Kenneth J. Kemphues

par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed

The first cleavage of C. elegans is asymmetric, generating daughter cells with different sizes, cytoplasmic components, and fates. Mutations in the par-1 gene disrupt this asymmetry. We report here that par-1 encodes a putative Ser/Thr kinase with similarity to kinases from yeasts and mammals. Two strong alleles have mutations in the kinase domain, suggesting that kinase activity is essential for par-1 function. PAR-1 protein is localized to the posterior periphery of the zygote and is distributed in a polar fashion preceding the asymmetric divisions of the germline lineage. Because PAR-1 distribution in the germline correlates with the distribution of germline-specific P granules, it is possible that PAR-1 functions in germline development as well as in establishing embryonic polarity.

Asymmetrically distributed PAR-3 protein contributes to cell polarity and spindle alignment in early C. elegans embryos

The par-3 gene is required for establishing polarity in early C. elegans embryos. Embryos from par-3 homozygous mothers show defects in segregation of cytoplasmic determinants and in positioning of the early cleavage spindles. We report here that the PAR-3 protein is asymmetrically distributed at the periphery of the zygote and asymmetrically dividing blastomeres of the germline lineage. The PAR-3 distribution is roughly the reciprocal of PAR-1, another protein required for establishing embryonic polarity in C. elegans. Analysis of the distribution of PAR-3 and PAR-1 in other par mutants reveals that par-2 activity is required for proper localization of PAR-3 and that PAR-3 is required for proper localization of PAR-1. In addition, the distribution of the PAR-3 protein correlates with differences in cleavage spindle orientation and suggests a mechanism by which PAR-3 contributes to control of cleavage pattern.

The C. elegans zyg-1 Gene Encodes a Regulator of Centrosome Duplication with Distinct Maternal and Paternal Roles in the Embryo

Centrosome duplication is a critical step in assembly of the bipolar mitotic spindle, but the molecular mechanisms regulating this process during the cell cycle and during animal development are poorly understood. Here, we report that the zyg-1 gene of Caenorhabditis elegans is an essential regulator of centrosome duplication. ZYG-1 is a protein kinase specifically required for daughter centriole formation that localizes transiently to centrosomes and acts at least one cell cycle prior to each spindle assembly event. In the embryo, ZYG-1 participates in a unique regulatory scheme whereby paternal ZYG-1 regulates duplication and bipolar spindle assembly during the first cell cycle, and maternal ZYG-1 regulates these processes thereafter. ZYG-1 is therefore a key molecular component of the centrosome/centriole duplication process.

