Deubiquitylation Machinery Is Required for Embryonic Polarity in Caenorhabditis elegans

The Caenorhabditis elegans one-cell embryo polarizes in response to a cue from the paternally donated centrosome and asymmetrically segregates cell fate determinants that direct the developmental program of the worm. We have found that genes encoding putative deubiquitylating enzymes (DUBs) are required for polarization of one-cell embryos. Maternal loss of the proteins MATH-33 and USP-47 leads to variable inability to correctly establish and maintain asymmetry as defined by posterior and anterior polarity proteins PAR-2 and PAR-3. The first observable defect is variable positioning of the centrosome with respect to the cell cortex and the male pronucleus. The severity of the polarity defects correlates with distance of the centrosome from the cortex. Furthermore, polarity defects can be bypassed by mutations that bring the centrosome in close proximity to the cortex. In addition we find that polarity and centrosome positioning defects can be suppressed by compromising protein turnover. We propose that the DUB activity of MATH-33 and USP-47 stabilizes one or more proteins required for association of the centrosome with the cortex. Because these DUBs are homologous to two members of a group of DUBs that act in fission yeast polarity, we tested additional members of that family and found that another C. elegans DUB gene, usp-46, also contributes to polarity. Our finding that deubiquitylating enzymes required for polarity in Schizosaccharomyces pombe are also required in C. elegans raises the possibility that these DUBs act through an evolutionarily conserved mechanism to control cell polarity.     

Different domains of C. elegans PAR-3 are required at different times in development

Polarity is a fundamental cellular feature that is critical for generating cell diversity and maintaining organ functions during development. In C. elegans, the one-cell embryo is polarized via asymmetric localization of the PAR proteins, which in turn are required to establish the future anterior-posterior axis of the embryo. PAR-3, a conserved PDZ domain-containing protein, acts with PAR-6 and PKC-3 (atypical protein kinase; aPKC) to regulate cell polarity and junction formation in a variety of cell types. To understand how PAR-3 localizes and functions during C. elegans development, we produced targeted mutations and deletions of conserved domains of PAR-3 and examined the localization and function of the GFP-tagged proteins in C. elegans embryos and larvae. We find that CR1, the PAR-3 self-oligomerization domain, is required for PAR-3 cortical distribution and function only during early embryogenesis and that PDZ2 is required for PAR-3 to accumulate stably at the cell periphery in early embryos and at the apical surface in pharyngeal and intestinal epithelial cells. We also show that phosphorylation at S863 by PKC-3 is not essential in early embryogenesis, but is important in later development. Surprisingly neither PDZ1 nor PDZ3 are essential for localization or function. Our results indicate that the different domains and phosphorylated forms of PAR-3 can have different roles during C. elegans development.     

A Semi-Dominant Mutation in the General Splicing Factor SF3a66 Causes Anterior-Posterior Axis Reversal in One-Cell Stage C. elegans Embryos

Establishment of anterior-posterior polarity in one-cell stage Caenorhabditis elegans embryos depends in part on astral microtubules. As the zygote enters mitosis, these microtubules promote the establishment of a posterior pole by binding to and protecting a cytoplasmic pool of the posterior polarity protein PAR-2 from phosphorylation by the cortically localized anterior polarity protein PKC-3. Prior to activation of the sperm aster, the oocyte Meiosis I and II spindles assemble and function, usually at the future anterior pole, but these meiotic spindle microtubules fail to establish posterior polarity through PAR-2. Here we show that a semi-dominant mutation in the general splicing factor SF3a66 can lead to a reversed axis of AP polarity that depends on PAR-2 and possibly on close proximity of oocyte meiotic spindles with the cell cortex. One possible explanation is that reduced levels of PKC-3, due to a general splicing defect, can result in axis reversal due to a failure to prevent oocyte meiotic spindle microtubules from interfering with AP axis formation.     

