Molecular symmetry breaking in the Frizzled-dependent planar polarity pathway.

The core planar polarity pathway consists of six proteins that form asymmetric intercellular complexes that segregate to opposite cell ends in developing tissues and specify polarized cell structures or behaviors. Within these complexes, the atypical cadherin Flamingo localizes on both sides of intercellular junctions, where it interacts homophilically in trans via its cadherin repeats, whereas the transmembrane proteins Frizzled and Strabismus localize to the opposite sides of apposing junctions. However, the molecular mechanisms underlying the formation of such asymmetric complexes are poorly understood. Using a novel tissue culture system, we determine the minimum requirements for asymmetric complex assembly in the absence of confounding feedback mechanisms. We show that complexes are intrinsically asymmetric and that an interaction of Frizzled and Flamingo in one cell with Flamingo in the neighboring cell is the key symmetry-breaking step. In contrast, Strabismus is unable to promote homophilic Flamingo trans binding and is only recruited into complexes once Frizzled has entered on the opposite side. This interaction with Strabismus requires intact intracellular loops of the seven-pass transmembrane domain of Flamingo. Once recruited, Strabismus stabilizes the intercellular complexes together with the three cytoplasmic core proteins. We propose a model whereby Flamingo exists in a closed conformation and binding of Frizzled in one cell results in a conformational change that allows its cadherin repeats to interact with a Flamingo molecule in the neighboring cell. Flamingo in the adjacent cell then undergoes a further change in the seven-pass transmembrane region that promotes the recruitment of Strabismus.

Distinct mechanisms of planar polarization by the core and Fat-Dachsous planar polarity pathways in the Drosophila wing.

Planar polarity describes the coordinated polarization of cells within a tissue plane, and in animals can be determined by the "core" or Fat-Dachsous pathways. Current models for planar polarity establishment involve two components: tissue-level "global" cues that determine the overall axis of polarity and cell-level feedback-mediated cellular polarity amplification. Here, we investigate the contributions of global cues versus cellular feedback amplification in the core and Fat-Dachsous pathways during Drosophila pupal wing development. We present evidence that these pathways generate planar polarity via distinct mechanisms. Core pathway function is consistent with strong feedback capable of self-organizing cell polarity, which can then be aligned with the tissue axis via weak or transient global cues. Conversely, generation of cell polarity by the Ft-Ds pathway depends on strong global cues in the form of graded patterns of gene expression, which can then be amplified by weak feedback mechanisms.

Selective function of the PDZ domain of Dishevelled in noncanonical Wnt signalling.

Dishevelled is a cytoplasmic hub that transduces Wnt signals to cytoplasmic effectors, which can be broadly characterised as canonical (ß-catenin dependent) and noncanonical, to specify cell fates and behaviours during development. To transduce canonical Wnt signals, Dishevelled binds to the intracellular face of Frizzled through its DEP domain and polymerises through its DIX domain to assemble dynamic signalosomes. Dishevelled also contains a PDZ domain, whose function remains controversial. Here, we use genome editing to delete the PDZ domain-encoding region from Drosophila dishevelled. Canonical Wingless signalling is entirely normal in these deletion mutants; however, they show defects in multiple contexts controlled by noncanonical Wnt signalling, such as planar polarity. We use nuclear magnetic resonance spectroscopy to identify bona fide PDZ-binding motifs at the C termini of different polarity proteins. Although deletions of these motifs proved aphenotypic in adults, we detected changes in the proximodistal distribution of the polarity protein Flamingo (also known as Starry night) in pupal wings that suggest a modulatory role of these motifs in polarity signalling. We also provide new genetic evidence that planar polarity relies on the DEP-dependent recruitment of Dishevelled to the plasma membrane by Frizzled.

Use of Fluorescence Recovery After Photobleaching (FRAP) to Measure In Vivo Dynamics of Cell Junction-Associated Polarity Proteins.

