochore upon microtubule attachment is converted to these and other chemical signals that control microtubule attachment and the SAC. Rapid progress in the field is revealing the existence of an intricate signaling network created right on the kinetochore. Here we review the current understanding of phosphorylation-mediated regulation of kinetochore functions and discuss how this signaling network generates an accurate switch that turns on and off the signaling output in response to kinetochore-microtubule attachment.

Genetic analysis of Caenorhabditis elegans Haspin-like genes shows that hasp-1 plays multiple roles in the germline.

Haspin is a histone kinase that promotes error-free chromosome segregation by recruiting the chromosomal passenger complex (CPC) to mitotic and meiotic chromosomes. Haspin remains less well studied than other M-phase kinases, and the models explaining Haspin function have been developed primarily in mitotic cells. Here, we generate strains containing new conditional or nonsense mutations in the Caenorhabditis elegans Haspin homologs hasp-1 and hasp-2 and characterize their phenotypes. We show that hasp-1 is responsible for all predicted functions of Haspin and that loss of function of hasp-1 using classical and conditional alleles produces defects in germline stem cell proliferation and spermatogenesis, and confirms its role in oocyte meiosis. Genetic analysis suggests that hasp-1 acts downstream of the Polo-like kinase plk-2 and shows synthetic interactions between hasp-1 and two genes expected to promote recruitment of the CPC by a parallel pathway that depends on the kinase Bub1. This work adds to the growing understanding of Haspin function by characterizing a variety of roles in an intact animal.

ZHP-3 acts at crossovers to couple meiotic recombination with synaptonemal complex disassembly and bivalent formation in C. elegans.

Crossover recombination and the formation of chiasmata normally ensure the proper segregation of homologous chromosomes during the first meiotic division. zhp-3, the Caenorhabditis elegans ortholog of the budding yeast ZIP3 gene, is required for crossover recombination. We show that ZHP-3 protein localization is highly dynamic. At a key transition point in meiotic prophase, the protein shifts from along the length of the synaptonemal complex (SC) to an asymmetric localization on the SC and eventually becomes restricted to foci that mark crossover recombination events. A zhp-3::gfp transgene partially complements a null mutation and reveals a separation of function; although the fusion protein can promote nearly wild-type levels of recombination, aneuploidy among the progeny is high, indicating defects in meiotic chromosome segregation. The structure of bivalents is perturbed in this mutant, suggesting that the chromosome segregation defect results from an inability to properly remodel chromosomes in response to crossovers. smo-1 mutants exhibit phenotypes similar to zhp-3::gfp mutants at higher temperatures, and smo-1; zhp-3::gfp double mutants exhibit more severe meiotic defects than either single mutant, consistent with a role for SUMO in the process of SC disassembly and bivalent differentiation. We propose that coordination of crossover recombination with SC disassembly and bivalent formation reflects a conserved role of Zip3/ZHP-3 in coupling recombination with SC morphogenesis.

Protein Immunodepletion and Complementation in Xenopus laevis Egg Extracts.

The Xenopus egg extract system has been widely used to study cell cycle events, including DNA replication, nuclear envelope formation, spindle assembly, chromosome condensation and kinetochore formation. The functional roles of the proteins involved in these processes can be determined by immunodepleting a protein of interest from the extract. As immunodepletion may result in co-depletion of other proteins, the protein of interest can be added back to the extract to verify its function. Additionally, proteins harboring point mutations or domain deletions may be added to assess their functions. Here we outline the immunodepletion procedure and two separate methods for restoring a protein of interest: addition of either a recombinant protein or an mRNA that supports translation in egg extracts. The tradeoffs between these two methods are discussed.

Dual recognition of chromatin and microtubules by INCENP is important for mitotic progression.

