Is a Persistent Global Bias Necessary for the Establishment of Planar Cell Polarity?

Planar cell polarity (PCP)–the coordinated polarisation of a whole field of cells within the plane of a tissue–relies on the interaction of three modules: a global module that couples individual cellular polarity to the tissue axis, a local module that aligns the axis of polarisation of neighbouring cells, and a readout module that directs the correct outgrowth of PCP-regulated structures such as hairs and bristles. While much is known about the molecular components that are required for PCP, the functional details of–and interactions between–the modules remain unclear. In this work, we perform a mathematical and computational analysis of two previously proposed computational models of the local module (Amonlirdviman et al., Science, 307, 2005; Le Garrec et al., Dev. Dyn., 235, 2006). Both models can reproduce wild-type and mutant phenotypes of PCP observed in the Drosophila wing under the assumption that a tissue-wide polarity cue from the global module persists throughout the development of PCP. We demonstrate that both models can also generate tissue-level PCP when provided with only a transient initial polarity cue. However, in these models such transient cues are not sufficient to ensure robustness of the resulting cellular polarisation.     

Establishment of global patterns of planar polarity during growth of the Drosophila wing epithelium

Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP. However, we recently identified epithelial morphogenesis as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally. Here, we use quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients.     

Mitotic internalization of planar cell polarity proteins preserves tissue polarity

Planar cell polarity (PCP) is the collective polarization of cells along the epithelial plane, a process best understood in the terminally differentiated Drosophila wing. Proliferative tissues such as mammalian skin also show PCP, but the mechanisms that preserve tissue polarity during proliferation are not understood. During mitosis, asymmetrically distributed PCP components risk mislocalization or unequal inheritance, which could have profound consequences for the long-range propagation of polarity. Here, we show that when mouse epidermal basal progenitors divide PCP components are selectively internalized into endosomes, which are inherited equally by daughter cells. Following mitosis, PCP proteins are recycled to the cell surface, where asymmetry is re-established by a process reliant on neighbouring PCP. A cytoplasmic dileucine motif governs mitotic internalization of atypical cadherin Celsr1, which recruits Vang2 and Fzd6 to endosomes. Moreover, embryos transgenic for a Celsr1 that cannot mitotically internalize exhibit perturbed hair-follicle angling, a hallmark of defective PCP. This underscores the physiological relevance and importance of this mechanism for regulating polarity during cell division.     

Intracellular trafficking of planar cell polarity proteins

Background

Planar cell polarity (PCP) is a phenomenon in which epithelial cells are polarized along the plane of a tissue. PCP is critical for a variety of developmental processes and is regulated by a set of evolutionarily conserved PCP signaling proteins. Many of the PCP proteins adopt characteristic asymmetric localizations on the opposing cellular boundaries. Currently, the molecular mechanisms that establish and maintain this PCP asymmetry remain largely unclear. Newly synthesized integral PCP proteins are transported along the secretory transport pathway to the plasma membranes. Once delivered to the plasma membranes, PCP proteins undergo endocytosis. Recent studies reveal insights into the intracellular trafficking of PCP proteins, suggesting that intracellular trafficking of PCP proteins contributes to establishing the PCP asymmetry.

Objective

To understand the intracellular trafficking of planar cell polarity proteins in the secretory transport pathway and endocytic transport pathway.

Methods

This review summarizes our current understanding of the intracellular trafficking of PCP proteins. We highlights the molecular mechanisms that regulate sorting of PCP proteins into transport vesicles and how the intracellular trafficking process regulates the asymmetric localizations of PCP proteins.

Results

Current studies reveal novel insights into the molecular mechanisms mediating intracellular trafficking of PCP proteins. This process is critical for delivering newly synthesized PCP proteins to their specific destinations, removing the unstable or mislocalized PCP proteins from the plasma membranes and preserving tissue polarity during proliferation of mammalian skin cells.

Conclusion

Understanding how PCP proteins are delivered in the secretory and endocytic transport pathway will provide mechanistic insights into how the asymmetric localizations of PCP proteins are established and maintained.

