The auxin-inducible degron 2 (AID2) system enables controlled protein knockdown during embryogenesis and development in Caenorhabditis elegans.

Targeted protein degradation using the auxin-inducible degron (AID) system is garnering attention in the research field of Caenorhabditis elegans, because of the rapid and efficient target depletion it affords, which can be controlled by treating the animals with the phytohormone auxin. However, the current AID system has drawbacks, i.e., leaky degradation in the absence of auxin and the requirement for high auxin doses. Furthermore, it is challenging to deplete degron-fused proteins in embryos because of their eggshell, which blocks auxin permeability. Here, we apply an improved AID2 system utilizing AtTIR1(F79G) and 5-phenyl-indole-3-acetic acid (5-Ph-IAA) to C. elegans and demonstrated that it confers better degradation control vs the previous system by suppressing leaky degradation and inducing sharp degradation using 1,300-fold lower 5-Ph-IAA doses. We successfully degraded the endogenous histone H2A.Z protein fused to an mAID degron and disclosed its requirement in larval growth and reproduction, regardless of the presence of maternally inherited H2A.Z molecules. Moreover, we developed an eggshell-permeable 5-Ph-IAA analog, 5-Ph-IAA-AM, that affords an enhanced degradation in laid embryos. Our improved system will contribute to the disclosure of the roles of proteins in C. elegans, in particular those that are involved in embryogenesis and development, through temporally controlled protein degradation.

Tumor suppressor APC is an attenuator of spindle-pulling forces during C. elegans asymmetric cell division.

The adenomatous polyposis coli (APC) tumor suppressor has dual functions in Wnt/ß-catenin signaling and accurate chromosome segregation and is frequently mutated in colorectal cancers. Although APC contributes to proper cell division, the underlying mechanisms remain poorly understood. Here we show that Caenorhabditis elegans APR-1/APC is an attenuator of the pulling forces acting on the mitotic spindle. During asymmetric cell division of the C. elegans zygote, a LIN-5/NuMA protein complex localizes dynein to the cell cortex to generate pulling forces on astral microtubules that position the mitotic spindle. We found that APR-1 localizes to the anterior cell cortex in a Par-aPKC polarity-dependent manner and suppresses anterior centrosome movements. Our combined cell biological and mathematical analyses support the conclusion that cortical APR-1 reduces force generation by stabilizing microtubule plus-ends at the cell cortex. Furthermore, APR-1 functions in coordination with LIN-5 phosphorylation to attenuate spindle-pulling forces. Our results document a physical basis for the attenuation of spindle-pulling force, which may be generally used in asymmetric cell division and, when disrupted, potentially contributes to division defects in cancer.

PIGN prevents protein aggregation in the endoplasmic reticulum independently of its function in the GPI synthesis.

Quality control of proteins in the endoplasmic reticulum (ER) is essential for ensuring the integrity of secretory proteins before their release into the extracellular space. Secretory proteins that fail to pass quality control form aggregates. Here we show the PIGN-1/PIGN is required for quality control in Caenorhabditis elegans and in mammalian cells. In C. elegans pign-1 mutants, several proteins fail to be secreted and instead form abnormal aggregation. PIGN-knockout HEK293 cells also showed similar protein aggregation. Although PIGN-1/PIGN is responsible for glycosylphosphatidylinositol (GPI)-anchor biosynthesis in the ER, certain mutations in C. elegans pign-1 caused protein aggregation in the ER without affecting GPI-anchor biosynthesis. These results show that PIGN-1/PIGN has a conserved and non-canonical function to prevent deleterious protein aggregation in the ER independently of the GPI-anchor biosynthesis. PIGN is a causative gene for some human diseases including multiple congenital seizure-related syndrome (MCAHS1). Two pign-1 mutations created by CRISPR/Cas9 that correspond to MCAHS1 also cause protein aggregation in the ER, implying that the dysfunction of the PIGN non-canonical function might affect symptoms of MCAHS1 and potentially those of other diseases.

Cortical Polarity of the RING Protein PAR-2 Is Maintained by Exchange Rate Kinetics at the Cortical-Cytoplasmic Boundary.

