C.elegans野生型和par突变体早期胚胎卵裂的观察

不对称分裂在动植物的发育中起到了非常重要的作用。Caenorhabditis elegans（C．elegans）胚胎最早的两次卵裂为研究控制不对称分裂的机制提供了很好的机会。用普通光学显微镜观察了野生型胚胎早期卵裂和par-1、par-2、par-3、par-4突变体胚胎的早期卵裂。野生型胚胎最早的分裂是不等的,产生了两个不同大小的子细胞。两个子细胞又以不同的方向进行第二次分裂。在C．elegans中任意一个par基因的缺失会使胚胎的第一次卵裂丧失不对称性。这会导致一些发育调控因子不能在特定的胚胎细胞中准确地定位,造成细胞分裂纺锤体方向的异常。par类基因参与不对称性的建立,这种不对称性决定了C．elegans身体的前后轴。     

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.     

An eIF4E-binding protein regulates katanin protein levels in C. elegans embryos

In Caenorhabditis elegans, the MEI-1–katanin microtubule-severing complex is required for meiosis, but must be down-regulated during the transition to embryogenesis to prevent defects in mitosis. A cullin-dependent degradation pathway for MEI-1 protein has been well documented. In this paper, we report that translational repression may also play a role in MEI-1 down-regulation. Reduction of spn-2 function results in spindle orientation defects due to ectopic MEI-1 expression during embryonic mitosis. MEL-26, which is both required for MEI-1 degradation and is itself a target of the cullin degradation pathway, is present at normal levels in spn-2 mutant embryos, suggesting that the degradation pathway is functional. Cloning of spn-2 reveals that it encodes an eIF4E-binding protein that localizes to the cytoplasm and to ribonucleoprotein particles called P granules. SPN-2 binds to the RNA-binding protein OMA-1, which in turn binds to the mei-1 3′ untranslated region. Thus, our results suggest that SPN-2 functions as an eIF4E-binding protein to negatively regulate translation of mei-1.     

Asymmetric distribution of PAR proteins in the mouse embryo begins at the 8-cell stage during compaction

In many organisms, like Caenorhabditis elegans and Drosophila melanogaster, establishment of spatial patterns and definition of cell fate are driven by the segregation of determinants in response to spatial cues, as early as oogenesis or fertilization. In these organisms, a family of conserved proteins, the PAR proteins, is involved in the asymmetric distribution of cytoplasmic determinants and in the control of asymmetric divisions. In the mouse embryo, it is only at the 8-cell stage during compaction that asymmetries, leading to cellular diversification and blastocyst morphogenesis, are first observed. However, it has been suggested that developmentally relevant asymmetries could be established already in the oocyte and during fertilization. This led us to study the PAR proteins during the early stages of mouse development. We observed that the homologues of the different members of the PAR/aPKC complex and PAR1 are expressed in the preimplantation mouse embryo. During the first embryonic cleavages, before compaction, PARD6b and EMK1 are observed on the spindle. The localization of these two proteins becomes asymmetric during compaction, when blastomeres flatten upon each other and polarize. PARD6b is targeted to the apical pole, whereas EMK1 is distributed along the baso-lateral domain. The targeting of EMK1 is dependent upon cell-cell interactions while the apical localization of PARD6b is independent of cell contacts. At the 16-cell stage, aPKCzeta colocalizes with PARD6b and a colocalization of the three proteins (PARD6b/PARD3/aPKCzeta can occur in blastocysts, only at tight junctions. This choreography suggests that proteins of the PAR family are involved in the setting up of blastomere polarity and blastocyst morphogenesis in the early mammalian embryo although the interactions between the different players differ from previously studied systems. Finally, they reinforce the idea that the first developmentally relevant asymmetries are set up during compaction.     