Binding to PKC-3, but not to PAR-3 or to a conventional PDZ domain ligand, is required for PAR-6 function in C. elegans

PAR-6 is a conserved protein important for establishment and maintenance of cell polarity in a variety of metazoans. PAR-6 proteins function together with PAR-3, aPKC and CDC-42. Mechanistic details of their interactions, however, are not fully understood. We studied the biochemical interactions between C. elegans PAR-6 and its binding partners and tested the requirements of these interactions in living worms. We show that PB1 domain-mediated binding of PAR-6 to PKC-3 is necessary for polarity establishment and PAR-6 cortical localization in C. elegans embryos. We also show that binding of PAR-6 and PAR-3 is mediated in vitro by a novel type of PDZ-PDZ interaction; the βC strand of PAR-6 PDZ binds the βD strand of PAR-3 PDZ1. However, this interaction is dispensable in vivo for PAR-6 function throughout the life of C. elegans. Mutations that specifically abolish conventional ligand binding to the PAR-6 PDZ domain also failed to affect PAR-6 function in vivo. We conclude that PAR-6 binding to PKC-3, but not to PAR-3 nor to a conventional PDZ ligand, is required for PAR-6 cortical localization and function in C. elegans.     

PAR-3 oligomerization may provide an actin-independent mechanism to maintain distinct par protein domains in the early Caenorhabditis elegans embryo

Par proteins establish discrete intracellular spatial domains to polarize many different cell types. In the single-cell embryo of the nematode worm Caenorhabditis elegans, the segregation of Par proteins is crucial for proper division and cell fate specification. Actomyosin-based cortical flows drive the initial formation of anterior and posterior Par domains, but cortical actin is not required for the maintenance of these domains. Here we develop a model of interactions between the Par proteins that includes both mutual inhibition and PAR-3 oligomerization. We show that this model gives rise to a bistable switch mechanism, allowing the Par proteins to occupy distinct anterior and posterior domains seen in the early C. elegans embryo, independent of dynamics or asymmetries in the actin cortex. The model predicts a sharp loss of cortical Par protein asymmetries during gradual depletion of the Par protein PAR-6, and we confirm this prediction experimentally. Together, these results suggest both mutual inhibition and PAR-3 oligomerization are sufficient to maintain distinct Par protein domains in the early C. elegans embryo.     

Co-evolution between an Endosymbiont and Its Nematode Host: Wolbachia Asymmetric Posterior Localization and AP Polarity Establishment

While bacterial symbionts influence a variety of host cellular responses throughout development, there are no documented instances in which symbionts influence early embryogenesis. Here we demonstrate that Wolbachia, an obligate endosymbiont of the parasitic filarial nematodes, is required for proper anterior-posterior polarity establishment in the filarial nematode B. malayi. Characterization of pre- and post-fertilization events in B. malayi reveals that, unlike C. elegans, the centrosomes are maternally derived and produce a cortical-based microtubule organizing center prior to fertilization. We establish that Wolbachia rely on these cortical microtubules and dynein to concentrate at the posterior cortex. Wolbachia also rely on PAR-1 and PAR-3 polarity cues for normal concentration at the posterior cortex. Finally, we demonstrate that Wolbachia depletion results in distinct anterior-posterior polarity defects. These results provide a striking example of endosymbiont-host co-evolution operating on the core initial developmental event of axis determination.     

PAR-6 levels are regulated by NOS-3 in a CUL-2 dependent manner in Caenorhabditiselegans

The PAR proteins have an essential and conserved function in establishing polarity in many cell types and organisms. However, their key upstream regulators remain to be identified. In C. elegans, regulators of the PAR proteins can be identified by their ability to suppress the lethality of par-2 mutant embryos. Here we show that a nos-3 loss of function mutant suppresses the lethality of par-2 mutants by regulating PAR-6 protein levels. The suppression requires the activity of the sex determination genes fem-1/2/3 and of the cullin cul-2. FEM-1 is a substrate-specific adaptor for a CUL-2-based ubiquitin ligase (CBC^FEM-1). Interestingly, we find that CUL-2 is required for the regulation of PAR-6 levels and that PAR-6 physically interacts with FEM-1. Our data strongly suggest that PAR-6 levels are regulated by the CBC^FEM-1 ubiquitin ligase thereby uncovering a novel role for the FEM proteins and cullin-dependent degradation in regulating PAR proteins and polarity processes.     

