CDK phosphorylation of SLD-2 is required for replication initiation and germline development in C. elegans

Cyclin-dependent kinase (CDK) plays a vital role in proliferation control across eukaryotes. Despite this, how CDK mediates cell cycle and developmental transitions in metazoa is poorly understood. In this paper, we identify orthologues of Sld2, a CDK target that is important for DNA replication in yeast, and characterize SLD-2 in the nematode worm Caenorhabditis elegans. We demonstrate that SLD-2 is required for replication initiation and the nuclear retention of a critical component of the replicative helicase CDC-45 in embryos. SLD-2 is a CDK target in vivo, and phosphorylation regulates the interaction with another replication factor, MUS-101. By mutation of the CDK sites in sld-2, we show that CDK phosphorylation of SLD-2 is essential in C. elegans. Finally, using a phosphomimicking sld-2 mutant, we demonstrate that timely CDK phosphorylation of SLD-2 is an important control mechanism to allow normal proliferation in the germline. These results determine an essential function of CDK in metazoa and identify a developmental role for regulated SLD-2 phosphorylation.     

Modeling the establishment of PAR protein polarity in the one-cell C. elegans embryo

At the one-cell stage, the C. elegans embryo becomes polarized along the anterior-posterior axis. The PAR proteins form complementary anterior and posterior domains in a dynamic process driven by cytoskeletal rearrangement. Initially, the PAR proteins are uniformly distributed throughout the embryo. After a cue from fertilization, cortical actomyosin contracts toward the anterior pole. PAR-3/PAR-6/PKC-3 (the anterior PAR proteins) become restricted to the anterior cortex. PAR-1 and PAR-2 (the posterior PAR proteins) become enriched in the posterior cortical region. We present a mathematical model of this polarity establishment process, in which we take a novel approach to combine reaction-diffusion dynamics of the PAR proteins coupled to a simple model of actomyosin contraction. We show that known interactions between the PAR proteins are sufficient to explain many aspects of the observed cortical PAR dynamics in both wild-type and mutant embryos. However, cytoplasmic PAR protein polarity, which is vital for generating daughter cells with distinct molecular components, cannot be properly explained within such a framework. We therefore consider additional mechanisms that can reproduce the proper cytoplasmic polarity. In particular we predict that cytoskeletal asymmetry in the cytoplasm, in addition to the cortical actomyosin asymmetry, is a critical determinant of PAR protein localization.     

Cell polarity in the early Caenorhabditis elegans embryo.

Beginning with the first mitotic division in a Caenorhabditis elegans embryo, asymmetric cleavages establish much of the body plan. Although they share a common axis of polarity, at least three kinds of asymmetric cell division occur: two are under intrinsic control, while a third requires an inductive signal and may operate repeatedly throughout development.     

Cell polarity and asymmetric cell division: the C. elegans early embryo

Cell polarity is crucial for many functions including cell migration, tissue organization and asymmetric cell division. In animal cells, cell polarity is controlled by the highly conserved PAR (PARtitioning defective) proteins. par genes have been identified in Caenorhabditis elegans in screens for maternal lethal mutations that disrupt cytoplasmic partitioning and asymmetric division. Although PAR proteins were identified more than 20 years ago, our understanding on how they regulate polarity and how they are regulated is still incomplete. In this chapter we review our knowledge of the processes of cell polarity establishment and maintenance, and asymmetric cell division in the early C. elegans embryo. We discuss recent findings that highlight new players in cell polarity and/or reveal the molecular details on how PAR proteins regulate polarity processes.     

Identification and characterization of factors required for microtubule growth and nucleation in the early C. elegans embryo

Microtubules (MTs) are dynamic polymers that undergo cell cycle and position-sensitive regulation of polymerization and depolymerization. Although many different factors that regulate MT dynamics have been described, to date there has been no systematic analysis of genes required for MT dynamics in a single system. Here, we use a transgenic EB1::GFP strain, which labels the growing plus ends of MTs, to analyze the growth rate, nucleation rate, and distribution of growing MTs in the Caenorhabditis elegans embryo. We also present the results from an RNAi screen of 40 genes previously implicated in MT-based processes. Our findings suggest that fast microtubule growth is dependent on the amount of free tubulin and the ZYG-9-TAC-1 complex. Robust MT nucleation by centrosomes requires AIR-1, SPD-2, SPD-5, and gamma-tubulin. However, we found that centrosomes do not nucleate MTs to saturation; rather, the depolymerizing kinesin-13 subfamily member KLP-7 is required to limit microtubule outgrowth from centrosomes.     