Here, we present a detailed protocol for fluorescence recovery after photobleaching (FRAP) to measure the dynamics of junctional populations of proteins in living tissue. Specifically, we describe how to perform FRAP in Drosophila pupal wings on fluorescently tagged core planar polarity proteins, which exhibit relatively slow junctional turnover. We provide a step-by-step practical guide to performing FRAP, and list a series of controls and optimizations to do before conducting a FRAP experiment. Finally, we describe how to present the FRAP data for publication.

How do the Fat-Dachsous and core planar polarity pathways act together and independently to coordinate polarized cell behaviours?

Planar polarity describes the coordinated polarization of cells within the plane of a tissue. This is controlled by two main pathways in Drosophila: the Frizzled-dependent core planar polarity pathway and the Fat-Dachsous pathway. Components of both of these pathways become asymmetrically localized within cells in response to long-range upstream cues, and form intercellular complexes that link polarity between neighbouring cells. This review examines if and when the two pathways are coupled, focusing on the Drosophila wing, eye and abdomen. There is strong evidence that the pathways are molecularly coupled in tissues that express a specific isoform of the core protein Prickle, namely Spiny-legs. However, in other contexts, the linkages between the pathways are indirect. We discuss how the two pathways act together and independently to mediate a diverse range of effects on polarization of cell structures and behaviours.

Molecular mechanisms mediating asymmetric subcellular localisation of the core planar polarity pathway proteins.

Planar polarity refers to cellular polarity in an orthogonal plane to apicobasal polarity, and is seen across scales from molecular distributions of proteins to tissue patterning. In many contexts it is regulated by the evolutionarily conserved 'core' planar polarity pathway that is essential for normal organismal development. Core planar polarity pathway components form asymmetric intercellular complexes that communicate polarity between neighbouring cells and direct polarised cell behaviours and the formation of polarised structures. The core planar polarity pathway consists of six structurally different proteins. In the fruitfly Drosophila melanogaster, where the pathway is best characterised, an intercellular homodimer of the seven-pass transmembrane protein Flamingo interacts on one side of the cell junction with the seven-pass transmembrane protein Frizzled, and on the other side with the four-pass transmembrane protein Strabismus. The cytoplasmic proteins Diego and Dishevelled are co-localised with Frizzled, and Prickle co-localises with Strabismus. Between these six components there are myriad possible molecular interactions, which could stabilise or destabilise the intercellular complexes and lead to their sorting into polarised distributions within cells. Post-translational modifications are key regulators of molecular interactions between proteins. Several post-translational modifications of core proteins have been reported to be of functional significance, in particular phosphorylation and ubiquitination. In this review, we discuss the molecular control of planar polarity and the molecular ecology of the core planar polarity intercellular complexes. Furthermore, we highlight the importance of understanding the spatial control of post-translational modifications in the establishment of planar polarity.

DAnkrd49 and Bdbt act via Casein kinase Iε to regulate planar polarity in Drosophila.

The core planar polarity proteins are essential mediators of tissue morphogenesis, controlling both the polarised production of cellular structures and polarised tissue movements. During development the core proteins promote planar polarisation by becoming asymmetrically localised to opposite cell edges within epithelial tissues, forming intercellular protein complexes that coordinate polarity between adjacent cells. Here we describe a novel protein complex that regulates the asymmetric localisation of the core proteins in the Drosophila pupal wing. DAnkrd49 (an ankyrin repeat protein) and Bride of Doubletime (Bdbt, a non-canonical FK506 binding protein family member) physically interact, and regulate each other's levels in vivo. Loss of either protein results in a reduction in core protein asymmetry and disruption of the placement of trichomes at the distal edge of pupal wing cells. Post-translational modifications are thought to be important for the regulation of core protein behaviour and their sorting to opposite cell edges. Consistent with this, we find that loss of DAnkrd49 or Bdbt leads to reduced phosphorylation of the core protein Dishevelled and to decreased Dishevelled levels both at cell junctions and in the cytoplasm. Bdbt has previously been shown to regulate activity of the kinase Discs Overgrown (Dco, also known as Doubletime or Casein Kinase Iε), and Dco itself has been implicated in regulating planar polarity by phosphorylating Dsh as well as the core protein Strabismus. We demonstrate that DAnkrd49 and Bdbt act as dominant suppressors of Dco activity. These findings support a model whereby Bdbt and DAnkrd49 act together to modulate the activity of Dco during planar polarity establishment.