Cell polarity arises through the spatial segregation of polarity regulators. PAR proteins are polarity regulators that localize asymmetrically to two opposing cortical domains. However, it is unclear how the spatially segregated PAR proteins interact to maintain their mutually exclusive partitioning. Here, single-molecule detection analysis in Caenorhabditis elegans embryos reveals that cortical PAR-2 diffuses only short distances, and, as a result, most PAR-2 molecules associate and dissociate from the cortex without crossing into the opposing domain. Our results show that cortical PAR-2 asymmetry is maintained by the local exchange reactions that occur at the cortical-cytoplasmic boundary. Additionally, we demonstrate that local exchange reactions are sufficient to maintain cortical asymmetry in a parameter-free mathematical model. These findings suggest that anterior and posterior PAR proteins primarily interact through the cytoplasmic pool and not via cortical diffusion.

The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans.

Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator's dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinase-dependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals.

HTZ-1/H2A.z and MYS-1/MYST HAT act redundantly to maintain cell fates in somatic gonadal cells through repression of ceh-22 in C. elegans.

The stable maintenance of acquired cell fates is important during development and for maintaining tissue homeostasis. Although histone modification is one of the major strategies used by cells to maintain their fates, the mechanisms by which histone variants maintain cell fates are not well understood. In C. elegans, the acetylated-histone-H4 (AcH4)-binding protein BET-1 acts downstream of the MYST family histone acetyltransferases MYS-1 and MYS-2 to establish and maintain cell fates in multiple cell lineages. Here we show that, in the bet-1 pathway, the histone H2A variant HTZ-1/H2A.z and MYS-1 are required for the maintenance of cell fates in a redundant manner. BET-1 controlled the subnuclear localization of HTZ-1. HTZ-1 and MYS-1 maintained the fates of the somatic gonadal cells (SGCs) through the repression of a target, ceh-22/Nkx2.5, which induced the formation of the leader cells of the gonad. H3K27 demethylase, UTX-1, had an antagonistic effect relative to HTZ-1 in the regulation of ceh-22. Nuclear spot assay revealed that HTZ-1 localized to the ceh-22 locus in SGCs in an utx-1-dependent manner. We propose that HTZ-1 and MYS-1 repress ceh-22 when UTX-1 removes its silencing mark, H3K27 methylation on the ceh-22 locus, thereby maintaining the fates of SGCs.

Power law relationship between cell cycle duration and cell volume in the early embryonic development of Caenorhabditis elegans.

Cell size is a critical factor for cell cycle regulation. In Xenopus embryos after midblastula transition (MBT), the cell cycle duration elongates in a power law relationship with the cell radius squared. This correlation has been explained by the model that cell surface area is a candidate to determine cell cycle duration. However, it remains unknown whether this second power law is conserved in other animal embryos. Here, we found that the relationship between cell cycle duration and cell size in Caenorhabditis elegans embryos exhibited a power law distribution. Interestingly, the powers of the time-size relationship could be grouped into at least three classes: highly size-correlated, moderately size-correlated, and potentially a size-non-correlated class according to C. elegans founder cell lineages (1.2, 0.81, and <0.39 in radius, respectively). Thus, the power law relationship is conserved in Xenopus and C. elegans, while the absolute powers in C. elegans were different from that in Xenopus. Furthermore, we found that the volume ratio between the nucleus and cell exhibited a power law relationship in the size-correlated classes. The power of the volume relationship was closest to that of the time-size relationship in the highly size-correlated class. This correlation raised the possibility that the time-size relationship, at least in the highly size-correlated class, is explained by the volume ratio of nuclear size and cell size. Thus, our quantitative measurements shed a light on the possibility that early embryonic C. elegans cell cycle duration is coordinated with cell size as a result of geometric constraints between intracellular structures.

The SWI/SNF chromatin remodeling complex selectively affects multiple aspects of serotonergic neuron differentiation.

