Aaron F. Severson

The Aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis

BACKGROUND The Aurora/Ipl1p-related kinase AIR-2 is required for mitotic chromosome segregation and cytokinesis in early Caenorhabditis elegans embryos. Previous studies have relied on non-conditional mutations or RNA-mediated interference (RNAi) to inactivate AIR-2. It has therefore not been possible to determine whether AIR-2 functions directly in cytokinesis or if the cleavage defect results indirectly from the failure to segregate DNA. One intriguing hypothesis is that AIR-2 acts to localize the mitotic kinesin-like protein ZEN-4 (also known as CeMKLP1), which later functions in cytokinesis. RESULTS Using conditional alleles, we established that AIR-2 is required at metaphase or early anaphase for normal segregation of chromosomes, localization of ZEN-4, and cytokinesis. ZEN-4 is first required late in cytokinesis, and also functions to maintain cell separation through much of the subsequent interphase. DNA segregation defects alone were not sufficient to disrupt cytokinesis in other mutants, suggesting that AIR-2 acts specifically during cytokinesis through ZEN-4. AIR-2 and ZEN-4 shared similar genetic interactions with the formin homology (FH) protein CYK-1, suggesting that AIR-2 and ZEN-4 function in a single pathway, in parallel to a contractile ring pathway that includes CYK-1. Using in vitro co-immunoprecipitation experiments, we found that AIR-2 and ZEN-4 interact directly. CONCLUSIONS AIR-2 has two functions during mitosis: one in chromosome segregation, and a second, independent function in cytokinesis through ZEN-4. AIR-2 and ZEN-4 may act in parallel to a second pathway that includes CYK-1.

Condensin and cohesin complexity: the expanding repertoire of functions

Condensin and cohesin complexes act in diverse nuclear processes in addition to their widely known roles in chromosome compaction and sister chromatid cohesion. Recent work has elucidated the contribution of condensin and cohesin to interphase genome organization, control of gene expression, metazoan development and meiosis. Despite these wide-ranging functions, several themes have come to light: both complexes establish higher-order chromosome structure by inhibiting or promoting interactions between distant genomic regions, both complexes influence the chromosomal association of other proteins, and both complexes achieve functional specialization by swapping homologous subunits. Emerging data are expanding the range of processes in which condensin and cohesin are known to participate and are enhancing our knowledge of how chromosome architecture is regulated to influence numerous cellular functions.

Centrosome Maturation and Mitotic Spindle Assembly in C. elegans Require SPD-5, a Protein with Multiple Coiled-Coil Domains

The maternally expressed C. elegans gene spd-5 encodes a centrosomal protein with multiple coiled-coil domains. During mitosis in mutants with reduced levels of SPD-5, microtubules assemble but radiate from condensed chromosomes without forming a spindle, and mitosis fails. SPD-5 is required for the centrosomal localization of gamma-tubulin, XMAP-215, and Aurora A kinase family members, but SPD-5 accumulates at centrosomes in mutants lacking these proteins. Furthermore, SPD-5 interacts genetically with a dynein heavy chain. We propose that SPD-5, along with dynein, is required for centrosome maturation and mitotic spindle assembly.

A Formin Homology Protein and a Profilin Are Required for Cytokinesis and Arp2/3-Independent Assembly of Cortical Microfilaments in C. elegans

BACKGROUND F-actin is enriched at the cortex of embryonic cells in the nematode Caenorhabditis elegans and is required for multiple processes that include the establishment of an anterior-posterior (A-P) axis and cytokinesis. However, the mechanisms that regulate cortical microfilament (MF) assembly remain poorly understood. RESULTS We show here that a profilin called PFN-1 accumulates at the cortex independent of the actin cytoskeleton and is required for the assembly or maintenance of cortical MFs and myosin. Reducing PFN-1 levels by RNAi results in cytokinesis and A-P polarity defects. PFN-1 binds to the Formin Homology (FH) protein CYK-1, which also is required for cortical MFs. In contrast to PFN-1 and CYK-1, the Arp2/3 complex appears to be dispensable for the assembly of cortical MFs, for A-P polarity, and for cytokinesis. Instead, the Arp2/3 complex is required for cell migrations that occur during gastrulation and may also be involved in cellular rearrangements required for epidermal enclosure prior to elongation of ovoid embryos into vermiform larvae. CONCLUSIONS We conclude that the FH protein CYK-1 and the profilin PFN-1 mediate the Arp2/3-independent assembly of MFs and are required for cytokinesis in the early embryo. These data suggest that CYK-1 and PFN-1 may nucleate MFs, as has recently been shown for an FH protein and a profilin in yeast.