     

Role of cell polarity and planar cell polarity (PCP) proteins in spermatogenesis

Abstract

Studies on cell polarity proteins and planar cell polarity (PCP) proteins date back to almost 40?years ago in Drosophila and C. elegans when these proteins were shown to be crucial to support apico-basal polarity and also directional alignment of polarity cells across the plane of an epithelium during morphogenesis. In adult mammals, cell polarity and PCP are most notable in cochlear hair cells. However, the role of these two groups of proteins to support spermatogenesis was not explored until a decade earlier when several proteins that confer cell polarity and PCP proteins were identified in the rat testis. Since then, there are several reports appearing in the literature to examine the role of both cell polarity and PCP in supporting spermatogenesis. Herein, we provide an overview regarding the role of cell polarity and PCP proteins in the testis, evaluating these findings in light of studies in other mammalian epithelial cells/tissues. Our goal is to provide a timely evaluation of these findings, and provide some thought provoking remarks to guide future studies based on an evolving concept in the field.     

The planar cell polarity pathway directs parietal endoderm migration

Parietal endoderm (PE) contributes to the yolk sac and is the first migratory cell type in the mammalian embryo. We can visualize PE migration in vitro using the F9 teratocarcinoma derived embryoid body outgrowth system and, show here that PE migration is directed by the non-canonical Wnt planar cell polarity (PCP) pathway via Rho/ROCK. Based on golgi apparatus localization and microtubule orientation, 68.6% of cells in control outgrowths are oriented in the direction of migration. Perturbation of Wnt signaling via sFRP treatment results in a loss of orientation coupled with an increase in cell migration. Inhibition of the PCP pathway at the level of Daam1 also results in a loss of cell orientation along with an increase in cell migration, as seen with sFRP treatment. Constitutively active Daam can inhibit the loss of orientation that occurs with sFRP treatment. We previously demonstrated that ROCK inhibition leads to an increase in cell migration, and we now show that these cells also lack oriented migration. Canonical Wnt signaling or the Rac arm of the PCP pathway does not appear to play a role in PE oriented migration. These data suggest the PCP pathway via Rho/ROCK modulates migration of PE.     

Planar polarity and tissue morphogenesis

Planar polarity is a global, tissue-level phenomenon that coordinates cell behavior in a two-dimensional plane. The Frizzled/planar cell polarity (PCP) and anterior-posterior (AP) patterning systems for planar polarity operate in a variety of cell types and provide direction to cells with different morphologies and behaviors. These two systems involve different sets of proteins but both use directional cues provided locally by communication between neighboring cells. This review describes our current understanding of the mechanisms that transmit directional signals from cell to cell and compares the strategies for generating global systems of spatial information in stationary and dynamic cell populations.     

Kermit Interacts with Gαo,Vang, and Motor Proteins in Drosophila Planar Cell Polarity

In addition to the ubiquitous apical-basal polarity, epithelial cells are often polarized within the plane of the tissue – the phenomenon known as planar cell polarity (PCP). In Drosophila, manifestations of PCP are visible in the eye, wing, and cuticle. Several components of the PCP signaling have been characterized in flies and vertebrates, including the heterotrimeric Go protein. However, Go signaling partners in PCP remain largely unknown. Using a genetic screen we uncover Kermit, previously implicated in G protein and PCP signaling, as a novel binding partner of Go. Through pull-down and genetic interaction studies, we find that Kermit interacts with Go and another PCP component Vang, known to undergo intracellular relocalization during PCP establishment. We further demonstrate that the activity of Kermit in PCP differentially relies on the motor proteins: the microtubule-based dynein and kinesin motors and the actin-based myosin VI. Our results place Kermit as a potential transducer of Go, linking Vang with motor proteins for its delivery to dedicated cellular compartments during PCP establishment.     