Multiple levels of regulation specify the polarity of an asymmetric cell division in C. elegans

Wnt signaling systems play important roles in the generation of cell and tissue polarity during development. We describe a Wnt signaling system that acts in a new way to orient the polarity of an epidermal cell division in C. elegans. In this system, the EGL-20/Wnt signal acts in a permissive fashion to polarize the asymmetric division of a cell called V5. EGL-20 regulates this polarization by counteracting lateral signals from neighboring cells that would otherwise reverse the polarity of the V5 cell division. Our findings indicate that this lateral signaling pathway also involves Wnt pathway components. Overexpression of EGL-20 disrupts both the asymmetry and polarity of lateral epidermal cell divisions all along the anteroposterior (A/P) body axis. Together our findings suggest that multiple, inter-related Wnt signaling systems may act together to polarize asymmetric cell divisions in this tissue.     

Revisiting the role of microtubules in C. elegans polarity

Cells must break symmetry to acquire polarity. Microtubules have been implicated in the induction of asymmetry in several cell types, but their role in the Caenorhabditis elegans zygote, a classic polarity model, has remained uncertain. One study (see Tsai and Ahringer on p. 397 of this issue) brings new light to this problem by demonstrating that severe loss of microtubules impairs polarity onset in C. elegans.     

Control of cell polarity by noncanonical Wnt signaling in C. elegans

The three Caenorhabditis elegans beta-catenin each function in distinct processes: BAR-1 in canonical Wnt signaling that controls cell fates and cell migrations, HMP-2 in cell adhesion and WRM-1 in Wnt signaling pathways that function in conjunction with a mitogen-activated kinase (MAPK) pathway to control the orientations, or cell polarities, of cells that undergo asymmetric cell divisions. In addition, WRM-1 does not interact with the canonical beta-catenin binding site in POP-1/Tcf. Thus, Wnt signaling through WRM-1 is noncanonical and, except for one division that might not include any of the three C. elegans beta-catenin, controls cell polarity in C. elegans.     

Regulatory mechanism of the initiation step of DNA replication by CDK in budding yeast

Cyclin-dependent kinases (CDKs) regulate the progression of the cell cycle in eukaryotes. At the onset of chromosomal DNA replication, CDKs phosphorylate two replication proteins, Sld2 and Sld3, in budding yeast. Phosphorylated Sld2 and Sld3 enhance the formation of complexes with the BRCT (BRCA1 C-terminal)-containing replication protein Dpb11. The formation of these complexes is essential and sufficient for the CDK-dependent activation of the initiation of chromosomal DNA replication. Multiple phosphorylation of Sld2 by CDKs fine-tunes the process of complex formation. Here, we discussed the regulation of the initiation step of chromosomal DNA replication via CDK-dependent phosphorylation.     

CRL2(LRR-1) targets a CDK inhibitor for cell cycle control in C. elegans and actin-based motility regulation in human cells

The Cip/Kip CDK inhibitor (CKI) p21(Cip1/WAF1) has?a critical role in the nucleus to limit cell proliferation by inhibiting CDK-cyclin complexes. In contrast, cytoplasmic p21 regulates cell survival and the actin cytoskeleton. These divergent functions for p21 in different cellular compartments suggest the necessity for complex regulation. In this study, we identify the CRL2(LRR-1) ubiquitin ligase as a conserved regulator of Cip/Kip CKIs that promotes the degradation of C. elegans CKI-1 and human p21. The nematode CRL2(LRR-1) complex negatively regulates nuclear CKI-1 levels to ensure G1-phase cell cycle progression in germ cells. In contrast, human CRL2(LRR1) targets cytoplasmic p21, acting as a critical regulator of cell motility that promotes a nonmotile stationary cell state by preventing p21 from inhibiting the Rho/ROCK/LIMK pathway. Inactivation of human CRL2(LRR1) leads to the activation of the actin-depolymerizing protein cofilin, dramatic reorganization of the actin cytoskeleton, and increased cell motility.     