The axial element protein HTP-3 promotes cohesin loading and meiotic axis assembly in C. elegans to implement the meiotic program of chromosome segregation

Faithful transmission of the genome through sexual reproduction requires reduction of genome copy number during meiosis to produce haploid sperm and eggs. Meiosis entails steps absent from mitosis to achieve this goal. When meiosis begins, sisters are held together by sister chromatid cohesion (SCC), mediated by the cohesin complex. Homologs then become linked through crossover recombination. SCC subsequently holds both sisters and homologs together. Separation of homologs and then sisters requires two successive rounds of chromosome segregation and the stepwise removal of Rec8, a meiosis-specific cohesin subunit. We show that HTP-3, a known component of the C. elegans axial element (AE), molecularly links these meiotic innovations. We identified HTP-3 in a genetic screen for factors necessary to maintain SCC until meiosis II. Our data show that interdependent loading of HTP-3 and cohesin is a principal step in assembling the meiotic chromosomal axis and in establishing SCC. HTP-3 recruits all known AE components to meiotic chromosomes and promotes cohesin loading, the first known involvement of an AE protein in this process. Furthermore, REC-8 and two paralogs, called COH-3 and COH-4, together mediate meiotic SCC, but they perform specialized functions. REC-8 alone is necessary and sufficient for the persistence of SCC after meiosis I. In htp-3 and rec-8 mutants, sister chromatids segregate away from one another in meiosis I (equational division), rather than segregating randomly, as expected if SCC were completely eliminated. AE assembly fails only when REC-8, COH-3, and COH-4 are simultaneously disrupted. Premature equational sister separation in rec8 mutants of other organisms suggests the involvement of multiple REC-8 paralogs, which may have masked a conserved requirement for cohesin in AE assembly.

Condensin restructures chromosomes in preparation for meiotic divisions

The production of haploid gametes from diploid germ cells requires two rounds of meiotic chromosome segregation after one round of replication. Accurate meiotic chromosome segregation involves the remodeling of each pair of homologous chromosomes around the site of crossover into a highly condensed and ordered structure. We showed that condensin, the protein complex needed for mitotic chromosome compaction, restructures chromosomes during meiosis in Caenorhabditis elegans. In particular, condensin promotes both meiotic chromosome condensation after crossover recombination and the remodeling of sister chromatids. Condensin helps resolve cohesin-independent linkages between sister chromatids and alleviates recombination-independent linkages between homologues. The safeguarding of chromosome resolution by condensin permits chromosome segregation and is crucial for the formation of discrete, individualized bivalent chromosomes.

Myosin and the PAR proteins polarize microfilament-dependent forces that shape and position mitotic spindles in Caenorhabditis elegans

In Caenorhabditis elegans, the partitioning proteins (PARs), microfilaments (MFs), dynein, dynactin, and a nonmuscle myosin II all localize to the cortex of early embryonic cells. Both the PARs and the actomyosin cytoskeleton are required to polarize the anterior-posterior (a-p) body axis in one-cell zygotes, but it remains unknown how MFs influence embryonic polarity. Here we show that MFs are required for the cortical localization of PAR-2 and PAR-3. Furthermore, we show that PAR polarity regulates MF-dependent cortical forces applied to astral microtubules (MTs). These forces, which appear to be mediated by dynein and dynactin, produce changes in the shape and orientation of mitotic spindles. Unlike MFs, dynein, and dynactin, myosin II is not required for the production of these forces. Instead, myosin influences embryonic polarity by limiting PAR-3 to the anterior cortex. This in turn produces asymmetry in the forces applied to MTs at each pole and allows PAR-2 to accumulate in the posterior cortex of a one-cell zygote and maintain asymmetry.

Divergent kleisin subunits of cohesin specify mechanisms to tether and release meiotic chromosomes

We show that multiple, functionally specialized cohesin complexes mediate the establishment and two-step release of sister chromatid cohesion that underlies the production of haploid gametes. In C. elegans, the kleisin subunits REC-8 and COH-3/4 differ between meiotic cohesins and endow them with distinctive properties that specify how cohesins load onto chromosomes and then trigger and release cohesion. Unlike REC-8 cohesin, COH-3/4 cohesin becomes cohesive through a replication-independent mechanism initiated by the DNA double-stranded breaks that induce crossover recombination. Thus, break-induced cohesion also tethers replicated meiotic chromosomes. Later, recombination stimulates separase-independent removal of REC-8 and COH-3/4 cohesins from reciprocal chromosomal territories flanking the crossover site. This region-specific removal likely underlies the two-step separation of homologs and sisters. Unexpectedly, COH-3/4 performs cohesion-independent functions in synaptonemal complex assembly. This new model for cohesin function diverges from that established in yeast but likely applies directly to plants and mammals, which utilize similar meiotic kleisins. DOI: http://dx.doi.org/10.7554/eLife.03467.001

CELL DIVISION : PLANT-LIKE PROPERTIES OF ANIMAL CELL CYTOKINESIS

Recent evidence that a syntaxin is required for cytokinesis in Caenorhabditis elegans embryos suggests that the mechanism of cell division in plant and animal cells may be more similar than previously imagined.

Oocyte Meiotic Spindle Assembly and Function

Gametogenesis in animal oocytes reduces the diploid genome content of germline precursors to a haploid state in gametes by discarding ¾ of the duplicated chromosomes through a sequence of two meiotic cell divisions called meiosis I and II. The assembly of the microtubule-based spindle structure that mediates this reduction in genome content remains poorly understood compared to our knowledge of mitotic spindle assembly and function. In this review, we consider the diversity of oocyte meiotic spindle assembly and structure across animal phylogeny, review recent advances in our understanding of how animal oocytes assemble spindles in the absence of the centriole-based microtubule-organizing centers that dominate mitotic spindle assembly, and discuss different models for how chromosomes are captured and moved to achieve chromosome segregation during oocyte meiotic cell division.