The Interactive Fly

Zygotically transcribed genes

Planar cell polarity/Tissue polarity genes

  • Signaling pathways involved in planar cell polarity
  • Jun mediates Frizzled-induced planar polarity determination in the Drosophila eye
  • Dissecting the molecular bridges that mediate the function of Frizzled in planar cell polarity
  • The proteins encoded by the Drosophila planar polarity effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of 'upstream' planar cell polarity proteins
  • The atypical cadherin Dachsous controls left-right asymmetry in Drosophila
  • Planar cell polarity in the larval epidermis of Drosophila and the role of microtubules
  • Microtubules provide directional information for core PCP function
  • Symmetry breaking in an edgeless epithelium by Fat2-regulated microtubule polarity
  • The clathrin adaptor AP-1 complex and Arf1 regulate planar cell polarity in vivo
  • Positioning of centrioles is a conserved readout of Frizzled planar cell polarity signalling
  • The PCP pathway regulates Baz planar distribution in epithelial cells
  • SEGGA: a toolset for rapid automated analysis of epithelial cell polarity and dynamics
  • Planar polarized Rab35 functions as an oscillatory ratchet during cell intercalation in the Drosophila epithelium
  • Meru couples planar cell polarity with apical-basal polarity during asymmetric cell division
  • The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dach
  • Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension
  • Vamana couples fat signaling to the Hippo pathway
  • Tissue tension and not interphase cell shape determines cell division orientation in the Drosophila follicular epithelium
  • Polarized microtubule dynamics directs cell mechanics and coordinates forces during epithelial morphogenesis
  • Epithelial cells release adenosine to promote local TNF production in response to polarity disruption
  • The Dachsous/Fat/Four-Jointed pathway directs the uniform axial orientation of epithelial cells in the Drosophila abdomen
  • Tissue flow regulates planar cell polarity independently of the Frizzled core pathway
  • Molecular mechanisms that regulate export of the planar cell-polarity protein Frizzled-6 out of the endoplasmic reticulum
  • A Genetic Screen Links the Disease-Associated Nab2 RNA-Binding Protein to the Planar Cell Polarity Pathway in Drosophila melanogaster
  • No Evidence that Wnt Ligands Are Required for Planar Cell Polarity in Drosophila
  • Frizzled-Dependent Planar Cell Polarity without Secreted Wnt Ligands
  • Girdin is a component of the lateral polarity protein network restricting cell dissemination
  • DAnkrd49 and Bdbt act via Casein kinase Iepsilon to regulate planar polarity
  • Hypoxia controls plasma membrane targeting of polarity proteins by dynamic turnover of PI4P and PI(4,5)P2
  • Cortical Cyclin A controls spindle orientation during asymmetric cell divisions in Drosophila
  • Parafibromin governs cell polarity and centrosome assembly in Drosophila neural stem cells
  • PTP10D-mediated cell competition is not obligately required for elimination of polarity-deficient clones
  • The Nab2 RNA-binding protein patterns dendritic and axonal projections through a planar cell polarity-sensitive mechanism
  • Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila

  • Prickle/spiny-legs isoforms control the polarity of the apical microtubule network in planar cell polarity
  • Clustering and negative feedback by endocytosis in planar cell polarity signaling is modulated by ubiquitinylation of Prickle
  • Rapid disruption of Dishevelled activity uncovers an intercellular role in maintenance of Prickle in core planar polarity protein complexes
  • Prickle isoforms determine handedness of helical morphogenesis
  • A Van Gogh/Vangl tyrosine phosphorylation switch regulates its interaction with core Planar Cell Polarity factors Prickle and Dishevelled

  • Genes involved in tissue polarity

    Signaling pathways involved in planar cell polarity

    Frizzled (Fz) signaling regulates the establishment of planar cell polarity (PCP). The PCP genes prickle and strabismus are thought to antagonize Fz signaling. They act in the same cell, R4, adjacent to that in which the Fz/PCP pathway is required in the Drosophila eye. Stbm and Pk interact physically; Stbm recruits Pk to the cell membrane. Through this interaction, Pk affects Stbm membrane localization and can cause clustering of Stbm. Pk is also known to interact with Dsh and is thought to antagonize Dsh by affecting its membrane localization. Thus the data suggest that the Stbm/Pk complex modulates Fz/Dsh activity, resulting in a symmetry-breaking step during polarity signaling (Jenny, 2003).

    pk function is required in the R4 precursor, as opposed to fz PCP signaling in R3, for control of polarity establishment. Stbm, a transmembrane protein also required in R4, interacts genetically and physically with Pk. This interaction is important for the recruitment of Pk to the plasma membrane. In Xenopus animal-cap explants, Stbm and Pk relocalize each other to subdomains of the membrane. A model is proposed of how Pk/Stbm might regulate Fz/Dsh signaling activity (Jenny, 2003).

    The in vitro molecular interaction between Pk and Stbm and their mutual relocalization in Xenopus animal caps suggest that they form multiprotein complexes. Several pieces of evidence indicate that the physical interaction is physiologically important: (1) correct membrane localization of Pk depends on stbm function because in stbm mutant tissue Pk staining is diffuse and absent (or strongly reduced) at the membrane; (2) Pk and Stbm interact genetically by mutually enhancing each other's GOF and LOF phenotypes in the eye; (3) pk is necessary for PCP signaling in the R4 precursor, the same cell in which stbm is required; (4) expression of the interacting domains of Pk or Stbm interferes with polarity establishment. In particular, a subfragment of 131 amino acids of the C-terminus of Pk, required for the molecular interaction, is sufficient to affect polarity (Jenny, 2003).

    Both Pk and Stbm act as if they antagonize Fz signaling. (1) In zebrafish, Stbm overexpression can prevent Wnt11 from rescuing a wnt11 mutation. (2) In the Drosophila wing, overexpression of Pk leads to wing hairs pointing towards the source of the overexpressed protein, behaving like a fz LOF clone (whereas overexpression of Fz leads to hairs pointing away from the Fz source). stbm LOF clones show the opposite behavior to fz LOF clones: wing hairs point away from the mutant patch, consistent with the mutant tissue having a higher Fz-activity (Jenny, 2003).

    In the Drosophila eye, evidence that pk acts antagonistically to fz comes from the fact that the Notch-signaling-responsive R4-specific reporter mdelta0.5-lacZ is expressed for a prolonged period in both R3/R4 precursors in a pksple1 mutant. This is explained if Fz activity in the R4 precursor is increased, resulting in higher levels of Dl there. This in turn leads to N activation and concomitant mdelta0.5-lacZ reporter expression in both cells of the R3/R4 pair. Conversely, in fz and dsh mutant eye discs (where Fz signaling is absent or reduced and thus Dl should not be upregulated) N-signaling activity and mdelta-lacZ expression is initially reduced in both cells. Fz activity is also antagonized by stbm in the eye. Mosaic analysis of stbm shows that it has the capability to instruct a cell to become R4 as long as the other cell of the R3/R4 pair is mutant for stbm. Therefore, in such an all-or-nothing situation, Stbm in the R3 precursor can override a positive signal of Fz, resulting in a cell fate switch to R4 fate (Jenny, 2003).

    In a wild-type situation with all PCP components present in both cells, it is crucial that Stbm activity is higher in R4 than in R3 to ensure proper Fz-signaling regulation. Therefore it is an intriguing possibility that a Pk/Stbm complex in the R4 precursor ensures such higher Stbm activity, and the associated higher Fz repression there is important for a proper R3/R4 cell fate decision (Jenny, 2003).

    How does the Stbm/Pk complex regulate Fz-signaling activity? During PCP establishment in the wing, Fz, Dsh, Dgo, Fmi and Pk are initially localized uniformly around the apical circumference of wing cells. During and after PCP signaling, these proteins relocalize and become differentially enriched: Pk concentrates on the proximal side of the cell, whereas Fz and Dsh become enriched distally. Fmi becomes enriched at both sides (Jenny, 2003).

    In the eye, the situation is analogous. During PCP establishment, signaling components at the R3/R4 cell border are relocalized from a uniform to a more restricted pattern. Stbm-YFP is localized on the R4 but not on the R3 side, and Fz-GFP ends up on the R3 but not the R4 side. The analogy between the R4/R3 and proximal/distal cell borders is corroborated by the genetic requirements in R3 and R4: the distally localized factors Dsh and Fz are required in R3, while proximally localized Pk is required in R4. Fmi is localized on both poles of each wing cell and also required in both cells of the R3 and R4 pair. The function of fmi has been linked to both proposed complexes, the 'Fz/Dsh side' (fmi is required for apical localization of both Fz and Dsh) and the Stbm/Pk complex. In addition to the genetic interactions between fmi and stbm or pk, a reduced membrane staining of Pk in fmi- clones in wing cells has been shown (Jenny, 2003).

    How do these changes in localization occur? Localization studies in Drosophila and Xenopus suggest that Pk and Stbm influence each other's localization and form clusters in subdomains of the cell membrane. Interestingly, such Stbm/Pk complexes also affect Fz-dependent Dsh membrane localization. Thus it is an intriguing possibility that the patches observed in animal cap cells upon coinjection of Stbm with Pk represent the result of a similar, though unpolarized, symmetry-breaking step during PCP signaling (Jenny, 2003).

    The PET/LIM domain of Pk can interact with the DEP-domain/C-terminus of Dsh. This interaction has been suggested to prevent Dsh membrane recruitment. Also, the C-terminus of Stbm can interact with Dsh as long as the PDZ domain is present. Since the data suggest that Pk regulates the activity and localization of Stbm, this regulation might promote or stabilize the interactions of Dsh with Stbm and/or Pk, thereby helping to pull Dsh away from a Fz-signaling complex. The Stbm/Pk complexes could then cause active release of Dsh from the membrane or target it for degradation, resulting in low levels of Dsh (and by inference Fz) at places where Pk and Stbm are enriched. Furthermore, in the R3 cell (or distally in the wing) an unknown factor might act to prevent either the formation of the Stbm/Pk complex or its effect on Dsh (Jenny, 2003).

    In conclusion, Pk and Stbm form a functional complex during PCP signaling in Drosophila and during convergent extension in Xenopus. Interestingly, in zebrafish, in addition to its function in convergent extension, Stbm is also required for the caudal migration of hindbrain motor neurons. This function of Stbm is independent of Dsh and the PCP genes tested so far. It will be interesting to determine whether Stbm and Pk function together in this context as well (Jenny, 2003).

    Jun mediates Frizzled-induced planar polarity determination in the Drosophila eye

    Jun acts as a signal-regulated transcription factor in many cellular decisions, ranging from stress response to proliferation control and cell fate induction. Genetic interaction studies have suggested that Jun and JNK signaling are involved in Frizzled (Fz)-mediated planar polarity generation in the Drosophila eye. However, simple loss-of-function analysis of JNK signaling components does not show comparable planar polarity defects. To address the role of Jun and JNK in Fz signaling, a combination of loss- and gain-of-function studies has been used. Like Fz, Jun affects the bias between the R3/R4 photoreceptor pair that is critical for ommatidial polarity establishment. Detailed analysis of jun- clones reveals defects in R3 induction and planar polarity determination, whereas gain of Jun function induces the R3 fate and associated polarity phenotypes. Affecting the levels of JNK signaling by either reduction or overexpression leads to planar polarity defects. Similarly, hypomorphic allelic combinations and overexpression of the negative JNK regulator Puckered causes planar polarity eye phenotypes, establishing that JNK acts in planar polarity signaling. The observation that Delta transcription in the early R3/R4 precursor cells is deregulated by Jun or Hep/JNKK activation, reminiscent of the effects seen with Fz overexpression, suggests that Jun is one of the transcription factors that mediates the effects of fz in planar polarity generation (Weber, 2000).

    Jun, as a member of the AP-1 family, is activated by many distinct extracellular stimuli and acts downstream of several signaling pathways. Besides its involvement in stress response, Jun has been implicated in the control of proliferation, apoptosis, morphogenesis and cell fate induction. In Drosophila, Jun is critical for the process of dorsal closure in embryogenesis acting downstream of the JNK module. It has also been implicated in cell fate induction downstream of Ras/ERK signaling in the eye. This analysis has shown that Jun also acts downstream of Fz in planar polarity signaling in the eye. It is the first transcription factor implicated in Fz/planar polarity signaling. Fz signaling also requires a JNK (or related kinase) module, and thus in the eye imaginal disc Jun acts downstream of both ERK and JNK. How does Jun achieve a specific response in this context? The S/T residues that are phosphorylated in Jun are the same for both ERK and JNK. Thus, although differences in phosphorylation level and/or preference for any of the serine/threonine target residues cannot be excluded in vivo, differential phosphorylation is unlikely to create specificity. A potential mechanism for specificity might be provided by other transcription factors that cooperate with Jun in the different processes. This is supported by the observation that the sev-JunAsp (expression of a constitutively active Jun) phenotype is a composite of two events, photoreceptor recruitment and ommatidial polarity generation. These two effects can, however, be separated by the reduction of specific interacting partners. In the process of Ras/ERK signaling in photoreceptor induction Jun interacts and synergizes with the ETS domain transcription factor Pointed (Pnt). Pnt has been characterized as a target of the ERK/Rl kinase in Drosophila in all ERK-dependent processes analyzed. However, it has not been linked to any JNK-mediated process. Removing one dose of pnt strongly suppresses the Ras/ERK-related extra photoreceptor phenotype of sev-JunAsp, whereas the polarity defects persist and thus are more prominent. This observation indicates that, in the absence of normal Pnt levels, sev-JunAsp specifically affects polarity, suggesting that the interaction with Pnt is important for its role in the ERK-mediated induction. It is likely that for its planar polarity function other specific transcription factors provide the specificity cues (Weber, 2000).

    Although all components of the JNK module tested genetically interact with sev-Fz and sev-Dsh, analysis of existing loss-of-function mutants did not show defects in planar polarity establishment, suggesting a redundant role. Even null alleles of the Drosophila homolog of JNKK hep have no effects on planar polarity (Weber, 2000).

    However, expression of a dominant negative (kinase dead) isoform of Bsk interferes with planar polarity, giving rise to typical polarity phenotypes, implying that Bsk and JNK signaling are important in this process. Consistently, homozygous mutant clones of the deficiency Df(2R)flp170B that removes bsk and other neighboring loci (a deficiency considered to be a true null for bsk), show a mild polarity phenotype in the eye, including the presence of symmetrical ommatidia (Weber, 2000).

    What are the redundant kinases in this process? Genetic interaction analysis with sev-Msn (Misshapen expressed in a Sevenless pattern) has shown that, besides hep and bsk, deficiencies affecting other MKKs and the Drosophila p38a and p38b loci suppress the sev-Msn phenotype. This suggested that the p38 kinase module [related to JNK and has been shown to have (at least partially) overlapping phosphorylation targets] might be responsible for the redundancy in this process. The analysis with the dominant negative (DN) Bsk isoform and the respective deficiencies suggests that the p38 kinase(s) are contributing to this redundancy, because they enhance the DN-Bsk phenotype in a manner very similar to that of the bsk deficiency. The identification of specific mutant alleles of p38a/b and double mutant analysis with bsk will be necessary to further clarify this issue (Weber, 2000).

    The available results indicate that the level of JNK/p38 signaling in planar polarity establishment is important, but that the removal of a single kinase does not significantly affect this level. In support, the observation that an allelic combination of hep and puc hypomorphic alleles can give rise to planar polarity eye phenotypes suggests that the balance between negative and positive regulators of JNK and related kinases is critical. Similarly, overexpression of the negative JNK regulator Puc, a dual specificity phosphatase, causes typical polarity defects similar to those of fz or dsh mutants. It is likely that this phosphatase negatively regulates all JNK-related kinases and thus reduces the overall signaling more than the lack of a single kinase (Weber, 2000).

    In summary, these data indicate that the transcriptional events downstream of Fz in R3 specification and chirality establishment (e.g. regulation of Dl) are mediated by Jun. The factors with which Jun is redundant in the imaginal discs are not yet identified. It is possible that other members of the AP-1 family are also involved in planar polarity signaling, since they are related to Jun and could dimerize with it via the leucine-zipper motif. A potential candidate is Fos, because like Jun, Fos is required downstream of JNK in the process of dorsal closure in the embryo. Similarly, the ETS domain protein Yan acts as a negative regulator in dorsal closure and is inactivated by JNK in the process. However, these factors do not show informative planar polarity phenotypes in clones and thus their involvement in this process remains unclear. Although AP-1 and ETS family members are attractive candidates, transcription factors belonging to other families cannot be excluded in this context (Weber, 2000).

    Dissecting the molecular bridges that mediate the function of Frizzled in planar cell polarity

    Many epithelia have a common planar cell polarity (PCP), as exemplified by the consistent orientation of hairs on mammalian skin and insect cuticle. One conserved system of PCP depends on Starry night (Stan, also called Flamingo), an atypical cadherin that forms homodimeric bridges between adjacent cells. Stan acts together with other transmembrane proteins, most notably Frizzled (Fz) and Van Gogh (Vang, also called Strabismus). In this study, using an in vivo assay for function, it was shown that the quintessential core of the Stan system is an asymmetric intercellular bridge between Stan in one cell and Stan acting together with Fz in its neighbour: such bridges are necessary and sufficient to polarise hairs in both cells, even in the absence of Vang. By contrast, Vang cannot polarise cells in the absence of Fz; instead, it appears to help Stan in each cell form effective bridges with Stan plus Fz in its neighbours. Finally, it was shown that cells containing Stan but lacking both Fz and Vang can be polarised to make hairs that point away from abutting cells that express Fz. It is deduced that each cell has a mechanism to estimate and compare the numbers of asymmetric bridges, made between Stan and Stan plus Fz, that link it with its neighbouring cells. It is proposed that cells normally use this mechanism to read the local slope of tissue-wide gradients of Fz activity, so that all cells come to point in the same direction (Struhl, 2012).

    In Drosophila and other animals, including vertebrates, there appear to be at least two conserved genetic systems responsible for planar cell polarity (PCP); this study is concerned with the Stan system. In Drosophila, epithelial cells become polarised by a multicellular gradient of Fz activity. To read this gradient, the Stan system builds intercellular bridges of Stan-Stan homodimers that allow neighbouring cells to compare their levels of Fz activity. Under this hypothesis, Fz and Stan are essential components, as without Fz there is nothing to compare and without Stan there is no means to make comparisons. The Stan system also depends on a third protein, Vang, which appears to act in a complementary way to Fz. This study has dissected the function of these proteins by confronting adjacent cells of different fz, Vang and stan genotypes and assaying the effects on PCP. The main finding is that, even in the absence of Vang, Fz can function to polarise cells if it is present in at least one of the two abutting cells. By contrast, Vang has no detectable function when Fz is absent. Based on these and on other results, it follows that, at the core of the Stan system, intercellular bridges form between Stan on its own and Stan complexed with Fz (StanFz), and these act to polarise cells on both sides. It is concluded that Vang acts as an auxiliary component, helping Stan bridge with StanFz. Furthermore, it is posited that the numbers and disposition of asymmetric Stan<<StanFz bridges linking each cell with its neighbours are the consequence of the Fz activity gradient and serve to polarise the cell (Struhl, 2012).

    A model has been built for how bridges between Stan and StanFz might determine the polarity of a cell (see The Stan system in PCP - a model). In the absence of Vang, expression of Fz in a sending cell can bias the polarity of a receiving cell that lacks Fz. Previous results indicate that within the receiving cell, Stan should accumulate only on the surface that faces the sending cell - because it is the only interface where it can form bridges with StanFz - and it is now proposed that it is this localised accumulation of Stan that biases the Vang- fz- receiving cell to make hairs on the other side, pointing away from the sending cell. A parsimonious hypothesis is that the apical membrane of each cell would have an unpolarised propensity to form hairs, and that an excess of Stan on one side locally inhibits this propensity, directing the production of hairs to the opposite side where there is least Stan. The response by a Vang- fz- cell eloquently suggests that the local accumulation of Stan bridged to StanFz in neighbouring cells is the main, and possibly the only, intracellular transducer of Stan system PCP (Struhl, 2012).

    Next consider the finding that Vang functions in receiving cells to help Stan interact productively with StanFz in sending cells. In the key experiment Vang is added to just the Vang- fz- receiving cell: this cell is now more strongly polarised by StanFz signal coming from the sending cell. Thus, Vang can act in the same cell as Stan to help it receive incoming StanFz signal. The model also explains why the polarising effect of the Fz-expressing cell propagates only one cell into the fz- surround, even when Vang activity is restored to the receiving cells - as Stan-Stan bridges do not form, and/or do not function, between neighbouring cells that lack Fz (Struhl, 2012).

    Last, consider the finding that cells lacking Fz can polarise cells devoid of Vang. In this case, only Stan<<StanFz and StanV<<StanFz bridges can form between the two cells, and as a consequence, only the StanFz form of Stan will accumulate on the surface of the Vang- receiving cell where it abuts the fz- sending cell. It is conjectured that Fz, when in a complex with Stan, acts to inhibit the normal action of Stan to block hair outgrowth. Therefore, the only place within the Vang- receiving cell where Stan can accumulate and block hair formation is on the far side, where it can form intercellular bridges with StanFz in the next Vang- cell. Accordingly, the receiving cell would be directed to make a hair on the near side, where it abuts the fz- sending cell. This reasoning also explains why the polarising effect of fz- sending cells on Vang- receiving cells appears to be limited mostly to the adjacent Vang- cell; because Stan<<StanFz and StanFz>>Stan bridges should form and/or function poorly between this cell and the next Vang- cell. Nevertheless, some imbalance between these two kinds of bridges probably does spread further than one cell; indeed fz- sending cells can polarise receiving cells up to two rows away in Vang- pupal wings (Struhl, 2012).

    All the many other experiments fit with the simple model, in which Stan accumulates at the cell surface only where it can form intercellular bridges with StanFz, and each cell is polarised by differences in the amounts of Stan that accumulate along each of its interfaces with adjacent cells. Vang is not essential for these bridges, but by acting on Stan it helps them form and/or makes them more effective (Struhl, 2012).

    How do wild-type cells acquire different numbers and dispositions of asymmetric bridges on opposite sides of the cell? In the Drosophila abdomen, in the anterior compartment of each segment, it has been argued that the Hh morphogen gradient drives a gradient of Fz activity. The slope of the vector of the Fz gradient would then be read by each cell via a comparison of the amount of Stan in its membranes. Within each cell, most Stan will accumulate on the cell surface that abuts the neighbour with most Fz activity, whereas most StanFz will accumulate on the opposite surface, where it confronts the neighbour with least Fz activity. This differential would then be amplified by feedback interactions both between and within cells. The result in each cell is a steep asymmetry in Stan activity that represses hair formation on one side, while allowing it at the other, directing all cells to make hairs that point 'down' the Fz gradient. The model differs in various and simplifying ways from the several and overlapping hypotheses published before. It makes Stan, rather than Fz, the main mediator of PCP, with differences in Fz activity between cells serving to regulate the local accumulation and transducing activity of Stan within cells (Struhl, 2012).

    A central premise of this model is that morphogen gradients do not act directly on each cell to polarise Fz activity, but rather indirectly, by first specifying stepwise differences in Fz activity between adjacent cells. Such an indirect mechanism is favored for two reasons. First, PCP in much of the abdominal epidermis is organised by Hh, which is transduced primarily by its effects on the transcription factor Cubitus interruptus (Ci). It is difficult to understand how graded extracellular Hh could act directly - without cell interactions and only through the regulation of transcription - to polarise Fz activity within each cell. In addition, previous studies used temperature to drive tissue-wide gradients of transcription of a fz transgene under the control of a heat shock promoter; these studies nicely establish that cell-by-cell differences in Fz activity generated by transcriptional regulation are sufficient to polarise cells. Second, it has been previously shown that the polarising action of Hh depends on the Stan system. Specifically, cells in which the Hh transduction pathway is autonomously activated by the removal of the negative regulator Patched require Stan to polarise neighbouring cells. That result adds to evidence that graded Hh creates differences in Fz activity between cells - presumably via transcriptional regulation - that lead to asymmetries in Fz and Stan activities within cells. The target gene could be either fz itself or any other gene whose activity might bias the formation of Stan<<StanFz versus StanFz>>Stan bridges (Struhl, 2012).

    Two previously developed staining experiments provide further support for this model with respect to Stan<<StanFz bridges. First, when Vang- clones are made in fz- flies (generating patches of Vang- fz- cells within fz- territory), a situation in which no Stan<<StanFz bridges can form, there is no accumulation of Stan near or at the border between the clone and the surround - and indeed this study now finds no polarisation of the fz- cells across the clone border. Second, and by contrast, when fz- clones are made in Vang- flies (generating patches of Vang- fz- cells within Vang- territory) Stan accumulates strongly along cell interfaces at the clone borders. Moreover, it is depleted from the cytoplasm of those cells of a clone that abut that border, indicating that Stan in Vang- fz- cells is accumulating at the apicolateral cell membrane where it can form stable intercellular Stan<<StanFz bridges. Previously, there was no evidence that this localisation of Stan within such Vang- fz- cells would polarise them. However, this study now shows that the Vang- fz- cells are polarised by their Fz-expressing neighbours and, also that the effect is reciprocal, their Fz-expressing neighbours are polarised in the same direction (Struhl, 2012).

    The molecular mechanisms by which Fz and Vang control the formation and activity of Stan bridges remain unknown. Consistent with a direct action of Fz on Stan, both in vivo and in vitro studies suggest a physical interaction between the two proteins. Thus, Fz might act in a StanFz complex to regulate both the bridging and transducing activities of Stan. There is no comparable evidence in Drosophila for direct interactions between Vang and Stan. However, their mammalian counterparts have been shown to interact with each other (Devenport, 2008). But Drosophila Vang does interact directly with Pk, while a different Pk-related protein, Espinas, appears to interact directly with Stan during Drosophila neuronal development. Hence, Vang and Pk might form a cis-complex with Stan in epidermal cells, allowing Vang to act directly on Stan and help it form intercellular bridges with StanFz. Intriguingly, there is some evidence that Vang in one cell can interact directly with Fz in adjacent cells. Such an interaction might enhance the capacity of Stan to bridge with StanFz by providing an additional binding surface between the two forms of Stan. Alternatively, Vang might affect the formation or stability of Stan<<StanFz bridges indirectly, consistent with evidence implicating it in the trafficking of proteins and lipids to the cell surface. For example, evidence has been presented that any Stan or StanFz on the cell surface that is not engaged in Stan<<StanFz bridges is rapidly endocytosed and recycled to other sites on the cell surface. Vang activity could bias this process in favour of Stan, thereby enhancing its capacity to form bridges with StanFz (Struhl, 2012).

    The results point to parallels between the Stan and Ds/Ft systems of PCP. First, both systems depend on the formation of asymmetric intercellular bridges between two distinct protocadherin-like molecules. For the Ds/Ft system, these are the Ds and Ft proteins themselves; for the Stan system, it is argued that these are two forms of Stan, either alone or in complex with Fz (StanFz). Second, morphogens may organise both systems by driving the graded transcription of target genes to create opposing gradients of bridging molecules. For the Ds/Ft system, at least two such target genes have been identified: ds itself and four-jointed (fj), a modulator of Ds/Ft interactions. For the Stan system, the existence of at least one such target gene induced by Hh is inferred. Third, for both systems, the two kinds of asymmetric bridges become distributed unequally on opposite faces of each cell, providing the information necessary to point all cells in the same direction. Thus for the Ds/Ft system, it is proposed that different amounts of Ds-Ft heterodimers would be distributed asymmetrically in the cell and this has been recently observed. Similarly, for the Stan system, there is plenty of evidence showing that Stan, Fz and Vang are unequally distributed within each cell. Finally, both systems have self-propagating properties: sharp disparities in Stan, Vang or Fz activity repolarise neighbouring cells over several cell diameters, even in the absence of the Ds/Ft syste, and the same is true of sharp disparities in Ds or Ft activity in the absence of the Stan system. Thus, the Stan and Ds/Ft systems may share a common logic that links morphogen gradients via the oriented assembly of asymmetric molecular bridges and feedback amplification, to cell polarisation (Struhl, 2012).

