C. elegans XMAP215/ZYG-9 and TACC/TAC-1 act at multiple times during oocyte meiotic spindle assembly and promote both spindle pole coalescence and stability.

The conserved two-component XMAP215/TACC modulator of microtubule stability is required in multiple animal phyla for acentrosomal spindle assembly during oocyte meiotic cell division. In C. elegans, XMAP215/zyg-9 and TACC/tac-1 mutant oocytes exhibit multiple and indistinguishable oocyte spindle assembly defects beginning early in meiosis I. To determine if these defects represent one or more early requirements with additional later and indirect consequences, or multiple temporally distinct and more direct requirements, we have used live cell imaging and fast-acting temperature-sensitive zyg-9 and tac-1 alleles to dissect their requirements at high temporal resolution. Temperature upshift and downshift experiments indicate that the ZYG-9/TAC-1 complex has multiple temporally distinct and separable requirements throughout oocyte meiotic cell division. First, we show that during prometaphase ZYG-9 and TAC-1 promote the coalescence of early pole foci into a bipolar structure, stabilizing pole foci as they grow and limiting their growth rate, with these requirements being independent of an earlier defect in microtubule organization that occurs upon nuclear envelope breakdown. Second, during metaphase, ZYG-9 and TAC-1 maintain spindle bipolarity by suppressing ectopic pole formation. Third, we show that ZYG-9 and TAC-1 also are required for spindle assembly during meiosis II, independently of their meiosis I requirements. The metaphase pole stability requirement appears to be important for maintaining chromosome congression, and we discuss how negative regulation of microtubule stability by ZYG-9/TAC-1 during oocyte meiotic cell division might account for the observed defects in spindle pole coalescence and stability.

Microtubules oppose cortical actomyosin-driven membrane ingression during C. elegans meiosis I polar body extrusion.

During C. elegans oocyte meiosis I cytokinesis and polar body extrusion, cortical actomyosin is locally remodeled to assemble a contractile ring that forms within and remains part of a much larger and actively contractile cortical actomyosin network. This network both mediates contractile ring dynamics and generates shallow ingressions throughout the oocyte cortex during polar body extrusion. Based on our analysis of requirements for CLS-2, a member of the CLASP family of proteins that stabilize microtubules, we recently proposed that a balance of actomyosin-mediated tension and microtubule-mediated stiffness limits membrane ingression throughout the oocyte during meiosis I polar body extrusion. Here, using live cell imaging and fluorescent protein fusions, we show that CLS-2 is part of a group of kinetochore proteins, including the scaffold KNL-1 and the kinase BUB-1, that also co-localize during meiosis I to structures called linear elements, which are present within the assembling oocyte spindle and also are distributed throughout the oocyte in proximity to, but appearing to underlie, the actomyosin cortex. We further show that KNL-1 and BUB-1, like CLS-2, promote the proper organization of sub-cortical microtubules and also limit membrane ingression throughout the oocyte. Moreover, nocodazole or taxol treatment to destabilize or stabilize oocyte microtubules leads to, respectively, excess or decreased membrane ingression throughout the oocyte. Furthermore, taxol treatment, and genetic backgrounds that elevate the levels of cortically associated microtubules, both suppress excess membrane ingression in cls-2 mutant oocytes. We propose that linear elements influence the organization of sub-cortical microtubules to generate a stiffness that limits cortical actomyosin-driven membrane ingression throughout the oocyte during meiosis I polar body extrusion. We discuss the possibility that this regulation of sub-cortical microtubule dynamics facilitates actomyosin contractile ring dynamics during C. elegans oocyte meiosis I cell division.

A germline-specific role for unconventional components of the γ-tubulin complex in Caenorhabditis elegans.

The γ-tubulin complex (γTuC) is a widely conserved microtubule nucleator, but some of its components, namely GCP4, GCP5 and GCP6 (also known as TUBGCP4, TUBGCP5 and TUBGCP6, respectively), have not been detected in Caenorhabditis elegans. Here, we identified two γTuC-associated proteins in C. elegans, GTAP-1 and GTAP-2, for which apparent orthologs were detected only in the genus Caenorhabditis. GTAP-1 and GTAP-2 were found to localize at centrosomes and the plasma membrane of the germline, and their centrosomal localization was interdependent. In early C. elegans embryos, whereas the conserved γTuC component MZT-1 (also known as MOZART1 and MZT1) was essential for the localization of centrosomal γ-tubulin, depletion of GTAP-1 and/or GTAP-2 caused up to 50% reduction of centrosomal γ-tubulin and precocious disassembly of spindle poles during mitotic telophase. In the adult germline, GTAP-1 and GTAP-2 contributed to efficient recruitment of the γTuC to the plasma membrane. Depletion of GTAP-1, but not of GTAP-2, severely disrupted both the microtubule array and the honeycomb-like structure of the adult germline. We propose that GTAP-1 and GTAP-2 are unconventional components of the γTuC that contribute to the organization of both centrosomal and non-centrosomal microtubules by targeting the γTuC to specific subcellular sites in a tissue-specific manner.