Evolution of early embryogenesis in rhabditid nematodes

The cell-biological events that guide early-embryonic development occur with great precision within species but can be quite diverse across species. How these cellular processes evolve and which molecular components underlie evolutionary changes is poorly understood. To begin to address these questions, we systematically investigated early embryogenesis, from the one- to the four-cell embryo, in 34 nematode species related to C. elegans. We found 40 cell-biological characters that captured the phenotypic differences between these species. By tracing the evolutionary changes on a molecular phylogeny, we found that these characters evolved multiple times and independently of one another. Strikingly, all these phenotypes are mimicked by single-gene RNAi experiments in C. elegans. We use these comparisons to hypothesize the molecular mechanisms underlying the evolutionary changes. For example, we predict that a cell polarity module was altered during the evolution of the Protorhabditis group and show that PAR-1, a kinase localized asymmetrically in C. elegans early embryos, is symmetrically localized in the one-cell stage of Protorhabditis group species. Our genome-wide approach identifies candidate molecules—and thereby modules—associated with evolutionary changes in cell-biological phenotypes.     

PAR proteins regulate microtubule dynamics at the cell cortex in C. elegans

BACKGROUND: The PAR proteins are known to be localized asymmetrically in polarized C. elegans, Drosophila, and human cells and to participate in several cellular processes, including asymmetric cell division and spindle orientation. Although astral microtubules are known to play roles in these processes, their behavior during these events remains poorly understood. RESULTS: We have developed a method that makes it possible to examine the residence time of individual astral microtubules at the cell cortex of developing embryos. Using this method, we found that microtubules are more dynamic at the posterior cortex of the C. elegans embryo compared to the anterior cortex during spindle displacement. We further observed that this asymmetry depends on the PAR-3 protein and heterotrimeric G protein signaling, and that the PAR-2 protein affects microtubule dynamics by restricting PAR-3 activity to the anterior of the embryo. CONCLUSIONS: These results indicate that PAR proteins function to regulate microtubule dynamics at the cortex during microtubule-dependent cellular processes.     

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.     

CDK-1 and Two B-Type Cyclins Promote PAR-6 Stabilization during Polarization of the Early C. elegans Embryo

In the C. elegans embryo, formation of an antero-posterior axis of polarity relies on signaling by the conserved PAR proteins, which localize asymmetrically in two mutually exclusive groups at the embryonic cortex. Depletion of any PAR protein causes a loss of polarity and embryonic lethality. A genome-wide RNAi screen previously identified two B-type cyclins, cyb-2.1 and cyb-2.2, as suppressors of par-2(it5ts) lethality. We found that the loss of cyb-2.1 or cyb-2.2 suppressed the lethality and polarity defects of par-2(it5ts) mutants and that these cyclins act in cell polarity with their cyclin-dependent kinase partner, CDK-1. Interestingly, cyb-2.1; cyb-2.2 double mutants did not show defects in cell cycle progression or timing of polarity establishment, suggesting that they regulate polarity independently of their typical role in cell cycle progression. Loss of both cyclin genes or of cdk-1 resulted in a decrease in PAR-6 levels in the embryo. Furthermore, the activity of the cullin CUL-2 was required to achieve suppression of par-2 lethality when both cyclins were absent. Our results support a model in which CYB-2.1/2/CDK-1 antagonize CUL-2 activity to promote stabilization of PAR-6 levels during polarization of the early C. elegans embryo. They also suggest that CYB-2.1 and CYB-2.2 contribute to the coupling of cell cycle progression and asymmetric segregation of cell fate determinants.     

CeLINC,a fluorescence-based protein–protein interaction assay in Caenorhabditis elegans

Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein’s function. We present Caenorhabditis elegans light-induced coclustering (CeLINC), an optical binary protein–protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein–protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Colocalization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome.     