C. elegans PAR-3 and PAR-6 are required for apicobasal asymmetries associated with cell adhesion and gastrulation

PAR proteins distribute asymmetrically across the anterior-posterior axis of the 1-cell-stage C. elegans embryo, and function to establish subsequent anterior-posterior asymmetries. By the end of the 4-cell stage, anteriorly localized PAR proteins, such as PAR-3 and PAR-6, redistribute to the outer, apical surfaces of cells, whereas posteriorly localized PAR proteins, such as PAR-1 and PAR-2, redistribute to the inner, basolateral surfaces. Because PAR proteins are provided maternally, distinguishing apicobasal from earlier anterior-posterior functions requires a method that selectively prevents PAR activity after the 1-cell stage. In the present study we generated hybrid PAR proteins that are targeted for degradation after the 1-cell stage. Embryos containing the hybrid PAR proteins had normal anterior-posterior polarity, but showed defects in apicobasal asymmetries associated with gastrulation. Ectopic separations appeared between lateral surfaces of cells that are normally tightly adherent, cells that ingress during gastrulation failed to accumulate nonmuscle myosin at their apical surfaces and ingression was slowed. Thus, PAR proteins function in both apicobasal and anterior-posterior asymmetry during the first few cell cycles of embryogenesis.     

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.     

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.     

Embryonic handedness choice in C. elegans involves the Galpha protein GPA-16

The mechanism by which polarity of the left-right (LR) axis is initially established with the correct handedness is not understood for any embryo. C. elegans embryos exhibit LR asymmetry with an invariant handedness that is first apparent at the six-cell stage and persists throughout development. We show here that a strong loss-of-function mutation in a gene originally designated spn-1 affects early spindle orientations and results in near randomization of handedness choice. This mutation interacts genetically with mutations in three par genes that encode localized cortical components. We show that the spn-1 gene encodes the Galpha protein GPA-16, which appears to be required for centrosomal association of a Gbeta protein. We will henceforth refer to this gene as gpa-16. These results demonstrate for the first time involvement of heterotrimeric G proteins in establishment of embryonic LR asymmetry and suggest how they might act.     

Symmetrically dividing cell specific division axes alteration observed in proteasome depleted C. elegans embryo

A fertilised Caenorhabditis elegans embryo shows an invariable pattern of cell division and forms a multicellular body where each cell locates to a defined position. Mitotic spindle orientation is determined by several preceding events including the migration of duplicated centrosomes on a nucleus and the rotation of nuclear-centrosome complex. Cell polarity is the dominant force driving nuclear-centrosome rotation and setting the mitotic spindle axis in parallel with the polarity axis during asymmetric cell division. It is reasonable that there is no nuclear-centrosome rotation in symmetrically dividing blastomeres, but the mechanism(s) which suppress rotation in these cells have been proposed because the rotations occur in some polarity defect embryos. Here we show the nuclear-centrosome rotation can be induced by depletion of RPN-2, a regulatory subunit of the proteasome. In these embryos, cell polarity is established normally and both asymmetrically and symmetrically dividing cells are generated through asymmetric cell divisions. The nuclear-centrosome rotations occurred normally in the asymmetrically dividing cell lineage, but also induced in symmetrically dividing daughter cells. Interestingly, we identified RPN-2 as a binding protein of PKC-3, one of critical elements for establishing cell polarity during early asymmetric cell divisions. In addition to asymmetrically dividing cells, PKC-3 is also expressed in symmetrically dividing cells and a role to suppress nuclear-centrosome rotation has been anticipated. Our data suggest that the expression of RPN-2 is involved in the mechanism to suppress nuclear-centrosome rotation in symmetrically dividing cells and it may work in cooperation with PKC-3.     

Cortical PAR polarity proteins promote robust cytokinesis during asymmetric cell division

Cytokinesis, the physical division of one cell into two, is thought to be fundamentally similar in most animal cell divisions and driven by the constriction of a contractile ring positioned and controlled solely by the mitotic spindle. During asymmetric cell divisions, the core polarity machinery (partitioning defective [PAR] proteins) controls the unequal inheritance of key cell fate determinants. Here, we show that in asymmetrically dividing Caenorhabditis elegans embryos, the cortical PAR proteins (including the small guanosine triphosphatase CDC-42) have an active role in regulating recruitment of a critical component of the contractile ring, filamentous actin (F-actin). We found that the cortical PAR proteins are required for the retention of anillin and septin in the anterior pole, which are cytokinesis proteins that our genetic data suggest act as inhibitors of F-actin at the contractile ring. Collectively, our results suggest that the cortical PAR proteins coordinate the establishment of cell polarity with the physical process of cytokinesis during asymmetric cell division to ensure the fidelity of daughter cell formation.     