Planar Cell Polarity Effector Proteins Inturned and Fuzzy Form a Rab23 GEF Complex.

A subset of Rab GTPases have been implicated in cilium formation in cultured mammalian cells [1-6]. Rab11 and Rab8, together with their GDP-GTP exchange factors (GEFs), TRAPP-II and Rabin8, promote recruitment of the ciliary vesicle to the mother centriole and its subsequent maturation, docking, and fusion with the cell surface [2-5]. Rab23 has been linked to cilium formation and membrane trafficking at mature cilia [1, 7, 8]; however, the identity of the GEF pathway activating Rab23, a member of the Rab7 subfamily of Rabs, remains unclear. Longin-domain-containing complexes have been shown to act as GEFs for Rab7 subfamily GTPases [9-12]. Here, we show that Inturned and Fuzzy, proteins previously implicated as planar cell polarity (PCP) effectors and in developmentally regulated cilium formation [13, 14], contain multiple longin domains characteristic of the Mon1-Ccz1 family of Rab7 GEFs and form a specific Rab23 GEF complex. In flies, loss of Rab23 function gave rise to defects in planar-polarized trichome formation consistent with this biochemical relationship. In cultured human and mouse cells, Inturned and Fuzzy localized to the basal body and proximal region of cilia, and cilium formation was compromised by depletion of either Inturned or Fuzzy. Cilium formation arrested after docking of the ciliary vesicle to the mother centriole but prior to axoneme elongation and fusion of the ciliary vesicle and plasma membrane. These findings extend the family of longin domain GEFs and define a molecular activity linking Rab23-regulated membrane traffic to cilia and planar cell polarity.

Reciprocal action of Casein Kinase Iε on core planar polarity proteins regulates clustering and asymmetric localisation.

The conserved core planar polarity pathway is essential for coordinating polarised cell behaviours and the formation of polarised structures such as cilia and hairs. Core planar polarity proteins localise asymmetrically to opposite cell ends and form intercellular complexes that link the polarity of neighbouring cells. This asymmetric segregation is regulated by phosphorylation through poorly understood mechanisms. We show that loss of phosphorylation of the core protein Strabismus in the Drosophila pupal wing increases its stability and promotes its clustering at intercellular junctions, and that Prickle negatively regulates Strabismus phosphorylation. Additionally, loss of phosphorylation of Dishevelled - which normally localises to opposite cell edges to Strabismus - reduces its stability at junctions. Moreover, both phosphorylation events are independently mediated by Casein Kinase Iε. We conclude that Casein Kinase Iε phosphorylation acts as a switch, promoting Strabismus mobility and Dishevelled immobility, thus enhancing sorting of these proteins to opposite cell edges.

Retromer Controls Planar Polarity Protein Levels and Asymmetric Localization at Intercellular Junctions.