Stabilization of cell polarity by the C. elegans RING protein PAR-2

Asymmetric localization of PAR proteins is a hallmark of polarized cells, but the mechanisms that create PAR asymmetry are not well understood. In the C. elegans zygote, PAR asymmetry is initiated by a transient actomyosin contraction, which sweeps the PAR-3/PAR-6/PKC-3 complex toward the anterior pole of the egg. The RING finger protein PAR-2 accumulates in a complementary pattern in the posterior cortex. Here we present evidence that PAR-2 participates in a feedback loop to stabilize polarity. PAR-2 is a target of the PKC-3 kinase and is excluded from the anterior cortex by PKC-3-dependent phosphorylation. The RING domain of PAR-2 is required to overcome inhibition by PKC-3 and stabilize PAR-2 on the posterior cortex. Cortical PAR-2 in turn prevents PAR-3/PAR-6/PKC-3 from returning to the posterior, in a PAR-1- and PAR-5-dependent manner. Our findings suggest that reciprocal inhibitory interactions among PAR proteins stabilize polarity by reinforcing an initial asymmetry in PKC-3.     

Cortical flows powered by asymmetrical contraction transport PAR proteins to establish and maintain anterior-posterior polarity in the early C. elegans embryo

The C. elegans PAR proteins PAR-3, PAR-6, and PKC-3 are asymmetrically localized and have essential roles in cell polarity. We show that the one-cell C. elegans embryo contains a dynamic and contractile actomyosin network that appears to be destabilized near the point of sperm entry. This asymmetry initiates a flow of cortical nonmuscle myosin (NMY-2) and F-actin toward the opposite, future anterior, pole. PAR-3, PAR-6, and PKC-3, as well as non-PAR proteins that associate with the cytoskeleton, appear to be transported to the anterior by this cortical flow. In turn, PAR-3, PAR-6, and PKC-3 modulate cortical actomyosin dynamics and promote cortical flow. PAR-2, which localizes to the posterior cortex, inhibits NMY-2 from accumulating at the posterior cortex during flow, thus maintaining asymmetry by preventing inappropriate, posterior-directed flows. Similar actomyosin flows accompany the establishment of PAR asymmetries that form after the one-cell stage, suggesting that actomyosin-mediated cortical flows have a general role in PAR asymmetry.     

Wnt-dependent spindle polarization in the early C. elegans embryo

Correct orientation of the mitotic spindle is crucial for the proper segregation of localized determinants and the correct spatial organization of cells in early embryos. The cues dividing cells use to orient their mitotic spindles are currently the subject of intensive investigation in a number of model systems. One of the cues that cells use during spindle orientation is provided by components of the Wnt signaling pathway. Because of its stereotypical cleavage divisions, the availability of Wnt pathway mutants and the ability to perform RNAi, and because cell-cell interactions can be studied in vitro, the C. elegans embryo continues to be a useful system for identifying specific cell-cell interactions in which Wnt-dependent signals polarize the mitotic spindle. This review discusses the evidence for involvement of Wnt signaling during spindle orientation in several contexts in the early C. elegans embryo, a process that involves upstream Wnt effectors but does not involve downstream nuclear effectors of Wnt signaling, and places this Wnt spindle orientation pathway in the larger context of other known modulators of spindle orientation in animal embryos.     

Acute drug treatment in the early C. elegans embryo

Genetic and genome-wide RNAi approaches available in C. elegans, combined with tools for visualizing subcellular events with high-resolution, have led to increasing adoption of the early C. elegans embryo as a model for mechanistic and functional genomic analysis of cellular processes. However, a limitation of this system has been the impermeability of the embryo eggshell, which has prevented the routine use of small molecule inhibitors. Here, we present a method to permeabilize and immobilize embryos for acute inhibitor treatment in conjunction with live imaging. To identify a means to permeabilize the eggshell, we used a dye uptake assay to screen a set of 310 candidate genes defined by a combination of bioinformatic criteria. This screen identified 20 genes whose inhibition resulted in >75% eggshell permeability, and 3 that permeabilized embryos with minimal deleterious effects on embryo production and early embryonic development. To mount permeabilized embryos for acute drug addition in conjunction with live imaging, we combined optimized inhibition of one of these genes with the use of a microfabricated chamber that we designed. We demonstrate that these two developments enable the temporally controlled introduction of inhibitors for mechanistic studies. This method should also open new avenues of investigation by allowing profiling and specificity-testing of inhibitors through comparison with genome-wide phenotypic datasets.