Displacement of basolateral Bazooka/PAR-3 by regulated transport and dispersion during epithelial polarization in Drosophila

Polarity landmarks guide epithelial development. In the early Drosophila ectoderm, the scaffold protein Bazooka (Drosophila PAR-3) forms apicolateral landmarks to direct adherens junction assembly. However, it is unclear how Bazooka becomes polarized. We report two mechanisms acting in concert to displace Bazooka from the basolateral membrane. As cells form during cellularization, basally localized Bazooka undergoes basal-to-apical transport. Bazooka requires its three postsynaptic density 95, discs large, zonula occludens-1 (PDZ) domains to engage the transport mechanism, but with the PDZ domains deleted, basolateral displacement still occurs by gastrulation. Basolateral PAR-1 activity appears to act redundantly with the transport mechanism. Knockdown of PAR-1 sporadically destabilizes cellularization furrows, but basolateral displacement of Bazooka still occurs by gastrulation. In contrast, basolateral Bazooka displacement is blocked with disruption of both the transport mechanism and phosphorylation by PAR-1. Thus Bazooka is polarized through a combination of transport and PAR-1–induced dispersion from basolateral membranes. Our work complements recent findings in Caenorhabditis elegans and thus suggests the coupling of transport and dispersion is a common protein polarization strategy.     

AMP-Activated Kinase Regulates Lipid Droplet Localization and Stability of Adipose Triglyceride Lipase in C. elegans Dauer Larvae

Animals have developed diverse mechanisms to adapt to their changing environment. Like many organisms the free-living nematode C. elegans can alternate between a reproductive mode or a diapause-like "dauer" stage during larval development to circumvent harsh environmental conditions. The master metabolic regulator AMP-activated protein kinase (AMPK) is critical for survival during the dauer stage, where it phosphorylates adipose triglyceride lipase (ATGL-1) at multiple sites to block lipid hydrolysis and ultimately protect the cellular triglyceride-based energy depot from rapid depletion. However, how the AMPK-mediated phosphorylation affects the function of ATGL-1 has not been characterised at the molecular level. Here we show that AMPK phosphorylation leads to the generation of 14-3-3 binding sites on ATGL-1, which are recognized by the C. elegans 14-3-3 protein orthologue PAR-5. Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation. In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation. Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress.     

An Atypical PKC Directly Associates and Colocalizes at the Epithelial Tight Junction with ASIP,a Mammalian Homologue of Caenorhabditis elegans Polarity Protein PAR-3

Cell polarity is fundamental to differentiation and function of most cells. Studies in mammalian epithelial cells have revealed that the establishment and maintenance of cell polarity depends upon cell adhesion, signaling networks, the cytoskeleton, and protein transport. Atypical protein kinase C (PKC) isotypes PKCζ and PKCλ have been implicated in signaling through lipid metabolites including phosphatidylinositol 3-phosphates, but their physiological role remains elusive. In the present study we report the identification of a protein, ASIP (atypical PKC isotype–specific interacting protein), that binds to aPKCs, and show that it colocalizes with PKCλ to the cell junctional complex in cultured epithelial MDCKII cells and rat intestinal epithelia. In addition, immunoelectron microscopy revealed that ASIP localizes to tight junctions in intestinal epithelial cells. Furthermore, ASIP shows significant sequence similarity to Caenorhabditis elegans PAR-3. PAR-3 protein is localized to the anterior periphery of the one-cell embryo, and is required for the establishment of cell polarity in early embryos. ASIP and PAR-3 share three PDZ domains, and can both bind to aPKCs. Taken together, our results suggest a role for a protein complex containing ASIP and aPKC in the establishment and/or maintenance of epithelial cell polarity. The evolutionary conservation of the protein complex and its asymmetric distribution in polarized cells from worm embryo to mammalian-differentiated cells may mean that the complex functions generally in the organization of cellular asymmetry.     