Multiscale analysis of pattern formation via intercellular signalling

Lateral inhibition, a juxtacrine signalling mechanism by which a cell adopting a particular fate inhibits neighbouring cells from doing likewise, has been shown to be a robust mechanism for the formation of fine-grained spatial patterns (in which adjacent cells in developing tissues diverge to achieve contrasting states of differentiation), provided that there is sufficiently strong feedback. The fine-grained nature of these patterns poses problems for analysis via traditional continuum methods since these require that significant variation takes place only over lengthscales much larger than an individual cell and such systems have therefore been investigated primarily using discrete methods. Here, however, we apply a multiscale method to derive systematically a continuum model from the discrete Delta-Notch signalling model of Collier et al. (J.R. Collier, N.A.M. Monk, P.K. Maini, J.H. Lewis, Pattern formation by lateral inhibition with feedback: a mathematical model of Delta-Notch intercellular signalling, J. Theor. Biol., 183, 1996, 429-446) under particular assumptions on the parameters, which we use to analyse the generation of fine-grained patterns. We show that, on the macroscale, the contact-dependent juxtacrine signalling interaction manifests itself as linear diffusion, motivating the use of reaction-diffusion-based models for such cell-signalling systems. We also analyse the travelling-wave behaviour of our system, obtaining good quantitative agreement with the discrete system.     

The wnt receptor ryk plays a role in Mammalian planar cell polarity signaling

Wnts are essential for a wide range of developmental processes, including cell growth, division, and differentiation. Some of these processes signal via the planar cell polarity (PCP) pathway, which is a β-catenin-independent Wnt signaling pathway. Previous studies have shown that Ryk, a member of the receptor tyrosine kinase family, can bind to Wnts. Ryk is required for normal axon guidance and neuronal differentiation during development. Here, we demonstrate that mammalian Ryk interacts with the Wnt/PCP pathway. In vitro analysis showed that the Wnt inhibitory factor domain of Ryk was necessary for Wnt binding. Detailed analysis of two vertebrate model organisms showed Ryk phenotypes consistent with PCP signaling. In zebrafish, gene knockdown using morpholinos revealed a genetic interaction between Ryk and Wnt11 during the PCP pathway-regulated process of embryo convergent extension. Ryk-deficient mouse embryos displayed disrupted polarity of stereociliary hair cells in the cochlea, a characteristic of disturbed PCP signaling. This PCP defect was also observed in mouse embryos that were double heterozygotes for Ryk and Looptail (containing a mutation in the core Wnt/PCP pathway gene Vangl2) but not in either of the single heterozygotes, suggesting a genetic interaction between Ryk and Vangl2. Co-immunoprecipitation studies demonstrated that RYK and VANGL2 proteins form a complex, whereas RYK also activated RhoA, a downstream effector of PCP signaling. Overall, our data suggest an important role for Ryk in Wnt/planar cell polarity signaling during vertebrate development via the Vangl2 signaling pathway, as demonstrated in the mouse cochlea.     

Mathematical Analysis of Spontaneous Emergence of Cell Polarity

Cell polarization, in which intracellular substances are asymmetrically distributed, enables cells to carry out specialized functions. While cell polarity is often induced by intracellular or extracellular spatial cues, spontaneous polarization (the so-called symmetry breaking) may also occur in the absence of spatial cues. Many computational models have been used to investigate the mechanisms of symmetry breaking, and it was proved that spontaneous polarization occurs when the lateral diffusion of inactive signaling molecules is much faster than that of active signaling molecules. This conclusion leaves an important question of how, as observed in many biological systems, cell polarity emerges when active and inactive membrane-bound molecules diffuse at similar rates while cycling between cytoplasm and membrane takes place. The recent studies of Rätz and Röger showed that, when the cytosolic and membrane diffusion are very different, spontaneous polarization is possible even if the membrane-bound species diffuse at the same rate. In this paper, we formulate a two-equation non-local reaction-diffusion model with general forms of positive feedback. We apply Turing stability analysis to identify parameter conditions for achieving cell polarization. Our results show that spontaneous polarization can be achieved within some parameter ranges even when active and inactive signaling molecules diffuse at similar rates. In addition, different forms of positive feedback are explored to show that a non-local molecule-mediated feedback is important for sharping the localization as well as giving rise to fast dynamics to achieve robust polarization.     