The chromosomal passenger complex (CPC), composed of inner centromere protein (INCENP), Survivin, Borealin, and the kinase Aurora B, contributes to the activation of the mitotic checkpoint. The regulation of CPC function remains unclear. Here, we reveal that in addition to Survivin and Borealin, the single α-helix (SAH) domain of INCENP supports CPC localization to chromatin and the mitotic checkpoint. The INCENP SAH domain also mediates INCENP's microtubule binding, which is negatively regulated by Cyclin-dependent kinase-mediated phosphorylation of segments flanking the SAH domain. The microtubule-binding capacity of the SAH domain is important for mitotic arrest in conditions of suppressed microtubule dynamics, and the duration of mitotic arrest dictates the probability, but not the timing, of cell death. Although independent targeting of INCENP to microtubules or the kinetochore/centromere promotes the mitotic checkpoint, it is insufficient for a robust mitotic arrest. Altogether, our results demonstrate that dual recognition of chromatin and microtubules by CPC is important for checkpoint maintenance and determination of cell fate in mitosis.

Heterogeneous architecture of vertebrate kinetochores revealed by three-dimensional superresolution fluorescence microscopy.

The kinetochore is often depicted as having a disk-like architecture in which the outer layer of proteins, which engage microtubules and control checkpoint signaling, are built on a static inner layer directly linked to CENP-A chromatin. Here, applying three-dimensional (3D) structural illumination microscopy (SIM) and stochastic optical reconstruction microscopy (STORM) to Xenopus egg extracts and tissue culture cells, we report various distribution patterns of inner and outer kinetochore proteins. In egg extracts, a configuration in which outer kinetochore proteins surround the periphery of CENP-A chromatin is common, forming an ∼200-nm ring-like organization that may engage a bundle of microtubule ends. Similar rings are observed in Xenopus tissue culture cells at a lower frequency but are enriched in conditions in which the spindle is disorganized. Although rings are rare in human cells, the distribution of both inner and outer kinetochore proteins elongates in the absence of microtubule attachment in a manner dependent on Aurora B. We propose a model in which the 3D organization of both the outer and inner kinetochore regions respond to the progression from lateral to end-on microtubule attachments by coalescing into a tight disk from less uniform distributions early in prometaphase.

Kinetochore function is controlled by a phospho-dependent coexpansion of inner and outer components.

It is widely accepted that the kinetochore is built on CENP-A-marked centromeric chromatin in a hierarchical order from inner to outer kinetochore. Recruitment of many kinetochore proteins depends on microtubule attachment status, but it remains unclear how their assembly/disassembly is orchestrated. Applying 3D structured illumination microscopy to Xenopus laevis egg extracts, here we reveal that in the absence of microtubule attachment, proteins responsible for lateral attachment and spindle checkpoint signaling expand to form micrometer-scale fibrous structures over CENP-A-free chromatin, whereas a core module responsible for end-on attachment (CENP-A, CENP-T, and Ndc80) does not. Both outer kinetochore proteins (Bub1, BubR1, Mad1, and CENP-E) and the inner kinetochore component CENP-C are integral components of the expandable module, whose assembly depends on multiple mitotic kinases (Aurora B, Mps1, and Plx1) and is suppressed by protein phosphatase 1. We propose that phospho-dependent coexpansion of CENP-C and outer kinetochore proteins promotes checkpoint signal amplification and lateral attachment, whereas their selective disassembly enables the transition to end-on attachment.

Dynein-dependent processive chromosome motions promote homologous pairing in C. elegans meiosis.

Meiotic chromosome segregation requires homologue pairing, synapsis, and crossover recombination, which occur during meiotic prophase. Telomere-led chromosome motion has been observed or inferred to occur during this stage in diverse species, but its mechanism and function remain enigmatic. In Caenorhabditis elegans, special chromosome regions known as pairing centers (PCs), rather than telomeres, associate with the nuclear envelope (NE) and the microtubule cytoskeleton. In this paper, we investigate chromosome dynamics in living animals through high-resolution four-dimensional fluorescence imaging and quantitative motion analysis. We find that chromosome movement is constrained before meiosis. Upon prophase onset, constraints are relaxed, and PCs initiate saltatory, processive, dynein-dependent motions along the NE. These dramatic motions are dispensable for homologous pairing and continue until synapsis is completed. These observations are consistent with the idea that motions facilitate pairing by enhancing the search rate but that their primary function is to trigger synapsis. This quantitative analysis of chromosome dynamics in a living animal extends our understanding of the mechanisms governing faithful genome inheritance.