    Prickle/spiny-legs isoforms control the polarity of the apical microtubule network in planar cell polarity

    Microtubules (MTs) are substrates upon which plus- and minus-end directed motors control the directional movement of cargos that are essential for generating cell polarity. Although centrosomal MTs are organized with plus-ends away from the MT organizing center, the regulation of non-centrosomal MT polarity is poorly understood. Increasing evidence supports the model that directional information for planar polarization is derived from the alignment of a parallel apical network of MTs and the directional MT-dependent trafficking of downstream signaling components. The Fat/Dachsous/Four-jointed (Ft/Ds/Fj) signaling system contributes to orienting those MTs. In addition to previously defined functions in promoting asymmetric subcellular localization of 'core' planar cell polarity (PCP) proteins, this study found that alternative Prickle (Pk-Sple) protein isoforms control the polarity of this MT network. This function allows the isoforms of Pk-Sple to differentially determine the direction in which asymmetry is established and therefore, ultimately, the direction of tissue polarity. Oppositely oriented signals that are encoded by oppositely oriented Fj and Ds gradients produce the same polarity outcome in different tissues or compartments, and the tissue-specific activity of alternative Pk-Sple protein isoforms has been observed to rectify the interpretation of opposite upstream directional signals. The control of MT polarity, and thus the directionality of apical vesicle traffic, by Pk-Sple provides a mechanism for this rectification (Olofsson, 2014).

    A model is proposed for coupling Ft/Ds/Fj to the core module. Gradients of Fj and Ds, by promoting asymmetric distribution of Ft/Ds heterodimers, align a parallel network of apical MTs. Vesicles containing Dsh are transcytosed towards MT plus-ends. In the presence of Pk, MT plus-ends are biased towards the high end of the Fj gradient and the low end of the Ds gradient, whereas in the presence of Sple, the MT plus-ends are biased towards areas with low levels of Fj and high levels of Ds expression. Predominance of Pk or Sple, therefore, determines how tissues differentially interpret, or rectify, the Ft/Ds/Fj signal to the core module. It is hypothesized that this signal serves to both orient the breaking of initial symmetry and to provide continual directional bias throughout polarization. Additional validation of this model would require the measurement of Eb1::GFP comet directions while controlling Pk-Sple isoform expression in wings bearing ectopic Ds and Fj gradients, an experiment that is beyond the technical capabilities with currently available reagents. However, further evidence in support of this model is found in the observation that, in Pk-predominant wings, MT polarity and hair polarity point from regions with high toward low Ds expression both in wild-type wings and in wings with ectopic reversed Ds gradients (Olofsson, 2014).

    It is noted that the distal plus-end bias of MTs is seen in much of the wild-type wing, but this bias decreases to equal proximal-distal plus-end distribution near to the most distal region of the wing. Thus, the mechanism described in this study might not affect the entirety of the wing; in contrast, plus-end bias was observed across the entire A-abd compartment (Olofsson, 2014).

    A model incorporating early Sple-dependent signaling and late Pk-dependent signaling has been proposed to explain PCP in the wing. The current observations and model are compatible with the data presented in support of that model; Sple expression, although always lower than Pk expression in wild-type wing, declined during pupal wing development, suggesting that, in pk mutants, polarity patterns might be set early in development, when Sple is still expressed and when Ds is present in a stripe through the central part of the wing, giving rise to anteroposterior oriented patterns (Olofsson, 2014).

    Pk (and presumably Sple, in Sple dependent compartments) is required for amplification of asymmetry by the core PCP mechanism (Tree, 2002; Amonlirdviman, 2005). These results indicate an additional, core module independent, function for these proteins in regulating the polarity of MTs. Furthermore, although the core function of Pk-Sple is not well defined, part of that function might include promoting the formation and movement along aligned apical microtubules of Fz-, Dsh- and Fmi-containing vesicles (Shimada, 2006). The relative abundance of transcytosing vesicles in Pk versus Sple tissues suggests that if Sple promotes MT-dependent trafficking, it does so less efficiently than Pk (Olofsson, 2014).

    These activities are remarkably similar to those that have been recently identified for Pk and Sple in fly axons, where Pk promotes or stabilizes MT minus-end orientation towards the cell body, and Sple promotes the orientation of minus-ends toward the synapse, which has effects on vesicle transport and neuronal activity. A common mechanism of differentially adapting the plus- and minus-ends of MT segments is proposed in both instances. In axons, similar to what was observed in this study, Pk also facilitates more robust cargo movement, whereas movement is less efficient when Sple is the dominantly expressed isoform. Furthermore, MT polarity defects might underlie the apical-basal polarity defects and early lethality of mouse prickle1 mutant embryos. As Ft and Ds are not known to regulate MTs in axons, these observations suggest that Pk and Sple are able to modify MT polarity independently of Ft/Ds. However, in wings, a consequence is only evident if MTs are first aligned by Ft/Ds activity (Olofsson, 2014).

    How Pk and Sple modulate the organization of MTs remains unknown, but possibilities include modifying the ability of Ft or Ds to capture or nucleate MTs, or altering plus-end dynamics to inhibit capture. These data also suggest the possibility of a more intimate link between the core PCP proteins and Ft/Ds than has been appreciated previously. Other concurrent signals, such as that proposed for Wnt4 and Wg at the wing margin, cannot be ruled out. However, the observations that (1) MTs correlate with the direction of core PCP polarization over space and time, (2) vesicle transcytosis is disrupted in ft clones in which MTs are randomized, (3) chemical disruption or stabilization of MTs disturbs polarity and (4) Pk and Sple isoform predominance rectifies signal interpretation by the core module in a fashion that follows both the wild-type and ectopic Ds gradients provide additional evidence for the model that a signal from the Ft/Ds/Fj system orients the core PCP system in substantial regions of the wing and abdomen (Olofsson, 2014).

    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. This study found 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. Pk was found to participate 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. these results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling (Cho, 2015).

    This study has shown that Cul1 complex-mediated ubiquitinylation of Pk is required for correct function of the core PCP signaling module, thereby ensuring proper alignment of hairs on the Drosophila wing. Ubiquitinylation by the Cul1 complex targets Pk for proteasome-dependent degradation, and in its absence, excess Pk accumulates, resulting in disruption of core PCP function. In several previous reports, ubiquitinylation has been recognized to regulate PCP signaling. In a mouse model, Smurf E3 ligases were shown to regulate PCP signaling by modulating Pk levels. However, mutation of Drosophila smurf failed to show PCP defects. In Drosophila, Cul3 E3 ligase-BTB protein-mediated regulation of Dsh ubiquitinylation modulates PCP signaling, as does the de-ubiqutinylating enzyme Faf, possibly acting on or upstream of Fmi, or more recently proposed to act on Pk. Loss of either activity shows subtle effects on final PCP outcomes in Drosophila. In no case is there a demonstrated mechanism for how these events impact the characteristic asymmetric subcellular localization of PCP proteins that underlies cell polarization (Cho, 2015).

    Slimb was found to be the F-box protein that mediates Pk and Cul1 complex association in vivo. It appears likely that the motif that mediates interaction between Pk and Slimb resides in the C-terminal half of the protein, as do the Vang interaction domain and the farnesylation (CaaX) motif. Of note, the amount of Slimb protein in the cell was also dependent on Pk. In previous cell culture studies, F-box proteins themselves were targeted for ubiquitinylation by their own Cul complexes when not bound by other substrates, and this appears to be the case here, as Slimb levels are increased in cul1 knock-down clones. Furthermore, this result supports the idea that Pk is the major target of the Cul1 complex during pupal wing development (Cho, 2015).

    If the Cul1-SkpA-Slimb complex targets Pk for degradation, why do Slimb and Pk accumulate together on the proximal side of wildtype cells? Pk is known to bind to Vang, and to localize with it in the proximal complex. Slimb adapts the Cul1 complex to Pk and is seen to colocalize with Vang on the proximal side, as well as with overexpressed Pk. However, this suggests that the Pk in this location is resistant to Cul1 complex-dependent degradation. Pk levels have long been known to be limited by a Vang-dependent activity. Recently, it has been shown that farnesylation of Pk is required for Pk to interact with Vang and promote its degradation, and that levels of Pk also depend on SkpA, leading to the suggestion that farnesylation-dependent Pk-Vang interaction results in SkpA-dependent Pk degradation. This study provides evidence suggesting that the Cul1-SkpA-Slimb E3 complex directly targets Pk for destruction, but in contrast, the finding that Pk with deleted CaaX domain accumulates to elevated levels in cul1 knock-down cells indicates that Cul1/SkpA/Slimb-dependent degradation is independent of farnesylation. Furthermore, the finding that Pk promotes internalization of Fmi-Vang-Pk during mutual exclusion of oppositely oriented core PCP complexes leads to a model, that is consistent with the shared observation that Pk associated with stable intercellular complexes ([Dsh-Fz-Fmi]-[Fmi-Vang-Pk]) is protected from degradation (Cho, 2015).

    In theory, generation of cell polarity requires the combination of a local self-enhancement of a cell polarity factor and a long range inhibition of the same factor. In isolated cells, likely the evolutionarily more ancient mechanism, intracellular local self-enhancement can arise through cooperativity among P proteins. Intracellular long range inhibition is most easily accomplished by limiting amounts of a component of the P complex, such that aggregation of P complexes in one location decreases the probability of aggregation elsewhere by depletion of that component (Cho, 2015).

    Cell polarization within a multicellular system introduces additional possible intercellular mechanisms for both the local self-enhancement and the long range inhibition (see Cell polarity establishment and the involvement of Pk-mediated endocytosis). If two polarity complexes, P and Q, exist, and can interact at junctions between adjacent cell boundaries, then both the local and long range effects can be mediated through these intercellular interactions. If P complexes recruit Q complexes to opposing sides of junctions, and if mutual antagonism between P and Q occurs, then long range inhibition can occur by P recruiting Q to the neighbor, where P is then excluded. Similarly, exclusion of P decreases Q in that region of the original cell, enabling the accretion of more P (in effect, cooperativity) (Cho, 2015).

    The peripheral membrane associated core PCP proteins Pk, Dsh and Dgo appear to mediate these polarization events, but how they do so is not known. They are not required for assembly of asymmetric [Fz-Fmi]-[Fmi-Vang] complexes, but were known to share the ability to induce clustering, and are all required for the feedback amplification that results in the asymmetric subcellular localization of PCP signaling complexes. While their action somehow promotes the assortment of proximal and distal core proteins to opposite sides of the cell, how they carry out this function, and in particular whether this is through intracellular or intercellular mechanisms, is unclear (Cho, 2015).

    To understand how excess Pk resulting from mutation of the Cul1 E3 complex disrupts PCP, Pk's role in establishment of core asymmetry was studied further. pk mutation causes symmetric distribution of other core proteins without substantially diminishing or enhancing their junctional recruitment. On the other hand, Pk overexpression causes both accumulation of higher levels of all proximal and distal core proteins and induces their clustering at apical membrane domains, generating discrete puncta. A Pk induced clustering of similarly oriented core complexes could explain both the aggregated punctate appearance and the increased levels of accumulated proteins if one assumes a steady state relationship between free asymmetric complexes and unassembled components as asymmetric complexes are sequestered into puncta (Cho, 2015).

    Pk over-expression study shows that Fz is not required for making Fmi clusters, but Vang is. This suggests an intracellular mechanism in which Pk interacts with Vang at the apical membrane to induce clustering. However, since Vang over-expression does not cause accumulation of other core proteins, a specific function for Pk beyond stabilization of Vang must be considered to explain the accumulation of other core proteins. Furthermore, the depletion of Fmi from the membrane achieved by the very high levels of Pk upon simultaneous Pk overexpression and Cul1 depletion argues for a function for Pk beyond clustering (Cho, 2015).

    Pk might stimulate amplification simply by promoting clustering, with long range inhibition mediated by other mechanisms, or perhaps by limiting amounts of Pk. However, the data suggest an alternative interpretation, as Pk-dependent mutual exclusion of oppositely oriented complexes is observed, forcing local accumulation of distal proteins induced the Pk-dependent removal of proximal proteins within the same cell. Exclusion is associated with Pk mediated internalization of Pk-Vang-Fmi complexes, suggesting that this exclusion involves endocytosis. The requirement for Vang in this internalization is consistent with a previous study showing that Vang contributes to Fmi internalization. It is therefore proposed that Pk is involved in an intercellular long range inhibition to promote feedback amplification (Cho, 2015).

    Like clustering, the Pk-induced routing of Fmi into intracellular vesicles was dependent on Vang, and Pk, Vang and Fmi colocalize in vesicles both apically and more basally, indicating that Fmi-Vang complex trafficking is regulated by associated Pk. However, unlike clustering, it is also dependent on Fz. This suggests a model for feedback inhibition in which oppositely oriented asymmetric complexes interact within clusters, leading to endocytosis and removal of Pk-Vang-Fmi. Competitive interaction between the proximal protein Pk and the distal protein Dgo for Dsh binding is known to occur, suggesting that these interactions might result in either of two alternative outcomes, one of which would be disruption of the proximal complex, and the other disruption of the distal complex. It is proposed that if the distal complex 'wins,' thus remaining stable, the proximal Pk-Vang-Fmi complex becomes internalized in a Pk-dependent step. Once there is a predominance of complex in a given orientation, Vang will be enriched on one side of the intercellular boundary with relatively little Fz present. Since Pk and Slimb associate with Vang, they too will be enriched, but the absence of competitive interactions from the Fz complex allows them to remain within clusters, accounting for the accumulation of Pk and Slimb on the proximal side of wildtype wing cells. According to this model, Pk and Slimb are observed primarily at sites where they are inactive and therefore not internalized (Cho, 2015).

    Modest levels of Pk overexpression both enhance accumulation of PCP protein complexes at the membrane and disrupt the normal orientation of polarization. This may be explained by enhanced feedback amplification that overwhelms the ability to interpret directional inputs. In contrast, the depletion of Fmi from the membrane observed with the very high levels of Pk induced by simultaneous Pk overexpression and Cul1 depletion suggests that sufficient Pk can induce internalization even without the competitive interactions from the Fz complex that normally stimulate internalization (Cho, 2015).

    The mechanism for Pk-dependent clustering is not known. As previously proposed, clustering may result from a scaffolding effect; the possibility of decreased endocytosis accounting for clustering was previously discounted. Whatever the mechanism, clustering by Pk must occur independent of Fz. Furthermore, Pk must enable the multimeric aggregation of complexes containing [Vang-Fmi]-[Fmi] or [Vang-Fmi]-[Fmi-Fz]. Induction of multimeric clustering would also provide a context for the dose-dependent competition that determines internalization of either the proximal or distal complex. Additional work will be required to determine how Pk facilitates clustering (Cho, 2015).

    Since Cul1 depletion increases the amount of Pk, and excess Pk produces clustering and amplification, how Cul1 might produce the observed phenotype is now considered. The simplest possibility is that in the Cul1 mutant, excess Pk produces excess clustering and amplification that overwhelms the directionality in the system. However, because Pk is associated with Slimb and yet stable in the polarized state, and because Pk degradation is dependent on Vang, the possibility is also entertained that Cul1-dependent degradation is somehow functionally coupled to Pk-mediated internalization. Additional studies will be required to distinguish these possibilities (Cho, 2015).

    In summary, a model is proposed in which Pk-dependent internalization of proximal complexes provides an intercellular long range inhibition that contributes to amplification of core protein asymmetric localization. At the same time, Pk provides a local cooperative effect by inducing clustering and accumulation of proximal complexes. The mechanism for clustering are not known, but a simple model is that Pk mediates closely related internalization events (Cho, 2015).

    It is noted that a similar intercellular long range inhibition was initially discussed long ago, except that [Vang-Pk] was proposed to disrupt [Fz-Dsh]. This interpretation was based largely on inference. The current study provides evidence that [Fz-Dsh] disrupts [Vang-Pk] (by promoting internalization). On theoretical grounds, either one would be sufficient to cause polarization, but the possibility cannot be excluded that both may occur. Indeed, vesicles containing Fz, Dsh and Fmi have been shown to be transcytosed in a microtubule-dependent fashion with a directional bias, and these vesicles appear to derive from apical junctions, where they may arise by exclusion (Cho, 2015).

    Although knock-down of smurf in flies reveals no function in PCP; the mechanism described in this study is similar to that inferred for Smurf in mouse PCP. Mice mutant for both Smurf1 and Smurf2 show PCP defects and lose asymmetric localization of core PCP proteins. Furthermore, biochemical evidence was provided that Smurfs, in the presence of the Dsh homolog Dvl2 (and Par6) mediated ubiquitinylation of mouse Pk1. From this, a model was proposed that proximal complexes containing Pk1, and presumably Vang and Celsr (Fmi), are disrupted upon proximity to distal complexes containing Fzd and Dvl2. This model is similar to the model of mutual exclusion, except that the mode of disruption was not directly addressed. While this study proposes disruption by internalization, perhaps coupled to degradation, the mouse stud was only able to address degradation. Furthermore, it is not known if, in mouse, Pk1 mediates clustering, perhaps by a related mechanism, as as is described in flies (Cho, 2015).

    The de-ubiquitinase USPX9 was recently identified as a regulator of Pk in the context of Pk's role in epilepsy in human, mouse, zebrafish and flies. Similarly, the orthologous Drosophila de-ubiquitinase Faf modulates the pksple dependent seizure phenotype in flies. These observations suggest that while the ubiquitinylating and de-ubiquitinylating activities of Smurf and USPX9 control the ubiquitinylation state of vertebrate Pk's, Cul1 and Faf may serve the analogous function to regulate ubiquitinylation of Drosophila Pk (Cho, 2015).

    The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of 'upstream' planar cell polarity proteins

    The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins, inturned, fuzzy and fritz, accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, this study has found that when over-expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. It was further shown that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. It was also found that over-expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group (Wang, 2014).

    The atypical cadherin Dachsous controls left-right asymmetry in Drosophila

    Left-right (LR) asymmetry is essential for organ development and function in metazoans, but how initial LR cue is relayed to tissues still remains unclear. This study proposes a mechanism by which the Drosophila LR determinant Myosin ID (MyoID) transfers LR information to neighboring cells through the planar cell polarity (PCP) atypical cadherin Dachsous (Ds). Molecular interaction between MyoID and Ds in a specific LR organizer controls dextral cell polarity of adjoining hindgut progenitors and is required for organ looping in adults. Loss of Ds blocks hindgut tissue polarization and looping, indicating that Ds is a crucial factor for both LR cue transmission and asymmetric morphogenesis. It was further shown that the Ds/Fat and Frizzled PCP pathways are required for the spreading of LR asymmetry throughout the hindgut progenitor tissue. These results identify a direct functional coupling between the LR determinant MyoID and PCP, essential for non-autonomous propagation of early LR asymmetry (Gonzalez-Morales, 2015).

    This work has revealed the existence of an hindgut-specific LR organizer having transient activity. LR information is transferred non-autonomously from this organizing center to the target tissue, involving a unique MyoID-Ds interaction taking place at a PCP signaling boundary (the H1/H2 boundary). Propagation of this initial LR information to the developing hindgut requires both Ds/Ft global and core Fz PCP signaling. Notably, these results suggest that MyoID can act as a directional cue to bias planar cell polarity (Gonzalez-Morales, 2015).

    So far, only a role for the core PCP pathway in cilia positioning and LR asymmetry had been reported in mouse, chick, and Xenopus. This study revealed a role of the Fat/Ds PCP pathway in LR asymmetry. The atypical cadherin Ds is essential for early LR planar polarization of hindgut precursors and later on for looping morphogenesis. Ds has a cell-non-autonomous function, allowing transfer of LR information from the H1 domain to H2 hindgut precursor cells. Ds, therefore, represents a critical relay factor acting at the boundary between, and linking, a LR organizer and its target tissue (Gonzalez-Morales, 2015).

    In addition to a MyoID-dependent function in H1, the mislooped phenotype induced upon Ds silencing in the H2 domain suggests that Ds also has a MyoID-independent activity in H2 cells, likely through interaction with other PCP genes. Indeed, reducing the activity of PCP global or core gene functions reveals that the two pathways are important in the H2 region for adult hindgut looping. However, the results reveal important differences in the way these pathways control hindgut asymmetry. First, although the adult phenotype is similar upon silencing of one or the other pathway, the early polarization of H2 cells in pupae (10 hr APF) is only affected when knocking down the activity of Ds, Ft, and Fj. These results show that the Ds/Ft pathway, but not the core pathway, is required for establishing early LR polarity. Second, the phenotype is quantitatively different, since silencing of the Ds, Ft, or Fj PCP gene led to a consistent and very strong phenotype, while reducing Fz PCP signaling had a significantly less penetrant one. These data suggest a partly overlapping function of both PCP signaling pathways for late hindgut morphogenesis. Therefore, the following sequential model is proposed: in H1 cells, MyoID interacts with the Ds intracellular domain, which becomes 'biased' toward dextral through a currently unknown mechanism. This initial LR bias is then transmitted across the H1/H2 boundary through Ds/Ft heterophilic interaction. Then, boundary H2 cells relay the initial bias and spread it to the remaining H2 cells through classical Ds/Ft PCP. It is interesting that the local signaling boundary suggested by this model is consistent with recent studies showing that Ds can propagate polarity information in a range of up to eight cells, a distance that is consistent with the size of the H2 domain at 10 hr APF. Once initial polarity has been set up through the Ds/Ft pathway, this is further relayed to and/or amplified by the core pathway. Notably, a similar two-step mechanism has also been proposed for the wing and could apply to other tissues (Gonzalez-Morales, 2015).

    The discovery of a coupling between the MyoID dextral factor and Ds is a nice example of crosstalk between existing signaling modules. In the simplest crosstalk model, the role of MyoID would just be to bias or tilt Ds function toward one side, possibly through Ds localization and/or activity polarization along the LR axis. Using both in vitro and in vivo assays, this study has shown that interaction between Ds and MyoID requires Ds intracellular domain, supporting a cytoplasmic interaction between the two proteins. These results, along with recent findings, suggest that Ds may represent a general platform for myosin function in different tissues. In particular, the intracellular domain of Ds was found to bind to the unconventional myosin Dachs, controlling Dachs polarized localization, which is important for subsequent cell rearrangements underlying thorax morphogenesis. However, in contrast to thoracic Dachs, MyoID is not obviously polarized in H1 cells, suggesting that the interaction between myosins and Ds may involve different mechanisms. Additionally, no LR polarized localization of MyoID or Ds was observed in H1 cells, although the existence of subtle asymmetries undetectable by available tools cannot be excluded. Nevertheless, alternative means to generate the LR bias in H1 include: (1) LR polarized expression of an unknown asymmetric factor or (2) LR asymmetric activity of Ds. These interesting possibilities are consistent with recent work showing that some type I myosins can generate directed spiral movement of actin filaments in vitro. It is tempting to speculate that, similarly, MyoID putative chiral activity could be translated into Ds asymmetrical function along the LR axis. Future work will explore this possibility as well as others to unravel the molecular basis of MyoID LR biasing activity in the H1 organizer (Gonzalez-Morales, 2015).

    The identification of the H1 domain as a specific adult tissue LR organizer demonstrates the existence of multiple independent tissue and stage-specific LR organizers in flies. This situation echoes what is known in other models, including vertebrates, in which at least two phases of asymmetry establishment can be distinguished. A first pre-gastrula phase, as early as the four-cell stage in Xenopus, involves the generation of asymmetric gradients of ions. Then, a second phase takes place at gastrulation and involves Nodal flow and asymmetric cell migration, eventually leading to asymmetric expression of the nodal gene in the left lateral plate mesoderm. In Drosophila, some interesting common and specific features can be drawn out by comparing the hindgut and terminalia organizers. The first major common feature is the fact that both organizers rely on MyoID function, showing the conserved role of this factor in Drosophila LR asymmetry. Second, the two organizers show temporal disconnection, acting much earlier than LR morphogenesis, which is expected of a structure providing directionality to tissues per se (24 hr for terminalia and ~72 hr for hindgut looping). Such temporal disconnection of MyoID function with late morphogenesis is also observed in the terminalia where a peak of MyoID activity precedes terminalia rotation by 24 hr. Time lag in MyoID function requires LR cue transmission and maintenance in developing tissues until directional morphogenesis. The finding of a role of Ds and PCP in hindgut LR asymmetry provides a simple mechanism by which initial LR information is maintained and transmitted across tissue through long-range PCP self-propagation (Gonzalez-Morales, 2015).

    Notably, the two organizers also show distinct features. In terminalia, MyoID has a cell-autonomous function in two adjacent domains. In addition, the terminalia organizer is permanent, developing as an integral component of the adult tissue. In contrast, MyoID in the imaginal ring has a cell-non-autonomous function. Indeed, a striking feature of the hindgut organizer is its transience as it detaches from the hindgut precursors 50 r before full looping morphogenesis prior to its degradation and elimination; hence, the need to transfer LR information to the H2 hindgut primordium. An interesting question then is whether the MyoID-Ds/PCP interaction is conserved in terminalia. This study has shown that the terminali rotation requires the activity of DE-cadherin; however, invalidation of the atypical cadherins Ds or Ft or core PCP signaling in the terminalia organizer did not affect asymmetry. The fact that PCP does not have a general role in Drosophila LR asymmetry is not altogether surprising, as MyoID cell-autonomous function in terminalia and organizer persistence does not require that LR information be transferred to and stored in other parts of the tissue, as is the case in the hindgut. Therefore, despite conservation of the MyoID-dependent upstream dextral cue, significant differences in downstream morphogenetic pathways imply alternative cellular mechanisms controlling cue transmission and maintenance (Gonzalez-Morales, 2015).

    The LR signaling module, comprising the dextral determinant MyoID and the still-unknown sinistral determinant, can therefore be coupled to distinct morphogenetic modules, including PCP, as shown in this study. It is suggested that coupling between LR asymmetry and PCP might be observed in processes requiring long-distance patterning of tissues and organ precursors, both in invertebrate and vertebrate models. Understanding organ LR morphogenesis clearly requires studying diverse and complementary models. In this context, the multiplicity of LR organizers discovered in Drosophila represents a powerful model to study the diversity in the coupling of LR organizers with downstream programs responsible for late tissue morphogenesis. In particular, the Drosophila hindgut represents an invaluable model for studying the genetic basis and molecular mechanisms coupling LR asymmetry with PCP patterning (Gonzalez-Morales, 2015).

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

    This study investigated 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. Whether these findings support the gradient model of Dachsous activity is discussed. 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. This proposition was tested in the larval cells; most microtubules grow perpendicularly to the axis of PCP. No meaningful bias was found in the polarity of microtubules aligned close to that axis. Published data from the pupal abdomen was reexamined, and no evidence was found supporting the hypothesis that microtubular orientation draws the arrow of PCP (Pietra, 2020).

    Microtubules provide directional information for core PCP function

    Planar cell polarity (PCP) signaling controls the polarization of cells within the plane of an epithelium. Two molecular modules composed of Fat(Ft) / Dachsous(Ds) / Four-jointed(Fj) and a 'PCP-core' including Frizzled(Fz) and Dishevelled(Dsh) contribute to polarization of individual cells. How polarity is globally coordinated with tissue axes is unresolved. Consistent with previous results, this study found that the Ft/Ds/Fj-module has an effect on a microtubule (MT)-cytoskeleton. Evidence is provided for the model that the Ft/Ds/Fj-module provides directional information to the core-module through this MT organizing function. Ft/Ds/Fj-dependent initial polarization of the apical MT-cytoskeleton is shown to occur prior to global alignment of the core-module, reveal that the anchoring of apical non-centrosomal MTs at apical junctions is polarized. Directional trafficking of vesicles containing Dsh was observed to depend on Ft. The feasibility of this model was demonstrated by mathematical simulation. Together, these results support the hypothesis that Ft/Ds/Fj provides a signal to orient core PCP function via MT polarization (Matis, 2014).

    Symmetry breaking in an edgeless epithelium by Fat2-regulated microtubule polarity

    Planar cell polarity (PCP) information is a critical determinant of organ morphogenesis. While PCP in bounded epithelial sheets is increasingly well understood, how PCP is organized in tubular and acinar tissues is not known. Drosophila egg chambers (follicles) are an acinus-like "edgeless epithelium" and exhibit a continuous, circumferential PCP that does not depend on pathways active in bounded epithelia; this follicle PCP directs formation of an ellipsoid rather than a spherical egg. This study uses an imaging algorithm to 'unroll' the entire 3D tissue surface and comprehensively analyze PCP onset. This approach traces chiral symmetry breaking to plus-end polarity of microtubules in the germarium, well before follicles form and rotate. PCP germarial microtubules provide chiral information that predicts the direction of whole-tissue rotation as soon as independent follicles form. Concordant microtubule polarity, but not microtubule alignment, requires the atypical cadherin Fat2, which acts at an early stage to translate plus-end bias into coordinated actin-mediated collective cell migration. Because microtubules are not required for PCP or migration after follicle rotation initiates, while dynamic actin and extracellular matrix are, polarized microtubules lie at the beginning of a handoff mechanism that passes early chiral PCP of the cytoskeleton to a supracellular planar polarized extracellular matrix and elongates the organ (Chen, 2016).

