Fluid flow dynamics in cellular patterning.

The development of complex forms of multicellular organisms depends on the spatial arrangement of cellular architecture and functions. The interior design of the cell is patterned by spatially biased distributions of molecules and biochemical reactions in the cytoplasm and/or on the plasma membrane. In recent years, a dynamic change in the cytoplasmic fluid flow has emerged as a key physical process of driving long-range transport of molecules to particular destinations within the cell. Here, recent experimental advances in the understanding of the generation of the various types of cytoplasmic flows and contributions to intracellular patterning are reviewed with a particular focus on feedback mechanisms between the mechanical properties of fluid flow and biochemical signaling during animal cell polarization.

Establishment of the PAR-1 cortical gradient by the aPKC-PRBH circuit.

Cell polarity is the asymmetric compartmentalization of cellular components. An opposing gradient of partitioning-defective protein kinases, atypical protein kinase C (aPKC) and PAR-1, at the cell cortex guides diverse asymmetries in the structure of metazoan cells, but the mechanism underlying their spatial patterning remains poorly understood. Here, we show in Caenorhabditis elegans zygotes that the cortical PAR-1 gradient is patterned as a consequence of dual mechanisms: stabilization of cortical dynamics and protection from aPKC-mediated cortical exclusion. Dual control of cortical PAR-1 depends on a physical interaction with the PRBH-domain protein PAR-2. Using a reconstitution approach in heterologous cells, we demonstrate that PAR-1, PAR-2, and polarized Cdc42-PAR-6-aPKC comprise the minimal network sufficient for the establishment of an opposing cortical gradient. Our findings delineate the mechanism governing cortical polarity, in which a circuit consisting of aPKC and the PRBH-domain protein ensures the local recruitment of PAR-1 to a well-defined cortical compartment.

Forceful patterning in mouse preimplantation embryos.

The generation of a functional organism from a single, fertilized ovum requires the spatially coordinated regulation of diverse cell identities. The establishment and precise arrangement of differentiated cells in developing embryos has, historically, been extensively studied by geneticists and developmental biologists. While chemical gradients and genetic regulatory networks are widely acknowledged to play significant roles in embryo patterning, recent studies have highlighted that mechanical forces generated by, and exerted on, embryos are also crucial for the proper control of cell differentiation and morphogenesis. Here we review the most recent findings in murine preimplantation embryogenesis on the roles of cortical tension in the coupling of cell-fate determination and cell positioning in 8-16-cell-stage embryos. These basic principles of mechanochemical coupling in mouse embryos can be applied to other pattern formation phenomena that rely on localized modifications of cell polarity proteins and actin cytoskeletal components and activities.

ImaEdge - a platform for quantitative analysis of the spatiotemporal dynamics of cortical proteins during cell polarization.

Cell polarity involves the compartmentalization of the cell cortex. The establishment of cortical compartments arises from the spatial bias in the activity and concentration of cortical proteins. The mechanistic dissection of cell polarity requires the accurate detection of dynamic changes in cortical proteins, but the fluctuations of cell shape and the inhomogeneous distributions of cortical proteins greatly complicate the quantitative extraction of their global and local changes during cell polarization. To address these problems, we introduce an open-source software package, ImaEdge, which automates the segmentation of the cortex from time-lapse movies, and enables quantitative extraction of cortical protein intensities. We demonstrate that ImaEdge enables efficient and rigorous analysis of the dynamic evolution of cortical PAR proteins during Caenorhabditis elegans embryogenesis. It is also capable of accurate tracking of varying levels of transgene expression and discontinuous signals of the actomyosin cytoskeleton during multiple rounds of cell division. ImaEdge provides a unique resource for quantitative studies of cortical polarization, with the potential for application to many types of polarized cells.This article has an associated First Person interview with the first authors of the paper.

A ZO-2 scaffolding mechanism regulates the Hippo signalling pathway.

Contact inhibition of proliferation is a critical cell density control mechanism governed by the Hippo signalling pathway. The biochemical signalling underlying cell density-dependent cues regulating Hippo signalling and its downstream effectors, YAP, remains poorly understood. Here, we reveal that the tight junction protein ZO-2 is required for the contact-mediated inhibition of proliferation. We additionally determined that the well-established molecular players of this process, namely Hippo kinase LATS1 and YAP, are regulated by ZO-2 and that the scaffolding function of ZO-2 promotes the interaction with and phosphorylation of YAP by LATS1. Mechanistically, YAP is phosphorylated when ZO-2 brings LATS1 and YAP together via its SH3 and PDZ domains, respectively, subsequently leading to the cytoplasmic retention and inactivation of YAP. In conclusion, we demonstrate that ZO-2 maintains Hippo signalling pathway activation by promoting the stability of LATS1 to inactivate YAP.