Tubulin isotype substitution revealed that isotype combination modulates microtubule dynamics in C. elegans embryos.

Microtubules (MTs) are polymers composed of α- and ß-tubulin heterodimers that are generally encoded by genes at multiple loci. Despite implications of distinct properties depending on the isotype, how these heterodimers contribute to the diverse MT dynamics in vivo remains unclear. Here, by using genome editing and depletion of tubulin isotypes following RNAi, we demonstrate that four tubulin isotypes (hereafter referred to as α1, α2, ß1 and ß2) cooperatively confer distinct MT properties in Caenorhabditis elegans early embryos. GFP insertion into each isotype locus reveals their distinct expression levels and MT incorporation rates. Substitution of isotype coding regions demonstrates that, under the same isotype concentration, MTs composed of ß1 have higher switching frequency between growth and shrinkage compared with MTs composed of ß2. Lower concentration of ß-tubulins results in slower growth rates, and the two α-tubulins distinctively affect growth rates of MTs composed of ß1. Alteration of ratio and concentration of isotypes distinctively modulates both growth rate and switching frequency, and affects the amplitude of mitotic spindle oscillation. Collectively, our findings demonstrate that MT dynamics are modulated by the combination (ratio and concentration) of tubulin isotypes with distinct properties, which contributes to create diverse MT behaviors in vivo.

Cortical microtubules oppose actomyosin-driven membrane ingression during C. elegans meiosis I polar body extrusion.

During C. elegans oocyte meiosis I, cortical actomyosin is locally remodeled to assemble a contractile ring near the spindle. In contrast to mitosis, when most cortical actomyosin converges into a contractile ring, the small oocyte ring forms within and remains part of a much larger and actively contractile cortical actomyosin network. This network both mediates contractile ring dynamics and generates shallow ingressions throughout the oocyte cortex during polar body extrusion. Based on our analysis of requirements for CLS-2, a member of the CLASP family of proteins that stabilize microtubules, we recently proposed that a balance of actomyosin-mediated tension and microtubule-mediated stiffness are required for contractile ring assembly within the oocyte cortical actomyosin network. Here, using live cell imaging and fluorescent protein fusions, we show that CLS-2 is part of a complex of kinetochore proteins, including the scaffold KNL-1 and the kinase BUB-1, that also co-localize to patches distributed throughout the oocyte cortex during meiosis I. By reducing their function, we further show that KNL-1 and BUB-1, like CLS-2, are required for cortical microtubule stability, to limit membrane ingression throughout the oocyte, and for meiotic contractile ring assembly and polar body extrusion. Moreover, nocodazole or taxol treatment to destabilize or stabilize oocyte microtubules, respectively, leads to excess or decreased membrane ingression throughout the oocyte and defective polar body extrusion. Finally, genetic backgrounds that elevate cortical microtubule levels suppress the excess membrane ingression in cls-2 mutant oocytes. These results support our hypothesis that CLS-2, as part of a sub-complex of kinetochore proteins that also co-localize to patches throughout the oocyte cortex, stabilizes microtubules to stiffen the oocyte cortex and limit membrane ingression throughout the oocyte, thereby facilitating contractile ring dynamics and the successful completion of polar body extrusion during meiosis I.

The ß-catenin HMP-2 functions downstream of Src in parallel with the Wnt pathway in early embryogenesis of C. elegans.

The Wnt and Src pathways are widely used signal transduction pathways in development. ß-catenin is utilized in both pathways, as a signal transducer and a component of the cadherin cell adhesion complex, respectively. A C. elegans ß-catenin HMP-2 is involved in cell adhesion, but its signaling role has been unknown. Here, we report that in early embryogenesis HMP-2 acts as a signaling molecule in the Src signal. During early embryogenesis in C. elegans, the Wnt and Src pathways are redundantly involved in endoderm induction at the four-cell stage and spindle orientation in an ABar blastomere. RNAi experiments demonstrated that HMP-2 functions in the Src pathway, but in parallel with the Wnt pathway in these processes. HMP-2 localized at the cell boundaries and nuclei, and its localization at cell boundaries was negatively regulated by SRC-1. In addition, HMP-2 was Tyr-phosphorylated in a SRC-1-dependent manner in vivo. Taken together, we propose that HMP-2 functions downstream of the Src signaling pathway and contribute to endoderm induction and ABar spindle orientation, in parallel with the Wnt signaling pathway.