The coordinated polarization of cells in the plane of a tissue, termed planar polarity, is a characteristic feature of epithelial tissues [1]. In the fly wing, trichome positioning is dependent on the core planar polarity proteins adopting asymmetric subcellular localizations at apical junctions, where they form intercellular complexes that link neighboring cells [1-3]. Specifically, the seven-pass transmembrane protein Frizzled and the cytoplasmic proteins Dishevelled and Diego localize to distal cell ends, the four-pass transmembrane protein Strabismus and the cytoplasmic protein Prickle localize proximally, and the seven-pass transmembrane spanning atypical cadherin Flamingo localizes both proximally and distally. To establish asymmetry, these core proteins are sorted from an initially uniform distribution; however, the mechanisms underlying this polarized trafficking remain poorly understood. Here, we describe the identification of retromer, a master controller of endosomal recycling [4-6], as a key component regulating core planar polarity protein localization in Drosophila. Through generation of mutants, we verify that loss of the retromer-associated Snx27 cargo adaptor, but notably not components of the Wash complex, reduces junctional levels of the core proteins Flamingo and Strabismus in the developing wing. We establish that Snx27 directly associates with Flamingo via its C-terminal PDZ binding motif, and we show that Snx27 is essential for normal Flamingo trafficking. We conclude that Wash-independent retromer function and the Snx27 cargo adaptor are important components in the endosomal recycling of Flamingo and Strabismus back to the plasma membrane and thus contribute to the establishment and maintenance of planar polarization.

A Dual Function for Prickle in Regulating Frizzled Stability during Feedback-Dependent Amplification of Planar Polarity.

The core planar polarity pathway coordinates epithelial cell polarity during animal development, and loss of its activity gives rise to a range of defects, from aberrant morphogenetic cell movements to failure to correctly orient structures, such as hairs and cilia. The core pathway functions via a mechanism involving segregation of its protein components to opposite cells ends, where they form asymmetric intracellular complexes that couple cell-cell polarity. This segregation is a self-organizing process driven by feedback interactions between the core proteins themselves. Despite intense efforts, the molecular pathways underlying feedback have proven difficult to elucidate using conventional genetic approaches. Here we investigate core protein function during planar polarization of the Drosophila wing by combining quantitative measurements of protein dynamics with loss-of-function genetics, mosaic analysis, and temporal control of gene expression. Focusing on the key core protein Frizzled, we show that its stable junctional localization is promoted by the core proteins Strabismus, Dishevelled, Prickle, and Diego. In particular, we show that the stabilizing function of Prickle on Frizzled requires Prickle activity in neighboring cells. Conversely, Prickle in the same cell has a destabilizing effect on Frizzled. This destabilizing activity is dependent on the presence of Dishevelled and blocked in the absence of Dynamin and Rab5 activity, suggesting an endocytic mechanism. Overall, our approach reveals for the first time essential in vivo stabilizing and destabilizing interactions of the core proteins required for self-organization of planar polarity.

Robust Asymmetric Localization of Planar Polarity Proteins Is Associated with Organization into Signalosome-like Domains of Variable Stoichiometry.

In developing epithelia, the core planar polarity proteins physically interact with each other and localize asymmetrically at opposite cell ends, forming intercellular complexes that link the polarity of neighboring cells. Using quantitative imaging to examine the composition of the core protein complex in vivo, we find that complex composition is unexpectedly plastic. The transmembrane proteins Frizzled and Flamingo form a stoichiometric nucleus in the complex, while the relative levels of the other four core proteins can vary independently. Exploring the functional consequences of this, we show that robust cell polarization is achieved over a range of complex stoichiometries but is dependent on maintaining appropriate levels of the components Frizzled and Strabismus. We propose that the core proteins assemble into signalosome-like structures, where stable association is not dependent on one-to-one interactions with binding partners, and signaling functions can act over a wide range of complex compositions.

Planar polarity: forcing cells into line.

Polarisation of tissues in the plane of an epithelium is fundamental for both animal morphogenesis and organ function. A new paper describes a role for mechanical cues in determining how such polarity is aligned with the body axes.

Strabismus promotes recruitment and degradation of farnesylated prickle in Drosophila melanogaster planar polarity specification.