Symmetry breaking and polarization of the C. elegans zygote by the polarity protein PAR-2.

Polarization of the C. elegans zygote is initiated by ECT-2-dependent cortical flows, which mobilize the anterior PAR proteins (PAR-3, PAR-6 and PKC-3) away from the future posterior end of the embryo marked by the sperm centrosome. Here, we demonstrate the existence of a second, parallel and redundant pathway that can polarize the zygote in the absence of ECT-2-dependent cortical flows. This second pathway depends on the polarity protein PAR-2. We show that PAR-2 localizes to the cortex nearest the sperm centrosome even in the absence of cortical flows. Once on the cortex, PAR-2 antagonizes PAR-3-dependent recruitment of myosin, creating myosin flows that transport the anterior PAR complex away from PAR-2 in a positive-feedback loop. We propose that polarity in the C. elegans zygote is initiated by redundant ECT-2- and PAR-2-dependent mechanisms that lower PAR-3 levels locally, triggering a positive-feedback loop that polarizes the entire cortex.

Sequential functioning of the ECT-2 RhoGEF, RHO-1 and CDC-42 establishes cell polarity in Caenorhabditis elegans embryos.

During development, the establishment of cell polarity is important for cells to undergo asymmetric cell divisions that give rise to diverse cell types. In C. elegans embryos, cues from the centrosome trigger the cortical flow of an actomyosin network, leading to the formation of anterior-posterior polarity. However, its precise mechanism is poorly understood. Here, we show that small GTPases have sequential and crucial functions in this process. ECT-2, a potential guanine nucleotide-exchange factor (GEF) for RHO-1, was uniformly distributed at the cortex before polarization, but was excluded from the posterior cortex by the polarity cue from the centrosomes. This local exclusion of ECT-2 led to an asymmetric RHO-1 distribution, which generated a cortical flow of the actomyosin that translocated PAR proteins and CDC-42 (Refs 4, 5) to the anterior cortex. Polarized CDC-42 was, in turn, involved in maintaining the established anterior-cortical domains. Our results suggest that a local change in the function of ECT-2 and RHO-1 links the centrosomal polarity cue with the polarization of the cell cortex.

Cell polarity: Adapting the PAR cascade to diverse cellular contexts.

Two new studies shed light on the intricacies of Caenorhabditis elegans embryo patterning, revealing how the conserved interaction and crosstalk of PAR proteins are adapted to perceive distinct cues, ultimately shaping unique asymmetries in form and function.

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.

Two phases of astral microtubule activity during cytokinesis in C. elegans embryos.

Microtubules of the mitotic spindle are believed to provide positional cues for the assembly of the actin-based contractile ring and the formation of the subsequent cleavage furrow during cytokinesis. In Caenorhabditis elegans, astral microtubules have been thought to inhibit cortical contraction outside the cleavage furrow. Here, we demonstrate by live imaging and RNA interference (RNAi) that astral microtubules play two distinct roles in initiating cleavage furrow formation. In early anaphase, microtubules are required for contractile ring assembly; in late anaphase, microtubules show different cortical behavior and seem to suppress cortical contraction at the poles, as suggested in previous studies. These two distinct phases of microtubule behavior depend on distinct regulatory pathways, one involving the gamma-tubulin complex and the other requiring aurora-A kinase. We propose that temporal and spatial regulation of two distinct phases of astral microtubule behavior is crucial in specifying the position and timing of furrowing.

Caenorhabditis elegans ortholog of the p24/p22 subunit, DNC-3, is essential for the formation of the dynactin complex by bridging DNC-1/p150(Glued) and DNC-2/dynamitin.

