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.

Protein Immunodepletion and Complementation in Egg Extracts.

The 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.

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.

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.

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.

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.

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.

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.

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.