Planar cell polarity in the larval epidermis of Drosophila and the role of microtubules

We investigate planar cell polarity (PCP) in the Drosophila larval epidermis. The intricate pattern of denticles depends on only one system of PCP, the Dachsous/Fat system. Dachsous molecules in one cell bind to Fat molecules in a neighbour cell to make intercellular bridges. The disposition and orientation of these Dachsous–Fat bridges allows each cell to compare two neighbours and point its denticles towards the neighbour with the most Dachsous. Measurements of the amount of Dachsous reveal a peak at the back of the anterior compartment of each segment. Localization of Dachs and orientation of ectopic denticles help reveal the polarity of every cell. We discuss whether these findings support our gradient model of Dachsous activity. Several groups have proposed that Dachsous and Fat fix the direction of PCP via oriented microtubules that transport PCP proteins to one side of the cell. We test this proposition in the larval cells and find that most microtubules grow perpendicularly to the axis of PCP. We find no meaningful bias in the polarity of microtubules aligned close to that axis. We also reexamine published data from the pupal abdomen and find no evidence supporting the hypothesis that microtubular orientation draws the arrow of PCP.     

Oligomerization of peripheral membrane proteins provides tunable control of cell surface polarity

Asymmetric distributions of peripheral membrane proteins define cell polarity across all kingdoms of life. Non-linear positive feedback on membrane binding is essential to amplify and stabilize these asymmetries, but how specific molecular sources of non-linearity shape polarization dynamics remains poorly understood. Here we show that the ability to oligomerize, which is common to many peripheral membrane proteins, can play a profound role in shaping polarization dynamics in simple feedback circuits. We show that size-dependent binding avidity and mobility of membrane-bound oligomers endow polarity circuits with several key properties. Size-dependent membrane binding avidity confers a form of positive feedback on the accumulation of oligomer subunits. Although insufficient by itself, this sharply reduces the amount of additional feedback required for spontaneous emergence and stable maintenance of polarized states. Size-dependent oligomer mobility makes symmetry breaking and stable polarity more robust with respect to variation in subunit diffusivities and cell sizes, and slows the approach to a final stable spatial distribution, allowing cells to “remember” polarity boundaries imposed by transient external cues. Together, these findings reveal how oligomerization of peripheral membrane proteins can provide powerful and highly tunable sources of non-linear feedback in biochemical circuits that govern cell surface polarity. Given its prevalence and widespread involvement in cell polarity, we speculate that self-oligomerization may have provided an accessible path to evolving simple polarity circuits.     

Clustering and Negative Feedback by Endocytosis in Planar Cell Polarity Signaling Is Modulated by Ubiquitinylation of Prickle

The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. We find that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. We show that Pk participates in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. Our results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.     

Transmembrane topology of mammalian planar cell polarity protein Vangl1

Vangl1 and Vangl2 are membrane proteins that play an important role in neurogenesis, and Vangl1/Vangl2 mutations cause neural tube defects in mice and humans. At the cellular level, Vangl proteins regulate the establishment of planar cell polarity (PCP), a process requiring membrane assembly of asymmetrically distributed multiprotein complexes that transmit polarity information to neighboring cells. The membrane topology of Vangl proteins and the protein segments required for structural and functional aspects of multiprotein membrane PCP complexes is unknown. We have used epitope tagging and immunofluorescence to establish the secondary structure of Vangl proteins, including the number, position, and polarity of transmembrane domains. Antigenic hemagglutinin A (HA) peptides (YYDVPDYS) were inserted in predicted intra- or extracellular loops of Vangl1 at positions 18, 64, 139, 178, 213, and 314, and individual mutant variants were stably expressed at the membrane of MDCK polarized cells. The membrane topology of the exofacial HA tag was determined by a combination of immunofluorescence in intact (extracellular epitopes) and permeabilized (intracellular epitopes) cells and by surface labeling. Results indicate that Vangl proteins have a four-transmembrane domain structure with the N-terminal portion (HA18 and HA64) and the large C-terminal portion (HA314) of the protein being intracellular. Topology mapping and hydropathy profiling show that the loop delineated by TMD1-2 (HA139) and TMD3-4 (HA213) is extracellular while the segment separating predicted TMD2-3 (HA178) is intracellular. This secondary structure reveals a compact membrane-associated portion with very short predicted extra- and intracellular loops, while the protein harbors a large intracellular domain.     