Regulatory programs that control the specification of serotonergic neurons have been investigated by genetic mutant screens in the nematode Caenorhabditis elegans. Loss of a previously uncloned gene, ham-3, affects migration and serotonin antibody staining of the hermaphrodite-specific neuron (HSN) pair. We characterize these defects here in more detail, showing that the defects in serotonin antibody staining are paralleled by a loss of the transcription of all genes involved in serotonin synthesis and transport. This loss is specific to the HSN class as other serotonergic neurons appear to differentiate normally in ham-3 null mutants. Besides failing to migrate appropriately, the HSNs also display axon pathfinding defects in ham-3 mutants. However, the HSNs are still generated and express a subset of their terminal differentiation features in ham-3 null mutants, demonstrating that ham-3 is a specific regulator of select features of the HSNs. We show that ham-3 codes for the C. elegans ortholog of human BAF60, Drosophila Bap60, and yeast Swp73/Rsc6, which are subunits of the yeast SWI/SNF and vertebrate BAF chromatin remodeling complex. We show that the effect of ham-3 on serotonergic fate can be explained by ham-3 regulating the expression of the Spalt/SALL-type Zn finger transcription factor sem-4, a previously identified regulator of serotonin expression in HSNs and of the ham-2 Zn transcription factor, a previously identified regulator of HSN migration and axon outgrowth. Our findings provide the first evidence for the involvement of the BAF complex in the acquisition of terminal neuronal identity and constitute genetic proof by germline knockout that a BAF complex component can have cell-type-specific roles during development.

Wnt signaling in C. elegans.

Wnt proteins are secreted lipid-modified glycoproteins that control many aspects of development in organisms ranging from sponges to vertebrates. Wnt proteins are also important regulators of C. elegans development, with functions in processes as diverse as cell fate specification, asymmetric cell division, cell migration and synapse formation. In this review, we will give an overview of what we currently know about the signaling mechanisms that mediate these different functions of Wnt.

Control of cell polarity and asymmetric division in C. elegans.

During development of Caenorhabditis elegans, most somatic cells divide asymmetrically to produce daughter cells with distinct fates. A Wnt signaling pathway called Wnt/ß-catenin asymmetry pathway controls both polarity of mother cells and distinct fates of daughter cells. Unlike the PCP pathway that regulates cell polarity in other organisms, this Wnt pathway in C. elegans requires ß-catenin. However, similar to the PCP pathway, signaling components including Dishevelled proteins are asymmetrically localized to the cell cortex. I will review current knowledge about the mechanism of this regulation and how the orientation of cell polarity is controlled by Wnt proteins.

Formation and functions of asymmetric microtubule organization in polarized cells.

Although microtubules are known to be essential for chromosome segregation during cell division, they also play important roles in the regulation and function of cell polarity. Cell polarization is fundamental to appropriate tissue patterning and the regulation of cellular diversity during animal development. In polarized cells, microtubules are often organized asymmetrically along the polarity axis. Recent studies show that such asymmetry in microtubule organization is important to connect a cell's polarization with its polarized functions. In some cases, asymmetrically organized microtubule arrays themselves induce cell polarity. Here we present an overview of the mechanisms and functions of asymmetric microtubule organization and discuss the possible role of microtubule asymmetry in the symmetry-breaking that leads to cell polarization.

Multiple functions of PBRM-1/Polybromo- and LET-526/Osa-containing chromatin remodeling complexes in C. elegans development.

The SWI/SNF-like chromatin remodeling complexes consist of two evolutionarily conserved subclasses, which are characterized by specific accessory components, the OSA/BAF250 and Polybromo proteins. These complexes regulate the expressions of distinct sets of target genes, with some overlap, and the regulatory components are thought to determine the target specificity for each complex. Here we isolated C. elegans mutants of the genes for the OSA/BAF250 homolog, LET-526, and the Polybromo homolog, PBRM-1, in a screen for the abnormal asymmetric cell division phenotype. In the asymmetric division of the T cell, both LET-526 and PBRM-1 regulated the asymmetric expression of psa-3/Meis between the T cell daughters, suggesting that the two subclasses share the same target. In the gonad, PBRM-1 regulated gonad primordium formation during embryogenesis, whereas LET-526 was required post-embryonically for distal tip cell (DTC) production from the gonad primordium, suggesting that these proteins have distinct targets for DTC development. Thus, the same cellular process is regulated by LET-526 and PBRM-1 in the asymmetric division of the T cell, but they regulate distinct cellular processes in the gonad morphogenesis. Although disruption of the core component PSA-1 or PSA-4 caused similar defects in the gonad and T cell, it also caused early embryonic arrest, which was not observed in the let-526, pbrm-1, or let-526 pbrm-1 double mutants, suggesting that some targets of SWI/SNF-like complexes do not require LET-526 or PBRM-1 for their transcription. Our results show that the target selection by SWI/SNF-like complexes during C. elegans development is intricately regulated by accessory components.