Dynactin is a multisubunit protein complex required for the activity of cytoplasmic dynein. In Caenorhabditis elegans, although 10 of the 11 dynactin subunits were identified based on the sequence similarities to their orthologs, the p24/p22 subunit has not been detected in the genome. Here, we demonstrate that DNC-3 (W10G11.20) is the functional counterpart of the p24/p22 subunit in C. elegans. RNAi phenotypes and subcellular localization of DNC-3 in early C. elegans embryos were nearly identical to those of the known dynactin components. All other dynactin subunits were co-immunoprecipitated with DNC-3, indicating that DNC-3 is a core component of dynactin. Furthermore, the overall secondary structure of DNC-3 resembles to those of the mammalian and yeast p24/p22. We found that DNC-3 is required for the localization of the DNC-1/p150(Glued) and DNC-2/dynamitin, the two components of the projection arm of dynactin, to the nuclear envelope of meiotic nuclei in the adult gonad. Moreover, DNC-3 physically interacted with DNC-1 and DNC-2 and significantly enhanced the binding ability between DNC-1 and DNC-2 in vitro. These results suggest that DNC-3 is essential for the formation of the projection arm subcomplex of dynactin.

A balance between antagonizing PAR proteins specifies the pattern of asymmetric and symmetric divisions in C. elegans embryogenesis.

Coordination between cell differentiation and proliferation during development requires the balance between asymmetric and symmetric modes of cell division. However, the cellular intrinsic cue underlying the choice between these two division modes remains elusive. Here, we show evidence in Caenorhabditis elegans that the invariable lineage of the division modes is specified by the balance between antagonizing complexes of partitioning-defective (PAR) proteins. By uncoupling unequal inheritance of PAR proteins from that of fate determinants during cell division, we demonstrate that changes in the balance between PAR-2 and PAR-6 can be sufficient to re-program the division modes from symmetric to asymmetric and vice versa in two daughter cells. The division mode adopted occurs independently of asymmetry in cytoplasmic fate determinants, cell-size asymmetry, and cell-cycle asynchrony between sister cells. We propose that the balance between PAR proteins represents an intrinsic self-organizing cue for the specification of the two division modes during development.

Mechanochemical Control of Symmetry Breaking in the Caenorhabditis elegans Zygote.

Cell polarity is the asymmetric organization of cellular components along defined axes. A key requirement for polarization is the ability of the cell to break symmetry and achieve a spatially biased organization. Despite different triggering cues in various systems, symmetry breaking (SB) usually relies on mechanochemical modulation of the actin cytoskeleton, which allows for advected movement and reorganization of cellular components. Here, the mechanisms underlying SB in Caenorhabditis elegans zygote, one of the most popular models to study cell polarity, are reviewed. A zygote initiates SB through the centrosome, which modulates mechanics of the cell cortex to establish advective flow of cortical proteins including the actin cytoskeleton and partitioning defective (PAR) proteins. The chemical signaling underlying centrosomal control of the Aurora A kinase-mediated cascade to convert the organization of the contractile actomyosin network from an apolar to polar state is also discussed.

Novel approaches to link apicobasal polarity to cell fate specification.

Understanding the development of apicobasal polarity (ABP) is a long-standing problem in biology. The molecular components involved in the development and maintenance of APB have been largely identified and are known to have ubiquitous roles across organisms. Our knowledge of the functional consequences of ABP establishment and maintenance is far less comprehensive. Recent studies using novel experimental approaches and cellular models have revealed a growing link between ABP and the genetic program of cell lineage. This mini-review describes some of the most recent advances in this new field, highlighting examples from Caenorhabditis elegans and mouse embryos, human pluripotent stem cells, and epithelial cells. We also speculate on the most interesting and challenging avenues that can be explored.

Myosin-II reorganization during mitosis is controlled temporally by its dephosphorylation and spatially by Mid1 in fission yeast.

Cytokinesis in many eukaryotes requires an actomyosin contractile ring. Here, we show that in fission yeast the myosin-II heavy chain Myo2 initially accumulates at the division site via its COOH-terminal 134 amino acids independently of F-actin. The COOH-terminal region can access to the division site at early G2, whereas intact Myo2 does so at early mitosis. Ser1444 in the Myo2 COOH-terminal region is a phosphorylation site that is dephosphorylated during early mitosis. Myo2 S1444A prematurely accumulates at the future division site and promotes formation of an F-actin ring even during interphase. The accumulation of Myo2 requires the anillin homologue Mid1 that functions in proper ring placement. Myo2 interacts with Mid1 in cell lysates, and this interaction is inhibited by an S1444D mutation in Myo2. Our results suggest that dephosphorylation of Myo2 liberates the COOH-terminal region from an intramolecular inhibition. Subsequently, dephosphorylated Myo2 is anchored by Mid1 at the medial cortex and promotes the ring assembly in cooperation with F-actin.