    This work identify a central symmetry-breaking role for microtubule polarity in PCP of an 'edgeless' epithelial organ. Microtubules are the earliest PCP molecule during follicle development, germarial microtubule polarity predicts the chirality of subsequent follicle PCP events, and disruption of either microtubule alignment or polarity in the germarium prevents all subsequent aspects of follicle PCP, including the coordinated cell motility that initiates follicle rotation. These requirements for microtubules are not due to secondary effects on actin, which retains its organization in germaria with disrupted microtubules. Importantly, unlike actin, which is required acutely and constantly for collective cell migration, microtubules are not strictly required for follicle cell motility once rotation has initiated. It is therefore proposed that microtubules provide the initial source of PCP information in the early follicle and that actin, through its role in promoting tissue rotation, serves to amplify and propagate PCP (Chen, 2016).

    The current data demonstrate that the microtubule polarity bias in the forming follicle predicts the chirality of PCP tissue rotation that initiates 10 hr later. It was recently reported that, in stage 4 follicles, microtubule plus-end orientation anticorrelates with rotation direction at stage 7. However, in that work, stage 4 and earlier follicles were thought to represent pre-rotation stages, contrary to the identification in this study of rotation initiation at stage 2 and that of another study that placed it at stage 1. Since both microtubule alignment and polarity are present in the germarium, the stage 5 correlation is not predictive and reflects pre-existing PCP information rather than revealing its source. In the absence of an independent and direct manipulation of microtubule plus-end orientation, it cannot be conclusively stated that the microtubule polarity that was document in the germarium is instructive for rotational direction. Nevertheless, the strong correlation between this chirality and the subsequent direction of follicle rotation, along with disruption of both in the absence of fat2, point to a model in which coordination of microtubule polarity in cells across the follicle is required for a unidirectional consensus among individual motile cells to initiate productive rotation (Chen, 2016).

    The data thus suggest that the atypical cadherin Fat2, a key regulator of follicle PCP, rotation, and elongation, acts through effects on early microtubule polarity. Through analyses of middle stages of follicle development, it has been argued that Fat2 regulates global PCP alignment of the cytoskeleton, as does the canonical PCP regulator Fat in the developing Drosophila wing. However, in fat2 germaria, actin and microtubule alignment are maintained; it is coordinated microtubule polarity that is lost. These phenotypes, along with its strong genetic interaction with CLASP, argue that Fat2 promotes rotation initiation and follicle PCP via its effects on microtubule polarity. An additional role in actin regulation other than polarized alignment has not been excluded; the requirement for Fat2 and actin, but not microtubules, to maintain rotation, as well as the direct binding of actin regulators by vertebrate Fat1, a possible ortholog of Drosophila Fat2, suggests such a role. Moreover, while this work was in revision, Squarr (2016) showed that Fat2 can directly influence the actin cytoskeleton via binding to the WAVE complex (Chen, 2016).

    As with PCP in the developing Drosophila wing disc, the initial PCP bias provided by microtubule polarity within the early follicle precursors is mild but becomes more robust as organogenesis progresses. A mechanism for amplification in the follicle involves whole-tissue rotation. Preventing rotation by disrupting actin or integrins causes a rapid loss of all PCP organization primed in the germarium. Interestingly, just as microtubules are largely dispensable for PCP after actin PCP becomes established, actin PCP is dispensable after circumferentially aligned ECM becomes established. Hence, PCP transitions from highly dynamic and intracellular microtubules, to longer-lasting and sometimes juxtacellular actin filaments, and then finally to the durable ECM fibrils that span multiple cells. PCP information in the follicle is therefore passed along by a 'handoff' mechanism to increasingly stable as well as larger-scale components that can ultimately biophysically influence the shape of the organ (Chen, 2016).

    Non-centrosomal microtubule arrays, and in particular their regulated polarized organization, have previously been implicated as central governors of global PCP in tissues such as Drosophila wings, zebrafish gastrulae, and mammalian airway epithelia. In 'edged' PCP tissues in Drosophila, a 'global PCP' module molecularly controlled by Fat is thought to use gradients of positional information from specific sources to bring individual cell PCP in alignment with overall body axes. In the circumferential 'edgeless' PCP axis of the follicle epithelium, where no such graded information is known, Fat2 seems to similarly coordinate the PCP of individual cells. That both contexts involve important roles for polarized microtubules and are controlled by related atypical cadherins raises the possibility of ancient links between the modes of epithelial PCP organization (Chen, 2016).

    The clathrin adaptor AP-1 complex and Arf1 regulate planar cell polarity in vivo

    A key step in generating a planar cell polarity (PCP) is the formation of restricted junctional domains containing Frizzled/Dishevelled/Diego (Fz/Dsh/Dgo) or Van Gogh/Prickle (Vang/Pk) complexes within the same cell, stabilized via Flamingo (Fmi) across cell membranes. Although models have been proposed for how these complexes acquire and maintain their polarized localization, the machinery involved in moving core PCP proteins around cells remains unknown. This study describes the AP-1 adaptor complex and Arf1 as major regulators of PCP protein trafficking in vivo. AP-1 and Arf1 disruption affects the accumulation of Fz/Fmi and Vang/Fmi complexes in the proximo-distal axis, producing severe PCP phenotypes. Using novel tools, a direct and specific Arf1 involvement was detected in Fz trafficking in vivo. Moreover, a conserved Arf1 PCP function was uncovered in vertebrates. These data support a model whereby the trafficking machinery plays an important part during PCP establishment, promoting formation of polarized PCP-core complexes in vivo (Carvajal-Gonzalez, 2015).

    This study demonstrates that the AP-1 complex and Arf1 are critical for PCP-core protein membrane localization during PCP establishment in multiple Drosophila tissues, and that it likely serves as a conserved function in zebrafish. Detailed analysis in Drosophila wings and zebrafish cells during gastrulation revealed that Arf1 function is required for polarized accumulation of core PCP components during PCP axis establishment. Defects in correct planar-polarized localization resulted in classical PCP defects in both animal models, and thus this study reveals a conserved function of the Arf1 protein network (Carvajal-Gonzalez, 2015).

    Arf1 and AP-1 act as the trafficking machinery for the core PCP components in epithelial cells during PCP establishment. A few studies suggested connections between PCP and trafficking before. First Rabenosyn, an endocytic-related protein, was found polarized in a PCP-type fashion following Fmi localization. Rabenosyn mutant cells display defects in cellular packing and accumulation of PCP-core components at the PM50. Second, the GTPase Rab23, which, similar to Rabenosyn, causes packing defects and multiple cellular hairs (mch), interacts with the cytosolic PCP-core protein Pk. And third, Gish/Rab11/nuf were recently shown to affect hair formation by either increasing the numbers of trichomes per cell (mch) or shortening the hairs (stunted hair phenotype). However, none of these proteins affected PCP-core component localization, including Fmi or Fz, or hair orientation in the adult, similar to how the Arf1/AP-1 network does (Carvajal-Gonzalez, 2015).

    Arf1 is known to act during trafficking from the Golgi in Drosophila and vertebrate cells. In the TGN, Arf1 aids in sorting of cargo proteins into carrier vesicles, regulating the binding of clathrin adaptor proteins such as GGAs (Golgi-localized, γ-adaptin ear-containing and ARF-binding proteins) or the clathrin adaptor complex AP-1. It was demonstrated that perturbation of Arf1 and AP-1 function reduced polarized accumulation of Fmi, Fz and Vang with minimal effects on non-planar-polarized membrane proteins (for example, DE-cadherin and FasIII). A distinct subpopulation of PCP-core component complexes with different turnover dynamics from unpolarized membrane junctional domains has recently been identified. These complexes are polarized, stable and restricted to large puncta, and also insensitive to manipulations of the endocytic/recycling machinery. Together with established Arf1/AP-1 functions at the Golgi, the current data support the notion that stable PCP complexes originate mainly from newly synthesized PM proteins. A hypothesis that is further supported by using the DmrD4-Fz-GFP fusion and manipulations to synchronize its ER release. Inhibition of Arf1 with BFA treatment prevented the arrival of Fz to the membrane in S2 cells. Importantly, Arf1 inhibition also inhibited Fz delivery to the apical junctional regions in vivo, where it must localize to participate in PCP establishment. A model is proposed whereby Arf1/AP-1 are involved in the specific transport of the PCP-core factor Fz, and most likely also Vang and Fmi, to the PM during PCP establishment, a process required for enrichment of these proteins in polarized complexes. Recently, it was shown in a mammalian non-polarized culture system that Vangl2 trafficking out of the Golgi depends on Arfaptin and the AP-1 complex, which is consistent with the current data and supports the evolutionary conserved task for these proteins in promoting PCP-core component localization (Carvajal-Gonzalez, 2015).

    Arf1 can also act through the COPI complex, and, for example, in flies, at the cis-Golgi, Arf1 together with its guanine nucleotide exchange factor/GEF Gartenzwerg regulate COPI retrograde transport. Thus attempts were made to test this possibility. However, the dsRNA-based KD in vivo of all COPI complex components is cell lethal and thus not informative in this context. Similarly, in cell culture COPI KD, depending on the level, was either too toxic or failed to show an effect on Fz trafficking. As such, although it cannot be completely excluded that COPI is also involved in the process, the comparable results of Arf1 and AP-1 indicate that Arf1 acts at least in part on AP-1 in PCP trafficking (Carvajal-Gonzalez, 2015).

    Several studies have identified genes that affect localized actin hair formation in Drosophila wing cells. Interestingly, apart from the PCP-core components, these usually fall into two cellular machinery categories: actin polymerization-related proteins and trafficking-related proteins. Importantly, both sets of factors have been linked to each other in diverse cellular environments, including carrier formation at the Golgi, yeast budding, formation of phagocytic cups or lamellipodia formation in migrating cells. Silencing of Arf1, Rab11, PI4KIIIβ and AP-1 leads to defects in actin-based hair formation in wing cells, notably leading to the formation of mch. Using Drosophila S2 cells, Arf1 is found not only in the Golgi but also at actin-rich cell edges, and that it is essential for lamellipodium biogenesis. In vertebrates, Arf1 is also required for actin polymerization, for example, in neuronal tissues during plasticity Arf1 regulates Arp2/3 through PICK1. Based on these data, a direct/parallel involvement of the Arf1 protein network is likely during hair formation, and indeed an enrichment of these proteins was observed in the growing actin hairs. Thus, in summary, the data on Arf1/AP-1 and its network suggest that it functions repeatedly in different PCP establishment processes, ranging from the initial PCP-core component localization to later restricting the foci of actin polymerization (Carvajal-Gonzalez, 2015).

    Positioning of centrioles is a conserved readout of Frizzled planar cell polarity signalling

    Planar cell polarity (PCP) signalling is a well-conserved developmental pathway regulating cellular orientation during development. An evolutionarily conserved pathway readout is not established and, moreover, it is thought that PCP mediated cellular responses are tissue-specific. A key PCP function in vertebrates is to regulate coordinated centriole/cilia positioning, a function that has not been associated with PCP in Drosophila. This study reports instructive input of Frizzled-PCP (Fz/PCP) signalling into polarized centriole positioning in Drosophila wings. It was shown that centrioles are polarized in pupal wing cells as a readout of PCP signalling, with both gain and loss-of-function Fz/PCP signalling affecting centriole polarization. Importantly, loss or gain of centrioles does not affect Fz/PCP establishment, implicating centriolar positioning as a conserved PCP-readout, likely downstream of PCP-regulated actin polymerization. Together with vertebrate data, these results suggest a unifying model of centriole/cilia positioning as a common downstream effect of PCP signalling from flies to mammals (Carvajal-Gonzalez, 2016).

    Taken together with observations that Fz/PCP signalling regulates basal body and cilia positioning in vertebrates (Song, 2010; Gray; 2011; Borovina, 2010), the current data on centriole positioning as a Fz/PCP readout in non-ciliated Drosophila wing cells indicate that centriole/MTOC (MT organizing centre)/basal body positioning is an evolutionarily conserved downstream effect of Fz/PCP signalling. Its link with actin polymerization (hair formation in Drosophila wing cells) suggests that actin polymerization effectors also affect cilia positioning, possibly through docking of the basal bodies to the apical membranes. Inturned, Fuzzy and Rho GTPases regulate apical actin assembly necessary for the docking of basal bodies to the apical membrane (Park, 2006; Pan, 2007) and this apical actin membrane accumulation is lost in Dvl1-3-depleted cells (Carvajal-Gonzalez, 2016).

    In left-right asymmetry establishment of the Drosophila hindgut, which is not a Fz/PCP-dependent process, asymmetric centriole positioning is observed. During this so-called planar cell shape chirality process, which affects gut-looping and thus embryonic left/right asymmetry, centriole positioning is however still dependent upon actin polymerization downstream of Rho GTPases (Rac and Rho), via MyoD and DE-cadherin control. As Rho GTPases (Rac, Cdc42 and Rho) are downstream effectors of Fz-Dsh/PCP complexes, and their mutants cause PCP-like phenotypes including multiple cellular hairs or loss of hairs in wing cells. It is thus tempting to infer that both processes, planar cell shape chirality and Fz/PCP, regulate centriole positioning through a common Rho GTPase-mediated actin polymerization pathway, initiated by an upstream cellular communication system, although this assumption will require experimental confirmation. In the mouse, Fz/PCP signalling regulates cilia movement/positioning in cochlear sensory cells via Rho GTPase-mediated processes, suggesting a similar mechanism in a representative mammalian PCP model system. In conclusion, the positioning of centrioles appears to be a key and an evolutionary conserved downstream readout of Fz/PCP signalling, ranging from flies to mammals in both ciliated and non-ciliated cells (Carvajal-Gonzalez, 2016).

    The PCP pathway regulates Baz planar distribution in epithelial cells

    The localisation of apico-basal polarity proteins along the Z-axis of epithelial cells is well understood while their distribution in the plane of the epithelium is poorly characterised. This study provides a systematic description of the planar localisation of apico-basal polarity proteins in the Drosophila ommatidial epithelium. The adherens junction proteins Shotgun and Armadillo, as well as the baso-lateral complexes, are bilateral, i.e. present on both sides of cell interfaces. In contrast, it is reported that other key adherens junction proteins, Bazooka and the myosin regulatory light chain (Spaghetti squash) are unilateral, i.e. present on one side of cell interfaces. Furthermore, planar cell polarity (PCP) and not the apical determinants Crumbs and Par-6 control Bazooka unilaterality in cone cells. Altogether, this work unravels an unexpected organisation and combination of apico-basal, cytoskeletal and planar polarity proteins that is different on either side of cell-cell interfaces and unique for the different contacts of the same cell (Aigouy, 2016).

    SEGGA: a toolset for rapid automated analysis of epithelial cell polarity and dynamics

    Epithelial remodeling determines the structure of many organs in the body through changes in cell shape, polarity and behavior and is a major area of study in developmental biology. Accurate and high-throughput methods are necessary to systematically analyze epithelial organization and dynamics at single-cell resolution. This study developed SEGGA, an easy-to-use software for automated image segmentation, cell tracking and quantitative analysis of cell shape, polarity and behavior in epithelial tissues. SEGGA is free, open source, and provides a full suite of tools that allow users with no prior computational expertise to independently perform all steps of automated image segmentation, semi-automated user-guided error correction, and data analysis. This study uses SEGGA to analyze changes in cell shape, cell interactions and planar polarity during convergent extension in the Drosophila embryo. These studies demonstrate that planar polarity is rapidly established in a spatiotemporally regulated pattern that is dynamically remodeled in response to changes in cell orientation. These findings reveal an unexpected plasticity that maintains coordinated planar polarity in actively moving populations through the continual realignment of cell polarity with the tissue axes (Farrell, 2017).

    Planar polarized Rab35 functions as an oscillatory ratchet during cell intercalation in the Drosophila epithelium

    The coordination between membrane trafficking and actomyosin networks is essential to the regulation of cell and tissue shape. This study examined Rab protein distributions during Drosophila epithelial tissue remodeling and show that Rab35 is dynamically planar polarized. Rab35 compartments are enriched at contractile interfaces of intercae

    The Rab family of small GTPase proteins is key mediators of membrane trafficking and cytoskeletal dynamics. Rab proteins regulate membrane compartment behaviors through their association with tethering and trafficking effectors, and mutations in Rab proteins are associated with a variety of diseases and developmental disorders. The Rab trafficking pathways that operate during cell intercalation in the early Drosophila gastrula have remained undefined, although the function of classic Clathrin and Dynamin-dependent early endocytic pathways has been explored. Indeed, it has been demonstrated that Formin and Myosin II proteins direct the endocytic uptake of dextran through specialized CIV (cortical immobile vesicle) structures. Additionally, in Drosophila, Rab35 has been shown to play a critical role in directing the morphogenesis of tracheal tube growth as well as synaptic vesicle sorting at the neuromuscular junction. In tissue culture models, Rab35 functions early in endosomal pathways to drive the generation of newborn endosomes and is essential for the terminal steps of cytokinesis and neurite outgrowth (Jewett, 2017).

    This study shows that Rab35 demonstrates remarkable compartmental behaviors at the plasma membrane. Rab35 compartments are initially contiguous with the cell surface and form dynamic structures that grow and shrink on the minute time scale. In the absence of Rab35 function, cell interfaces undergo contractile steps, but these steps rapidly reverse themselves, consistent with Rab35 mediating an essential 'ratcheting' function that directs progressive interface contraction. Rab35 compartments form independently of Myosin II function, but require actomyosin forces to terminate. These compartments further function as endocytic hubs that have transient interactions late in their formation with Rab5 and Rab11 endosomes (Jewett, 2017).

    The current results have shown that a trafficking network centered on Rab35 acts as a ratcheting mechanism to ensure that interface contraction is progressive and irreversible. Rab35 functions upstream of junctional actomyosin forces during interface contraction to provide a membranous ratchet, and suggests that cytoskeletal-derived forces are required to terminate compartmental behaviors. Rab35 compartments are more numerous and possess longer compartmental times at contractile interfaces of actively intercalating cells. Rab35 compartments form at the plasma membrane and rapidly grow in size during periods of interface contraction, before shrinking through endocytic-dependent processes. Rab35 compartmental behaviors therefore represent a critical point of convergence at which cytoskeletal and membrane trafficking pathways function to drive changes in cell shape (Jewett, 2017).

    Changes in cell shape in many systems are driven by pulsatile processes that initiate directed movement before cycling through periods in which the force generating network reforms. Previous work on ratcheting function has concentrated on different actomyosin regimes governed by Myosin II regulators such as Rho1 and Rok that trigger contraction and cell ratcheting. Importantly, when Rab35 function is disrupted, apical cell areas maintain oscillatory behaviors and AP interface lengths still undergo brief periods of contraction. However, contractile periods are followed by reversals and interfaces re-lengthen, producing a failure in interface shortening. This 'wobble' behavior is consistent with Rab35 functioning as a ratchet ensuring unidirectional movement during interface contraction. These results also suggest that junctional actomyosin-driven behaviors may be responding to an upstream trafficking apparatus centered on Rab35. Indeed, junctional Myosin II localization is disrupted in Rab35 knockdown embryos, and accumulates intracellularly along with integral membrane proteins (Neurotactin) and F-actin. In tissue culture cells, it has been shown that disruption of Rab35 similarly leads to an accumulation of F-actin during abscission, which provides another link between Rab35 and cytoskeletal dynamics. Finally, and again consistent with expectations for a compartment-driven ratcheting function, it is also striking that Rab35 compartments are not present uniformly during cell intercalation, but begin to form specifically as interface contraction occurs (Jewett, 2017).

    The AP patterning system is responsible for directing Rab35 compartment formation away from apical/medial sites and functionally engaging Rab35 at AP interfaces. It is interesting to note that high levels of anterior patterning information, such as is present in the head region, inhibit planar polarity, cell intercalation, and Rab35 compartment dynamics, while maintaining interfacial Rab35. The common appearance of apically localized Rab35 compartments in the ventral furrow and in bnt mutant embryos (embryos derived from mothers homozygous for the genes bicoid, nanos and torso-like, 'bnt') suggests a model in which ventral fate genes could mediate apical constriction by re-directing Rab35 compartment formation through the inhibition of AP patterning cues (Jewett, 2017).

    Myosin II is found in several different populations within intercalating cells. Transient, web-like Myosin II localizations are found in highly dynamic structures in the apical/medial regions of epithelial cells, while more stable, cable-like structures are present at cell junctions. Junctional Myosin II enrichment is dependent on Rab35 function, and is required for the termination of Rab35 compartments. However, the role of apical/medial actomyosin forces and cell oscillations on Rab35 compartmental behaviors is less clear. It appears that Rab35 compartments may require Myosin II medial 'flows' to function as a polarizing cue, as Rab35 compartments become less planar polarized in the absence of Myosin II function. It is hypothesize that cell oscillations produce cycles of high and low tension during which Rab35 compartments can form as infoldings of slackened plasma membrane that, when internalized, prevents interface length from rebounding to the same length as was present prior to area contraction. It is important to note that the termination of Rab35 compartments and the internalization of membrane is critical to ratcheting and productive interface contraction, as merely driving more plasma membrane into Rab35 compartments (as is observed in Y-27632, PitStop2, or chlorpromazine-injected embryos) in the absence of compartment termination does not direct lasting changes in cell topologies (Jewett, 2017).

    These results also suggest how oscillatory area contractions can be linked to productive movements. In worm and fly embryos14, 47, actomyosin contractions show both productive and non-productive periods. The functional engagement of area oscillations may depend on the presence of the Rab35 ratchet, as immediately prior to intercalation Rab35 compartments are absent from cell interfaces. However, it is intriguing that Rab35 compartments can form when Myosin II function is compromised in Y-27632-injected embryos. It may be that the small amount of Myosin II function left after Y-27632 injection is enough to drive small oscillations in area, or that thermal fluctuations in cell area are enough for compartment formation. Regardless, this will be an interesting area for further studies (Jewett, 2017).

    What is the nature of Rab35 compartments? While there are punctate Rab35 compartments present in the apical cytoplasm that partially colocalize with endosomal markers, the major population of Rab35 during early gastrulation and cell intercalation is present in tubular compartments that are contiguous with the cell surface. The extremely elongated Rab35 plasma membrane tubules observed in either Myosin II-disrupted or endocytosis-inhibited embryos, as well as the immediate filling of Rab35 compartments at the cell surface, are consistent with Rab35 marking endocytic infoldings of the plasma membrane. Additionally, Rab35 compartments are positive for a plasma membrane PIP2 species, PtdIns(4,5)P2, and Rab35 immunogold TEM imaging and the quantification of immunogold particle localization demonstrates that Rab35 is largely present in compartments at the cell surface. Indeed, open infoldings marked by Rab35 immunogold form in subapical zones near the adherens junctions. It is interesting to note that similar open, tubular structures have also been observed by TEM near electron dense junctions during cell intercalation18. When Rab35 function is compromised, there is a shift of endocytic, dextran uptake away from AP interfaces and a dramatic increase in failed endocytic events. In tissue culture, Rab35 has been shown to function early in endosomal pathways and immediately after vesicle scission, to drive the generation of newborn endosomes. The data suggest a subtle shift in Rab35 function to an earlier time point occurs during early Drosophila morphogenesis, but is broadly consistent with a Rab35-driven function in directing the delivery of membrane from the cell surface to endosomal pathways. However, the results do not exclude an added role for Rab35 at endosomal compartments, as F-actin and Myosin II accumulate intracellularly in Rab35 compromised embryos. This accumulation is at substantially lower levels than occurs at the cell cortex in wild-type embryos, but could contribute to the observed cell intercalation dynamics, especially the ~17% decrease in oscillatory area amplitudes (Jewett, 2017).

    The association of Rab35 compartments with markers for early (Rab5) or recycling (Rab11) endosomes was also examined. While the large majority of Rab35 compartments do not localize with early or recycling endosomes, there was a small fraction of compartments that do (15%). Interestingly, when observed under live imaging conditions, it became apparent that Rab35 compartments do often have transient interactions with endosomal compartments (98%) that occur specifically toward the end of Rab35 lifetimes, consistent with Rab35 feeding endocytosed membrane through a specialized plasma membrane contiguous compartment. These results are consistent with the broader conclusion of this work that endocytic uptake of plasma membrane components is essential to ensuring irreversible changes in interface length that drive cell neighbor exchange. This endocytic uptake could function through two potential mechanisms: (1) E-cadherin adhesion complex removal, or (2) general membrane removal, which would require that individual interfaces possess isolated membrane domains. Embryos that expressed fluorescently labeled Rab35 and E-cadherin did not express E-cadherin at levels sufficient to be resolvable by confocal microscopy, thus making it difficult to distinguish between these models, but this will be an important open question going forward. Previous work has proposed that MyoII function clusters E-cad complexes for endocytosis, while the current results show that Rab35 functions upstream of junctional Myosin. Thus, Rab35 compartments may serve to direct Myosin II clustering of E-cadherin adhesion complexes for endocytosis and junctional remodeling (Jewett, 2017).

    An interesting feature of Rab35 behaviors is the absence of paired compartments at contracting interfaces. Approximately 50% of Rab35 compartments form without the presence of any Rab35 compartments on the opposing side of an interface, and 93% of compartments are present without a matching, synchronous Rab35 compartment. This monopolarity of Rab35 function is the first evidence of asymmetric behaviors across a common, shared interface during early germ-band extension (GBE). As Rab35 compartmental behaviors reflect Myosin II activity, this further suggests that there are likely anisotropic forces on opposing sides of individual interfaces. This anisotropy could drive the generation of shear forces that would cause the dissociation of homophilic extracellular E-cadherin bonds. It is also intriguing that, in terms of area oscillations, Rab35 compartmental formation is best correlated with cycles of area contractions in the opposing cell at a shared interface. This would be consistent with the above model in which changes in apical cell area are transmitted through E-cadherin junctions to a neighboring cell that may be in a differentially tensioned state. This may then again permit slackened plasma membrane to be taken up by Rab35-dependent endocytic processes (Jewett, 2017).

    Meru couples planar cell polarity with apical-basal polarity during asymmetric cell division

    Polarity is a shared feature of most cells. In epithelia, apical-basal polarity often coexists, and sometimes intersects with planar cell polarity (PCP), which orients cells in the epithelial plane. From a limited set of core building blocks (e.g. the Par complexes for apical-basal polarity and the Frizzled/Dishevelled complex for PCP), a diverse array of polarized cells and tissues are generated. This suggests the existence of little-studied tissue-specific factors that rewire the core polarity modules to the appropriate conformation. In Drosophila sensory organ precursors (SOPs), the core PCP components initiate the planar polarization of apical-basal determinants, ensuring asymmetric division into daughter cells of different fates. This study shows that Meru, a RASSF9/RASSF10 homologue, is expressed specifically in SOPs, recruited to the posterior cortex by Frizzled/Dishevelled, and in turn polarizes the apical-basal polarity factor Bazooka (Par3). Thus, Meru belongs to a class of proteins that act cell/tissue-specifically to remodel the core polarity machinery (Banerjee, 2017).

    Polarity is a fundamental feature of most cells and tissues. It is evident both at the level of individual cells and groups of cells (e.g. planar cell polarity (PCP) in epithelia. However, despite the fact that different cell types use a common set of molecules to establish and maintain polarity (Par complexes, Fz-PCP pathway), the organization of polarized cells and cell assemblies varies dramatically across different species and tissues. This implies the existence of factors that act in a cell or tissue-specific manner to modulate/rewire the core polarity machinery into the appropriate organization. Despite many advances in understanding of polarity in unicellular and multicellular contexts, little is known about the identity or function of such factors (Banerjee, 2017).

    An example of polarity remodeling is the process of asymmetric cell division (ACD), where cells need to rearrange their polarity determinants into a machinery capable of asymmetrically segregating cell fate determinants, vesicles and organelles, as well as controlling the orientation of the mitotic spindle. ACDs result in two daughter cells of different fates and occur in numerous cell types and across species. Well-studied examples include budding in Saccharomyces cerevisiae, ACD in the early embryo of Caenorhabditis elegans, or ACD of progenitor cells in the mammalian stratified epidermis and neural stem cells in the mammalian neocortex. In Drosophila melanogaster, the study of germline stem cells, neuroblasts (neural stem cells) and sensory organ precursors (SOPs) has greatly contributed to understanding of the cell biology and molecular mechanisms of ACD (Banerjee, 2017).

