PAR proteins, embryonic polarity and cell polarity.
The research interest of the lab is early embryogenesis in the nematode Caenorhabditis elegans. Our major focus for the past several years has been the analysis of a set of genes (the par genes) that are required for proper cytoplasmic partitioning in early embryos. Mutations in any of these six par genes lead to a maternal effect on the pattern and timing of the early cleavages (see movies). These loci define components of a maternally derived system for localization of cytoplasmic constituents during early cleavages. Failure to localize these constituents results in improper pattern and timing of cleavages, and leads to the ultimate death of the embryos. The protein products of four of the par genes are distributed asymmetrically at the periphery of early blastomeres.
Several aspects of the Par mutant phenotypes are illustrated in the figure below. In wild-type embryos the first cleavage spindle is positioned toward the posterior such that cleavage results in two cells of different sizes; the anterior cell, AB, is larger than the posterior cell, P1. In par mutant embryos (except for par-4) the anterior and posterior cells are of nearly equal size (first column, below). In the figure, par-5 and par-6 are not shown; par-5 is similar in phenotype to par-2 and par-6 is similar to par-3.
The AB cell divides before P1 and the AB cleavage spindle is oriented perpendicular to the long axis of the embryo, while the P1 spindle is oriented parallel to the long axis of the wild-type embryo. The tubulin and DAPI stained embryos in the middle column, below, illustrate the synchrony of these two cells in par mutants and characteristic cleavage spindle orientation defects.
P granules are a component of the egg cytoplasm segregated to the posterior pole during the first cell cycle and to the germline P lineage in subsequent divisions. P granules are not properly segregated in par mutant embryos (third column: P granule distributions in four-cell embryos).
The par genes were identified in screens for maternal effect lethal (Mel) mutations that had defects in early cleavages (Kemphues et al., 1988). Additional alleles were identified as Mels that arrested as amorphous cellular masses missing intestine or with excess pharynx. These alleles also exhibited early cleavage defects. Listing of par mutations.
We have also identified a nonmuscle myosin, NMY-2, that interacts with PAR-1. RNAi with nmy-2 sequences produces a Par phenotype in embryos (Guo and Kemphues, 1996). Another gene, pkc-3, encodes an atypical protein kinase C and gives a Par phenotype in the embryos of RNAi-treated worms, very similar to par-3 and par-6 mutant phenotypes (Tabuse et al. 1998).
The products of several of the par genes are localized asymmetrically in the one-cell embryo. PAR-1 and PAR-2 are localized to the posterior cortex during the first cell cycle, while PAR-3, PAR-6 and PKC-3 are localized to the anterior periphery of the cell. Binding studies indicate that these anterior proteins can form a complex (based on our unpublished results that are consistent with biochemical results from studies of mammalian and Drosophila proteins). PAR-4 is present throughout the cytoplasm and all around the cortex of early embryos. PAR-5 is present throughout the cytoplasm of early embryos and NMY-2 is present all around the egg cortex. For immunofluorescence images of the PAR proteins in one-cell C. elegans embryos as well as more information about these proteins, go to the PAR proteins page.
PAR proteins also have roles in later stages of development. We have learned that PAR-1 is required for morphogenesis of the vulva (Hurd and Kemphues, 2003) and that the anterior complex genes (PAR-3/PAR-6/PKC-3) play a role in establishing or maintaining cell polarity in the spermatheca (Aono et al., 2004).
Recently we have focused on understanding how the anterior PAR proteins are localized asymmetrically and how they interact with each other. We learned that the interaction of CDC-42 with PAR-6 is required for maintenance of polarity but at least to some extent, is not required for the initial establishment of anterior PAR asymmetry (Aceto et al., 2006). We also learned, from a study of cdc-37(RNAi) embryos, that PAR-6 accumulates at the cortex via two different modes. One mode is dependent upon CDC-42, does not involve co-localization with PAR-3 and is normally blocked in the posterior by the action of CDC-37. The other mode is independent of CDC-42, involves co-localization with PAR-3 and is dependent on the action of CDC-37 (Beers and Kemphues, 2006). CDC-37 is a co-chaperone of HSP90 with an apparent bias for stabilizing protein kinases.