PARsing Embryonic Polarity

Thus, in spite of widely divergent developmental mechanisms, worms and flies both use a member of the PAR-1/MARK/KIN1 family during an early step in polarity establishment. What does this mean? One possibility is that this is a case of a conserved function being independently co-opted by evolution for use in polarity. That is, conserved features of this family of kinases (e.g., a regulatable kinase activity and a C-terminal domain with a hypothetical role in localization) have been selected independently for a role in polarity during evolution of the two animals. In this case, the method of activation, the substrates, and the output would be expected to be different. The more interesting possibility, however, is that this family of kinases is part of a biochemical pathway that has been conserved for its role in setting polarity in a variety of organisms and cell types. If so, then we clearly have much to learn about this new pathway in both systems.Several observations are consistent with co-option. First is the failure to identify other conserved components after extensive genetic analysis in each system. Second is the difference in the apparent output of kinase activity. Although Drosophila PAR-1 seems to most directly affect microtubule organization, there is no evidence that C. elegans PAR-1 affects microtubule organization. In fact, most aspects of C. elegans polarity are resistant to microtubule-inhibiting drugs and sensitive to microfilament-inhibiting drugs. Third, although PAR-1 localization depends upon PAR-3, the PAR-3 homolog, Bazooka (see below), does not have a role in localizing Drosophila PAR-1. Similarly, although Drosophila PAR-1 localization depends upon oskar, there is no recognizable oskar homolog in worms.On the other hand, none of these observations rule out conservation of a core cassette of interacting proteins with a fundamental role in polarity. It is possible that the direct activators and targets of PAR-1/MARK/KIN1 family members are conserved but have fundamental roles throughout development and therefore, like Drosophila PAR-1, will only be discovered by reverse genetic methods. In addition, the output of PAR-1 in the two systems may not be as different as it appears; the role of microtubules in C. elegans polarity is still an open question. Other observations more positively support a conserved cassette. Conservation of PAR-1 fits well with the apparent conservation of function of germline granules in flies and worms. Perhaps most compelling is the consistent coincidence of PAR-1/MARK/KIN1 family kinases with polarity systems in other organisms and cell types. KIN1 has a clear role in polarity in yeast and Drosophila PAR-1, in addition to its polar distribution in oocytes, is restricted basolaterally in follicle cells, a distribution that has also been seen for mammalian PAR-1 in polarized epithelial cells (see Shulman et al. 2000xShulman, J.M, Benton, R, and St Johnston, D. Cell. 2000; 101: 377–388Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesShulman et al. 2000).Possible conservation of PAR function in polarity is not limited to PAR-1. Homologs of PAR-3 have been identified and are associated with polarity in flies and in mammals. A PAR-3 homolog in Drosophila, Bazooka, has recently been found to be localized apically in embryonic epithelial cells, is required for maintenance of the epithelium, and plays a key role in cell polarity during the asymmetric divisions of neuroblasts (See Jan and Jan 2000xJan, Y.-N and Jan, L.Y. Cell. 2000; 100: 599–602Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesJan and Jan 2000). Although the function of mammalian PAR-3 is not clearly known, it is also localized apically in epithelial cells and like PAR-3, biochemically interacts with an atypical protein kinase C (Izumi et al. 1998xIzumi, Y, Hirose, T, Tamai, Y, Hirai, S, Nagashima, Y, Fujimoto, T, Tabuse, Y, Kemphues, K, and Ohno, S. J. Cell Biol. 1998; 143: 95–106Crossref | PubMed | Scopus (375)See all ReferencesIzumi et al. 1998).Although the extent to which PAR function is conserved between animals remains to be seen, it seems pretty clear that PARs are polar and that understanding the function of this interesting class of proteins promises to yield insights into establishment of polarity in a wide range of systems.*E-mail: kjk1@cornell.edu

RNAi analysis of genes expressed in the ovary of Caenorhabditis elegans

As a step towards comprehensive functional analysis of genomes, systematic gene knockout projects have been initiated in several organisms [1]. In metazoans like C. elegans, however, maternal contribution can mask the effects of gene knockouts on embryogenesis. RNA interference (RNAi) provides an alternative rapid approach to obtain loss-of-function information that can also reveal embryonic roles for the genes targeted [2,3]. We have used RNAi to analyze a random set of ovarian transcripts and have identified 81 genes with essential roles in embryogenesis. Surprisingly, none of them maps on the X chromosome. Of these 81 genes, 68 showed defects before the eight-cell stage and could be grouped into ten phenotypic classes. To archive and distribute these data we have developed a database system directly linked to the C. elegans database (Wormbase). We conclude that screening cDNA libraries by RNAi is an efficient way of obtaining in vivo function for a large group of genes. Furthermore, this approach is directly applicable to other organisms sensitive to RNAi and whose genomes have not yet been sequenced.

A Drosophila melanogaster homologue of Caenorhabditis elegans par-1 acts at an early step in embryonic-axis formation.

A Drosophila melanogaster homologue of Caenorhabditis elegans par-1 acts at an early step in embryonic-axis formation

Molecular genetics of asymmetric cleavage in the early Caenorhabditis elegans embryo.