    SOPs (or pI cells) divide asymmetrically within the plane of the epithelium into pIIa and pIIb daughter cells. pIIa and pIIb themselves divide asymmetrically to give rise to the different cell types of the external sensory organs (bristles), which are part of the peripheral nervous system and allow the adult fly to sense mechanical or chemical stimuli. Individual SOPs are selected by Notch-dependent lateral inhibition from multicellular clusters of epithelial cells expressing proneural genes (proneural clusters) (Banerjee, 2017).

    The unequal segregation of cell fate determinants (the Notch pathway modulators Numb and Neuralized), which specifies the different fates of the daughter cells, requires their asymmetric localization on one side of the cell cortex prior to mitosis. This is achieved by remodeling the PCP and apical-basal polarity systems in the SOP, and by orienting the spindle relative to the tissue axis. The epithelial sheet that forms the pupal notum (dorsal thorax), where the best-studied SOPs are located, is planar polarized along the anterior-posterior tissue axis, with the transmembrane receptor Frizzled (Fz) and its effector Dishevelled (Dsh) localizing to the posterior side of the cell cortex, while the transmembrane protein Van Gogh (Vang, also known as Strabismus) and its interactor Prickle (Pk) are found anteriorly. The apical-basal polarity determinants central to SOP polarity are the PDZ domain-containing scaffold protein Bazooka (Baz, or Par3), atypical Protein Kinase C (aPKC) and Partitioning defective 6 (Par6), which localize apically in epithelial cells and the basolaterally localized membrane-associated guanylate kinase homologues (MAGUK) protein Discs-large (Dlg). In most epithelial cells, these proteins localize uniformly around the cell cortex, whereas in SOPs they show a striking asymmetric localization during mitosis: the Baz-aPKC-Par6 complex is found at the posterior cell cortex, opposite an anterior complex consisting of Dlg, Partner of Inscuteable (Pins) and the G-protein subunit Gαi. The Fz-Dsh complex provides the spatial information for the Baz-aPKC-Par6 complex, while Vang-Pk positions the Dlg-Pins-Gαi complex (likely through direct interaction between Vang and Dlg). The asymmetric distribution of the polarity determinants then directs the positioning of cell fate determinants at the anterior cell cortex. Additionally, Fz-Dsh and Pins orient the spindle along the anterior-posterior axis by anchoring it on both sides of the cell via Mushroom body defective (Mud, mammalian NuMA) and Dynein (Banerjee, 2017).

    The planar symmetry of the Baz-aPKC-Par6 complex in SOPs is initially broken in interphase via Fz-Dsh, and is independent of the Dlg-Pins-Gαi complex. Once this initial asymmetry is established, the core PCP components become dispensable for Par complex polarization at metaphase due to the mutual antagonism between the opposing polarity complexes, which then maintains asymmetry during cell division. Indeed, Baz is still polarized in fz mutants during mitosis, but losing both pins and fz results in Baz spreading uniformly around the cortex. Crucially, it is unclear how Fz-Dsh can transmit planar information to the Baz-aPKC-Par6 complex in SOPs but not in neighboring epithelial cells. The cell-type dependent coupling between PCP and apical-basal polarity suggests the involvement of unknown SOP-specific factors in this process (Banerjee, 2017).

    The four N-terminal RASSFs (Ras association domain family) in humans (RASSF7-10) have been associated with various forms of cancer, but the exact processes in which these scaffolding proteins act remain mostly elusive. Drosophila RASSF8, the homologue of human RASSF7 and RASSF8, is required for junctional integrity via Baz. Interestingly, human RASSF9 and RASSF10 were found in an interaction network with Par3 (the mammalian Baz homologue) and with several PCP proteins. The Drosophila genes CG13875 and CG32150 are believed to be homologues of human RASSF9 and RASSF10, respectively and remarkably, CG32150 mRNA is highly enriched in SOPs (Banerjee, 2017).

    This study shows that Meru, encoded by CG32150, is an SOP-specific factor, capable of linking PCP and apical-basal polarity. Meru localizes asymmetrically in SOPs based on the polarity information provided by Fz/Dsh, and is able to recruit Baz to the posterior cortex (Banerjee, 2017).

    PCP provides the spatial information for the initial polarization of SOPs at interphase, resulting in the planar polarization of Baz, which is uniformly localized prior to SOP differentiation. How Fz/Dsh communicate with Baz and enable its asymmetric enrichment was unknown. Based on the current results and previous findings, the following model is proposed for the role of Meru in SOP polarization. Upon selection and specification of SOPs, Meru expression is transcriptionally activated by the AS-C transcription factors (Reeves and Posakony, 2005). At interphase, planar-polarized Fz/Dsh recruit Meru to the membrane and hence direct its polarization. Meru in turn positions and asymmetrically enriches Baz, promoting the asymmetry of aPKC-Par6. Upon entry into mitosis, Meru is also required to retain laterally localized Baz, thus supporting the antagonism between the opposing Dlg-Pins-Gαi and Baz-aPKC-Par6 complexes, ultimately enabling the correct positioning of cell fate determinants (Banerjee, 2017).

    The meru mutant cell fate phenotype (bristle duplication or loss) is weaker than the baz loss-of-function phenotype, which results in loss of entire SOPs. This is likely due to two factors: (1) unlike meru mutants, the full baz mutant phenotype is the result of a complete loss of Baz in all cells of the SOP lineage, which is known to cause multiple defects including apoptosis of many sensory organ cells as well as cell fate transformations; (2) since a small amount of Baz is retained at the cortex of some meru mutant cells, it is likely that this residual Baz can still be polarized through the antagonistic activity of Pins at metaphase and thus partially rescues SOP polarization. Indeed, it was observed that reduction of pins or baz levels by RNAi strongly enhanced the meru cell specification phenotype. Conversely, supplying excess levels of Baz in a meru mutant background presumably restores sufficient Baz at the cortex to rescue the meru specification defect, as long as Pins is present to drive asymmetry at mitosis. (Banerjee, 2017).

    While a decrease in cortical Baz can account for the cell specification defects in meru mutants, it does not explain the spindle orientation phenotypee. This abnormal spindle alignment could either be due to a decrease in Fz/Dsh levels/activity, or a decrease in the ability of Dsh to recruit the spindle-tethering factor Mud. No gross abnormalities were detected in Fz levels in meru mutants, though the presence of Fz in all neighboring cells would make it difficult to detect subtle decreases in SOPs. Further work will be required to understand Meru's role in spindle orientation (Banerjee, 2017).

    Analysis of Meru in Drosophila is in agreement with the association of human RASSF9 and RASSF10 with both Par3 and PCP proteins previously reported. However, while the interaction with Dsh is conserved between the fly and human proteins, the transmembrane protein Vangl1 (the mammalian homologue of Vang), rather than its antagonist Fz was recovered in the mammalian proteomic analysis. This could reflect species-specific differences or altered polarity in the transformed human embryonic kidney 293 cells used for the mammalian work. Although Meru (CG32150) was classified as a potential homologue of RASSF10, alignment of the protein sequences showed similar sequence identities for both human RASSF9 (31%) and RASSF10 (26%). Thus, further functional work on Meru, its Drosophila paralogue CG13875, as well as mammalian RASSF9 and RASSF10 is required to understand the evolutionary and functional relationships between these proteins (Banerjee, 2017).

    Little is known about the in vivo functions of either RASSF9 or RASSF10 in other species. Xenopus RASSF10 is prominently expressed in the brain and other neural tissues of tadpoles, potentially indicating a function in neurogenesis, a process where ACDs are known to take place. Interestingly, mouse RASSF9 shows a cell-specific expression in keratinocytes of the skin and loss of RASSF9 results in differentiation defects of the stratified epidermis. Considering that Par3 is required for ACD of basal layer progenitors of the stratified epidermis this raises the exciting prospect that RASSF9 might regulate ACD in the mammalian skin (Banerjee, 2017).

    The polarization of cells and tissues is essential for their architecture and ultimately allows them to fulfill their function. The polarity machinery can be considered as a series of modules that are combined in a cell or tissue-specific manner, and hence requires specific factors that can create a polarity network appropriate to each tissue and cell type. This study has identified Meru as an SOP-specific factor, which is able to link PCP (Fz-Dsh) with apical-basal polarity (Baz). The PCP proteins Vang and Pk promote the positioning of the opposing Dlg-Pins-Gαi complex. Although Vang can directly bind to Dlg, the SOP and neuroblast-specific factor, Banderuola (aka Wide Awake) was recently shown to be required for Dlg localization and could thus constitute a link between the two polarity systems on the opposite side of the cortex (Banerjee, 2017).

    There is increasing evidence that cell-type specific rewiring of the polarity modules may be a widespread phenomenon. For instance, in different parts of the embryonic epidermis, Baz is planar polarized by Rho-kinase or by the Fat-PCP pathway, while in the retina, Vang is responsible for Baz polarization. Apical-basal polarity can also operate upstream of PCP in some systems, as in Drosophila photoreceptor specification, where aPKC restricts Fz activity by inhibitory phosphorylation in a subset of photoreceptor precursors. Thus, tissue-specific factors are likely to operate in a number of different contexts (Banerjee, 2017).

    The interplay between PCP and apical-basal polarity is also evident in other species, as Dishevelled has been reported to promote axon differentiation in rat hippocampal neurons by stabilizing aPKC, while Xenopus Dishevelled is required for Lethal giant larvae (Lgl) basal localization in the ectoderm. Interestingly, both mammalian Par3 and the Vang homologue Vangl2 are required for progenitor cell ACD in the developing mouse neocortex, raising the question as to whether PCP and apical-basal polarity are also connected in mammalian ACDs. It is therefore proposed that tissue-specific factors such as Meru might enable the diversity and plasticity observed across different polarized cells and tissues by rewiring the core polarity systems (Banerjee, 2017).

    The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dach

    Much of the Hippo and planar cell polarity (PCP) signaling mediated by the Drosophila protocadherin Fat depends on its ability to change the subcellular localization, levels and activity of the unconventional myosin Dachs. To better understand this process, a structure-function analysis of Dachs was performed, and this was used to identify a novel and important mediator of Fat and Dachs activities, a Dachs-binding SH3 protein that has been named Dlish (Dachs ligand with SH3s). Dlish was found to be regulated by Fat and Dachs. Dlish also binds Fat and the Dachs regulator Approximated, and Dlish is required for Dachs localization, levels and activity in both wild type and fat mutant tissue. The evidence supports dual roles for Dlish. Dlish tethers Dachs to the subapical cell cortex, an effect partly mediated by the palmitoyltransferase Approximated under the control of Fat. Conversely, Dlish promotes the Fat-mediated degradation of Dachs (Zhang, 2016).

    Heterophilic binding between the giant Drosophila protocadherins Fat and Dachsous (Ds) both limits organ growth, via regulation of the Hippo pathway, and orients planar cell polarity (PCP), through cell-by-cell polarization of Fat, Ds and their downstream effectors. Loss of Fat and, to a lesser extent, Ds, leads to the profound overgrowth of the Drosophila imaginal discs that give rise to adult appendages, and loss of either disorders the polarity of cell divisions, hairs and other morphological features in a variety of Drosophila tissues. But while players and pathways have been defined that are genetically downstream of Fat-Ds binding, only a little is known about the biochemical links between these and their most powerful regulator, the intracellular domain (ICD) of Fat (Zhang, 2016).

    A good deal of the recent work on Fat effectors has focused on the regulation of unconventional type XX myosin Dachs. Dachs is critical first because it provides the only known marker specifically sensitive to changes in the Fat/Ds branches of both the Hippo and PCP pathways. Dachs is normally concentrated in the subapical cell cortex, overlapping subapically-concentrated Fat and Ds. Loss of Fat greatly increases subapical Dachs levels, and polarization of Fat and Ds to opposite cell faces can in turn polarize Dachs to the face with less Fat. Fat thus inhibits or destabilizes subapical Dachs, while Ds may do the opposite. Downstream changes in Hippo or PCP activities do not affect Dachs (Zhang, 2016).

    Dachs changes are also critical because they play a major role downstream of Fat. Dachs binds to and inhibits the activity of the kinase Warts (the Drosophila Lats1/2 ortholog), both reducing Warts levels and changing its conformation. Warts is concentrated in the subapical cell cortex, and thus the increased cortical Dachs of fat mutants should reduce the phosphorylation of Yorkie by Warts, allowing Yorkie to move into the nucleus to drive the transcription of growth-promoting target genes. Indeed, Dachs is necessary for the overgrowth and increased Yorkie target gene expression of fat mutants. Dachs overexpression also causes overgrowth, although more weakly than the overgrowth caused by the loss of Fat, indicating that Dachs is partly sufficient (Zhang, 2016).

    Dachs can also bind to the core PCP pathway component Spiny legs (Sple) and alter its localization, thus influencing PCP in the subset of tissues that rely on Sple. The increased levels of unpolarized Dachs in fat mutants may misdirect Sple, accounting for at least some of the PCP defects; fat mutant hair PCP defects are improved, although not eliminated, by loss of dachs (Zhang, 2016).

    Dachs has not been shown to interact directly with Fat's ICD, and only three other proteins are known to affect Dachs accumulation in the subapical cell cortex, the casein kinase ε Discs overgrown (Dco), Approximated (App) and F-box-like 7 (Fbxl7). Dco may act through Fat itself: Dco binds and phosphorylates the Fat ICD, and loss of Dco function causes strong overgrowth and increases subapical Dachs, similar to loss of Fat (Zhang, 2016).

    App suggests a mechanism in which Fat inhibits the tethering of Dachs to protein complexes in the subapical domain. app mutants decrease subapical Dachs levels and reduce Dachs activity. Thus, like dachs mutants, app mutants reverse the overgrowth and increased Yorkie target gene expression normally observed in fat mutants, and improve hair PCP. App is one of 20 Drosophila DHHC palmitoyltransferases, transmembrane proteins responsible for adding palmitates to cytoplasmic proteins and thereby anchoring them to cell membranes. App is also concentrated in the subapical cell membrane and can bind both Dachs and the Fat ICD. Thus, in the simplest model App palmitoylates or tethers Dachs, concentrating it in the cell cortex, and Fat works in part by sequestering or inhibiting App. However, Dachs is not detectably palmitoylated (Zhang, 2016).

    This study describes the function of a novel Dachs binding protein, and shows that its effects provide strong evidence for both the palmitoylation-dependent and degradation-dependent regulation of Dachs. Structure-function analysis of Dachs found regions required for its normal subapical localization, and this information was used as the basis for a screen for novel Dachs binding partners. A direct binding partner was found for the Dachs C-terminus, the previously uncharacterized SH3 domain protein CG10933, which has been renamed Dachs ligand with SH3s, or Dlish. The activity and subapical concentration of Dlish are regulated by Fat, Dco and Dachs, and Dlish in turn is required for the subapical concentration and full activity of Dachs in both wild type and fat mutant cells. Dlish localization also depends on App; furthermore Dlish binds to and is palmitoylated by App, and palmitoylation can be suppressed by Fat. Loss of Dlish also increases the total levels of Dachs, likely by blocking Fat-mediated destabilization of Dachs. It is proposed that Dlish targets Dachs to subapical protein complexes in part via Fat-regulated, App-mediated palmitoylation. Dlish thereby concentrates Dachs where it can efficiently inhibit subapical Warts, and conversely links Dachs to the machinery for Fat-dependent destabilization (Zhang, 2016).

    The unconventional myosin Dachs is an important effector Fat/Ds-regulated Hippo signaling, as its heightened subapical levels in fat mutants inhibit and destabilize Warts, freeing Yorkie to increase the expression of growth-promoting genes. A structure-function analysis of Dachs was used as a springboard to search for new binding partners that are critical for Dachs localization and function, and have found Dlish (CG10933), a novel SH3 domain protein. Dlish binds directly to the Dachs C-terminus; loss of Dlish disrupts Dachs localization, levels and function: subapical accumulation of Dachs is reduced and cytoplasmic and total levels increase, both in wild type and fat mutant tissue, while activity is lost. Importantly, Dlish is regulated by Fat, as loss of Fat greatly increases Dlish levels in the subapical cell cortex and, like Dachs, Dlish is needed for much of the fat mutant overgrowth (Zhang, 2016).

    Dlish also binds the ICD of Fat and other Fat-binding proteins, including two that likely mediate part of its function: the palmitoyltransferase App and the F-box protein Fbxl7. Thus Dlish provides a new biochemical link from the Fat ICD to Dachs regulation. Evidence indicates that Dlish plays two different and opposing roles (see Models of Fat-mediated regulation of subapical Dachs by Fat-inhibited subapical tethering and Fat-stimulated destabilization). First, it helps tether Dachs in the subapical cell membrane, in part via Fat-regulated, App-dependent palmitoylation, so that Dachs can more efficiently inhibit Warts. Second, it links Dachs to Fat-organized machinery for Dachs destabilization, including Fbxl7, and thus helps reduce Dachs levels (Zhang, 2016).

    Dlish and Dachs cooperate to target or tether the Dlish-Dachs complex, as each is necessary, and to a weaker extent sufficient, for the subapical concentration of the other. The Dachs contribution is likely through tethering the complex to the cortical cytoskeleton, as this study found that loss of the F-Actin-binding myosin head blocks the subapical localization of Dachs. This would agree with recent biochemical analyses that suggest that Dachs has no motor function, acting rather as an F-Actin-binding scaffolding protein (Zhang, 2016).

    The Dlish contribution, on the other hand, depends at least in part on its ability to bind the transmembrane DHHC palmitoyltransferase App. Loss of App and Dlish have very similar effects on Dachs localization and activity. This study found that loss of App disrupts the subapical accumulation of Dlish in vivo and that App can stimulate palmitoylation of Dlish in vitro. Thus, palmitoylation of Dlish likely stimulates membrane association of both Dlish and its binding partner Dachs (Zhang, 2016).

    App also has additional effects on Fat pathway activity. First, App has palmitoyltransferase-independent activity and can co-IP Dachs in vitro. Thus, while palmitoylation of Dlish may mediate some of App's activity, subapical App may simultaneously help localize the Dlish-Dachs complex by physical tethering. And while both palmitoylation and tethering of the Dlish-Dachs complex is likely critical for the fat mutant phenotype, App also has a function that depends on the presence of Fat, as App can bind, palmitoylate and inhibit the activity of Fat's ICD (Zhang, 2016).

    An important question is whether the absence of Fat regulates the App-dependent tethering of the Dlish-Dachs complex. The Fat ICD can complex with both Dlish and App. Dlish and App can bind not only the C-terminal region of the Fat ICD where Fat is palmitoylated, but also the PH and Hippo domains which was shown to played the strongest role in Dachs regulation. An attractive mechanism is that Fat inhibits the interaction between App and Dlish, reducing App's ability to palmitoylate and tether the Dlish-Dachs complex. In the absence of Fat, App and Dlish are freed to tether Dachs, and Dachs now inhibits and destabilizes Warts, causing overgrowth. In support of this model, it was found that overexpression of Fat's ICD in vitro can reduce App-stimulated palmitoylation of Dlish (Zhang, 2016).

    The evidence further indicates that Dlish targets Dachs for Fat-dependent destabilization. Loss of Fat increases not only subapical Dachs, but also total Dachs levels. In the presence of Dlish the increased Dachs remains subapical. Loss of Dlish also increases the total levels of Dachs, but now that increase is cytoplasmic, and much less effective at inhibiting Warts. These Dachs increases are unlikely to have independent causes, as they are not additive; total Dachs levels are similar after loss of Fat, Dlish or both (Zhang, 2016).

    It is proposed that in wild type cells there is a flux of the Dachs-Dlish complex from the cytoplasm to the subapical cell cortex, where a Fat-dependent complex destabilizes Dachs. Normally the tethering effects of Dlish predominate over the Fat-dependent destabilization, and moderate levels of subapical Dachs are maintained. Destabilization is lost without Fat; this combines with Dlish-mediated tethering to increase subapical Dachs. Without Dlish the subapical tethering of Dachs is disrupted, access to Fat-dependent destabilization is lost and the now cytoplasmic Dachs increases. The model thus explains why the excess, largely cytoplasmic Dachs caused by reduced Dlish function is not greatly influenced by the presence or absence of Fat (Zhang, 2016).

    In addition to any effects caused by changing the subcellular localization of Dachs, Dlish may also provide a direct link to the machinery for protein ubiquitination, as Dlish can co-IP with the E3 ubiquitin ligase Fbxl7, as well as the related F-box ubiquitin ligase Slimb. Fbxl7 is particularly intriguing, as it binds to and is regulated by Fat's ICD, and reduces subapical Dachs, perhaps via ubiquitination. Slimb can bind and ubiquitinate Expanded, a subapical regulator of Hippo signaling with links to Fat and Dachs function. But while it was found that loss of Fbxl7 or Slimb increases subapical Dachs and Dlish, these effects are weak, and the large increase in total Dachs levels caused by loss of Dlish or Fat must involve additional partners (Zhang, 2016).

    Mutations in Fat’s closest mammalian homolog Fat4 (FatJ) and its Ds-like ligands strongly disrupt PCP-like processes, and have in humans been associated with the multisystem defects of Hennekam and Van Maldergem syndromes. There has been some debate, however, about whether the mammalian proteins retain direct regulation of Hippo activity. Nonetheless, Fat4 has been linked to Hippo changes in both normal development and tumors, mutations in Fat4 or Dachsous1 change the balance of precursors and mature neurons in the developing neuroepithelium of both humans and mice, and the mouse defect can be reversed by knockdown of the Yki homolog Yap. But the mechanisms underlying these effects are unknown, and Fat4 cannot regulate Hippo signaling in Drosophila (Zhang, 2016).

    It is therefore important to note that while homologs of Dachs and Dlish are found throughout the animal kingdom, they are apparently absent from vertebrates. This suggests that the Dachs-Dlish branch of the Fat-Ds pathway, with its powerful effect on Warts activity, is also lacking. Nonetheless, it has been suggested that Drosophila Fat and Ds can affect Hippo pathway activity in a Dachs-independent manner. It is also clear that Drosophila Fat has Dachs-independent effects on PCP; indeed the N-terminal 'PCP' domain of the Fat ICD that did not affect Dachs in this study is sufficient to improve the PCP defects of fat mutants. These or alternative pathways may still be present in mammals (Zhang, 2016).

    Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension

    Actomyosin contraction generates mechanical forces that influence cell and tissue structure. During convergent extension in Drosophila, the spatially regulated activity of the myosin activator Rho-kinase promotes actomyosin contraction at specific planar cell boundaries to produce polarized cell rearrangement. The mechanisms that direct localized Rho-kinase activity are not well understood. This study shows that Rho GTPase recruits Rho-kinase to adherens junctions and is required for Rho-kinase planar polarity. Shroom, an asymmetrically localized actin- and Rho-kinase-binding protein, amplifies Rho-kinase and myosin II planar polarity and junctional localization downstream of Rho signaling. In Shroom mutants, Rho-kinase and myosin II achieve reduced levels of planar polarity, resulting in decreased junctional tension, a disruption of multicellular rosette formation, and defective convergent extension. These results indicate that Rho GTPase activity is required to establish a planar polarized actomyosin network, and the Shroom actin-binding protein enhances myosin contractility locally to generate robust mechanical forces during axis elongation (Simoes, 2014).

    Rho-kinase is an essential regulator of actomyosin contractility, but the mechanisms that generate Rho-kinase asymmetry to produce spatially regulated forces during development are not well understood. This study shows that Rho GTPase signaling is required for the planar polarized localization of Rho-kinase and myosin II during Drosophila axis elongation. Direct interaction between Rho and Rho-kinase recruits Rho-kinase to adherens junctions but is not sufficient for full Rho-kinase planar polarity, suggesting that other mechanisms amplify the effects of Rho signaling. This study provides evidence that the actin-binding protein Shroom regulates Rho-kinase localization and planar polarized actomyosin contractility to promote sustained cell rearrangements during axis elongation. Shroom is present in a planar polarized distribution at adherens junctions in intercalating cells, consistent with a direct and localized function. Shroom planar polarity requires Rho activity, indicating that Shroom is an effector of Rho signaling. In Shroom mutants, Rho-kinase and myosin II junctional localization and planar polarity initiate normally but fail to be amplified and maintained during axis elongation. Consequently, planar polarized contractile forces and multicellular rosette rearrangements are reduced in Shroom mutants, resulting in decreased convergent extension. These results support a role for Shroom in regulating planar polarized actomyosin contractility and junctional remodeling during convergent extension, expanding the morphogenetic functions of this highly conserved protein beyond its known role in apical constriction (Simoes, 2014).

    The data support a model in which Rho GTPase and Shroom have distinct functions in regulating Rho-kinase localization and planar polarized myosin contractility during convergent extension. Rho GTPase recruits Rho-kinase to adherens junctions and initiates planar polarity, and Shroom plays a modulatory role in enhancing and maintaining planar polarized myosin contractility downstream of Rho signaling. Rho GTPase binds to Rho-kinase and could regulate its localization directly. Rho does not bind to Shroom but may regulate Shroom planar polarity indirectly through its effect on the actin cytoskeleton. Rho-kinase, usually viewed as a downstream effector of Shroom, feeds back to maintain Shroom planar polarity and its own planar polarized localization. Rho-kinase could directly phosphorylate Shroom to reinforce planar cell polarity. Alternatively, Rho-kinase could promote Shroom localization through remodeling of the actin cytoskeleton, as the Shroom actin-binding domain is necessary and sufficient for targeting to planar junctions, and Rho-kinase can phosphorylate known regulators of actin (Simoes, 2014).

    These findings may be relevant to neural tube development in vertebrates, which involves a combination of apical constriction, polarized junctional remodeling, and cell shape changes. Shroom3 is required for neural tube closure in the mouse, frog, and chick, and disrupting the interaction between Shroom and Rho-kinase reduces the number of rosettes in the chick neural plate. Unlike mutants that have disrupted rosette-based movements caused by defects in cell adhesion, the defects in Shroom mutants are likely a result of reduced myosin II activity. Rosette behaviors in Drosophila predominate midway through elongation at stage 8, coinciding with the stage when myosin becomes mislocalized in Shroom mutants. A failure to reinforce actomyosin contractility during elongation in Shroom mutants could selectively disrupt later-onset, higher-order cell rearrangements, with no effect on local neighbor exchange events that are more frequent at earlier stages. Alternatively, rosette formation may require more force, as rosettes form through the contraction of multicellular actomyosin cables that are under a higher level of tension and accumulate more myosin. In Shroom mutants, defects in myosin junctional localization may prevent contractile forces from reaching the levels necessary to produce rosette-based convergent extension movements. It will be interesting to explore whether planar polarized Shroom activity plays a general role in promoting junctional remodeling and enhancing mechanical force generation in processes that require strong actomyosin contractility during development (Simoes, 2014).

    Rho GTPase signaling is an excellent candidate to break planar symmetry, as a small fraction of active Rho protein can trigger rapid and dramatic changes in the actin cytoskeleton. In one model, a subtle increase in Rho activity at AP cell boundaries could provide an instructive cue, guiding planar cell polarity by recruiting Rho-kinase, modifying the actin cytoskeleton, and facilitating the cortical association of the Rho-kinase regulator Shroom. Alternatively, Rho could regulate Rho-kinase planar polarity indirectly through its role in promoting Rho-kinase apical localization. Although it is challenging to visualize a small and highly dynamic population of active Rho protein in vivo, several findings support the idea that localized Rho activity could play an instructive role in planar polarity. First, myosin planar polarity and directional cell rearrangements occur normally at early stages in Shroom mutants, suggesting that other signals are able to generate localized myosin activity. The partial planar asymmetry of a fragment containing the RB domain of Rho-kinase, which is predicted to interact with the active pool of Rho GTPase, suggests that Rho could contribute to this asymmetry. Second, Rho is required for the planar polarized localization of Shroom, raising the possibility that Rho signaling could provide an essential source of Shroom asymmetry. Third, the upstream Rho activator RhoGEF2 in Drosophila and PDZ-RhoGEF in the chick display a subtle planar asymmetry during epithelial bending and elongation. Multiple activators and inhibitors of Rho could act together to generate a spatially localized pattern of Rho activity, as is the case for apical constriction. Notably, although Rho GTPase activity is necessary to establish Rho-kinase and myosin planar polarity, it is not sufficient to maintain their activity at high enough levels to allow sustained force generation and rosette rearrangements in Shroom mutants. It is proposed that Rho promotes the recruitment of Shroom as part of a positive feed-forward mechanism that reinforces planar polarized actomyosin contractility during convergent extension (Simoes, 2014).

