Loss of the puromycin-sensitive aminopeptidase, PAM-1, triggers the spindle assembly checkpoint during the first mitotic division in Caenorhabditis elegans.

Puromycin-sensitive aminopeptidases have long been implicated in cell-cycle regulation, but the mechanism remains unknown. Here we show that mutations in the gene encoding the C. elegans puromycin-sensitive aminopeptidase, PAM-1 , cause chromosome segregation defects and an elongated mitosis in the one-cell embryo. Depleting a known regulator of the spindle assembly checkpoint (SAC), MDF-2 (MAD2 in humans), restores normal mitotic timing to pam-1 mutants but exacerbates the chromosome segregation defects. Thus, PAM-1 is required for proper attachment of chromosomes to the mitotic spindle and its absence triggers the SAC.

Interactions between the WEE-1.3 kinase and the PAM-1 aminopeptidase in oocyte maturation and the early C. elegans embryo.

Puromycin-sensitive aminopeptidases are found across phyla and are known to regulate the cell-cycle and play a protective role in neurodegenerative disease. PAM-1 is a puromycin-sensitive aminopeptidase important for meiotic exit and polarity establishment in the one-cell Caenorhabditis elegans embryo. Despite conservation of this aminopeptidase, little is known about its targets during development. In order to identify novel interactors, we conducted a suppressor screen and isolated four suppressing mutations in three genes that partially rescued the maternal-effect lethality of pam-1 mutants. Suppressed strains show improved embryonic viability and polarization of the anterior-posterior axis. We identified a missense mutation in wee-1.3 in one of these suppressed strains. WEE-1.3 is an inhibitory kinase that regulates maturation promoting factor. Although the missense mutation suppressed polarity phenotypes in pam-1, it does so without restoring centrosome-cortical contact or altering the cortical actomyosin cytoskeleton. To see if PAM-1 and WEE-1.3 interact in other processes, we examined oocyte maturation. Although depletion of wee-1.3 causes sterility due to precocious oocyte maturation, this effect was lessened in pam-1 worms, suggesting that PAM-1 and WEE-1.3 interact in this process. Levels of WEE-1.3 were comparable between wild-type and pam-1 strains, suggesting that WEE-1.3 is not a direct target of the aminopeptidase. Thus, we have established an interaction between PAM-1 and WEE-1.3 in multiple developmental processes and have identified suppressors that are likely to further our understanding of the role of puromycin-sensitive aminopeptidases during development.

Sustained centrosome-cortical contact ensures robust polarization of the one-cell C. elegans embryo.

In C. elegans, the anterior-posterior axis is established at the one-cell stage when the embryo polarizes along its long axis. One model suggests that a cue from the centrosome triggers symmetry breaking and is then dispensable for further steps in the process. In the absence of the initial centrosome cue, a redundant mechanism, reliant on the centrosome's microtubules, can polarize the cell. Despite this model, data from multiple sources suggest that direct centrosome-contact with the cortex may play a role in ensuring robust polarization. Some of this past work includes analysis of pam-1 mutants, which lack a functional puromycin-sensitive aminopeptidase and have aberrant centrosome positioning and variable polarization defects. To better understand the role of centrosome dynamics in polarization, we looked in detail at centrosome behavior in relation to key polarity landmarks in pam-1 mutants as well as those lacking cortical flows. We provide evidence for a model in which sustained direct contact between the centrosome and the cortex acts to reinforce both the actomyosin and the microtubule-dependent pathways. This contact is necessary for polarization when flows are inhibited.

A survey of new temperature-sensitive, embryonic-lethal mutations in C. elegans: 24 alleles of thirteen genes.

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci.

The PAM-1 aminopeptidase regulates centrosome positioning to ensure anterior-posterior axis specification in one-cell C. elegans embryos.