Caenorhabditis elegans Aurora A kinase is required for the formation of spindle microtubules in female meiosis.

In many animals, female meiotic spindles are assembled in the absence of centrosomes, the major microtubule (MT)-organizing centers. How MTs are formed and organized into meiotic spindles is poorly understood. Here we report that, in Caenorhabditis elegans, Aurora A kinase/AIR-1 is required for the formation of spindle microtubules during female meiosis. When AIR-1 was depleted or its kinase activity was inhibited in C. elegans oocytes, although MTs were formed around chromosomes at germinal vesicle breakdown (GVBD), they were decreased during meiotic prometaphase and failed to form a bipolar spindle, and chromosomes were not separated into two masses. Whereas AIR-1 protein was detected on and around meiotic spindles, its kinase-active form was concentrated on chromosomes at prometaphase and on interchromosomal MTs during late anaphase and telophase. We also found that AIR-1 is involved in the assembly of short, dynamic MTs in the meiotic cytoplasm, and these short MTs were actively incorporated into meiotic spindles. Collectively our results suggest that, after GVBD, the kinase activity of AIR-1 is continuously required for the assembly and/or stabilization of female meiotic spindle MTs.

Cell polarity: centrosomes release signals for polarization.

New findings reveal that, in Caenorhabditis elegans embryos, the centrosome provides signals that induce cell polarization, independently of its function as the microtubule-organizing center.

C. elegans CPB-3 interacts with DAZ-1 and functions in multiple steps of germline development.

Cytoplasmic polyadenylation element-binding proteins (CPEBs) are well-conserved RNA-binding proteins, which regulate mRNA translation mainly through control of poly(A) elongation. Here, we show that CPB-3, one of the four CPEB homologs in C. elegans, positively regulates multiple aspects of oocyte production. CPB-3 protein was highly expressed in early meiotic regions of the hermaphrodite gonad. Worms deficient in cpb-3 were apparently impaired in germ cell proliferation and differentiation including sperm/oocyte switching and progression of female meiosis. We also show that cpb-3 is likely to promote the meiotic entry in parallel with gld-3, a component of one of the redundant but essential genetic pathways for the entry to and progression through meiosis. Taken together, CPEB appears to have a conserved role in the early phase of meiosis and in the sperm/oocyte specification, in addition to its reported function during meiotic progression.

Protein phosphatase 4 is required for centrosome maturation in mitosis and sperm meiosis in C. elegans.

The centrosome consists of two centrioles surrounded by the pericentriolar material (PCM). In late G2 phase, centrosomes enlarge by recruiting extra PCM, and concomitantly its microtubule nucleation activity increases dramatically. The regulatory mechanisms of this dynamic change of centrosomes are not well understood. Protein phosphatase 4 (PP4) is known to localize to mitotic centrosomes in mammals and Drosophila. An involvement of PP4 in the mitotic spindle assembly has been implicated in Drosophila, but in vivo functions of PP4 in other organisms are largely unknown. Here we characterize two Caenorhabditis elegans PP4 genes, named pph-4.1 and pph-4.2. Inhibition of the function of each gene by RNA-mediated interference (RNAi) revealed that PPH-4.1 was essential for embryogenesis but PPH-4.2 was not. More specifically, PPH-4.1 was required for the formation of spindles in mitosis and sperm meiosis. However, this phosphatase was apparently dispensable for female meiotic divisions, which do not depend on centrosomes. In the cell depleted of pph-4.1 activity, localization of gamma-tubulin and a Polo-like kinase homologue to the centrosome was severely disturbed. Immunofluorescence staining revealed that PPH-4.1 was present at centrosomes from prophase to telophase, but not during interphase. These results indicate that PPH-4.1 is a centrosomal protein involved in the recruitment of PCM components to the centrosome, and is essential for the activation of microtubule nucleation potential of the centrosome. Furthermore, chiasmata between homologous chromosomes were often absent in oocytes that lacked pph-4.1 activity. Thus, besides promoting spindle formation, PPH-4.1 appears to play a role in either the establishment or the maintenance of chiasmata during meiotic prophase I.