    Planar polarized cell rearrangements require the active maintenance of cell polarity in large populations of dynamically moving cells. This study shows that Shroom and Rho GTPase signaling play distinct roles in the establishment and maintenance of polarized actomyosin contractility during convergent extension. The upstream spatial cues that localize actomyosin contractility to specific planar cellular domains are not known. An asymmetry in the organization of the actin cytoskeleton is the earliest evidence of planar polarity in the Drosophila embryo. Distinct actin-binding domains in different Shroom isoforms have been proposed to target Shroom protein and its effectors to different regions of the cell. Moreover, the actin-binding domain is critical for Shroom planar polarity. These findings support the idea that an asymmetry in the actin cytoskeleton is an essential spatial input that regulates the localization of Shroom, the contractile machinery, and ultimately the forces that control cell rearrangement and tissue structure. The upstream spatial cues that generate these asymmetries could involve an asymmetry in Rho signaling, perhaps through the local activation of upstream signaling proteins that regulate Rho GTPase activity. Alternatively, the critical event in the establishment of planar cell polarity could be a Rho-independent reorganization of the actin cytoskeleton that biases the activity of Shroom, Rho-kinase, and myosin, which in turn modify the cytoskeleton to allow robust and sustained cell polarization. Elucidation of the upstream spatial cues that regulate actomyosin localization and dynamics will provide insight into the mechanisms that direct polarized cell behavior (Simoes, 2014).

    Vamana couples fat signaling to the Hippo pathway

    The protocadherins Dachsous and Fat initiate a signaling pathway that controls growth and planar cell polarity by regulating the membrane localization of the atypical myosin Dachs. How Dachs is regulated by Fat signaling has remained unclear. This study identified the vamana gene (CG10933: Dachs ligand with SH3s or Dlish) as playing a crucial role in regulating membrane localization of Dachs and in linking Fat and Dachsous to Dachs regulation. Vamana, an SH3-domain-containing protein, physically associates with and co-localizes with Dachs and promotes its membrane localization. Vamana also associates with the Dachsous intracellular domain and with a region of the Fat intracellular domain that is essential for controlling Hippo signaling and levels of Dachs. Epistasis experiments, structure-function analysis, and physical interaction experiments argue that Fat negatively regulates Dachs in a Vamana-dependent process. These findings establish Vamana as a crucial component of the Dachsous-Fat pathway that transmits Fat signaling by regulating Dachs (Misra, 2016).

    Coordinated growth and morphogenesis is critical to the development of tissues of specific size and shape. Dachsous (Ds)-Fat signaling (henceforth, Fat signaling) controls both growth, through regulation of Hippo signaling, and morphogenesis, through regulation of planar cell polarity (PCP). Fat signaling regulates Hippo signaling and PCP by controlling the membrane localization of the atypical myosin protein Dachs. Many studies have provided important insights into both how Dachs influences Hippo signaling, and how it influences PCP. In contrast, the mechanism by which Fat signaling actually controls Dachs has remained less well understood (Misra, 2016).

    Fat and Ds are atypical cadherins with novel intracellular domains (ICD), which localize to the plasma membrane just apical to the adherens junctions. Fat and Ds bind to each other in a heterophilic manner, and this interaction is modulated by the Golgi-resident kinase, Four-jointed (Fj), which phosphorylates their extracellular domains. This heterophilic binding, together with the graded expression of Ds and Fj, contribute to polarization of Ds and Fat localization within cells. Three different ways by which Fat signaling influences Hippo signaling have been described: Fat signaling influences the membrane localization of Expanded (Ex) , the levels of Wts protein, and the interaction of Wts with its cofactor Mats. Each of these effects on Hippo signaling depends upon Dachs. Fat signaling affects PCP in at least two ways: through an influence on junctional tension, and by regulating the Spiny-legs (Sple) isoform of the prickle locus. Both of these effects also involve Dachs (Misra, 2016).

    Dachs was identified as a key downstream effector of Fat signaling because mutations in dachs completely suppress the overgrowth induced by fat mutations, and partially suppress the PCP defects induced by fat mutations. Dachs localizes to the cell membrane just apical to the adherens junction in a polarized manner; in the developing wing Dachs is localized to the distal sides of the cell, in response to the proximal-distal gradients of Ds and Fj expression. Dachs membrane localization requires a palmitoyltransferase encoded by approximated (app), but how App influences Dachs localization is unknown. In fat or ds mutants increased levels of Dachs are observed at the apical membrane and Dachs is no longer polarized. Forcing Dachs membrane localization by fusing it to Zyxin phenocopies fat mutants. Conversely, overexpression of full-length Fat or even just the Fat intracellular domain (ICD) displaces Dachs from the membrane into the cytoplasm. These and other observations have indicated that Fat regulates growth by modulating the levels of Dachs at apical membranes, and regulates Dachs-dependent PCP by directing Dachs asymmetry (Misra, 2016).

    To understand how Fat functions, several studies have examined the roles of different regions of the Fat ICD. These studies identified two regions that mediate its growth-suppressive function. One, the D region, around amino acids 4,975 to 4,993, makes a modest contribution to Hippo pathway regulation, as when this region is deleted flies are viable but their wings are approximately 30% larger than normal, and also rounder than normal. The D region is required for interaction with the ubiquitin ligase, Fbxl7, which reduces Dachs membrane levels, and mutation of which results in phenotypes similar to deletion of the D region. A second region, which has been referred to as HM, Hpo, or H2, is defined by observations that deletions within this region block the ability of Fat to activate Hippo signaling. Two alleles of fat, fat61 and fatsum, have also been identified that harbor mutations within this region, and are associated with tissue overgrowth comparable with that caused by fat null mutations. However, the mechanism by which this region, which for simplicity is referred to as the H region, regulates the Hippo pathway, and whether it affects Dachs, are unknown (Misra, 2016).

    This study reports the isolation and characterization of the Src homology 3 (SH3)-domain-containing protein encoded by vamana (vam). Loss of vam function decreases growth, whereas overexpression of vam promotes growth. These effects are mediated through regulation of the Hippo pathway, and vam functions genetically downstream of fat, as vam mutations can suppress both growth and PCP phenotypes of fat. Vam localizes to the apical region of epithelial cells in a polarized manner, co-localizing with Dachs, and is required for normal membrane localization of Dachs. Vam physically associates with the carboxy-terminal domain of Dachs and the ICDs of Ds and Fat, and is regulated by the H region of the Fat ICD. These observations identify Vam as a key mediator of signaling from Fat to Dachs (Misra, 2016).

    These studies identified the C-terminal region of Dachs as sufficient to mediate its interaction with Vam. Interestingly, the original dachs allele, described almost a century ago by Bridges and Morgan (1919), is a hypomorphic allele associated with insertion of a blood transposon just upstream of the C-terminal region. Hence this allele likely encodes a truncated protein that lacks the Vam-interaction domain. Consistent with this inference, the vam null phenotype appears similar to the dachs1 phenotype. Thus, a requirement for interaction with Vam can explain the basis for the original identification of dachs (Misra, 2016).

    Vam is evolutionarily conserved among insects but with no close homologs in vertebrates. This is consistent with the fact that Dachs is also only found in insects, and the sequence of the H region is not conserved in vertebrate Fat genes. Nonetheless, Vam is structurally related to a broad family of SH2- and SH3-domain-containing proteins exemplified by CRK, Grb2, Myd88, and NCK. These proteins are referred to as signal-transducing adapter proteins and facilitate formation of protein complexes that play key roles in signal transduction. Vam is composed of just three SH3 domains; this domain organization is most similar to that of the NCK family of adapters, which contain three SH3 domains along with one SH2 domain. The finding that Vam uses both SH3-1 and SH3-3 to interact with Fat and Ds is also reminiscent of NCK family adapters, as they engage effectors using multiple SH3 domains. The Drosophila ortholog of NCK, dreadlocks (dock), interacts with cell-adhesion molecules encoded by hibris, kirre, roughest, and sticks and stones (sns) to regulate actin polymerization and growth cone migration, and functional redundancy of SH3 domains has been observed for dock. Multiple SH3 domains are also commonly observed in proteins involved in vesicular trafficking. The observation that in vam mutants Dachs accumulates in cytoplasmic puncta that could be vesicular structures suggests that Vam might influence the trafficking of Dachs (Misra, 2016).

    Fat and Ds proteins are conserved in vertebrates, where they play important roles in controlling PCP, and have also been proposed to influence Hippo signaling. In the absence of a Dachs homolog, however, it has been unclear how downstream signaling is mediated in vertebrate Ds-Fat pathways. The discovery that Vam links Ds and Fat to downstream signaling raises the possibility that a different member of the signal-transducing adapter proteins could mediate downstream Ds-Fat signaling in vertebrates (Misra, 2016).

    The H region of the Fat ICD plays a crucial role in Hippo pathway regulation. This analysis of Fat ICD truncations revealed that the H region inhibits Vam and Dachs membrane accumulation, the influence of Fat ICD deletions on Hippo signaling correlates with their influence on Vam and Dachs membrane localization, and the H region of Fat can associate with Vam. Together with observations that Vam associates with and regulates Dachs, these observations lead to the inferrence that the H region normally functions to promote Hippo signaling through its association with, and regulation of, Vam. Fat also influences growth and Dachs accumulation through a second region of the ICD, the D region, which interacts with Fbxl7. Because mutation of the D region, or mutations in Fbxl7, have weaker phenotypes than mutations in the H region, the H region appears to play the larger role in Dachs regulation, but nonetheless it is expected that both regions normally act in parallel to regulate membrane levels of Dachs and thus, ultimately, Hippo signaling (Misra, 2016).

    The localization of Vam in different genotypes, together with its physical interactions, suggests models for how Vam regulates Dachs localization. Since Vam and Dachs are reciprocally required for each other's membrane localization, it is inferred that a complex between these two proteins is required for their stable localization to apical junctions, where Dachs regulates PCP (via interactions with Sple) and Hippo signaling (via interactions with Zyxin and Warts). The observations that Fat promotes removal of Vam and Dachs from the subapical membrane, associates with Vam, yet does not visibly co-localize with Vam at apical junctions, suggests that Fat normally removes Vam-Dachs complexes from the subapical membrane. One mechanism by which this might occur is through binding of Fat to Vam, followed by endocytosis of Fat-Vam-Dachs complexes. Alternatively, Fat binding might disrupt Vam-Dachs binding, as these proteins normally do not localize to the membrane in isolation (Misra, 2016).

    It was also observed that Vam can interact with the Ds ICD, and that it does so through the same SH3 domains as it uses to interact with the Fat ICD. This suggests that these interactions are likely to be competitive. In this case, interaction of Vam with the Ds ICD could promote Vam and Dachs membrane localization by opposing the influence of Fat on Vam. For example, by competing with Fat for binding to Vam, Ds could prevent Fat from disrupting Vam-Dachs interactions, or promoting endocytosis of a Vam-Dachs complex. Consistent with this suggestion that the Ds ICD stabilizes Vam and Dachs at apical junctions, Vam, Ds, and Dachs normally all co-localize in puncta on the distal side of wing cells. The ability of Vam to associate with the ICDs of both Fat and Ds could thus provide a simple mechanism explaining how the ICD of Ds seems to promote Dachs membrane localization, whereas the ICD of Fat inhibits it (Misra, 2016).

    Tissue tension and not interphase cell shape determines cell division orientation in the Drosophila follicular epithelium

    This study investigated the cell behaviors that drive morphogenesis of the Drosophila follicular epithelium during expansion and elongation of early-stage egg chambers. Cell division was found not to be required for elongation of the early follicular epithelium, but drives the tissue toward optimal geometric packing. The orientation of cell divisions with respect to the planar tissue axis was examined, and a bias toward the primary direction of tissue expansion was found. However, interphase cell shapes demonstrate the opposite bias. Hertwig's rule, which holds that cell elongation determines division orientation, is therefore broken in this tissue. This observation cannot be explained by the anisotropic activity of the conserved Pins/Mud spindle-orienting machinery, which controls division orientation in the apical-basal axis and planar division orientation in other epithelial tissues. Rather, cortical tension at the apical surface translates into planar division orientation in a manner dependent on Canoe/Afadin, which links actomyosin-to-adherens junctions. These findings demonstrate that division orientation in different axes-apical-basal and planar-is controlled by distinct, independent mechanisms in a proliferating epithelium (Finegan, 2018).

    The results demonstrate that planar cell division orientation in the FE is independent of Pins/Mud. Instead, it relies on tissue tension and Canoe, which links actomyosin-based cortical tension to the cell. These findings show that division orientation occurs independently in the apical-basal and planar tissue axes. Although this study has not investigated the effects of tension modulation or Cno disruption in the embryonic ectoderm, the data suggest that bimodal spindle orientation is common to immature epithelia (Finegan, 2018).

    It is proposed that on the tissue scale, tension is oriented toward the elongation axis, but local anisotropies in the cortex, which could be stochastic or the consequence of cell behaviors, cause local differences in the tension exerted on individual cells. This is consistent with a model whereby division orientation is not dictated by an intrinsic genetic or developmental program, but is instead mediated by extrinsic physical factors. This bias in division orientation is a granular property of the epithelium; the division angle of any one cell cannot be predicted based on tissue-level tension, but a population bias exists such that cells will have bias to divide toward the axis of tissue elongation (Finegan, 2018).

    This study raises the question of whether biphasic spindle orientation is special to the FE, or a broad feature of immature epithelia. In other tissues, cell shape reflects tissue-level tension and cell shape cues direct the orientation of the spindle to establish the cell division angle. It was previously observed that dividing cells can move apically, detaching from the basement membrane. A potential explanation for this movement is that it allows dividing cells to escape the migration force that drives cell shape. This raises the question of how, if not through cell shape, apical tension is communicated to the spindle-orienting machinery in the FE. This question will form the basis of future work (Finegan, 2018).

    Polarized microtubule dynamics directs cell mechanics and coordinates forces during epithelial morphogenesis

    Coordinated rearrangements of cytoskeletal structures are the principal source of forces that govern cell and tissue morphogenesis. However, unlike for actin-based mechanical forces, knowledge about the contribution of forces originating from other cytoskeletal components remains scarce. This study has establish microtubules as central components of cell mechanics during tissue morphogenesis. Individual cells were found to be mechanically autonomous during early Drosophila wing epithelium development. Each cell contains a polarized apical non-centrosomal microtubule cytoskeleton that bears compressive forces, whereby acute elimination of microtubule-based forces leads to cell shortening. It was further established that the Fat planar cell polarity (Ft-PCP) signalling pathway couples microtubules at adherens junctions (AJs) and patterns microtubule-based forces across a tissue via polarized transcellular stability, thus revealing a molecular mechanism bridging single cell and tissue mechanics. Together, these results provide a physical basis to explain how global patterning of microtubules controls cell mechanics to coordinate collective cell behaviour during tissue remodelling. These results also offer alternative paradigms towards the interplay of contractile and protrusive cytoskeletal forces at the single cell and tissue levels (Singh, 2018).

    During development individual cells assemble into complex tissues and organs with specialized forms and functions. Tissue morphogenesis is driven by mechanical forces that are generated by the cytoskeleton within cells and transmitted in a coordinated manner through adhesion molecules across neighbouring cells. The best-studied cytoskeletal component is actin, which, together with other proteins, forms protrusive and contractile arrays, a fundamental constituent of rearrangements on the single cell and tissue levels. Recent work has suggested that microtubules, similar to actin, can also generate forces in cells. However, understanding of the contribution of microtubules to cell mechanics, cell shape changes and force coordination during morphogenesis remains poor. This is mainly due to the fact that many current models describing the mechanical state of tissues during shape changes focus on actomyosin dynamics and/or rely on continuum mechanics. These studies, which are based on coarse-grain observations of cell movements or cell shape changes, reveal only part of the physical mechanisms that drive morphogenesis and do not directly investigate the physicomechanical context of tissue remodelling. To understand the relationship between cell mechanics, force patterning and molecular structure, this study investigated the mechanical properties of microtubules at high spatiotemporal resolution using wing development in Drosophila melanogaster as a paradigm (Singh, 2018).

    During pupal wing development, non-centrosomal microtubules form an apical array of parallel microtubule bundles that are globally aligned along the proximal-distal (P-D) axis. Patterning of the microtubule cytoskeleton depends on the Ft-PCP signalling pathway and occurs during the early phase of wing reshaping (that is, between 14 and 18 h after puparium formation, or APF). This patterning is associated with extensive changes in cell shape, cell divisions and cell-cell rearrangements. In the Drosophila wing, the Ft-PCP pathway further orients cell elongation and cell divisions along the P-D axis to induce wing tissue elongation. Intriguingly, rescue of the Hippo pathway in Ft-PCP mutant animals, in which microtubule alignment is impaired, aberrant development results in perturbed cell elongation and an abnormal rounded wing shape, suggesting that there is an interdependence between these events. Therefore this study explored the possibility that microtubule-based cell mechanics control cell and tissue shape during early wing development between 16 and 18 h APF (Singh, 2018).

    Tissue remodelling is driven by intrinsic and extrinsic mechanisms, and it has previously been shown that extrinsic mechanical forces act during the late phase of wing reshaping (starting 18 h APF). These forces are generated by hinge contraction of the wing that is attached to the cuticle on the distal side. This study evaluated the mechanical autonomy of individual cells before hinge contraction at an earlier developmental stage (that is, between 16 and 18 h APF). This was done by isolating a single cell (or a small patch of cells) using a single-pulse multipoint procedure to cut AJs, thus mechanically uncoupling individual cells from their surrounding. Strikingly, the shape of individual isolated cells did not change significantly upon laser ablation at 17-18 h APF, when cells in the wing are already elongated. The same result was obtained when patches of cells were isolated. Additional analyses of the Feret's diameter before and after ablation showed a small isotropic decrease in cell size, providing evidence that at this early stage, individual cells are not influenced by the neighbouring cells or by tissue-scale forces in a polarized fashion. Consistently, analysis of animals expressing a mutant form of dumpy protein, an extracellular matrix protein associated with tissue remodelling at later developmental stages, showed no substantial differences in wing shape compared to wild-type wings at 18 h APF. Together, these experiments argue that unlike later stages, cell autonomous forces are the major drivers of initial cell shape changes between 16 and 18 h APF (Singh, 2018).

    To identify the molecular mechanism underlying cell autonomous shape formation, the distribution and dynamics of two cytoskeletal force-generators were investigated: microtubules and non-muscle myosin II (MyoII) as a component of the actomyosin cytoskeleton. MyoII was detected at the apical cell cortex at the level of AJs. A subsequent analysis of the signal distribution within single cells revealed a planar polarized distribution of MyoII along the P-D axis, which correlated with increased tension along the same junctions. As MyoII provides contractile forces, this should result in P-D junctional shortening upon laser ablation. However, this is inconsistent with the current ablation experiments, suggesting that there is an opposing force present. Interestingly, staining of microtubules showed planar polarized apical microtubules along the P-D axis at the level of AJs. Microtubules are the stiffest cytoskeletal filaments, with a persistence length on the order of millimetres. Microtubules are therefore well suited to balance the tension generated by actomyosin contraction. Consistently, the distribution of microtubules and MyoII in mechanically isolated cells remain polarized. In addition, microtubule and MyoII polarity was preserved in dumpy mutant wings at 18 h APF, indicating that they are polarized in a cell autonomous fashion. The possible role of the atypical myosin Dachs, a downstream component of the Fat signalling pathway, was also analyzed. Dachs mutant wings showed no change in cell elongation or microtubule polarity, which is consistent with recent work reporting that recombinant Dachs does not have ATPase activity and can therefore not function as a molecular motor. Together, these observations argue that planar polarized microtubules may balance actomyosin tensional forces that pull on P-D junctions and stabilize cell shape (Singh, 2018).

    To validate this hypothesis, and to elucidate the dynamic and functional role of microtubules in cell mechanics, their properties were investigated during wing development. Live cell imaging of EOS-α-tubulin (EOS-Tub) showed that microtubules were not static but engaged in complex and dynamic rearrangements. An analysis of microtubule straightness showed that in wing cells, virtually all microtubules along the P-D axis were bent, consistently undergoing short wavelength buckling (~3 μm) near the cell cortex. It was further observed that growing microtubules remain straight and only start to buckle after they reach the cell cortex, exhibiting local short wavelength buckling near these sites. This result indicates that microtubule polymerization can generate considerable compressive forces to induce microtubule buckling (Singh, 2018).

    Next, whether buckling of microtubules in Drosophila wing epithelium is indeed a result of forces acting on microtubules was also investigated, as suggested by the current experiments and in vitro studies, or whether the cellular environment yields more flexible microtubules. This is important, as buckling of flexible microtubules would rule out a role in balancing actomyosin contractility. To probe the forces of single microtubule filaments in vivo, individual microtubules were cut by laser ablation and the subsequent relaxation was monitored using live imaging. Previously curved microtubules rapidly straighten out, thus verifying that microtubules are indeed loaded with compressive forces. Finally, it was also observed that local ablation of microtubules triggers a rapid translocation of the adjacent junction. This finding supports the idea that non-centrosomal microtubules continuously generate pushing forces via polymerization that may then be stored as compressive forces in a polarized fashion to balance contractile forces generated by junctional actomyosin (Singh, 2018).

    How are microtubules polarized along the P-D axis? While the molecular mechanism has remained elusive, previous work has established that the Ft-PCP signalling pathway aligns the apical microtubule network along the P-D axis by regulating association sites of microtubules with AJs. Considering the observed stability of aligned microtubules, whether directional differences in microtubule dynamics could serve as a mechanism for the planar polarization of microtubules was tested. Monitoring of EB1 tagged with green fluorescent protein (EB1-GFP) revealed two populations of microtubule-plus ends: fast growing microtubules with a growth velocity of 24.43 ± 0.43 7mi;m min-1 (mean ± s.e.m.), and slow growing microtubules with a velocity of 17.06 ± 0.26 7mi;m min-1. A further analysis showed that the microtubule growth rates depended on relative localization within cells as well as the growth angle relative to the P-D axis. Microtubule growth rates in the cell interior were higher compared to the cell cortex. Similarly, microtubules along the P-D axis grew faster than microtubules growing perpendicular to the P-D axis along the A-P axis, establishing a spatial gradient in microtubule growth velocity. The lower growth rate along the A-P axis close to the cell periphery suggests that there is more frequent pausing and switching between polymerization and depolymerization of microtubules, thus indicating a decreased stability of A-P oriented microtubules (Singh, 2018).

    It was reasoned that over time, such differences in dynamics and stability may result in predominantly P-D aligned microtubules. To test this hypothesis, the cortical residence time was analyzed of microtubules as a function of their angle with respect to the P-D axis. Intriguingly, it found that microtubules that interact with the P-D cell cortex have a longer lifetime than microtubules interacting with the A-P cortex. Upon closer inspection, dynamic cycles of short-lived interactions of microtubules with A-P junctions were noted followed by depolymerization. Importantly, A-P oriented microtubules do not show buckling behaviour, which is in contrast to P-D oriented microtubules, but rather undergo catastrophe soon after interaction with A-P oriented cell junctions. This result suggests that microtubule-plus ends are less stable at these sites and thus cannot sustain long-lasting interactions with the cell cortex, which are required to generate compressive forces. Building on these observations, in silico probing was performed to see whether the angular difference in lifetime may indeed be sufficient for microtubule polarization. Assuming a random orientation for de novo formed microtubules, the lifetime of each microtubule was defined as a function of the angle with a maximal lifetime along the P-D axis. Upon expiration, individual microtubules were re-introduced into the system at random angles, therefore keeping the total number of microtubules constant. Consistent with the in vivo observations, the simulation reached a steady-state at which a constant fraction of microtubules polarized along the P-D axis. Taken together, these observations point to a mechanism whereby microtubule stability regulates the planar alignment of the microtubule cytoskeleton along the P-D axis, which in turn directs cell mechanics along this axis. These data place directional microtubule stability upstream of proposed mechanisms of how cell shape influences microtubule alignment. Furthermore, these results are consistent with previous findings that microtubule association with P-D oriented AJs during the initial stage of wing development depends on Ft-PCP signalling (Singh, 2018).

    Having established that planar polarized microtubule-based forces shape single cells, their mechanical coupling and integration into tissue-level mechanics were investigated. In a first round of experiments, transcellular coupling of microtubules were investigated on the ultrastructural level using transmission electron microscopy (TEM). In agreement with previous work, AJs were juxtaposed in neighbouring cells associated with microtubule filaments that span across cells in wild-type wings, forming supracellular cables analogous to myosin cables. Notably, no such association was observed in ftl(2)fdd1 / ftl(2)fd dGC13 (ft d) and ftl2 fd/ftGRV;ActP-Gal4/UAS-FtΔECDΔN-1 (N1) mutant wings, in which microtubules are randomly oriented in wing cells, therefore providing structural support for the Ft-PCP-dependent stabilization of microtubule-based forces at P-D oriented AJs. Consistently, ft mutant clones showed a fragmented microtubule cytoskeleton, arguing that there is Ft-PCP-dependent stabilization of microtubules via coupling at AJs (Singh, 2018).

    To further validate the role of polarized transcellular microtubule stability in tissue mechanics and organization, tissue shape changes were observed upon acute perturbation of microtubule-based forces. To control microtubule dynamics in a precise spatial and temporal manner, he recently developed photostatin (PST1)35, a photo-switchable analogue of combretastatin A-4 (CA4)36 was used. The drug was applied to directly test the requirement of microtubules for cell shape maintenance. Notably, it was found that the exposed wing area contracted along the P-D axis upon microtubule inhibition. Quantitative cell shape analysis showed a small but significant reduction in the elongation index (EI) in selective regions where the drug was activated, arguing that polarized tissue stabilization is via microtubule-based forces. Finally, overexpression of the microtubule-severing protein Spastin increased cell shape heterogeneity. These results are consistent with the hypothesis that an intact polarized microtubule cytoskeleton is not only required for the maintenance of anisotropic cell shape but also critically involved in shaping the whole tissue during morphogenesis via polarized transcellular force stability (Singh, 2018).

    Understanding the role of microtubules during animal development has so far been limited, especially because of a shortage of methods suitable to demonstrate causality in vivo. Taking advantage of complementary genetic, chemical, numerical and microscopy approaches, these experiments unveil polarized microtubule-based compressive forces as an alternative principle for stabilizing and maintaining cell and tissue shape during morphogenesis. Alignment of microtubules along the P-D axis was found to be based on increased longevity and polymerization of microtubules interacting with P-D oriented AJs compared to non-polarized microtubules. The result of this microtubule patterning along the P-D axis is an asymmetric distribution of protruding forces, which are stored in a polarized fashion via compressive loads on microtubules. Considering that actomyosin and microtubules are both planar polarized, it is plausible to envision that the observed compressive load on microtubules plays an active role in balancing actin-based contractile forces, resulting in the cell mechanical autonomy observed in the laser ablation experiments. Intriguingly, it was recently shown that acetylation of microtubules increases their mechanical resistance and that microtubules undergo self-repair upon damage. These important features support the role of the microtubule cytoskeleton as a site of long-term compressive force storage. Finally, evidence is provided that planar polarized microtubules are coupled at AJs across individual cells, bridging forces on the tissue level via polarized transcellular stability. Although the molecular identity remains elusive, the data suggests an involvement of AJ-associated proteins organized by the Ft-PCP pathway in this process (Singh, 2018).

    Collectively, this work provides evidence that PCP-based planar patterning of the microtubule cytoskeleton not only results in polarized cell-autonomous forces but also coordinates global force patterning during tissue morphogenesis. The proposed mechanism establishes the Ft-PCP pathway at the onset of cell and wing elongation, before shape changes, due to extrinsic mechanical forces. Consistently, in a Ft-PCP mutant, in which initial elongation fails, consecutive remodelling by extrinsic tensile forces cannot rescue these length defects, therefore leading to shorter and rounder adult wings. Considering that the Ft-PCP signalling pathway controls a variety of dynamic cell population in vertebrates, the microtubule-based mechanism described in this study is likely to be physiologically relevant beyond wing development (Singh, 2018).

    Epithelial cells release adenosine to promote local TNF production in response to polarity disruption

    Disruption of epithelial integrity contributes to chronic inflammatory disorders through persistent activation of stress signalling. This study uncovered a mechanism whereby disruption of apico-basal polarity promotes stress signalling. Depletion of Scribbled (Scrib), a baso-lateral determinant, causes epithelial cells to release adenosine through equilibrative channels into the extracellular space. Autocrine activation of the adenosine receptor leads to transcriptional upregulation of TNF, which in turn boosts the activity of JNK signalling. Thus, disruption of cell polarity feeds into a well-established stress pathway through the intermediary of an adenosine signalling branch. Although this regulatory input could help ensuring an effective response to acute polarity stress, it is suggested that it becomes deleterious in situations of low-grade chronic disruption by provoking a private inflammatory-like TNF-driven response within the polarity-deficient epithelium (Poernbacher, 2018).