Multiple Wnts redundantly control polarity orientation in Caenorhabditis elegans epithelial stem cells.

During development, cell polarization is often coordinated to harmonize tissue patterning and morphogenesis. However, how extrinsic signals synchronize cell polarization is not understood. In Caenorhabditis elegans, most mitotic cells are polarized along the anterior-posterior axis and divide asymmetrically. Although this process is regulated by a Wnt-signaling pathway, Wnts functioning in cell polarity have been demonstrated in only a few cells. We analyzed how Wnts control cell polarity, using compound Wnt mutants, including animals with mutations in all five Wnt genes. We found that somatic gonadal precursor cells (SGPs) are properly polarized and oriented in quintuple Wnt mutants, suggesting Wnts are dispensable for the SGPs' polarity, which instead requires signals from the germ cells. Thus, signals from the germ cells organize the C. elegans somatic gonad. In contrast, in compound but not single Wnt mutants, most of the six seam cells, V1-V6 (which are epithelial stem cells), retain their polarization, but their polar orientation becomes random, indicating that it is redundantly regulated by multiple Wnt genes. In contrast, in animals in which the functions of three Wnt receptors (LIN-17, MOM-5, and CAM-1) are disrupted--the stem cells are not polarized and divide symmetrically--suggesting that the Wnt receptors are essential for generating polarity and that they function even in the absence of Wnts. All the seam cells except V5 were polarized properly by a single Wnt gene expressed at the cell's anterior or posterior. The ectopic expression of posteriorly expressed Wnts in an anterior region and vice versa rescued polarity defects in compound Wnt mutants, raising two possibilities: one, Wnts permissively control the orientation of polarity; or two, Wnt functions are instructive, but which orientation they specify is determined by the cells that express them. Our results provide a paradigm for understanding how cell polarity is coordinated by extrinsic signals.

Wnt regulates spindle asymmetry to generate asymmetric nuclear ß-catenin in C. elegans.

Extrinsic signals received by a cell can induce remodeling of the cytoskeleton, but the downstream effects of cytoskeletal changes on gene expression have not been well studied. Here, we show that during telophase of an asymmetric division in C. elegans, extrinsic Wnt signaling modulates spindle structures through APR-1/APC, which in turn promotes asymmetrical nuclear localization of WRM-1/ß-catenin and POP-1/TCF. APR-1 that localized asymmetrically along the cortex established asymmetric distribution of astral microtubules, with more microtubules found on the anterior side. Perturbation of the Wnt signaling pathway altered this microtubule asymmetry and led to changes in nuclear WRM-1 asymmetry, gene expression, and cell-fate determination. Direct manipulation of spindle asymmetry by laser irradiation altered the asymmetric distribution of nuclear WRM-1. Moreover, laser manipulation of the spindles rescued defects in nuclear POP-1 asymmetry in wnt mutants. Our results reveal a mechanism in which the nuclear localization of proteins is regulated through the modulation of microtubules.

Distinct and mutually inhibitory binding by two divergent ß-catenins coordinates TCF levels and activity in C. elegans.

Wnt target gene activation in C. elegans requires simultaneous elevation of ß-catenin/SYS-1 and reduction of TCF/POP-1 nuclear levels within the same signal-responsive cell. SYS-1 binds to the conserved N-terminal ß-catenin-binding domain (CBD) of POP-1 and functions as a transcriptional co-activator. Phosphorylation of POP-1 by LIT-1, the C. elegans Nemo-like kinase homolog, promotes POP-1 nuclear export and is the main mechanism by which POP-1 nuclear levels are lowered. We present a mechanism whereby SYS-1 and POP-1 nuclear levels are regulated in opposite directions, despite the fact that the two proteins physically interact. We show that the C terminus of POP-1 is essential for LIT-1 phosphorylation and is specifically bound by the diverged ß-catenin WRM-1. WRM-1 does not bind to the CBD of POP-1, nor does SYS-1 bind to the C-terminal domain. Furthermore, binding of WRM-1 to the POP-1 C terminus is mutually inhibitory with SYS-1 binding at the CBD. Computer modeling provides a structural explanation for the specificity in WRM-1 and SYS-1 binding to POP-1. Finally, WRM-1 exhibits two independent and distinct molecular functions that are novel for ß-catenins: WRM-1 serves both as the substrate-binding subunit and an obligate regulatory subunit for the LIT-1 kinase. Mutual inhibitory binding would result in two populations of POP-1: one bound by WRM-1 that is LIT-1 phosphorylated and exported from the nucleus, and another, bound by SYS-1, that remains in the nucleus and transcriptionally activates Wnt target genes. These studies could provide novel insights into cancers arising from aberrant Wnt activation.