Diego interacts with Prickle and Strabismus/Van Gogh to localize planar cell polarity complexes

Planar cell polarity (PCP) in the Drosophila eye is established by the distinct fate specifications of photoreceptors R3 and R4, and is regulated by the Frizzled (Fz)/PCP signaling pathway. Before the PCP proteins become asymmetrically localized to opposite poles of the cell in response to Fz/PCP signaling, they are uniformly apically colocalized. Little is known about how the apical localization is maintained. We provide evidence that the PCP protein Diego (Dgo) promotes the maintenance of apical localization of Flamingo (Fmi), an atypical Cadherin-family member, which itself is required for the apical localization of the other PCP factors. This function of Dgo is redundant with Prickle (Pk) and Strabismus (Stbm), and only appreciable in double mutant tissue. We show that the initial membrane association of Dgo depends on Fz, and that Dgo physically interacts with Stbm and Pk through its Ankyrin repeats, providing evidence for a PCP multiprotein complex. These interactions suggest a positive feedback loop initiated by Fz that results in the apical maintenance of other PCP factors through Fmi.     

Drosophila ATP6AP2/VhaPRR functions both as a novel planar cell polarity core protein and a regulator of endosomal trafficking

Planar cell polarity (PCP) controls the orientation of cells within tissues and the polarized outgrowth of cellular appendages. So far, six PCP core proteins including the transmembrane proteins Frizzled (Fz), Strabismus (Stbm) and Flamingo (Fmi) have been identified. These proteins form asymmetric PCP domains at apical junctions of epithelial cells. Here, we demonstrate that VhaPRR, an accessory subunit of the proton pump V‐ATPase, directly interacts with the protocadherin Fmi through its extracellular domain. It also shows a striking co‐localization with PCP proteins during all pupal wing stages in Drosophila. This localization depends on intact PCP domains. Reversely, VhaPRR is required for stable PCP domains, identifying it as a novel PCP core protein. VhaPRR performs an additional role in vesicular acidification as well as endolysosomal sorting and degradation. Membrane proteins, such as E‐Cadherin and the Notch receptor, accumulate at the surface and in intracellular vesicles of cells mutant for VhaPRR. This trafficking defect is shared by other V‐ATPase subunits. By contrast, the V‐ATPase does not seem to have a direct role in PCP regulation. Together, our results suggest two roles for VhaPRR, one for PCP and another in endosomal trafficking. This dual function establishes VhaPRR as a key factor in epithelial morphogenesis.     

Plant cell polarity: sterols enter into action after cytokinesis

In yeast and animal cells, the sterol composition of membranes is a key factor that controls the polarity of membrane proteins by regulating their intracellular trafficking or lateral diffusion. A recent study in Nature Cell Biology demonstrates that plant sterols play a major role in the acquisition of cell polarity by modulating endocytosis after cell division.     

Seven-Pass Transmembrane Cadherins: Roles and Emerging Mechanisms in Axonal and Dendritic Patterning

The Flamingo/Celsr seven-transmembrane cadherins represent a conserved subgroup of the cadherin superfamily involved in multiple aspects of development. In the developing nervous system, Fmi/Celsr control axonal blueprint and dendritic morphogenesis from invertebrates to mammals. As expected from their molecular structure, seven-transmembrane cadherins can induce cell–cell homophilic interactions but also intracellular signaling. Fmi/Celsr is known to regulate planar cell polarity (PCP) through interactions with PCP proteins. In the nervous system, Fmi/Celsr can function in collaboration with or independently of other PCP genes. Here, we focus on recent studies which show that seven-transmembrane cadherins use distinct molecular mechanisms to achieve diverse functions in the development of the nervous system.     

Crosstalk of cell polarity signaling pathways

Cell polarity, the asymmetric organization of cellular components along one or multiple axes, is present in most cells. From budding yeast cell polarization induced by pheromone signaling, oocyte polarization at fertilization to polarized epithelia and neuronal cells in multicellular organisms, similar mechanisms are used to determine cell polarity. Crucial role in this process is played by signaling lipid molecules, small Rho family GTPases and Par proteins. All these signaling circuits finally govern the cytoskeleton, which is responsible for oriented cell migration, cell shape changes, and polarized membrane and organelle trafficking. Thus, typically in the process of cell polarization, most cellular constituents become polarized, including plasma membrane lipid composition, ion concentrations, membrane receptors, and proteins in general, mRNA, vesicle trafficking, or intracellular organelles. This review gives a brief overview how these systems talk to each other both during initial symmetry breaking and within the signaling feedback loop mechanisms used to preserve the polarized state.