In the one-cell Caenorhabditis elegans embryo, the anterior-posterior (A-P) axis is established when the sperm donated centrosome contacts the posterior cortex. While this contact appears to be essential for axis polarization, little is known about the mechanisms governing centrosome positioning during this process. pam-1 encodes a puromycin sensitive aminopeptidase that regulates centrosome positioning in the early embryo. Previously we showed that pam-1 mutants fail to polarize the A-P axis. Here we show that PAM-1 can be found in mature sperm and in cytoplasm throughout early embryogenesis where it concentrates around mitotic centrosomes and chromosomes. We provide further evidence that PAM-1 acts early in the polarization process by showing that PAR-1 and PAR-6 do not localize appropriately in pam-1 mutants. Additionally, we tested the hypothesis that PAM-1's role in polarity establishment is to ensure centrosome contact with the posterior cortex. We inactivated the microtubule motor dynein, DHC-1, in pam-1 mutants, in an attempt to prevent centrosome movement from the cortex and restore anterior-posterior polarity. When this was done, the aberrant centrosome movements of pam-1 mutants were not observed and anterior-posterior polarity was properly established, with proper localization of cortical and cytoplasmic determinants. We conclude that PAM-1's role in axis polarization is to prevent premature movement of the centrosome from the posterior cortex, ensuring proper axis establishment in the embryo.

The puromycin-sensitive aminopeptidase PAM-1 is required for meiotic exit and anteroposterior polarity in the one-cell Caenorhabditis elegans embryo.

In the nematode Caenorhabditis elegans, sperm entry into the oocyte triggers the completion of meiosis and the establishment of the embryonic anteroposterior (AP) axis. How the early embryo makes the transition from a meiotic to a mitotic zygote and coordinates cell cycle changes with axis formation remains unclear. We have discovered roles for the C. elegans puromycin-sensitive aminopeptidase PAM-1 in both cell cycle progression and AP axis formation, further implicating proteolytic regulation in these processes. pam-1 mutant embryos exhibit a delay in exit from meiosis: thus, this peptidase is required for progression to mitotic interphase. In addition, the centrosomes associated with the sperm pronucleus fail to closely associate with the posterior cortex in pam-1 mutants, and the AP axis is not specified. The meiotic exit and polarity defects are separable, as inactivation of the B-type cyclin CYB-3 in pam-1 mutants rescues the meiotic exit delay but not the polarity defects. Thus PAM-1 may regulate CYB-3 during meiotic exit but presumably targets other protein(s) to regulate polarity. We also show that the pam-1 gene is expressed both maternally and paternally, providing additional evidence that sperm-donated gene products have important roles during early embryogenesis in C. elegans. The degradation of proteins through ubiquitin-mediated proteolysis has been previously shown to regulate the cell cycle and AP axis formation in the C. elegans zygote. Our analysis of PAM-1 requirements shows that a puromycin-sensitive aminopeptidase is also required for proteolytic regulation of the oocyte to embryo transition.

Conditional dominant mutations in the Caenorhabditis elegans gene act-2 identify cytoplasmic and muscle roles for a redundant actin isoform.

Animal genomes each encode multiple highly conserved actin isoforms that polymerize to form the microfilament cytoskeleton. Previous studies of vertebrates and invertebrates have shown that many actin isoforms are restricted to either nonmuscle (cytoplasmic) functions, or to myofibril force generation in muscle cells. We have identified two temperature-sensitive and semidominant embryonic-lethal Caenorhabditis elegans mutants, each with a single mis-sense mutation in act-2, one of five C. elegans genes that encode actin isoforms. These mutations alter conserved and adjacent amino acids predicted to form part of the ATP binding pocket of actin. At the restrictive temperature, both mutations resulted in aberrant distributions of cortical microfilaments associated with abnormal and striking membrane ingressions and protrusions. In contrast to the defects caused by these dominant mis-sense mutations, an act-2 deletion did not result in early embryonic cell division defects, suggesting that additional and redundant actin isoforms are involved. Accordingly, we found that two additional actin isoforms, act-1 and act-3, were required redundantly with act-2 for cytoplasmic function in early embryonic cells. The act-1 and -3 genes also have been implicated previously in muscle function. We found that an ACT-2::GFP reporter was expressed cytoplasmically in embryonic cells and also was incorporated into contractile filaments in adult muscle cells. Furthermore, one of the dominant act-2 mutations resulted in uncoordinated adult movement. We conclude that redundant C. elegans actin isoforms function in both muscle and nonmuscle contractile processes.