Extracellular control of PAR protein localization during asymmetric cell division in the C. elegans embryo.

The axis of asymmetric cell division is controlled to determine the future position of differentiated cells during animal development. The asymmetric localization of PAR proteins in the Drosophila neuroblast and C. elegans embryo are aligned with the axes of the embryo. However, whether extracellular or intracellular signals determine the orientation of the localization of PAR proteins remains controversial. In C. elegans, the P0 zygote and germline cells (P1, P2, and P3) undergo a series of asymmetric cell divisions. Interestingly, the axis of the P0 and P1 divisions is opposite to that of the P2 and P3 divisions. PAR-2, a ring-finger protein, and PAR-1, a kinase, relocalize to the anterior side of the P2 and P3 germline precursors at the site of contact with endodermal precursors. Using an in vitro method, we have found that the PAR-2 protein is distributed asymmetrically in the absence of extracellular signals, but the orientation of the protein localization in the P2 and P3 cells is determined by contact with endodermal precursor cells. Our mutant analyses suggest that mes-1 and src-1, which respectively encode a transmembrane protein and a tyrosine kinase, were not required to establish the asymmetric distribution of PAR-2, but were required to determine its orientation at the site of contact with the endodermal precursors. The PAR-2 localization during the asymmetric P2 and P3 divisions is controlled by extracellular signals via MES-1/SRC-1 signaling. Our findings suggest that Src functions as an evolutionarily conserved molecular link that coordinates extrinsic cues with PAR protein localization.

Regulation of asymmetric positioning of nuclei by Wnt and Src signaling and its roles in POP-1/TCF nuclear asymmetry in Caenorhabditis elegans.

In various polarized cells, positions of nuclei are often off-center. However, extrinsic signals regulating nuclear off-centering and its biologic roles remain to be elucidated. In Caenorhabditis elegans, polarity of the EMS cell undergoing asymmetric division is regulated by the MOM-2/Wnt and MES-1 signals from its posterior neighbor P2 cell. We show that after divisions of different cells including EMS, the nuclei of the posterior but not anterior daughter cells are anchored to the posterior cell cortex via centrosomes. We also show that this nuclear anchoring is regulated by components of the Wnt pathway and SRC-1 that functions in MES-1 signaling. To understand the biologic roles of nuclear anchoring, we analyzed its effects on asymmetric nuclear localization of POP-1/TCF that is also regulated by Wnt and Src signaling. We found that in mom-2 mutants where the nuclear anchoring and POP-1 asymmetry is partially inhibited, the proximity of the nucleus to the cell cortex correlated with POP-1 asymmetry. Furthermore, in mutants of mom-2, the defect in the anchoring is clearly correlated with that of asymmetric fate determination. These results suggest that the asymmetric nuclear anchoring functions in asymmetric division by enhancing POP-1 asymmetry.

Double bromodomain protein BET-1 and MYST HATs establish and maintain stable cell fates in C. elegans.

The maintenance of cell fate is important for normal development and tissue homeostasis. Epigenetic mechanisms, including histone modifications, are likely to play crucial roles in cell-fate maintenance. However, in contrast to the established functions of histone methylation, which are mediated by the polycomb proteins, the roles of histone acetylation in cell-fate maintenance are poorly understood. Here, we show that the C. elegans acetylated-histone-binding protein BET-1 is required for the establishment and maintenance of stable fate in various lineages. In most bet-1 mutants, cells adopted the correct fate initially, but at later stages they often transformed into a different cell type. By expressing BET-1 at various times in development and examining the rescue of the Bet-1 phenotype, we showed that BET-1 functions both at the time of fate acquisition, to establish a stable fate, and at later stages, to maintain the established fate. Furthermore, the disruption of the MYST HATs perturbed the subnuclear localization of BET-1 and caused bet-1-like phenotypes, suggesting that BET-1 is recruited to its targets through acetylated histones. Our results therefore indicate that histone acetylation plays a crucial role in cell-fate maintenance.