    This study has shown that adenosine acts as a warning signal in response to sub-apoptotic perturbation of polarity in epithelial cells. Adenosine released in the extracellular space triggers the local production of TNF, which in turn activates JNK, a well-established stress mediator. Because extracellular adenosine (e-Ado) is short-lived, it is unlikely to act at a long range. In fact, it is suggested that a large number of contiguous cells need to be disrupted for them to collectively release adenosine at a level that is sufficient to elicit a response. This community effect would ensure that a stress response is only mounted when a significant amount of tissue is affected, as would occur in a situation of low-grade but widespread stress. In this model of perturbation of cell polarity, adenosine promotes TNF production within the affected epithelium itself, eliciting a private inflammatory-like response without the involvement of immune cells. Such a local, e-Ado-stimulated, response could be an important contributor to pathologies associated with chronic epithelial damage. The above scenario is in contrast with the orthodox view that it is the activated monocytes/macrophages that normally produce TNF and that this is inhibited by adenosine. It is speculated that the type of response elicited by tissue disruption depends on the severity of insult or type of pathology, a consideration that must be kept in mind while designing therapeutic strategies (Poernbacher, 2018).

    Rapid disruption of Dishevelled activity uncovers an intercellular role in maintenance of Prickle in core planar polarity protein complexes

    Planar polarity, the coordinated polarization of cells in the plane of a tissue, is important for normal tissue development and function. Proteins of the core planar polarity pathway become asymmetrically localized at the junctions between cells to form intercellular complexes that coordinate planar polarity between cell neighbors. This study combined tools to rapidly disrupt the activity of the core planar polarity protein Dishevelled, with quantitative measurements of protein dynamics and levels, and mosaic analysis, to investigate Dishevelled function in maintenance of planar polarity. Mechanistic insight is provided into the hierarchical relationship of Dishevelled with other members of the core planar polarity complex. Notably, it was shown that removal of Dishevelled in one cell causes rapid release of Prickle into the cytoplasm in the neighboring cell. This release of Prickle generates a self-propagating wave of planar polarity complex destabilization across the tissue. Thus, Dishevelled actively maintains complex integrity across intercellular junctions (Ressurreicao, 2018).

    Defining the roles of individual components in signaling networks can be a significant challenge. This is particularly so when the network is not a simple linear pathway, if components play more than one role in the cell (pleiotropy), and if there is 'adaptation' such that over time, the pathway adjusts to the effects of experimental manipulations. However, in many cases, these difficulties can be bypassed through methods that rapidly alter protein activities (Ressurreicao, 2018).

    Consistent with this, it has been recently shown that spatiotemporal activation of gene expression is an effective tool for dissecting feedback interactions during planar polarity patterning in the Drosophila wing (Warrington, 2017). The current work used methods for rapidly disrupting protein function to probe the role of the Dsh protein in planar polarity. The main finding is that Dsh regulates Pk membrane association in core planar polarity complexes, acting cell-non-autonomously to prevent its relocalization to the cytoplasm. Notably, this role for Dsh is specifically revealed when Dsh is rapidly depleted from core protein complexes but not in the simple dsh loss-of-function situation, when instead a largely mobile fraction of Pk is seen associated with cell junctions. It is speculated that a Dsh-dependent signal normally passes between cells via the core protein complexes to maintain Pk recruitment. When this signal is disrupted, Pk rapidly leaves the junctions. However, in the long-term absence of Dsh, Pk can return to cell junctions, where it is speculated to weakly associates with cell membranes by virtue of being prenylated (Ressurreicao, 2018).

    What might be the nature of the intercellular signal from Dsh to Pk? It is suggested that it passes via the Fmi homodimers that form between cells, as numerous lines of evidence indicate these are essential for cell-cell signaling in planar polarity. A simple possibility is that Dsh binding to Fz induces a conformational change in the complex that passes via the Fmi homodimers to alter the conformation of bound Stbm, thus creating a Pk binding site. Such molecular signaling events mediated by allostery are common features of ligand-receptor interactions. In support of the model that the Dsh signal is transduced via a change in Fz behavior, it is noted that following Dsh disruption, Fz distribution and stability is altered faster than those of Fmi and Stbm (Ressurreicao, 2018).

    A related mechanism is suggested by recent observations that the core proteins incorporate into intercellular complexes non-stoichiometrically and that all components contribute to complex stability. These findings are interpreted as suggesting that core complex stability is dependent on a phase transition mediated by multivalent interactions between the core proteins, with Dsh playing a critical role. The rapid destabilization of Fz after Dsh depletion may be a result of loss of multivalent binding interactions mediated between the different domains of Dsh, as also occurs in Wnt signalosome assembly, over time leading to a reduction in multivalent binding interactions between Fmi and Stbm. This would thus produce a gradual 'loosening' of the complex that would result in release of Pk from its binding interactions with Fmi and Stbm. Some support for this model comes from the observation that super-resolution microscopy immediately after Dsh disruption shows subtle changes in the size and distribution of Fmi in junctional puncta (Ressurreicao, 2018).

    A striking observation is that if Dsh fails to maintain Pk recruitment in intercellular complexes, free Pk can destabilize Dsh in the same cell, leading to release of Pk in the neighboring cell and a wave of core planar polarity complex destabilization. This observation both supports previous work showing that physiological levels of Pk can effectively destabilize Fz-Dsh complexes at cell junctions (Warrington, 2017) and highlights the importance of sequestering Pk into 'proximal' complexes, to prevent unregulated activity of Pk within the cell. It is proposed that Fmi mediates essential intercellular signals from Fz-Dsh in 'distal' core complexes that actively maintain Pk in 'proximal' core protein complexes. In turn, this promotes the effective segregation of distal and proximal complexes to opposite cell ends, driven in part by destabilizing feedback interactions between Pk and Dsh in the same cell (Ressurreicao, 2018).

    The Dachsous/Fat/Four-Jointed pathway directs the uniform axial orientation of epithelial cells in the Drosophila abdomen

    The achievement of the final form of an individual requires not only the control of cell size and differentiation but also integrative directional cues to instruct cell movements, positions, and orientations. In Drosophila, the adult epidermis of the abdomen is created de novo by histoblasts. As these expand and fuse, they uniformly orient along the anteroposterior axis. The Dachsous/Fat/Four-jointed (Ds/Ft/Fj) pathway is key for their alignment. The refinement of the tissue-wide expression of the atypical cadherins Ds and Ft result in their polarization and directional adhesiveness. Mechanistically, the axially oriented changes in histoblasts respond to the redesign of the epithelial field. It is suggested that the role of Ds/Ft/Fj in long-range oriented cell alignment is a general function and that the regulation of the expression of its components will be crucial in other morphogenetic models or during tissue repair (Mangione, 2018).

    PCP pathways act as key coordinators in the makeover of planar epithelial tissues during development by modulating adhesive interactions and mechanical forces. However, the regulatory means that these pathways use to direct the topographical organization of epithelial cells are far from being clear. By applying in vivo analyses to the morphogenesis of the adult abdominal epidermis of Drosophila, this study has characterized a new mechanism for the stepwise long-range cobblestone organization of the tissue. The organization of the abdominal epithelial landscape was found to be the result of an axially oriented adhesiveness mediated by the Ds/Ft/Fj pathway. The directional cues dictated by this pathway put the epithelial cells on the right track, orienting their otherwise changing shapes along the A/P axis (Mangione, 2018).

    Global tissue changes may involve many different activities: coordinated cell-cell rearrangements triggering tissue reorientation or convergent extension,or spatially controlled proliferation and growth, division orientation, and death. The uniform axially oriented alignment of histoblasts emerges in a precise spatiotemporal manner through coordinated changes in cell shape orientation. Histoblasts constitute a highly proliferative tissue with much room for expansion. In this scenario, which is very different from that of epithelial tissues constrained in their dimensions like the fly notum, to reach uniformity in cell alignment orientations, changes in cell shape and area would be preferred over cell intercalations. When the expressions of ds, ft, or fj are affected, the relative orientations of cell alignments are severely disturbed without alterations in tissue differentiation (Mangione, 2018).

    It is not known which positional cues the Ds/Ft/Fj pathway interprets to dictate the stereotyped uniform anisotropic polarization of the abdominal epidermis along the A/P axis. Some tips may come from analyses on the regulation of the components of the core Fz-PCP system. This system is a required partner of the Ds/Ft/Fj pathway for proper planar polarity acquisition. Both pathways rely on primary long-range global cues to coordinate short-range cell polarity. During the development of the eye and wing in Drosophila, the secreted factor Wingless (Wg/Wnt1) modulates intercellular interactions in both systems. In the Ds/Ft/Fj pathway, Ds and Ft form heterodimers, whereas in the Fz-PCP system, there are Fz-Vang heterodimers and Stan-Stan homodimers. In the eye and the wing discs, Wg binds to Fz, which affects Fz-Vang interactions and the activity of the Fz-PCP system. As a result, cells orient toward the source of Wnt expression (Wg and Wnt4) at the compartment margins. Wg/Wnt4 could be playing an equivalent role during the axial uniform alignment of oriented cells in the abdomen. While Wg specifies the tergite and sternite cell fates, how it could regulate the graded expression of ds, ft, or fj or influence the uniform axial orientation of histoblasts remains poorly explored (Mangione, 2018).

    It is known that differential adhesive properties between neighboring cells prevent intermingling, as they tend to minimize their contacts. In clones, this leads to smooth borders. Major differences were found in roughness, perimeter, and, to a lesser extent, roundness in mutant clones for members of the Ds/Ft/Fj pathway. These differences strongly support a role for the Ds/Ft/Fj pathway and, in particular, the opposing graded expressions of the atypical cadherins Fat and Ds, in generating directional information at cell contacts. Mutant clones generate planar conflicts between cells with different adhesive properties and smooth borders at specific edges, and the directional information is lost (Mangione, 2018).

    The evolving functional pattern delineated by the Ds/Ft/Fj pathway arises as an elegant and efficient way to dictate directional order across developmental fields. Several pieces of evidence point to it as a key element modulating similar processes in different organisms. Mitral valve prolapse (MVP) is a common cardiac valve disease, the genetic etiology of which has remained elusive. Recently, it has been shown that MVP could be traced back, both in mice and fish models, to developmental errors in valve morphogenesis. The epicardial-derived cells fail to uniformly align into the posterior leaflet, and this failure correlates to a missense mutation in the DCHS1 gene, the human homolog of ds. Similarly, the establishment of polarized arrays of aligned chondrocytes in the zebrafish developing pharyngeal arch requires Fat3 and Dchs2 cadherins. During postnatal stages in mammals, hair follicles progressively get in line, both locally and globally, precisely with the A/P axis. This process of postnatal refinement has been suggested, although not proved, to be the result of instructive functions originating from the Ds/Ft/Fj pathway. Whether vertebrate Ds/Ft/Fj signaling is essential to propagate polarity at a distance, is linked to molecular gradients, or may interact with other polarizing signals remains unknown (Mangione, 2018).

    The subcellular polarization of the atypical myosin Dachs (D) in response to the Ds/Ft/Fj pathway appears to be uncoupled from the uniform orientation of cell alignments; D polarity is reoriented but sustained in ds, and its loss did not affect cell alignments uniformity. These data led to a hypothesis that a bias in contractility mediated by D at the cell cortex may be not critical for uniform cell orientation. Moreover, contractile anisotropy as a factor directing the axial uniformity of histoblasts is unsupported by the observed isotropic distribution of vinculin at cell vertices. In this scenario, asymmetric adhesiveness through heterodimeric interactions between Ds and Ft appears to be a more plausible element directing the uniform orientation of histoblasts (Mangione, 2018).

    Assuming that cells and tissues tend to minimize their surface free energy, contacts through adhesion molecules and contractile activities at the cell cortex would be key determinants of cell and tissue shape. Adhesiveness will promote cells to spread their shared surface, while contractility will counterbalance the adhesive forces. Differential adhesive properties within histoblasts would introduce anisotropic tension affecting cell-cell contacts and the capacity to coordinately orient shape changes in the tissue plane. This anisotropic tension, which is revealed by laser microsurgery, is lost in ds mutants. Anisotropic tension is also uncovered in clones, in which contact angles and lengths between histoblasts adjust to their conflicting genotypes and their relative location. Differential adhesiveness at cell junctions would have direct input into surface tension. Tensile patterns rather than cell positions would therefore play instructive roles in the acquisition of uniform order. At any given point in time, they will reflect the recent developmental history of the tissue. Along this line, during the expansion and remodeling of the histoblasts, the expression pattern of Ft modulated by Ds and fj evolves into an A/P gradient spanning whole compartments. This expression refinement will result in the spreading throughout the epithelium of a counterbalanced adhesion share between Ft and Ds that will delineate the axially oriented surface tension landscape that will instruct uniform cell alignments (Mangione, 2018).

    Will the final arrangement of the cells be physically stable (minimal energy) upon completion or will a secondary event be necessary to stabilize it? In Caenorhabditis elegans, the epidermal cells elongate during development and subsequently attach to the cuticle to fix their shape. Thus, the collagenous exoskeleton secreted by the apical surface of the epidermis seems to be indispensable. The histoblasts use their apical surface to attach to the overlaying pupal cuticle very early on. These contacts are very dynamic during the period of expansion and become stabilized by the end of tissue remodeling. Whether they fulfill a hardening role on the tissue landscape is an open question (Mangione, 2018).

    The role this study has uncovered for the Ds/Ft/Fj pathway implementing the uniform orientation of the alignment of histoblasts does not relate to any developmental function in patterning, cell specification, and/or differentiation. The directional evolution of the expression of the different Ds/Ft/Fj pathway elements sets a spatially and temporally controlled directional adhesive partnership between Ds and Ft. This provides the basis for the establishment of a dynamic adhesiveness pattern unfolding over time that directs cell shape changes, ultimately guiding the uniform alignment of the epithelial cells across the tissue (Mangione, 2018).

    Tissue flow regulates planar cell polarity independently of the Frizzled core pathway

    Planar cell polarity (PCP) regulates the orientation of external structures. A core group of proteins that includes Frizzled forms the heart of the PCP regulatory system. Other PCP mechanisms that are independent of the core group likely exist, but their underlying mechanisms are elusive. This study shows that tissue flow is a mechanism governing core group-independent PCP on the Drosophila notum. Loss of core group function only slightly affects bristle orientation in the adult central notum. This near-normal PCP results from tissue flow-mediated rescue of random bristle orientation during the pupal stage. Manipulation studies suggest that tissue flow can orient bristles in the opposite direction to the flow. This process is independent of the core group and implies that the apical extracellular matrix functions like a "comb" to align bristles. These results reveal the significance of cooperation between tissue dynamics and extracellular substances in PCP establishment (Ayukawa, 2022).

    Prickle isoforms determine handedness of helical morphogenesis

    Subcellular asymmetry directed by the planar cell polarity (PCP) signaling pathway orients numerous morphogenetic events in both invertebrates and vertebrates. This paper describes a morphogenetic movement in which the intertwined socket and shaft cells of the Drosophila anterior wing margin mechanosensory bristles undergo PCP-directed apical rotation, inducing twisting that results in a helical structure of defined chirality. The Frizzled/Vang PCP signaling module coordinates polarity among and between bristles and surrounding cells to direct this rotation. Furthermore, it was shown that dynamic interplay between two isoforms of the Prickle protein determines right- or left-handed bristle morphogenesis. Evidence is provided that, Frizzled/Vang signaling couples to the Fat/Dachsous PCP directional signal in opposite directions depending on whether Pk(pk) or Pk(sple) predominates. Dynamic interplay between Pk isoforms is likely to be an important determinant of PCP outcomes in diverse contexts. Similar mechanisms may orient other lateralizing morphogenetic processes (Cho, 2020).

    Work in Drosophila indicates that at least two molecular modules contribute to PCP signaling. The core module acts both to amplify molecular asymmetry, and to coordinate polarization between neighboring cells, producing a local alignment of polarity. Proteins in the core module, including the serpentine protein Frizzled (Fz), the seven-pass atypical cadherin Flamingo (Fmi; a.k.a. Starry night), the 4-pass protein Van Gogh (Vang; a.k.a. Strabismus), and the cytosolic/peripheral membrane proteins Dishevelled (Dsh), Diego (Dgo), and the PET/Lim domain protein Prickle (Pk) adopt asymmetric subcellular localizations that predict the morphological polarity pattern such as hairs in the fly wing. These proteins communicate at cell boundaries, recruiting one group to the distal side of cells, and the other to the proximal side, through the function of an incompletely understood feedback mechanism, thereby aligning the polarity of adjacent cells. A global module serves to provide directional information to the core module by converting tissue level expression gradients to asymmetric subcellular Fat (Ft) / Dachsous (Ds) heterodimer localization. The atypical cadherins Ft and Ds form heterodimers which may orient in either of two directions at cell-cell junctions. The Golgi resident protein Four-jointed (Fj) acts on both Ft and Ds as an ectokinase to make Ft a stronger ligand, and Ds a weaker ligand, for the other. Graded Fj and Ds expression therefore result in the conversion of transcriptional gradients to subcellular gradients, producing a larger fraction of Ft-Ds heterodimers in one orientation relative to the other. Other less well defined sources of global directional information appear to act in partially overlapping, tissue dependent ways (Wu et al., 2013; Sharp and Axelrod, 2016) (Cho, 2020).

    Various Drosophila tissues depend primarily on either of two isoforms of Pk, Prickleprickle (Pkpk) and Pricklespiny-legs (Pksple). These isoforms have been proposed to determine the direction in which core PCP signaling responds to directional information provided by the Ft/Ds/Fj system. Pksple binds directly to Ds, orienting Pksple-dependent core signaling with respect to the Ds and Fj gradients, while Pkpk-dependent core signaling has been proposed to couple less directly through a mechanism in which the Ft/Ds/Fj module directs the polarity of an apical microtubule cytoskeleton on which vesicles containing core proteins Fz, Dsh and Fmi undergo directionally biased trafficking. Others, however, have argued that Pkpk-dependent core signaling is instead uncoupled from the Ft/Ds/Fj signal (Cho, 2020).

    The most intensively studied morphogenetic responses to PCP signaling in Drosophila occur in epithelia such as wing and abdomen, in which cellular projections called trichomes (hairs) grow in a polarized fashion from the apical surface, and in ommatidia of the eye, in which photoreceptor clusters achieve chirality via directional cell fate signaling. Chaete (bristles), which serve as sensory organs, are also polarized by PCP signaling. Bristles comprise the 4-5 progeny of a sensory mother cell (SMC), one of which, the shaft, extends a process above the epithelium such that it tilts in a defined direction with respect to the tissue. In mechanosensory microchaete on the notum, one daughter of the SMC divides to produce the shaft and a socket cell that surrounds the shaft where it emerges from the epithelial surface; the other SOP daughter divides to produce a glial cell, a sheath cell and a neuron. Studies of microchaete polarity have shown that the initial division of the SMC is polarized by PCP in the epithelium from which it derives, such that the two daughters are born in defined positions with respect to each other. However, subsequent events that ultimately determine the direction of shaft polarity have not been described (Cho, 2020).

    This study bristle chose polarity on the anterior margin of the wing (AWM) for study. A row of stout mechanosensory bristles and a row of curved chemosensory bristles are on the dorsal surface, and a mixed row of mechano and chemosensory bristles is on the ventral side. All of these bristles tilt toward the distal end of the wing in wildtype. In pkpk mutant wings, and in wings overexpressing Pksple, a large fraction of the AWM bristles point proximally rather than distally; the pkpk phenotype is suppressed by mutation of dsh, implicating the core PCP signaling mechanism in this process. However, the morphogenetic process resulting in polarity and the genetics of its apparent control by PCP signaling have not been explored. Here, focusing on the dorsal mechanosensory bristles, this report describes analysis of the underlying morphogenesis leading to AWM bristle polarization and shows that polarization results from a corkscrew-like helical morphogenetic process involving the shaft and socket cells. Furthermore, the results reveal how interplay between Pkpk and Pksple control the handedness of the helical growth, and how the Ft/Ds/Fj system directs it in opposite orientations depending on whether the core PCP mechanism operates in a Pkpk- or Pksple-dependent mode (Cho, 2020).

    Producing structures of defined chirality requires directional information on three Cartesian axes. The results of this study indicate that in determining bristle chirality, PCP provides directional information along the proximal-distal axis. The apical-basal axis is defined by the epithelium, while the dorsal-ventral axis is likely defined by the dorsal-ventral compartment boundary (Cho, 2020).

    This study has shown that the polarity of wing margin bristles (proximal or distal tilt) is determined by controlling the handedness of helical growth. The entwined shaft and socket cells undergo an apical clockwise or counterclockwise rotation that results in a left-handed or right-handed helical structure, placing the shaft to the proximal (wildtype) or distal (pkpk mutant) side of the socket cell. The direction of rotation depends on PCP signaling among and between margin and socket cells. Helical cellular structures of defined handedness, such as the bristles resulting from properly directed rotation, have been noted in bacterial and plant species, but few examples have been described in animals (Cho, 2020).

    The entwined twisting of the shaft and socket is a coordinated morphogenetic event, and the apparent stereotyped junctional rearrangement of additional margin cells suggests that at least some other cells are involved as well. It is not known in which cell or cells mechanics are regulated to drive this morphogenesis. One possibility is that an internal cytoskeletal mechanism induces the helical growth of the socket and/or shaft cells. Another possibility is that the side of the socket cell crescent marked by Pk at 24 hr is anchored, while the other side grows to wrap around the shaft, inducing junctional rearrangements and propelling the rotation of the apical portion of the shaft relative to the socket cell. Apical rotation could then cause twisting of the more basal portions of the socket cell, and could in turn direct the shaft to the corresponding side of the socket cell (Cho, 2020).

    The precise location at which the PCP signal is required to determine rotation direction is unclear. Because asymmetric core complexes were observed at margin-socket cell junctions, but very little at shaft-margin or shaft-socket junctions, it is hypothesized that interaction between the socket and surrounding margin cells is the essential determinant of rotation. PCP proteins at these junctions could control junctional dynamics, as is known to occur in other systems. This will require further investigation (Cho, 2020).

    A Van Gogh/Vangl tyrosine phosphorylation switch regulates its interaction with core Planar Cell Polarity factors Prickle and Dishevelled

    Epithelial tissues can be polarized along two axes: in addition to apical-basal polarity they are often also polarized within the plane of the epithelium, known as planar cell polarity (PCP). PCP depends upon the conserved Wnt/Frizzled (Fz) signaling factors, including Fz itself and Van Gogh (Vang/Vangl in mammals). In this study, taking advantage of the complementary features of Drosophila wing and mouse skin PCP establishment, how Vang/Vangl phosphorylation on a specific conserved tyrosine residue affects its interaction with two cytoplasmic core PCP factors, Dishevelled (Dsh/Dvl1-3 in mammals) and Prickle (Pk/Pk1-3) was dissected. Pk and Dsh/Dvl were shown to bind to Vang/Vangl in an overlapping region centered around this tyrosine. Strikingly, Vang/Vangl phosphorylation promotes its binding to Prickle, a key effector of the Vang/Vangl complex, and inhibits its interaction with Dishevelled. Thus phosphorylation of this tyrosine appears to promote the formation of the mature Vang/Vangl-Pk complex during PCP establishment and conversely it inhibits the Vang interaction with the antagonistic effector Dishevelled. Intriguingly, the phosphorylation state of this tyrosine might thus serve as a switch between transient interactions with Dishevelled and stable formation of Vang-Pk complexes during PCP establishment (Humphries, 2023).

    This study identified through mass spectrometry analyses with mouse skin epidermis samples phosphorylation on mouse Vangl2 Y308 residue (equivalent to Y374 in Drosophila Vang). This tyrosine lies within with the overlapping binding regions of Pk and Dsh in Vang/Vangl, and, importantly, its charge/phosphorylation status regulates selective binding between Pk and Dsh, with phosphorylation tipping the balance towards Pk binding. It was demonstrated in vivo that binding of Vang to both cytoplasmic core PCP factors is physiologically important (which is the first in vivo evidence for a Vang-Dsh binding requirement). This study provides novel insight into the critical importance of Vang tyrosine phosphorylation and reveals mechanistic features of how regulation of the binding of antagonistic PCP factors to Vang/Vangl during the process of PCP complex segregation and polarity establishment is achieved (Humphries, 2023).

    While previous work defined a broad region within the C-tail of Vang to interact with both Pk and Dsh, the mechanistic regulation and physiological significance of these interactions remained unresolved. Importantly, the defined region is conserved between Drosophila Vang and mammalian Vangl1/2 genes. These data reveal that a small conserved stretch of amino acids within this broader region is both necessary (as shown in the whole Vang protein) and sufficient (as deduced from the in vitro peptide assays) to interact with both cytoplasmic core PCP factors. This region is well conserved between all Vang family members and centered on the tyrosine, which can be phosphorylated, as our mass spec data reveal. Mutational studies define that Pk binding is mediated by tyrosine phosphorylation and associated negative charge, while Dsh requires the aromatic ring found in tyrosine (and also phenylalanine) for its binding to Vang. It is worth noting that this Vang region, shared by both Pk and Dsh for binding, is specific for these two factors, as other Vang associated cytoplasmic PCP proteins, for example, Dgo and Scrib, are not affected by mutations within this domain (Humphries, 2023).

    The importance of the Vang-Pk complex has been well documented in vivo and is also the core of one of the two stable PCP 'core complexes' that result from PCP factor interactions and signaling. In contrast, an interaction between Vang/Vangl and Dsh/Dvl family members has only been documented biochemically. The dissection of binding requirements allowed us to generate single point mutations in Vang that separate binding to only one of the cytoplasmic factors, either Pk or Dsh. The associated in vivo rescue experiments provided the possibility for physiological testing of a functional requirement of the individual interactions between Vang and Dsh or Pk. While all three point mutations display partial rescue, their function is reduced and thus the respective amino acids are physiologically required. Interfering with binding of Vang to both factors (VangY374A) shows the weakest rescue, with the mutant displaying defects that are more similar to the Vang- null phenotype than the other point mutants. Nevertheless, as the point mutations affecting individual Vang-Pk or Vang-Dsh interactions also displayed only partial rescue of the Vang-/- defects, these data indicate that interactions with either cytoplasmic PCP factor, Pk and Dsh, are critical for in vivo Vang function in PCP core complex localization and hence PCP establishment. The phenotypic defects with the Vang-V376A mutant seen in adult wings suggest that it might behave as a mild neomorph, although no dominant effect was observed when heterozygous over Vang-WT. Importantly, this is the first physiological in vivo evidence demonstrating that Vang has a requirement to interact with Dsh during PCP complex segregation. Of all point mutants tested, Vang-Y374F, showed the strongest partial rescue (appeared closest to wild-type). This is consistent with the notion that Pk does not strictly require binding to Vang for membrane association, and that formation of a Vang-Pk complex is much more complcated than a single interaction between the two proteins. Of note, each single point mutant affects the formation of the stable polarized core PCP complexes, as evident in the protein localization studies in 28h APF pupal wings, suggesting that interfering with any interaction among the core PCP factors causes a significant disruption to the PCP interaction cascade and network, needed for normal asymmetric complex polarizations (Humphries, 2023).

    It is intriguing to think about how phosphorylation, and lack thereof, affects the formation of stable core PCP complexes. The data indicate that binding of Dsh/Dvl to Vang/Vangl is physiological, and yet in standard co-staining studies Vang and Dsh do not co-localize. How does Dsh binding to an unphosphorylated Y374 region affect core PCP complex formation (Humphries, 2023)?

    There are a few potential scenarios, and importantly Vang is not a major membrane recruiter of Pk, if at all and thus other factors likely contribute to this. First, a Vang-Pk association, which is assumed to be stable in wing cells in the proximal junctional membrane region, should likely not form in other areas of the cell membrane. As such a Vang-Dsh transient/intermediary interaction might serve a function to prevent Vang-Pk binding. If the kinase in question is asymmetrically localized or active, for example in the proximal area, then-and only then-a switch from Vang-Dsh to a Vang-Pk interaction would occur. If the kinase in question is not asymmetrically active or localized, Dsh binding to this Vang region might be required to prevent the kinase to act on Vang in cell membrane domains, where formation of a Vang-Pk complex should not form, for example the distal vertex of a wing cell. In such a mechanistic scenario Dsh would keep Vang/Vangl 'flexible' to find the right cellular context, where/when the presence of the kinase would initiate the switch to a Vang-P at Y374 (Y308 in mVangl2) and thus support an interaction with Pk and its local effectors. While these are intriguing mechanistic models, they remain speculative (Humphries, 2023).