Aurora-A Breaks Symmetry in Contractile Actomyosin Networks Independently of Its Role in Centrosome Maturation.

Cell polarity is facilitated by a rearrangement of the actin cytoskeleton at the cell cortex. The program triggering the asymmetric remodeling of contractile actomyosin networks remains poorly understood. Here, we show that polarization of Caenorhabditis elegans zygotes is established through sequential downregulation of cortical actomyosin networks by the mitotic kinase, Aurora-A. Aurora-A accumulates around centrosomes to locally disrupt the actomyosin contractile activity at the proximal cortex, thereby promoting cortical flows during symmetry breaking. Aurora-A later mediates global disassembly of cortical actomyosin networks, which facilitates the initial polarization through suppression of centrosome-independent cortical flows. Translocation of Aurora-A from the cytoplasm to the cortex is sufficient to interfere with the cortical actomyosin networks independently of its roles in centrosome maturation and cell-cycle progression. We propose that Aurora-A activity serves as a centrosome-mediated cue that breaks symmetry in actomyosin contractile activity, and facilitates the initial polarization through global suppression of cortical actomyosin networks.

TPXL-1 activates Aurora A to clear contractile ring components from the polar cortex during cytokinesis.

During cytokinesis, a signal from the central spindle that forms between the separating anaphase chromosomes promotes the accumulation of contractile ring components at the cell equator, while a signal from the centrosomal microtubule asters inhibits accumulation of contractile ring components at the cell poles. However, the molecular identity of the inhibitory signal has remained unknown. To identify molecular components of the aster-based inhibitory signal, we developed a means to monitor the removal of contractile ring proteins from the polar cortex after anaphase onset. Using this assay, we show that polar clearing is an active process that requires activation of Aurora A kinase by TPXL-1. TPXL-1 concentrates on astral microtubules coincident with polar clearing in anaphase, and its ability to recruit Aurora A and activate its kinase activity are essential for clearing. In summary, our data identify Aurora A kinase as an aster-based inhibitory signal that restricts contractile ring components to the cell equator during cytokinesis.

Importance of a myosin II-containing progenitor for actomyosin ring assembly in fission yeast.

An actomyosin-based contractile ring provides the forces necessary for cell cleavage in several organisms [1-3]. Myosin II is an essential component of the actomyosin ring and has also been detected as a "spot" in interphase Schizosaccharomyces pombe cells [4-5]. It is currently unknown if this myosin II-containing spot is important for cytokinesis. In this study, we characterize this myosin II-containing spot using a combination of genetic and cell biological analyses. Whereas myosin II at the actomyosin ring undergoes rapid turnover, myosin II at the spot does not. Maintenance of the myosin II-containing spot is independent of F-actin function. Interestingly, maintenance of this myosin II spot in interphase requires the function of Rng3p, a UCS domain-containing protein, the Caenorhabditis elegans homolog of which has recently been shown to be a cochaperone for myosin II assembly [6]. Disassembly of the spot in interphase prevents actomyosin ring formation in the subsequent mitosis, implying that the spot might represent a progenitor that is important for assembly of the actomyosin ring. Given that mitosis represents a short period of the fission yeast cell cycle, organization of this progenitor structure in interphase might ensure proper assembly of the actomyosin ring and successful cell division.

Cortical forces and CDC-42 control clustering of PAR proteins for Caenorhabditis elegans embryonic polarization.

Cell polarization enables zygotes to acquire spatial asymmetry, which in turn patterns cellular and tissue axes during development. Local modification in the actomyosin cytoskeleton mediates spatial segregation of partitioning-defective (PAR) proteins at the cortex, but how mechanical changes in the cytoskeleton are transmitted to PAR proteins remains elusive. Here we uncover a role of actomyosin contractility in the remodelling of PAR proteins through cortical clustering. During embryonic polarization in Caenorhabditis elegans, actomyosin contractility and the resultant cortical tension stimulate clustering of PAR-3 at the cortex. Clustering of atypical protein kinase C (aPKC) is supported by PAR-3 clusters and is antagonized by activation of CDC-42. Cortical clustering is associated with retardation of PAR protein exchange at the cortex and with effective entrainment of advective cortical flows. Our findings delineate how cytoskeleton contractility couples the cortical clustering and long-range displacement of PAR proteins during polarization. The principles described here would apply to other pattern formation processes that rely on local modification of cortical actomyosin and PAR proteins.

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.