The core planar polarity proteins are required to specify the orientation of structures that are polarised in the plane of the epithelium. In the Drosophila melanogaster wing, the core proteins localise asymmetrically at either proximal or distal cell edges. Asymmetric localisation is thought to be biased by long-range cues, causing asymmetric complexes to become aligned with the tissue axes. Core proteins are then thought to participate in feedback interactions that are necessary to amplify asymmetry, and in order for such feedback interactions to operate correctly, the levels of the core proteins at junctions must be tightly regulated. We have investigated regulation of the core protein Prickle (Pk) in the pupal wing. The core protein Strabismus (Stbm) is required to recruit Pk into asymmetric complexes at proximal cell ends, and we report here that it also promotes proteasomal degradation of excess Pk, probably via a Cullin-1 dependent process. We also show for the first time that Pk is farnesylated in vivo, and this is essential for Pk function in the wing. Notably, farnesylation of Pk is necessary for it to be recruited into asymmetric complexes and function in feedback amplification, probably by reinforcing weak direct interactions between Stbm and Pk. Furthermore, farnesylation is also required for Stbm to promote proteasomal degradation of Pk. We propose that Stbm recruits farnesylated Pk into asymmetric complexes, but also promotes degradation of excess Pk that would otherwise perturb feedback amplification.

A Cul-3-BTB ubiquitylation pathway regulates junctional levels and asymmetry of core planar polarity proteins.

The asymmetric localisation of core planar polarity proteins at apicolateral junctions is required to specify cell polarity in the plane of epithelia. This asymmetric distribution of the core proteins is proposed to require amplification of an initial asymmetry by feedback loops. In addition, generation of asymmetry appears to require the regulation of core protein levels, but the importance of such regulation and the underlying mechanisms is unknown. Here we show that ubiquitylation acts through more than one mechanism to control core protein levels in Drosophila, and that without this regulation cellular asymmetry is compromised. Levels of Dishevelled at junctions are regulated by a Cullin-3-Diablo/Kelch ubiquitin ligase complex, the activity of which is most likely controlled by neddylation. Furthermore, activity of the deubiquitylating enzyme Fat facets is required to maintain Flamingo levels at junctions. Notably, ubiquitylation does not alter the total cellular levels of Dishevelled or Flamingo, but only that of the junctional population. When junctional core protein levels are either increased or decreased by disruption of the ubiquitylation machinery, their asymmetric localisation is reduced and this leads to disruption of planar polarity at the tissue level. Loss of asymmetry by altered core protein levels can be explained by reference to feedback models for amplification of asymmetry.

The Frizzled-dependent planar polarity pathway locally promotes E-cadherin turnover via recruitment of RhoGEF2.

Polarised tissue elongation during morphogenesis involves cells within epithelial sheets or tubes making and breaking intercellular contacts in an oriented manner. Growing evidence suggests that cell adhesion can be modulated by endocytic trafficking of E-cadherin (E-cad), but how this process can be polarised within individual cells is poorly understood. The Frizzled (Fz)-dependent core planar polarity pathway is a major regulator of polarised cell rearrangements in processes such as gastrulation, and has also been implicated in regulation of cell adhesion through trafficking of E-cad; however, it is not known how these functions are integrated. We report a novel role for the core planar polarity pathway in promoting cell intercalation during tracheal tube morphogenesis in Drosophila embryogenesis, and present evidence that this is due to regulation of turnover and levels of junctional E-cad by the guanine exchange factor RhoGEF2. Furthermore, we show that core pathway activity leads to planar-polarised recruitment of RhoGEF2 and E-cad turnover in the epidermis of both the embryonic germband and the pupal wing. We thus reveal a general mechanism by which the core planar polarity pathway can promote polarised cell rearrangements.

Dynamics of core planar polarity protein turnover and stable assembly into discrete membrane subdomains.