Pod-2, along with pod-1, defines a new class of genes required for polarity in the early Caenorhabditis elegans embryo

The asymmetric division of the one-cell Caenorhabditis elegans zygote gives rise to two cells of different size and fate, thereby establishing the animal's anterior--posterior (a-p) axis. Through genetics, a number of genes required for this polarity have been characterized, but many components remain unidentified. Recently, our laboratory discovered a mutation in the pod-1 gene (for polarity and osmotic defective) that uniquely perturbed polarity and osmotic protection. Here, we describe a new C. elegans polarity gene identified during screens for conditional embryonic lethals. Embryos in which this gene has been mutated show a loss of physical and developmental asymmetries in the one-cell embryo, including the mislocalization of PAR and POD-1 proteins required for early polarity. Furthermore, mutant embryos are osmotically sensitive, allowing us to designate this gene pod-2. Thus, pod-2, along with pod-1, defines a new class of C. elegans polarity genes. Genetic analyses indicate that pod-2 functions in the same pathway as pod-1. Temperature-shift studies indicate that pod-2 is required during oogenesis, indicating that aspects of embryonic polarization may precede fertilization. pod-2 mutant embryos also exhibit a unique germline inheritance defect in which germline identity localizes to the wrong spot in the one-cell embryo and is therefore inherited by the wrong cell at the four-cell stage. Our data suggest that pod-2 may be required to properly position an a-p polarity cue.     

MEX-5 and MEX-6 function to establish soma/germline asymmetry in early C. elegans embryos

An asymmetrical network of cortically localized PAR proteins forms shortly after fertilization of the C. elegans egg. This network is required for subsequent asymmetries in the expression patterns of several proteins that are encoded by nonlocalized, maternally expressed mRNAs. We provide evidence that two nearly identical genes, mex-5 and mex-6, link PAR asymmetry to those subsequent protein asymmetries. MEX-5 is a novel, cytoplasmic protein that is localized through PAR activities to the anterior pole of the 1-cell stage embryo. MEX-5 localization is reciprocal to that of a group of posterior-localized proteins called germline proteins. Ectopic expression of MEX-5 is sufficient to inhibit the expression of germline proteins, suggesting that MEX-5 functions to inhibit anterior expression of the germline proteins.     

CDC-42 controls early cell polarity and spindle orientation in C. elegans.

BACKGROUND: Generation of asymmetry in the one-cell embryo of C. elegans establishes the anterior--posterior axis (A-P), and is necessary for the proper identity of early blastomeres. Conserved PAR proteins are asymmetrically distributed and are required for the generation of this early asymmetry. The small G protein Cdc42 is a key regulator of polarity in other systems, and recently it has been shown to interact with the mammalian homolog of PAR-6. The function of Cdc42 in C. elegans had not yet been investigated, however. RESULTS: Here, we show that C. elegans cdc-42 plays an essential role in the polarity of the one-cell embryo and the proper localization of PAR proteins. Inhibition of cdc-42 using RNA interference results in embryos with a phenotype that is nearly identical to par-3, par-6, and pkc-3 mutants, and asymmetric localization of these and other PAR proteins is lost. We further show that C. elegans CDC-42 physically interacts with PAR-6 in a yeast two-hybrid system, consistent with data on the interaction of human homologs. CONCLUSIONS: Our results show that CDC-42 acts in concert with the PAR proteins to control the polarity of the C. elegans embryo, and provide evidence that the interaction of CDC-42 and the PAR-3/PAR-6/PKC-3 complex has been evolutionarily conserved as a functional unit.     

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.     

Multiple maternal proteins coordinate to restrict the translation of C. elegans nanos-2 to primordial germ cells

Although germ cell formation has been relatively well understood in worms and insects, how germ cell-specific developmental programs are initiated is not clear. In Caenorhabditis elegans, translational activation of maternal nos-2 mRNA is the earliest known molecular event specific to the germline founder cell P(4). Cis-elements in nos-2 3'UTR have been shown to mediate translational control; however, the trans-acting proteins are not known. Here, we provide evidence that four maternal RNA-binding proteins, OMA-1, OMA-2, MEX-3 and SPN-4, bind nos-2 3'UTR to suppress its translation, and POS-1, another maternal RNA-binding protein, relieves this suppression in P(4). The POS-1: SPN-4 ratio in P(4) increases significantly over its precursor, P(3); and POS-1 competes with SPN-4 for binding to nos-2 RNA in vitro. We propose temporal changes in the relative concentrations of POS-1 and SPN-4, through their effect on the translational status of maternal mRNAs such as nos-2, initiate germ cell-specific developmental programs in C. elegans.     