    In general, the function of Vang/Vangl proteins in PCP establishment remains unresolved. While Vang family proteins are critical for the process and they can physically interact with all other core PCP factors, their contribution to the stability of the intercellular junctional complexes remains unclear, which seem to mainly require Fz-Fmi::Fmi interactions, although Vang/Vangl proteins are part of these asymmetric complexes and bind to Fz intercellularly. Moreover, the non-stoichiometric manner in which the stable PCP complexes form, with for example one single Pk molecule per 6 Vang molecules, suggests complicated mechanistic scenarios that do not rely on one-to-one protein interactions. Importantly, the formation and maintenance of stable PCP complexes requires also Dgo (Diversin in vertebrates) and extends beyond interactions among the core factors, including Scribble and CK1ε and many regulatory interactions are still to be discovered. Complex in vivo experiments will be necessary to better understand the mechanistic sequence of events (Humphries, 2023).

    It is currently unclear which tyrosine kinase(s) act on Vang to mediate its phosphorylation on the Y374 residue (Y308 in mVangl2) and this is one of the regulatory interactions to be still discovered. Sequence motif searches in the Vang Y374 flaking region suggest that Src family kinases could be involved, with no other kinase family having a higher probability (by sequence alignment searches). It is however technically difficult to prove that Src kinases indeed act on this Vang residue in a physiological context, and unfortunately in vitro kinase assays have proven uninformative, as most tyrosine kinases tested could phosphorylate Vang in such assays on multiple residues. Redundancy of Src kinases is an issue in in vivo studies in both the systems (mouse skin and Drosophila wing epithelia), as there are several Src family kinases in both Drosophila and mice, for example. Moreover, in addition to cell survival requirements, many cellular functions are associated with Src family kinases. For example, in Drosophila the two main Src family members are either viable with no overt developmental phenotypes in imaginal discs (Src64, redundant with Src42) or are largely cell lethal (Src42) when analyzed in vivo. They have also been linked to a vast variety of cellular functions, ranging from cytoskeletal regulation and cell adhesion, to synaptic plasticity, proliferation, cell death, and others. Src kinases remain nonetheless likely candidate(s), as (i) GOF phenotypes were observed consistent with a PCP function and (ii) genetic interactions with these Src GOF defects suggest that Vang is required in these contexts. However, again, a loss-of-function scenario to really demonstrate a Src function in PCP establishment remains elusive and should be the focus of future studies (Humphries, 2023).

    Molecular mechanisms that regulate export of the planar cell-polarity protein Frizzled-6 out of the endoplasmic reticulum

    Planar cell polarity (PCP) is a process during which cells are polarized along the plane of the epithelium and is regulated by several transmembrane signaling proteins. After their synthesis, these PCP proteins are delivered along the secretory transport pathway to the plasma membrane, where they perform their physiological functions. This study found that the delivery of a PCP protein, Frizzled-6, to the cell surface is regulated by two conserved polybasic motifs: one located in its first intracellular loop and the other in its C-terminal cytosolic domain. The polybasic motif of Frizzled is also important for its surface localization in the Drosophila wing. Frizzled-6 packaging into vesicles at the endoplasmic reticulum (ER) is regulated by a direct interaction between the polybasic motif and the Glu-62 and Glu-63 residues on the secretion-associated Ras-related GTPase 1A (SAR1A) subunit of coat protein complex II (COPII). Moreover, it was found that newly synthesized Frizzled-6 is associated with another PCP protein, cadherin EGF LAG seven-pass G-type receptor 1 (CELSR1), in the secretory transport pathway and that this association regulates their surface delivery. These results reveal insight into the molecular machinery that regulates the ER export of Frizzled-6. They also suggest that the association of CELSR1 with Frizzled-6 is important, enabling efficient Frizzled-6 delivery to the cell surface (Tang, 2020).

    A Genetic Screen Links the Disease-Associated Nab2 RNA-Binding Protein to the Planar Cell Polarity Pathway in Drosophila melanogaster

    Mutations in the gene encoding the ubiquitously expressed RNA-binding protein ZC3H14 result in a non-syndromic form of autosomal recessive intellectual disability in humans. Studies in Drosophila have defined roles for the ZC3H14 ortholog, Nab2 (aka Drosophila Nab2 or dNab2), in axon guidance and memory due in part to interaction with a second RNA-binding protein, the fly Fragile X homolog Fmr1, and coregulation of shared Nab2-Fmr1 target mRNAs. Despite these advances, neurodevelopmental mechanisms that underlie defective axonogenesis in Nab2 mutants remain undefined. Nab2 null phenotypes in the brain mushroom bodies (MBs) resemble defects caused by alleles that disrupt the planar cell polarity (PCP) pathway, which regulates planar orientation of static and motile cells via a non-canonical arm of the Wnt/Wg pathway. A kinked bristle phenotype in surviving Nab2 mutant adults additionally suggests a defect in F-actin polymerization and bundling, a PCP-regulated processes. To test for Nab2-PCP genetic interactions, a collection of PCP mutant alleles was screened for modification of a rough-eye phenotype produced by Nab2 overexpression in the eye (GMR>Nab2) and, subsequently, for modification of a viability defect among Nab2 nulls. Multiple PCP alleles dominantly modify GMR>Nab2 eye roughening and a subset rescue low survival and thoracic bristle kinking in Nab2 zygotic nulls. Collectively, these genetic interactions identify the PCP pathway as a potential target of the Nab2 RNA-binding protein in developing eye and wing tissues and suggest that PCP signaling could contribute to neurological defects that result from loss of Drosophila Nab2 or its vertebrate ortholog ZC3H14 (Lee, 2020).

    No Evidence that Wnt Ligands Are Required for Planar Cell Polarity in Drosophila

    The frizzled (fz) and dishevelled (dsh) genes are highly conserved members of both the planar cell polarity (PCP) pathway and the Wnt signaling pathway. Given these dual functions, several studies have examined whether Wnt ligands provide a tissue-scale orientation cue for PCP establishment during development, and these studies have reached differing conclusions. This issue was reexamined in the Drosophila melanogaster wing and notum using split-Gal4 co-expression analysis, multiplex somatic CRISPR, and double RNAi experiments. Pairwise loss-of-function experiments targeting wg together with other Wnt genes, via somatic CRISPR or RNAi, do not produce PCP defects in the wing or notum. In addition, somatic CRISPR against evi (aka wntless), which is required for the secretion of Wnt ligands, did not produce detectable PCP phenotypes. Altogether, these results do not support the hypothesis that Wnt ligands contribute to PCP signaling in the Drosophila wing or notum (Ewen-Campen, 2020).

    Frizzled-Dependent Planar Cell Polarity without Secreted Wnt Ligands

    Planar cell polarity (PCP) organizes the orientation of cellular protrusions and migratory activity within the tissue plane. PCP establishment involves the subcellular polarization of core PCP components. It has been suggested that Wnt gradients could provide a global cue that coordinates local PCP with tissue axes. This study dissected the role of Wnt ligands in the orientation of hairs of Drosophila wings, an established system for the study of PCP. PCP was normal in quintuple mutant wings that rely solely on the membrane-tethered Wingless for Wnt signaling, suggesting that a Wnt gradient is not required. A nanobody-based approach to was used to trap Wntless in the endoplasmic reticulum, and hence prevent all Wnt secretion, specifically during the period of PCP establishment. PCP was still established. It is concluded that, even though Wnt ligands could contribute to PCP, they are not essential, and another global cue must exist for tissue-wide polarization (Yu, 2020).

    Girdin is a component of the lateral polarity protein network restricting cell dissemination

    Epithelial cell polarity defects support cancer progression. It is thus crucial to decipher the functional interactions within the polarity protein network. This study shows that Drosophila Girdin (girders of actin filaments) and its human ortholog GIRDIN or GIV (Galpha-interacting vesicle associated protein) sustain the function of crucial lateral polarity proteins by inhibiting the apical kinase aPKC. Loss of GIRDIN expression is also associated with overgrowth of disorganized cell cysts. Moreover, cell dissemination was observed from GIRDIN knockdown cysts and tumorspheres, thereby showing that GIRDIN supports the cohesion of multicellular epithelial structures. Consistent with these observations, alteration of GIRDIN expression is associated with poor overall survival in subtypes of breast and lung cancers. Overall, this study discovered a core mechanism contributing to epithelial cell polarization from flies to humans. These data also indicate that GIRDIN has the potential to impair the progression of epithelial cancers by preserving cell polarity and restricting cell dissemination (Biehler, 2020).

    The ability of epithelia to form physical barriers is provided by specialized cell-cell junctions, including the zonula adherens (ZA). The latter is a belt-like adherens junction composed primarily of the transmembrane homotypic receptor E-cadherin, which is linked indirectly to circumferential F-actin bundles through adaptor proteins such as β-catenin and α-catenin. In Drosophila embryonic epithelia, the protein Girdin stabilizes the ZA by reinforcing the association of the cadherin-catenin complex with the actin cytoskeleton (Ha, 2015). This function in cell-cell adhesion is preserved in mammals, and supports collective cell migration (Wang, 2018; Wang, 2015). Fly and human Girdin also contribute to the coordinated movement of epithelial cells through the organization of supracellular actin cables (Biehler, 2020).

    In addition to creating barriers, epithelial tissues generate vectorial transport and spatially oriented secretion. The unidirectional nature of these functions requires the polarization of epithelial cells along the apical-basal axis. In Drosophila, the scaffold protein Bazooka (Baz) is crucial to the early steps of epithelial cell polarization, and for proper assembly of the ZA. Baz recruits atypical Protein Kinase C (aPKC) together with its regulator Partitioning defective protein 6 (Par-6) to the apical membrane. The small GTPase Cdc42 contributes to the activation of aPKC and p21-activated kinase (Pak1), thereby acting as a key regulator of cell polarity. Baz also contributes to apical positioning of the Crumbs (Crb) complex, which is composed mainly of Crb, Stardust (Sdt), and PALS1-associated Tight Junction protein (Patj). Once properly localized, the aPKC-Par-6 and Crb complexes promote the apical exclusion of Baz, which is then restricted to the ZA. The apical exclusion of Baz is essential to the positioning of the ZA along the apical-basal axis, and for full aPKC activation (Biehler, 2020).

    The function of aPKC is evolutionarily preserved, and human atypical PKCλ (PKClambda in other mammals) and PKCζ PKCzeta)contribute to epithelial cell polarization. aPKC maintains the identity of the apical domain through phospho-dependent exclusion of lateral polarity proteins such as Yurt (Yrt) and Lethal (2) giant larvae (Lgl). In return, these proteins antagonize the Crb- and aPKC-containing apical machinery to prevent the spread of apical characteristics to the lateral domain. In combination with the function of Baz, these feedback mechanisms provide a fine-tuning of aPKC activity in addition to specifying its subcellular localization. This is crucial, as both over- and under-activation of aPKC is deleterious to epithelial polarity in fly and mammalian cells, and ectopic activation of aPKC can lead to cell transformation (Biehler, 2020).

    Cell culture work has established that mammalian GIRDIN interacts physically with PAR3 -the ortholog of Baz-and PKCλ. Depletion of GIRDIN in Madin-Darby Canine Kidney (MDCK) epithelial cells delays the formation of tight junctions in Ca2+ switch experiments. GIRDIN is also an effector of AMP-activated protein kinase (AMPK) in the maintenance of tight junction integrity under energetic stress. Moreover, mammalian GIRDIN is required for the formation of epithelial cell cysts with a single lumen, supporting a role for this protein in epithelial morphogenesis as reported in flies. As cyst morphogenesis is linked to epithelial cell polarity, these studies suggest that GIRDIN is involved in establishing the apical-basal axis. However, further studies are required to clarify the role of GIRDIN in apical-basal polarity per se, as other cellular processes could explain the phenotype associated with altered GIRDIN expression. For instance, spindle orientation defects impair the formation of epithelial cysts. Of note, PAR3, aPKC, and AMPK are all required for proper spindle positioning in dividing epithelial cells. The molecular mechanisms sustaining the putative role of GIRDIN in epithelial cell polarity also need to be better deciphered. This study investigated the role of fly and human Girdin proteins in the regulation of epithelial cell polarity, and showed that these proteins are part of the lateral polarity protein network. One crucial function of Girdin proteins is to repress aPKC function. It was also discovered that loss of Girdin proteins promotes overgrowth of cell cysts, and cell dissemination from these multicellular structures. Consistent with these data, it was found that low GIRDIN expression correlates with poor overall survival in subtypes of breast and lung cancers (Biehler, 2020).

    Using classical genetics in flies, this study has shown that mutation in Girdin exacerbates the polarity defects in zygotic lgl or yrt mutant embryos and concludes that Girdin is part of the lateral polarity network. It was also found that Girdin opposes the function of aPKC, which plays a crucial role in the establishment and maintenance of the apical domain by antagonizing lateral proteins such as Lgl and Yrt. Thus a model is proposed in which Girdin supports the activity of Yrt and Lgl by restricting the activity of aPKC. This work demonstrates that the role of Girdin in restricting aPKC activity is evolutionarily conserved. This function confers on human GIRDIN the ability to maintain apical-basal polarity in Caco-2 cells, and to support epithelial cyst morphogenesis. These results are in line with previous studies suggesting a role for GIRDIN in polarity and cystogenesis in MDCK and MCF10A epithelial cells. It was shown that PKCλ enhances GIRDIN expression in MDCK cells. Moreover, knockdown of aPKC or GIRDIN gives a similar phenotype characterized by defects in tight junction integrity and cyst formation. It was thus proposed that GIRDIN is an effector of PKCλ. Although cell-type-specific mechanisms may exist, the current data suggest that this hypothesis needs to be revisited in favor of a model in which the induction of GIRDIN expression by PKClambda in MDCK cells initiates a negative feedback loop instead of cooperation between these proteins. The fact that both overactivation of aPKC or inhibition of its activity is deleterious to epithelial cell polarity and cyst morphogenesis may underlie the conflicting interpretations of the data in the literature. GIRDIN is also known to modulate heterotrimeric G protein signaling-a role that seems to contribute to the formation of normal cysts by MDCK cells (Sasaki, 2015). In addition, it was demonstrated recently that GIRDIN acts as an effector of AMP-activated protein kinase (AMPK) under energetic stress to maintain tight junction function (Aznar, 2016). Of note, these two functions are not shared by fly Girdin (Ghosh, 2017; Garcia-Marcos, 2009; Ghosh, 2017), and were thus acquired by GIRDIN during evolution to fulfill specialized functions. In contrast, the discovery in this study of the Girdin-dependent inhibition of aPKC reveals a core mechanism contributing to epithelial cell polarization from flies to humans (Biehler, 2020).

    GIRDIN is considered to be an interesting target in cancer due to its role in cell motility, and high levels of GIRDIN have been reported to correlate with a poor prognosis in some human cancers. Notwithstanding that GIRDIN may favor tumor cell migration, the current study indicates that inhibition of GIRDIN function in the context of cancer would be a double-edged sword for many reasons. Indeed, this study showed that knockdown of GIRDIN exacerbates the impact of aPKC overexpression, and leads to overgrowth and lumen filling of Caco-2 cell cysts. Of note, overexpression of aPKC can lead to cell transformation, and was associated with a poor outcome in several epithelial cancers. This study thus establishes that inhibiting GIRDIN in patients showing increased aPKC expression levels could worsen their prognosis. According to the data, abolishing GIRDIN function in tumor cells with decreased levels of the human Lgl protein LLGL1, as reported in many cancers, could also support the progression of the disease by altering the polarity phenotype. Cell detachment and dissemination was observed from GIRDIN knockdown cysts, thus showing that GIRDIN is required for the cohesion of multicellular epithelial structures. Of note, cells, either individually or as clusters, detaching from cysts are alive and some of them remain viable. This is analogous to what was reported in Girdin mutant Drosophila embryos in which cell cysts detach from the ectoderm and survive outside of it. Other phenotypes in Girdin mutant embryos are consistent with a role for Girdin in epithelial tissue cohesion, including rupture of the ventral midline and fragmentation of the dorsal trunk of the trachea. Mechanistically, Girdin strengthens cell-cell adhesion by promoting the association of core adherens junction components with the actin cytoskeleton. A recent study established that this molecular function is evolutionarily conserved, and that GIRDIN favors the association of β-CATENIN with F-ACTIN. Since knockdown of GIRDIN results in cell dispersion from Caco-2 cell cysts, and since weakening of E-CADHERIN-mediated cell-cell adhesion contributes to cancer cell dissemination and metastasis, it is plausible that reduced GIRDIN expression contribute to the formation of secondary tumors and cancer progression. This may explain why this study found that low mRNA expression levels of GIRDIN correlates with decreased survival in more aggressive breast cancer subtypes and lung adenocarcinoma. Future studies using xenograft in mice, and investigating the expression of GIRDIN protein in cancer patients will help validating whether GIRDIN can repress the progression of certain types of epithelial cancers (Biehler, 2020).

    In conclusion, using a sophisticated experimental scheme combining in vivo approaches in D. melanogaster with 3D culture of human cells, this study defined a conserved core mechanism of epithelial cell polarity regulation. Specifically, Girdin was shown to repress the activity of aPKC to support the function of Lgl and Yrt, and ensure stability of the lateral domain. This is of broad interest in cell biology, as proper epithelial cell polarization is crucial for the morphogenesis and physiology of most organs. In addition, the maintenance of a polarized epithelial architecture is crucial to prevent various pathological conditions such as cancer progression. Importantly, this study showed that normal GIRDIN function potentially impairs the progression of epithelial cancers by preserving cell polarity whilst restricting cell growth and cell dissemination. Thus, these results place a caveat on the idea that GIRDIN could be an interesting target to limit cancer cell migration, and indicate that inhibition of GIRDIN in the context of cancer could be precarious. Potential drugs targeting GIRDIN would thus be usable only in the context of precision medicine where a careful analysis of aPKC, LLGL1, and E-CAD expression, as well as the polarity status of tumor cells would be analyzed prior to treatment. Inhibition of GIRDIN in patients carrying tumors with altered expression of these proteins would likely worsen the prognosis (Biehler, 2020).

    DAnkrd49 and Bdbt act via Casein kinase Iepsilon 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. This study describes 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, it was found 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. This study demonstrates 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 (Strutt, 2020).

    Planar polarity describes the phenomenon whereby cells coordinate their polarity in the plane of a tissue: for example the hairs on the skin point in the same direction, cilia coordinate their beating, and cells coordinate their movements during tissue morphogenesis. Understanding the mechanisms by which this coordinated polarisation occurs is of prime importance, as disruption of polarity can have diverse consequences, including neural tube closure defects, hydrocephalus and defects in neuronal migration (Strutt, 2020).

    The fly wing is a well-characterised model system in which to study planar polarity. Each cell within the adult wing produces a single hair, or trichome, which points towards the distal end of the wing. Furthermore, viable mutations that cause characteristic swirling of the trichomes have been identified, and the genes associated with these mutations were subsequently found to be highly conserved, and to regulate planar polarity throughout the animal kingdom (Strutt, 2020).

    The core planar polarity proteins (hereafter known as the core proteins) are the best characterised group of proteins that regulate planar polarity. In the pupal wing, the core proteins adopt asymmetric subcellular localisations prior to trichome emergence, and in their absence trichomes emerge from the centre of the cell rather than at the distal cell edge. The core proteins comprise the atypical cadherin Flamingo (Fmi, also known as Starry Night [Stan]), the transmembrane proteins Frizzled (Fz) and Strabismus (Stbm, also known as Van Gogh [Vang]), and three cytoplasmic proteins Dishevelled (Dsh), Prickle (Pk) and Diego (Dgo). Fmi localises to proximal and distal cell edges in the pupal wing, but is excluded from lateral cell edges, while Fz, Dsh and Dgo localise to distal cell edges and Stbm and Pk to proximal cell edges. These proteins form intercellular complexes at cell junctions, that couple neighbouring cells and allow them to coordinate their polarity (Strutt, 2020).

    The mechanisms by which the core proteins become asymmetrically localised are poorly understood. The overall direction of polarity is thought to be determined by tissue-specific 'global' cues: these may include gradients of morphogens or Fat/Dachsous cadherin activity, or other cues such as mechanical tension. In the wing these global cues may directly regulate core protein localisation or act indirectly via effects on growth and tissue morphogenesis. Global cues are thought to lead to subtle biases in core protein localisation within cells that are subsequently amplified by feedback between the core proteins, in which positive (stabilising) interactions between complexes of the same orientation are coupled with negative (destabilising) interactions between complexes of opposite orientation. In mathematical models, such feedback interactions have been demonstrated to be sufficient to amplify weak biases in protein localisation, leading to sorting of complexes and robust asymmetry (Strutt, 2020).

    Experimental evidence for feedback is only beginning to be elucidated. Cell culture experiments have demonstrated competitive binding between several of the core proteins, which may be important for feedback. Furthermore, it has recently been shown that the core protein Pk acts through Dsh to destabilise Fz in the same cell, while stabilising Fz across cell junctions via Stbm (Strutt, 2020).

    In order for feedback to operate, cells must utilise the general cellular machinery: for example active endocytosis is necessary for Pk to destabilise Fz. Furthermore, post-translational modifications of the core proteins are likely to be key mediators of feedback. For example loss of ubiquitination pathway components and some protein kinases have been shown to disrupt planar polarity. In flies, a Cullin-3/Diablo/Kelch ubiquitin ligase complex regulates Dsh levels at cell junctions, while the de-ubiquitinase Fat Facets regulates Fmi levels. Stbm also negatively regulates Pk levels, and ubiquitination of Pk by Cullin-1/SkpA/Slimb promotes internalisation of Fmi-Stbm-Pk complexes. Similarly, in vertebrates, the Stbm homologue Vangl2 may promote local degradation of Pk via ubiquitination by Smurf E3 ubiquitin ligases. Furthermore, Drosophila Fz phosphorylation is mediated by atypical Protein Kinase C, and Dsh is a target of phosphorylation by the Discs Overgrown (Dco, also known as Doubletime [Dbt] or Casein Kinase Iε [CKIε]) and Abelson kinases. Dco/CKIε has also been implicated in phosphorylation of Stbm in both flies and vertebrates (Strutt, 2020).

    This study describes the identification of two new regulators of planar polarity in the Drosophila wing. Bride of Doubletime (Bdbt) and DAnkrd49 were shown to be binding partners that regulate each other's levels. Loss of either protein disrupts asymmetric localisation of the core proteins. Furthermore, they regulate overall levels of Dsh and Dsh phosphorylation in the pupal wing, and evidence is provided that they act by modulating the activity of the kinase Dco (Strutt, 2020).

    This study present evidence for a novel protein complex regulating planar polarity, consisting of the ankyrin repeat protein DAnkrd49 and the non-canonical FKBP family member Bdbt. DAnkrd49 and Bdbt physically interact in vitro and regulate each other's levels in vivo. This suggests that they may act in a complex in which each is required to stabilise the other, a model supported by the phenotypic similarity in mutant clones. Their activity is required for core protein asymmetry in the pupal wing, and for normal levels and phosphorylation of the cytoplasmic core protein Dsh (Strutt, 2020).

    Previous work on circadian rhythms has established a physical interaction between Bdbt and the kinase Dco, and that Dco activity is regulated by Bdbt (Fan, 2013). Notably, Dco has previously been implicated in promoting both Dsh and Stbm phosphorylation in planar polarity signalling. Thus, the data are consistent with both DAnkrd49 and Bdbt acting to regulate Dco activity in planar polarity. Firstly, loss of function clones of dco are seen, and DAnkrd49/Bdbt share a common phenotype: in their absence reduced overall levels of Dsh and reduced levels of Dsh phosphorylation. Secondly, strong genetic interactions are seen between dco and DAnkrd49/Bdbt. Although a physical interaction has been observed between Dco and Bdbt, no direct interaction was seen between Dco and DAnkrd49. Therefore, a simple model is that the role of DAnkrd49 is to stabilise Bdbt, while Bdbt directly regulates Dco activity. Thus this study defines a regulatory cascade, whereby DAnkrd49 and Bdbt promote Dco activity, which in turn regulates phosphorylation of core proteins and asymmetric localisation (Strutt, 2020).

    An alternative model is that DAnkrd49 and Bdbt act to stabilise Dsh, independently of Dco. Proteins of the FKBP family are known to regulate the stability of target proteins. In canonical FKBP family members this stabilisation is a result of PPIase activity, which assists protein folding; whereas other family members stabilise their target proteins by direct binding. The catalytic sites for PPIase are not conserved in Bdbt. A binding interaction between Dsh and either DAnkrd49 or Bdbt was not seen, arguing that Bdbt is unlikely to directly stabilise Dsh (Strutt, 2020).

    In addition to defects in planar polarity, other pleiotropic defects are seen in DAnkrd49 clones and Bdbt clones, including cell size defects, reduced proliferation and poor viability. These could plausibly be explained by regulation of Dco activity by DAnkrd49 and Bdbt. Dco acts in multiple signalling pathways. It phosphorylates the tumour suppressor Fat, which regulates cell growth and survival via the Hippo signalling pathway. Interestingly, a hypomorphic mutation in dco causes tissue overgrowth and increased activity of the caspase inhibitor DIAP1, phenocopying loss of Hippo signalling pathway components, while cells completely lacking Dco activity have reduced expression of the caspase inhibitor DIAP1 and reduced proliferation. Dco may also act in additional signalling pathways, and these are thought to include Hedgehog signalling and canonical Wnt signalling. Hence, the multitude of signalling pathways regulated by Dco may explain the complex phenotypes seen in the absence of DAnkrd49 and Bdbt. Alternatively, it is possible that DAnkrd49 and Bdbt may regulate other downstream targets in addition to Dco (Strutt, 2020).

    As Dco has also been implicated in phosphorylating Stbm, loss of DAnkrd49 and Bdbt is also expected to reduce Stbm phosphorylation in pupal wings. However this could not be verified directly, as Stbm mobility in SDS-PAGE is only marginally increased when Dco activity is reduced, and is not noticeably altered in extracts from animals with reduced activity of DAnkrd49 or Bdbt. Nevertheless, recent work has shown that phosphorylation of both Stbm and Dsh by Dco is functionally important in establishing correct planar polarity, making the regulation of Dco activity of great interest (Strutt, 2020).

    Interestingly, protein interactions studies in human cell lines have identified a STRING network between Ankrd49, CKIδ/CKIε and FKBP family members. Although the FKBP proteins identified in these studies are not the closest orthologues to Drosophila Bdbt, this nevertheless suggests conservation of a regulatory cascade of Ankrd49/FKBP promoting Dco activity, which in turn might phosphorylate core planar polarity proteins. Furthermore, the DISEASES online resource, which integrates results from text mining, manually curated disease-gene associations and genome-wide association studies, has linked Ankrd49 with brachydactyly subtypes. Brachydactyly is a key feature of Robinow syndrome, a disease closely associated with core planar polarity mutations; thus understanding this regulatory cascade may be of importance for human health (Strutt, 2020).

    Hypoxia controls plasma membrane targeting of polarity proteins by dynamic turnover of PI4P and PI(4,5)P2

    Phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate (PIP2) are key phosphoinositides that determine the identity of the plasma membrane (PM) and regulate numerous key biological events there. To date, mechanisms regulating the homeostasis and dynamic turnover of PM PI4P and PIP2 in response to various physiological conditions and stresses remain to be fully elucidated. This study reports that hypoxia in Drosophila induces acute and reversible depletion of PM PI4P and PIP2 that severely disrupts the electrostatic PM targeting of multiple polybasic polarity proteins. Genetically encoded ATP sensors confirmed that hypoxia induces acute and reversible reduction of cellular ATP levels which showed a strong real-time correlation with the levels of PM PI4P and PIP2 in cultured cells. By combining genetic manipulations with quantitative imaging assays this study showed that PI4KIIIα, as well as Rbo/EFR3 and TTC7 that are essential for targeting PI4KIIIα to PM, are required for maintaining the homeostasis and dynamic turnover of PM PI4P and PIP2 under normoxia and hypoxia. These results revealed that in cells challenged by energetic stresses triggered by hypoxia, ATP inhibition and possibly ischemia, dramatic turnover of PM PI4P and PIP2 could have profound impact on many cellular processes including electrostatic PM targeting of numerous polybasic proteins (Lu, 2022).