The core planar polarity proteins localize asymmetrically to the adherens junctions of epithelial cells, where they have been hypothesized to assemble into intercellular complexes. Here, we show that the core proteins are preferentially distributed to discrete membrane subdomains ("puncta"), where they form asymmetric contacts between neighboring cells. Using an antibody internalization assay and fluorescence recovery after photobleaching in prepupal and pupal wings, we have investigated the turnover of two key core proteins, Flamingo and Frizzled, and find that the localization of both within puncta is highly stable. Furthermore, the transmembrane core proteins, Flamingo, Frizzled, and Strabismus, are necessary for stable localization of core proteins to junctions, whereas the cytoplasmic core proteins are required for their concentration into puncta. Thus, we define the distinct roles of specific core proteins in the formation of asymmetric contacts between cells, which is a key event in the generation of coordinated cellular asymmetry.

Asymmetric localisation of planar polarity proteins: Mechanisms and consequences.

Planar polarisation of tissues is essential for many aspects of developmental patterning. It is regulated by a conserved group of core planar polarity proteins, which localise asymmetrically within cells prior to morphological signs of polarisation. A subset of these core proteins also interact across cell boundaries, mediating intercellular communication that co-ordinates polarity between neighbouring cells. Core protein localisation subsequently mediates changes in the actin cytoskeleton which lead to overt polarisation. In this review we discuss the mechanisms by which the core planar polarity proteins become asymmetrically localised, and the significance of this subcellular localisation for both intercellular communication and downstream effects on the cytoskeleton.

Differential stability of flamingo protein complexes underlies the establishment of planar polarity.

BACKGROUND: The planar polarization of developing tissues is controlled by a conserved set of core planar polarity proteins. In the Drosophila pupal wing, these proteins adopt distinct proximal and distal localizations in apicolateral junctions that act as subcellular polarity cues to control morphological events. The core polarity protein Flamingo (Fmi) localizes to both proximal and distal cell boundaries and is known to have asymmetric activity, but the molecular basis of this asymmetric activity is unknown. RESULTS: We examine the role of Fmi in controlling asymmetric localization of polarity proteins in pupal wing cells. We find that Fmi interacts preferentially with distal-complex components, rather than with proximal components, and present evidence that there are different domain requirements for Fmi to associate with distal and proximal components. We further show that distally and proximally localized proteins cooperate to allow stable accumulation of Fmi at apicolateral junctions and present evidence that the rates of endocytic trafficking of Fmi are increased when Fmi is not in a stable asymmetric complex. Finally, we provide evidence that Fmi is trafficked from junctions via both Dishevelled-dependent and Dishevelled-independent mechanisms. CONCLUSIONS: We present a model in which the primary function of Fmi is to participate in the formation of inherently stable asymmetric junctional complexes: Removal from junctions of Fmi that is not in stable complexes, combined with directional trafficking of Frizzled and Fmi to the distal cell edge, drives the establishment of cellular asymmetry.

Differential activities of the core planar polarity proteins during Drosophila wing patterning.

During planar polarity patterning of the Drosophila wing, a "core" group of planar polarity genes has been identified which acts downstream of global polarity cues to locally coordinate cell polarity and specify trichome production at distal cell edges. These genes encode protein products that assemble into asymmetric apicolateral complexes that straddle the proximodistal junctional region between adjacent cells. We have carried out detailed genetic analysis experiments, analysing the requirements of each complex component for planar polarity patterning. We find that the three transmembrane proteins at the core of the complex, Frizzled, Strabismus and Flamingo, are required earliest in development and are the only components needed for intercellular polarity signalling. Notably, cells that lack both Frizzled and Strabismus are unable to signal, revealing an absolute requirement for both proteins in cell-cell communication. In contrast the cytoplasmic components Dishevelled, Prickle and Diego are not needed for intercellular communication. These factors contribute to the cell-cell propagation of polarity, most likely by promotion of intracellular asymmetry. Interestingly, both local polarity propagation and trichome placement occur normally in mutant backgrounds where asymmetry of polarity protein distribution is undetectable, suggesting such asymmetry is not an absolute requirement for any of the functions of the core complex.