A spindle checkpoint functions during mitosis in the early Caenorhabditis elegans embryo.

During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.

Roles for two partially redundant alpha-tubulins during mitosis in early Caenorhabditis elegans embryos.

The Caenorhabditis elegans genome encodes multiple isotypes of alpha-tubulin and beta-tubulin. Roles for a number of these tubulins in neuronal development have been described, but less is known about the isoforms that function during early embryonic development. Microtubules are required for multiple events after fertilization produces a one-cell zygote in C. elegans, including pronuclear migration, mitotic spindle assembly and function, and proper spindle positioning. Here we describe a conditional and dominant mis-sense mutation in the C. elegans alpha-tubulin gene tba-1 that disrupts pronuclear migration and positioning of the first mitotic spindle, and results in a highly penetrant embryonic lethality, at the restrictive temperature of 26 degrees C. Our analysis of the dominant tba-1 (or346ts) allele suggests that TBA-1 assembles into microtubules in early embryonic cells. However, we also show that reduction of tba-1 function using RNA interference results in defects much less severe than those caused by the dominant or346ts mutation, due to partial redundancy of TBA-1 and another alpha-tubulin called TBA-2. Reducing the function of both TBA-1 and TBA-2 results in severe defects in microtubule-dependent processes. We conclude that microtubules in the early C. elegans embryo are composed of both TBA-1 and TBA-2, and that the dominant tba-1(or346ts) mutation disrupts MT assembly or stability. Cell Motil.

Maternally expressed and partially redundant beta-tubulins in Caenorhabditis elegans are autoregulated.

The mitotic spindle, which partitions replicated chromosomes to daughter cells during cell division, is composed of microtubule assemblies of alpha/beta-tubulin heterodimers. Positioning of the mitotic spindle influences the size and location of daughter cells, and can be important for the proper partitioning of developmental determinants. We describe two semi-dominant mis-sense mutations in tbb-2, one of two C. elegans beta-tubulin genes that are maternally expressed and together are required for microtubule-dependent processes in the early embryo. These mutations result in a posteriorly displaced and misoriented mitotic spindle during the first cell division. In contrast, a probable tbb-2 null allele is recessive, and when homozygous results in less severe spindle positioning defects and only partially penetrant embryonic lethality. Two of the tbb-2 mutations result in reduced levels of TBB-2 protein, and increased levels of a second maternally expressed beta-tubulin, TBB-1. However, levels of TBB-1 are not increased in a tbb-2 mutant with an allele that does not result in reduced levels of TBB-2 protein. We conclude that feedback regulation influences maternal beta-tubulin expression in C. elegans, but cannot fully restore normal microtubule function in the absence of one beta-tubulin isoform.

Heads or tails: cell polarity and axis formation in the early Caenorhabditis elegans embryo.

In C. elegans, the first embryonic axis is established shortly after fertilization and requires both the microtubule and microfilament cytoskeleton. Cues from sperm-donated centrosomes result in a cascade of events that polarize the distribution of widely conserved PAR proteins at the cell cortex. The PAR proteins in turn polarize the cytoplasm and position mitotic spindles. Lessons learned from C. elegans should improve our understanding of how cells become polarized and divide asymmetrically during development.

The anaphase-promoting complex and separin are required for embryonic anterior-posterior axis formation.

Polarization of the one-cell C. elegans embryo establishes the animal's anterior-posterior (a-p) axis. We have identified reduction-of-function anaphase-promoting complex (APC) mutations that eliminate a-p polarity. We also demonstrate that the APC activator cdc20 is required for polarity. The APC excludes PAR-3 from the posterior cortex, allowing PAR-2 to accumulate there. The APC is also required for tight cortical association and posterior movement of the paternal pronucleus and its associated centrosome. Depletion of the protease separin, a downstream target of the APC, causes similar pronuclear and a-p polarity defects. We propose that the APC/separin pathway promotes close association of the centrosome with the cortex, which in turn excludes PAR-3 from the posterior pole early in a-p axis formation.