Asymmetric cleavage plays an important role in Caenorhabditis elegans embryogenesis. In addition to generating cellular diversity, several early asymmetric cleavages contribute to the spatial organization of the embryo. Genetic and molecular analyses of several genes, including six par genes and the mex-1 and mes-1 genes, together with experimental embryological studies, have provided insights into mechanisms controlling polarity and spindle orientations during these cleavages. In particular, these studies focus attention on microfilament-based motility and changing protein distributions at the cell cortex.

Mutations in the par genes of Caenorhabditis elegans affect cytoplasmic reorganization during the first cell cycle.

A dramatic reorganization of cytoplasm occurs during the first cell cycle in embryos of the nematode, Caenorhabditis elegans. We present here the results of a quantitative study of some of the events during this reorganization in wild-type embryos and in par mutant embryos. The par mutations define a set of genes required for cytoplasmic localization in early embryos. We show that par mutations lead to defects in several events of the reorganization. Mutations in all four of the par genes we studied lead to defects in pseudocleavage and asymmetric redistribution of cortical microfilaments. In addition, some of the par mutations affect streaming of cytoplasm, migration of the pronuclei, and asymmetric shortening of the embryo. We propose that the major function of the par genes might be to orchestrate this initial reorganization of cytoplasm.

The C. elegans homolog of Drosophila Lethal giant larvae functions redundantly with PAR-2 to maintain polarity in the early embryo

Polarity is essential for generating cell diversity. The one-cell C. elegans embryo serves as a model for studying the establishment and maintenance of polarity. In the early embryo, a myosin II-dependent contraction of the cortical meshwork asymmetrically distributes the highly conserved PDZ proteins PAR-3 and PAR-6, as well as an atypical protein kinase C (PKC-3), to the anterior. The RING-finger protein PAR-2 becomes enriched on the posterior cortex and prevents these three proteins from returning to the posterior. In addition to the PAR proteins, other proteins are required for polarity in many metazoans. One example is the conserved Drosophila tumor-suppressor protein Lethal giant larvae (Lgl). In Drosophila and mammals, Lgl contributes to the maintenance of cell polarity and plays a role in asymmetric cell division. We have found that the C. elegans homolog of Lgl, LGL-1, has a role in polarity but is not essential. It localizes asymmetrically to the posterior of the early embryo in a PKC-3-dependent manner, and functions redundantly with PAR-2 to maintain polarity. Furthermore, overexpression of LGL-1 is sufficient to rescue loss of PAR-2 function. LGL-1 negatively regulates the accumulation of myosin (NMY-2) on the posterior cortex, representing a possible mechanism by which LGL-1 might contribute to polarity maintenance.

Depletion of the co-chaperone CDC-37 reveals two modes of PAR-6 cortical association in C. elegans embryos.

PAR proteins play roles in the establishment and maintenance of polarity in many different cell types in metazoans. In C. elegans, polarity established in the one-cell embryo determines the anteroposterior axis of the developing animal and is essential to set the identities of the early blastomeres. PAR-1 and PAR-2 colocalize at the posterior cortex of the embryo. PAR-3, PAR-6 and PKC-3 (aPKC) colocalize at the anterior cortex of the embryo. A process of mutual exclusion maintains the anterior and posterior protein domains. We present results indicating that a homolog of the Hsp90 co-chaperone Cdc37 plays a role in dynamic interactions among the PAR proteins. We show that CDC-37 is required for the establishment phase of embryonic polarity; that CDC-37 reduction allows PAR-3-independent cortical accumulation of PAR-6 and PKC-3; and that CDC-37 is required for the mutual exclusion of the anterior and posterior group PAR proteins. Our results indicate that CDC-37 acts in part by maintaining PKC-3 levels and in part by influencing the activity or levels of other client proteins. Loss of the activities of these client proteins reveals that there are two sites for PAR-6 cortical association, one dependent on CDC-42 and not associated with PAR-3, and the other independent of CDC-42 and co-localizing with PAR-3. We propose that, in wild-type embryos, CDC-37-mediated inhibition of the CDC-42-dependent binding site and PAR-3-mediated release of this inhibition provide a key mechanism for the anterior accumulation of PAR-6.