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.     

Breaking the epithelial polarity barrier in cancer: the strange case of LKB1/PAR-4

The PAR clan of polarity regulating genes was initially discovered in a genetic screen searching for genes involved in asymmetric cell divisions in the Caenorhabditis elegans embryo. Today, investigations in worms, flies and mammals have established PAR proteins as conserved and fundamental regulators of animal cell polarization in a broad range of biological phenomena requiring cellular asymmetries. The human homologue of invertebrate PAR-4, a serine–threonine kinase LKB1/STK11, has caught attention as a gene behind Peutz–Jeghers polyposis syndrome and as a bona fide tumour suppressor gene commonly mutated in sporadic cancer. LKB1 functions as a master regulator of AMP-activated protein kinase (AMPK) and 12 other kinases referred to as the AMPK-related kinases, including four human homologues of PAR-1. The role of LKB1 as part of the energy sensing LKB1-AMPK module has been intensively studied, whereas the polarity function of LKB1, in the context of homoeostasis or cancer, has gained less attention. Here, we focus on the PAR-4 identity of LKB1, discussing the weight of evidence indicating a role for LKB1 in regulation of cell polarity and epithelial integrity across species and highlight recent investigations providing new insight into the old question: does the PAR-4 identity of LKB1 matter in cancer?     

PAR proteins direct asymmetry of the cell cycle regulators Polo-like kinase and Cdc25

Cell cycle lengths vary widely among different cells within an animal, yet mechanisms of cell cycle length regulation are poorly understood. In the Caenorhabditis elegans embryo, the first cell division produces two cells with different cell cycle lengths, which are dependent on the conserved partitioning-defective (PAR) polarity proteins. We show that two key cell cycle regulators, the Polo-like kinase PLK-1 and the cyclin-dependent kinase phosphatase CDC-25.1, are asymmetrically distributed in early embryos. PLK-1 shows anterior cytoplasmic enrichment and CDC-25.1 shows PLK-1-dependent enrichment in the anterior nucleus. Both proteins are required for normal mitotic progression. Furthermore, these asymmetries are controlled by PAR proteins and the muscle excess (MEX) proteins MEX-5/MEX-6, and the latter is linked to protein degradation. Our results support a model whereby the PAR and MEX-5/MEX-6 proteins asymmetrically control PLK-1 levels, which asymmetrically regulates CDC-25.1 to promote differences in cell cycle lengths. We suggest that control of Plk1 and Cdc25 may be relevant to regulation of cell cycle length in other developmental contexts.     

The asymmetric division of the Arabidopsis zygote: from cell polarity to an embryo axis

During plant embryogenesis, a simple body plan consisting of shoot and root meristem that are connected by the embryo axis is set up by the first few rounds of cell divisions after fertilization. Postembryonically, the elaborate architecture of plants is created from stem cell populations of both meristems. Here, we address how the main axis (apical-basal) of the plant embryo is established from the single-celled zygote and the role that the asymmetric division of the zygote plays in this process. We will mainly draw on examples from the model plant Arabidopsis, for which several key regulators have been identified during the last years.     

OOC-3, a novel putative transmembrane protein required for establishment of cortical domains and spindle orientation in the P(1) blastomere of C. elegans embryos

Asymmetric cell divisions require the establishment of an axis of polarity, which is subsequently communicated to downstream events. During the asymmetric cell division of the P(1) blastomere in C. elegans, establishment of polarity depends on the establishment of anterior and posterior cortical domains, defined by the localization of the PAR proteins, followed by the orientation of the mitotic spindle along the previously established axis of polarity. To identify genes required for these events, we have screened a collection of maternal-effect lethal mutations on chromosome II of C. elegans. We have identified a mutation in one gene, ooc-3, with mis-oriented division axes at the two-cell stage. Here we describe the phenotypic and molecular characterization of ooc-3. ooc-3 is required for the correct localization of PAR-2 and PAR-3 cortical domains after the first cell division. OOC-3 is a novel putative transmembrane protein, which localizes to a reticular membrane compartment, probably the endoplasmic reticulum, that spans the whole cytoplasm and is enriched on the nuclear envelope and cell-cell boundaries. Our results show that ooc-3 is required to form the cortical domains essential for polarity after cell division.