C. elegans Brat homologs regulate PAR protein-dependent polarity and asymmetric cell division

The evolutionary conserved PAR proteins control polarization and asymmetric division in many organisms. Recent work in Caenorhabditis elegans demonstrated that nos-3 and fbf-1/2 can suppress par-2(it5ts) lethality, suggesting that they participate in cell polarity by regulating the function of the anterior PAR-3/PAR-6/PKC-3 proteins. In Drosophila embryos, Nanos and Pumilio are homologous to NOS-3 and FBF-1/2 respectively and control cell polarity by forming a complex with the tumor suppressor Brat to inhibit Hunchback mRNA translation. In this study, we investigated the possibility that Brat could control cell polarity and asymmetric cell division in C. elegans. We found that disrupting four of the five C. elegans Brat homologs (Cebrats) individually results in suppression of par-2(it5ts) lethality, indicating that these genes are involved in embryonic polarity. Two of the Cebrats, ncl-1 and nhl-2, partially restore the localization of PAR proteins at the cortex. While mutations in the four Cebrat genes do not severely impair polarity, they display polarity-associated defects. Surprisingly, these defects are absent from nos-3 mutants. Similarly, while nos-3 controls PAR-6 protein levels, this is not the case for any of the Cebrats. Our results, together with results from Drosophila, indicate that Brat family members function in generating cellular asymmetries and suggest that, in contrast to Drosophila embryos, the C. elegans homologs of Brat and Nanos could participate in embryonic polarity via distinct mechanisms.     

PAR-4/LKB1 regulates DNA replication during asynchronous division of the early C. elegans embryo

Regulation of cell cycle duration is critical during development, yet the underlying molecular mechanisms are still poorly understood. The two-cell stage Caenorhabditis elegans embryo divides asynchronously and thus provides a powerful context in which to study regulation of cell cycle timing during development. Using genetic analysis and high-resolution imaging, we found that deoxyribonucleic acid (DNA) replication is asymmetrically regulated in the two-cell stage embryo and that the PAR-4 and PAR-1 polarity proteins dampen DNA replication dynamics specifically in the posterior blastomere, independently of regulators previously implicated in the control of cell cycle timing. Our results demonstrate that accurate control of DNA replication is crucial during C. elegans early embryonic development and further provide a novel mechanism by which PAR proteins control cell cycle progression during asynchronous cell division.     

PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division

During asymmetric cell division, the mitotic spindle and polarized myosin can both determine the position of the cytokinetic furrow. However, how cells coordinate signals from the spindle and myosin to ensure that cleavage occurs through the spindle midzone is unknown. Here, we identify a novel pathway that is essential to inhibit myosin and coordinate furrow and spindle positions during asymmetric division. In Caenorhabditis elegans one-cell embryos, myosin localizes at the anterior cortex whereas the mitotic spindle localizes toward the posterior. We find that PAR-4/LKB1 impinges on myosin via two pathways, an anillin-dependent pathway that also responds to the cullin CUL-5 and an anillin-independent pathway involving the kinase PIG-1/MELK. In the absence of both PIG-1/MELK and the anillin ANI-1, myosin accumulates at the anterior cortex and induces a strong displacement of the furrow toward the anterior, which can lead to DNA segregation defects. Regulation of asymmetrically localized myosin is thus critical to ensure that furrow and spindle midzone positions coincide throughout cytokinesis.     

Detecting Amyloid-β Aggregation with Fiber-Based Fluorescence Correlation Spectroscopy

Soluble aggregates critically influence the chemical and biological aspects of amyloid protein aggregation, but their population is difficult to measure, especially in vivo. We take an optical fiber-based fluorescence correlation spectroscopy (FCS) approach to characterize a solution of aggregating amyloid-β molecules. We find that this technique can easily resolve aggregate particles of size 100 nm or greater in vitro, and the size distribution of these particles agrees well with that obtained by conventional FCS techniques. We propose fiber FCS as a tool for studying aggregation in vivo.