    The inner leaflet of the plasma membrane (PM) is the most negatively charged membrane surface due to its enrichment of phospholipids including phosphatidylserine and phosphoinositides (PPIns) PI4P (phosphatidylinositol (PtdIns) 4-phosphate) and PIP2 (PtdIns 4,5-biphosphate (PI(4,5)P2)). The unique combination of PI4P and PIP2 is crucial to determine the PM identity by regulating many key biological events in the PM including cell signaling, endocytosis, and channel activation. Moreover, for proteins with positively charged domains/surfaces, electrostatic binding to the PM is a fundamental mechanism underlying the regulation of their subcellular localization and biological activity. One typical example can be found in polarity proteins that play essential and conserved roles in regulating various types of cell polarity such as apical-basal polarity in epithelial cells. Recent discoveries showed that multiple polarity proteins such as Lgl, aPKC, and Dlg contain positively charged polybasic motifs that electrostatically bind the negatively charged inner surface of PM (Dong, 2020; Dong, 2015; Lu, 2021), and such electrostatic PM targeting has now emerged as a mechanism essential for regulating their subcellular localization and biological activities in cell polarity (Lu, 2022).

    While mechanisms regulating the interaction between polybasic motifs and PM have been relatively well studied, much less is known how the homeostasis and turnover of PM PI4P and PIP2 may impact the electrostatic PM targeting. Although sophisticated mechanisms exist to maintain the steady state levels of PM PI4P and PIP2 under normal conditions, previous live imaging experiments in Drosophila showed a striking phenomenon that hypoxia induces acute and reversible loss of PM localization of polybasic polarity proteins Lgl, aPKC, and Dlg in epithelial cells (Dong, 2020; Dong, 2015; Lu, 2021), likely through reducing intracellular ATP levels (Dong, 2015). Previous studies also showed that PM PIP2 could be reversibly depleted under hypoxia (Dong, 2015), suggesting that a potential connection from hypoxia to ATP inhibition to PM phospholipids depletion to loss of electrostatic PM targeting of polybasic proteins. However, to date how PM PI4P levels are regulated under hypoxia is unknown. Even less is known about the mechanisms through which hypoxia and ATP inhibition impact PM PI4P and PIP2 levels, and consequently the electrostatic PM targeting of numerous proteins (Lu, 2022).

    For this study, quantitative live imaging experiments were carried out in Drosophila and cultured mammalian cells using multiple genetically encoded sensors to show that acute hypoxia induces dramatic but reversible depletion of PM PI4P and PIP2, accompanied by concurrent loss of PM localization of polybasic polarity protein Lgl. Using genetically encoded ATP sensors, a real-time correlation was confimed between the intracellular ATP levels and PM levels of PI4P and PIP2 in cultured cells. This study further identified that PI4KIIIα (PtdIns-4 kinase IIIα) and its PM targeting machinery are required for the proper dynamic turnover of PM PI4P and PIP2 under hypoxia and ATP inhibition, as well as for properly restoring the post-hypoxia electrostatic PM targeting of Lgl. These studies reveal a potential regulatory mechanism that dynamically controls PM PI4P and PIP2 levels in response to hypoxia and ATP inhibition. The results suggest that genetic deficiencies in regulating such dynamic turnover of PM PI4P and PIP2 could have profound impact on cell physiology including polarity, when cells are challenged by energetic stresses triggered by hypoxia, ischemia and ATP inhibition (Lu, 2022).

    It is speculated that the reduction of intracellular ATP levels, through either hypoxia or drug inhibition, triggers acute loss of PM PI4P and PIP2 by two possible mechanisms. PI4P and PIP2 could be maintained at slow turnover rates on the PM, but reduction of ATP activates a specific cellular response to acutely deplete PM PI4P and PIP2. Alternatively, a more parsimonious mechanism would be that PM PI4P and PIP2 are constantly under fast turnover, which requires high activity of PI and PIP kinases. ATP reduction, which directly inhibits the activity of these kinases, pivots the equilibrium to the dephosphorylation process which converts the PIP2 to PI4P and PI4P to PI (Lu, 2022).

    Consistent with the critical role of PI4P in maintaining PM identity and its biological activity, the data revealed that cells undergoing hypoxia/ATP inhibition consistently prioritize the maintenance and recovery of PM PI4P over the intracellular PI4P pool in a PI4KIIIα-dependent manner. However, while PI4KIIIα is well characterized for its essential role in generating the PI4P on the PM, KmATP values of PI4KIIIα (500-700 μM) and PI4KIIβ(Fwd) (~400 μM) are about one or two orders higher than that of PI4KIIα (10-50 μM). Such KmATP differences would suggest that, in contrast to the current results, the intracellular PI4P pool should deplete more slowly and recover more quickly than the PM PI4P in cells undergoing hypoxia/ATP inhibition, as PI4KIIIα would be the first PI4K to lose activity under hypoxia and the last to become active under reoxygenation (Lu, 2022).

    One possible reason behind such a discrepancy could be that KmATP of PI4KIIIα was measured decades ago using purified PI4KIIIα enzymes from tissues such as bovine brains and uterus. Recent data showed that PI4KIIIα forms a highly ordered multi-protein membrane targeting complex essential for its activity. It is thus possible that PI4KIIIα in the complex may have much lower KmATP in vivo, and/or has dramatically increased enzymatic activity to produce sufficient PI4P at the PM even when ATP levels are much lower than the measured Km. Alternatively, the KmATP of PI4KIIIα is indeed high and live imaging results actually highlight a prioritized transfer of PI4P from the intracellular pool to maintain or replenish the PM PI4P levels. Phosphatidylinositol (PI) is abundant on intracellular membranes, but not the PM. Therefore, during the early phase of reoxygenation when intracellular ATP levels are low, PI4P is first synthesized at the intracellular pool by PI4KIIα but is immediately transferred to the PM. Only after the full replenishment of PM PI4P is the intracellular PI4P pool filled. Supporting this transfer PI4P from intracellular pools to PM pools, the data show loss of PM PI4P recovery in PI4K-3KD cells, in which the maintenance of intracellular pool of PI4P is supposedly impaired (Lu, 2022).

    The data are consistent with the view that in wild type cells under hypoxia/ATP inhibition, PM PIP2 depletes and recovers through direct inter-conversion with PI4P on the PM. Interestingly, in both PI4KIIIα-RNAi and PI4K-3KD cells, PM recovery of PIP2 is preceded with transient intracellular PIP2-positive puncta which were not seen in recovering wild-type cells. It is possible that in the absence or delay of PM PI4P recovery, enzymes such as PIP5K are instead electrostatically recruited to the intracellular PI4P-positive puncta to convert PI4P to PIP2. It is unclear, however, in PI4K knock down cells whether the delayed PM PIP2 recovery originates from the PIP2 generated in these puncta. Additional sensors are necessary to confirm the co-localization of PI4P and PIP2 on these transient puncta. Notably, MEF cells from PI4KIIIα knock-out mice also showed increased PIP2-positive intracellular vesicles (Lu, 2022 and references therein).

    The existence of intracellular P4M x 2::GFP puncta in PI4K-3KD cells suggest that the knock down of PI4KIIα and fwd is unlikely complete, but the severe reduction of PM PI4P confirms the knock down is strong enough to greatly enhance the defects in PI4KIIIα-RNAi cells. Such partial knock-down by PI4K-3KD is actually necessary for imaging assays, as completely blocking PI4P synthesis is cell lethal. It is striking that PM PIP2 is well maintained in the near absence of PM PI4P in PI4K-3KD cells. Synthetic biology-based evidence suggested that PIP5K can be sufficient to make PIP2 from PI in E. coli by phosphorylating both its fourth and fifth positions in the absence of PI4Ks, and it is possible that similar pathway maintains the steady state PM PIP2 levels in PI4K-3KD cells. Nonetheless, imaging experiments showed that PI4K activity is essential for cells to maintain PM PIP2 levels when cells are subject to hypoxia. In this regard, study of PI4K-compromised cells repeatedly revealed deficiencies in PI4P/PIP2 turnover and electrostatic PM targeting that can only be observed when cells are subject to energetic stress conditions (Lu, 2022).

    PM targeting of PI4KIIIα strictly depends on its formation of an obligate superassembly with TTC7 (YPP1), FAM126 (Hycin) and EFR3 (Rbo). A recent study also showed that RNAi knock-downs of PI4KIIIα, TTC7 and Rbo yielded similar phenotypes in Drosophila wing discs, such as moderately reduced PM PI4P but no obvious changes of PM PIP2 (Basu, 2020). Same RNAi knock-downs in Drosophila photoreceptors also showed similar phenotypes such as reduced PI4P levels and impaired light response, although PIP2 levels also appear to be reduced (Balakrishnan, 2018). Moreover, the data showed that knocking down TTC7 also reduced PM localization of Rbo, supporting that components in PI4KIIIα complex may act interdependently for proper PM targeting in vivo (Lu, 2022).

    The hypoxia-resistant PM localization of Rbo/dEFR3 suggests that under hypoxia/ATP inhibition PI4KIIIα maintains its PM localization, which should be essential for its role in recovering the PM PI4P. The core complex of PI4KIIIα/TTC7/FAM126 forms a collective basic surface that electrostatically binds to the acidic inner leaflet of the PM which could be sensitive to the loss of PM PI4P and PIP2. However, TTC7 also interacts with the C-terminus of EFR3. PM targeting of yeast EFR3 requires a basic patch that interacts with general acidic phospholipids but is not disrupted by the loss of PM PI4P and PIP2. Mammalian EFR3 contains an additional N-terminal Cys-rich palmitoylation site that is also required for the PM targeting. Such dual and PI4P/PIP2-independent mechanisms are supported by the hypoxia-resistant PM localization of Rbo as was observed. Future studies will be needed to directly investigate the PM targeting of PI4KIIIα, TTC7 and FAM126 in vivo under hypoxia/ATP inhibition (Lu, 2022).

    While previous studies showed that genetically reducing PM PIP2 levels disrupts the PM localization of several polarity proteins including Lgl, it is difficult to conclude whether such loss of PM targeting is the direct consequence PIP2 reduction. This study was able to quantitatively and qualitatively demonstrate that in cells undergoing hypoxia-reoxygenation the acute and reversible loss of PM targeting of Lgl directly correlates with the turnover of PM PI4P and PIP2. Consistent with the idea that Lgl appears to depend more on PIP2 for its PM targeting, Lgl closely follows the dynamic turnover and relocation of PIP2 during hypoxia and reoxygenation. In particular, ectopic and transient puncta of Lgl::GFP seen in PI4KIIIα-RNAi or PI4K-3KD cells under reoxygenation appear to be strikingly similar to PIP2-positive puncta in these cells, although due to the limited array of biosensors it has not been possible to directly confirm the co-localization of Lgl::GFP and PIP2 in these transient puncta. Additional genetically encoded biosensors for PI4P and PIP2 (e.g. P4M x 2::iRFP and PLC-PH::iRFP) are in development for such experiments (Lu, 2022).

    It is notable that in rbo-RNAi cells, Lgl::GFP formed very few transient puncta prior to PM recovery during reoxygenation, even though PLC-PH::GFP showed plenty of prominent puncta. The reason for such difference between Lgl::GFP and PLC-PH::GFP in rbo-RNAi cells is unclear, though likely derives from the requirement of polybasic motif proteins for additional anionic lipids at the plasma membrane, specifically high molar fractions of phosphatidylserine in addition to lower concentrations of polyanionic phosphoinositides (Lu, 2022).

    In summary, this study revealed an acute and reversible loss of PI4P and PIP2 from PM under hypoxia/ATP inhibition in both Drosophila and human cultured cells. Such dynamic turnover of PM PI4P and PIP2 explains the dramatic loss of the PM targeting of polybasic polarity proteins such as Lgl under hypoxia. How cells meticulously maintain steady state PM PI4P and PIP2 levels under normal physiological conditions has been extensively studied; these studies highlight the importance of understanding mechanisms controlling this homeostasis and dynamics of phosphoinositides under energetic stresses triggered by hypoxia, ATP inhibition and ischemia, and the critical role of the interplay between polarity proteins and PM phosphoinositides in controlling cell polarity under normal and disease conditions (Lu, 2022).

    Cortical Cyclin A controls spindle orientation during asymmetric cell divisions in Drosophila

    The coordination between cell proliferation and cell polarity is crucial to orient the asymmetric cell divisions to generate cell diversity in epithelia. In many instances, the Frizzled/Dishevelled planar cell polarity pathway is involved in mitotic spindle orientation, but how this is spatially and temporally coordinated with cell cycle progression has remained elusive. Using Drosophila sensory organ precursor cells as a model system, this study shows that Cyclin A, the main Cyclin driving the transition to M-phase of the cell cycle, is recruited to the apical-posterior cortex in prophase by the Frizzled/Dishevelled complex. This cortically localized Cyclin A then regulates the orientation of the division by recruiting Mud, a homologue of NuMA, the well-known spindle-associated protein. The observed non-canonical subcellular localization of Cyclin A reveals this mitotic factor as a direct link between cell proliferation, cell polarity and spindle orientation (Darnat, 2022).

    Parafibromin governs cell polarity and centrosome assembly in Drosophila neural stem cells

    Neural stem cells (NSCs) divide asymmetrically to balance their self-renewal and differentiation, an imbalance in which can lead to NSC overgrowth and tumor formation. The functions of Parafibromin, a conserved tumor suppressor, in the nervous system are not established. This study demonstrated that Drosophila Parafibromin/Hyrax (Hyx) inhibits ectopic NSC formation by governing cell polarity. Hyx is essential for the asymmetric distribution and/or maintenance of polarity proteins. hyx depletion results in the symmetric division of NSCs, leading to the formation of supernumerary NSCs in the larval brain. Importantly, human Parafibromin was shown to rescue the ectopic NSC phenotype in Drosophila hyx mutant brains. This study also discovered that Hyx is required for the proper formation of interphase microtubule-organizing center and mitotic spindles in NSCs. Moreover, Hyx is required for the proper localization of 2 key centrosomal proteins, Polo and AurA, and the microtubule-binding proteins Msps and D-TACC in dividing NSCs. Furthermore, Hyx directly regulates the polo and aurA expression in vitro. Finally, overexpression of polo and aurA could significantly suppress ectopic NSC formation and NSC polarity defects caused by hyx depletion. These data support a model in which Hyx promotes the expression of polo and aurA in NSCs and, in turn, regulates cell polarity and centrosome/microtubule assembly. This new paradigm may be relevant to future studies on Parafibromin/HRPT2-associated cancers (Deng, 2022).

    PTP10D-mediated cell competition is not obligately required for elimination of polarity-deficient clones

    Animal organs maintain tissue integrity and ensure removal of aberrant cells through several types of surveillance mechanisms. One prominent example is the elimination of polarity-deficient mutant cells within developing Drosophila imaginal discs. This has been proposed to require heterotypic cell competition dependent on the receptor tyrosine phosphatase PTP10D within the mutant cells. This study reports experiments to test this requirement in various contexts and found that PTP10D is not obligately required for the removal of scribble (scrib) mutant and similar polarity-deficient cells. These experiments used identical stocks with which another group can detect the PTP10D requirement, and the results do not vary under several husbandry conditions including high and low protein food diets. Although it was not possible to identify the source of the discrepant results, it is suggested that the role of PTP10D in polarity-deficient cell elimination may not be absolute (Gerlach, 2022).

    The Nab2 RNA-binding protein patterns dendritic and axonal projections through a planar cell polarity-sensitive mechanism

    RNA-binding proteins support neurodevelopment by modulating numerous steps in post-transcriptional regulation, including splicing, export, translation, and turnover of mRNAs that can traffic into axons and dendrites. One such RNA-binding protein is ZC3H14, which is lost in an inherited intellectual disability. The Drosophila melanogaster ZC3H14 ortholog, Nab2, localizes to neuronal nuclei and cytoplasmic ribonucleoprotein granules and is required for olfactory memory and proper axon projection into brain mushroom bodies. Nab2 can act as a translational repressor in conjunction with the Fragile-X mental retardation protein homolog Fmr1 and shares target RNAs with the Fmr1-interacting RNA-binding protein Ataxin-2. However, neuronal signaling pathways regulated by Nab2 and their potential roles outside of mushroom body axons remain undefined. This study presents an analysis of a brain proteomic dataset that indicates that multiple planar cell polarity proteins are affected by Nab2 loss, and couple this with genetic data that demonstrate that Nab2 has a previously unappreciated role in restricting the growth and branching of dendrites that elaborate from larval body-wall sensory neurons. Further analysis confirms that Nab2 loss sensitizes sensory dendrites to the genetic dose of planar cell polarity components and that Nab2-planar cell polarity genetic interactions are also observed during Nab2-dependent control of axon projection in the central nervous system mushroom bodies. Collectively, these data identify the conserved Nab2 RNA-binding protein as a likely component of post-transcriptional mechanisms that limit dendrite growth and branching in Drosophila sensory neurons and genetically link this role to the planar cell polarity pathway. Given that mammalian ZC3H14 localizes to dendritic spines and controls spine density in hippocampal neurons, these Nab2-planar cell polarity genetic data may highlight a conserved path through which Nab2/ZC3H14 loss affects morphogenesis of both axons and dendrites in diverse species (Corgiat, 2022).

    Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila

    A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. This study shows that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, these data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions (Koca, 2023).

    This study demonstrates that dAbl regulates cell motility during OR. Although loss of Abl function interferes with multiple aspects of photoreceptor development and morphogenesis, overexpression of dAbl in developing ommatidial clusters in eye discs affects specifically OR, suggesting that dAbl has a defined function in rotation. During OR, dAbl appears to have an inhibitory role, as ommatidial clusters with increased dAbl levels under-rotate, whereas dAbl mutant ommatidia tend to rotate faster (Koca, 2023).

    The localization pattern of dAbl posterior to the MF provides further insight about its role in OR. dAbl becomes apically localized in photoreceptors R8, R2/R5, and R4, following a steady phase of rotation, at the time when clusters slow down and refine their motility until the completion of the 90° angle. Prominent Abl localization within the apical plane of specific photoreceptors suggests that Abl is likely to have a local function in the apical junctional domain. Under-rotation features observed upon dAbl overexpression are consistent with the notion that dAbl becomes apically localized in specific R cells, toward the later stages of OR, to slow down the process. Interestingly, there is a differential localization of dAbl between R3 and R4 in the apical junctional domain. Considering the role of the R3/R4 pair and associated signaling pathways in OR, it is tempting to speculate that this differential dAbl localization is comparable to the requirement of the Nmo kinase within R3/R4, with Nmo providing a directional impulse to rotation in R433 and dAbl regulating its slowing down. The data argue that dAbl activity within R3/R4 pairs is indeed important for fine-tuning rotation. Knockdown and overexpression of dAbl in R3/R4 pairs lead to over-rotation and under-rotation, respectively, during the active rotation process in eye discs, suggesting that Abl activity negatively regulates rotation. Specifically, knockdown of Abl in R3/R4 leads to over-rotation of ommatidia, which, taken together with the WT localization of Abl being restricted to the R4 apical junctional domain, suggests that Abl is required in R4 within the apical region to slow down rotation. In the case of under-rotation caused by m Δ0.5>Abl overexpression, apical dAbl was detected in both cells of the R3/R4 pair and, importantly, temporally earlier in this background compared with WT, suggesting that early dAbl expression in both cells causes an under-rotation phenotype by interfering with rotation. Taken together, these observations are consistent with the hypothesis that the timing and specificity of apical localization of dAbl in R4 is critical for its normal function in OR (Koca, 2023).

    Notably, Abl overexpression does not appear to affect ommatidial chirality and the localization of PCP factors, as Fmi expression and localization remain intact. Furthermore, Abl overexpression causes a specific and severe under-rotation defect, unlikely resulting from deregulation of core PCP factors, which are commonly associated with random ommatidial chirality and rotation. It is most likely that Abl overexpression, under sev- or m Δ0.5-Gal4 drivers, is temporally too late to interfere with Fz/PCP signaling-mediated R3/R4 cell fate decisions, and thus specifically affects OR (Koca, 2023).

    Fz/PCP signaling appears dispensable for the R4-specific apical dAbl localization, as the pattern is maintained in core PCP mutant ommatidia. Yet dAbl does synergize with Fmi, when co-overexpressed in the R3/R4 pair, in a rotation specific manner. This OR-associated functional interaction of Abl with membrane-associated core PCP factors, along with the localization pattern of Abl in the apical domain further suggests that dAbl activity is important in R4 in the apical junctional domain. The results identify Notch and Notch signaling in R4 as critical for apical dAbl localization. Notch over-activation within the R3/R4 pair (via expression of stable isoforms of the receptor) induces apical dAbl localization in both cells of the pair. In contrast, expression in R3/R4 pairs of a version of Notch deficient in Delta binding, the key Notch ligand in the eye, and thus interference with ligand induced Notch activation, leads to a loss of apical dAbl in R4. Similarly, reduction of Notch levels in R3/R4 cells (via RNAi-mediated knockdown) also causes a marked decrease in apical dAbl levels in R4. As Notch-dependent transcription is still active in these backgrounds, the combination of these results suggests that Notch-mediated dAbl apical localization is rather direct, and not via a secondary mechanism through transcriptional regulation. This conclusion is corroborated by the co-immunoprecipitation experiments (Koca, 2023).

    Several experimental lines support the hypothesis that the Notch receptor physically recruits dAbl to the membrane. In salivary glands, Notch overexpression augments junctional dAbl localization, leaving total dAbl levels unaffected. dAbl co-immunoprecipitates with Notch in third-instar larval eye disc extracts, supporting a membrane-associated Notch-Abl interaction in vivo, independent of nuclear Notch signaling activity. The sev>Abl GOF rotation phenotype is markedly suppressed upon removal of one copy of Notch, further supporting the idea that a functional N-Abl signaling module in the apical domain of R4 regulates OR (Koca, 2023).

    dAbl localization appears to be within the apical region and not restricted to the apical membrane ring. There may be multiple reasons for this. As the Notch receptor is cleaved upon ligand binding and its intracellular domain is released to the cytoplasm, distribution of Abl molecules in the apical region may be broader than restricted to the transmembrane fraction of Notch. Abl-Notch interactions likely last after Notch cleavage, considering efficient Abl co-immunoprecipitation with the Notch ICD. Abl can also interact with actomyosin cytoskeletal elements, which are apically enriched in R cells (Koca, 2023).

    As the apical diameter of R cells in this region is less than 2 μm, the imaging resolution does not separate the membrane Abl signal from the juxta-membrane cytoplasmic signal. Notably, in Notch overexpression contexts, Abl signal is often detected as a ring at the apical membrane, likely attributable to the presence of more uncleaved membrane-associated Notch. Furthermore, it is possible to detect and quantify Abl at junctions in salivary glands, and thus document the increased levels of membrane-associated Abl upon higher Notch levels. All these data are consistent with the notion that Abl is specifically recruited to the apical junctional membrane domain by Notch (Koca, 2023).

    In Drosophila, dAbl has been suggested to act downstream of Notch during axonal pathfinding in embryos. Compelling evidence suggests that a non-canonical Notch signaling branch, which does not entail nuclear Notch activity, instructs axonal pathfinding and axon-guidance-specific genetic interactions between dAbl and Notch argue that a non-canonical Notch signaling pathway via dAbl may be at work in this context (Koca, 2023).

    The results are in accordance with these observations and provide further evidence for a non-canonical Notch-Abl signaling module during morphogenesis. Recently, a non-canonical Notch pathway has been reported in the regulation of adherens junction organization during human vascular barrier formation, with the transmembrane domain of Notch forming complexes with the tyrosine phosphatase LAR, vascular endothelial cadherin, and Rac1GEF Trio to confer barrier function in human engineered microvessels. The Notch transmembrane domain requires the cleavage of the Notch extracellular and intracellular domains in this context. The data during OR indicate that apical dAbl recruitment in R4 similarly requires Notch activation by Delta. Whether the transmembrane domain of Notch is an essential component of dAbl recruitment and/or regulation remains to be confirmed. There is a growing body of evidence that Notch uses alternative downstream signaling events to regulate cellular morphogenesis and organization, besides canonical transcriptional target gene regulation. (Koca, 2023).

    Abl appears to affect junctional N-cad and Arm levels in the R3/R4 pair. N-cad mutants show OR defects. Although the mechanism of N-cad involvement remains unclear, N-cad and/or Arm at the R3/R4 boundary could mediate the communication between these cells to determine relative force generation or other directional behavior to give the rotation direction or impulse/force. Such mechanisms have been suggested in border cell migration through E-cad (Koca, 2023).

    N-cad mutant ommatidia appear to over-rotate unlike Abl-overexpressing ommatidia (in which N-cad is downregulated at the R3/R4 border). Although this seems like a discrepancy, Abl overexpression by m Δ0.5-Gal4 (unlike N-cad mutations) is spatially and temporally restricted to R3/R4s, possibly accounting for the differences observed in these backgrounds. Furthermore, Abl likely affects OR via regulating several downstream effectors, including cytoskeletal regulators, in parallel to N-cad and thus has a more complex impact on OR than N-cad alone (Koca, 2023).

    The observation that the non-phosphorylatable isoform of Arm/β-catenin, ArmY667F, rescues the Abl GOF defects, supports the idea that Arm is a key and direct target of dAbl in the OR context. dAbl is involved in the regulation of multi-cellular reorganization in the context of Drosophila germband elongation through the phosphorylation of Arm/β-catenin on tyrosine 667 (Y667), by which it controls adherens junction turnover to promote convergent extension cell movements (Koca, 2023).

    The data argue that dAbl may similarly be involved in regulating Arm/β-catenin dynamics through the same residue during the OR process. The under-rotation phenotype associated with the dAbl GOF (sev>Abl) showed a trend toward rescue by co-(over)expression of Arm-WT and ArmY667E, which is likely due to the fact that exogenously overexpressed Arm isoforms compete with endogenous Arm for dAbl binding. Further experiments will be needed to test these hypotheses (Koca, 2023).

    The requirement of Abl in R4 for accurate rotation suggests that it acts antagonistically to Nemo which is enriched at junctions in R4 early via core PCP factors and its function is to promote rotation (Koca, 2023).

    There is a temporal sequence of apical plane enrichment of factors in R4 with Nemo first to initiate rotation, and Abl a few hours later to slow it down. It was originally proposed that OR is a two-step process, with an initial fast rotation to 45° and a subsequent slower step to achieve the full 90°. However, this idea goes back to the identification of the original allele of nemo, which is a hypomorph, and only affected the rotation process partially (Koca, 2023).

    Recent live imaging studies documenting OR dynamics have established that rotation is continuous with comparable speed throughout. Similarly, there is growing evidence that for rotation to occur correctly, adherens junctions need to be dynamically regulated at the interface between all photoreceptors and the non-rotating inter-ommatidial cells, and possibly between individual inter-ommatidial cells. It is thus very likely that Abl overexpression with m Δ0.5 and sev drivers interferes with rotation by affecting adherens junction regulation and dynamics in all or multiple R cells, like Nemo (Koca, 2023).

    Localization of Abl within the apical plane of R4, as well as R2/R5, is detected at late stages of rotation (from rows 7 and 8 onward), when rotation needs to be slowed down and stopped at 90°, indicating that Abl has a role at the late phases of the process, to terminate rotation. There are additional cues that appear to signal within ommatidia to stop rotation. For example, EGFR signaling via Argos (the original allele of argos being 'roulette/rlt') certainly feeds into slowing down rotation, as without the inhibitory EGFR ligand, argosrlt mutant clusters rotate beyond 90° (as the name 'roulette' indicates). Similarly, Scabrous (Sca), a secreted fibrinogen-like factor, has been suggested to regulate the properties of the extracellular matrix to create a barrier to rotation (Koca, 2023).

    Although the mechanism of Sca function remains unknown, a direct involvement of the ECM in rotation has been reported with a specific link of Integrin signaling and ECM in the OR process. A model is thus emerging that suggests the degree of rotation depends on an interplay between multiple signaling pathways, including Notch-Abl signaling, and their regulatory input to cell adhesion and cytoskeletal elements (Koca, 2023).

    Notch signaling in R3/R4 pairs is critical to coordinate OR via its feeding into the transcriptional regulation of argos, with Notch signaling directly promoting the transcription of argos, the inhibitory ligand to EGFR, required to fine-tune EGFR signaling activity during OR. This study shows that Notch signaling regulates OR via apical junctional recruitment of dAbl in R4, linking Notch activity to non-canonical, Abl-mediated Notch signaling and associated local cellular processes, with Abl modulating cadherin/β-catenin-based junctional complexes. Involvement of Notch signaling in cellular morphogenesis has been suggested in various contexts, including Drosophila oogenesis and neuronal pathfinding, zebrafish sensory organ development and human vascular barrier formation among others (Koca, 2023).

    Besides the reported Notch signaling-mediated transcriptional inputs into adhesion and cytoskeletal dynamics a direct link from the Notch receptor to cell adhesion has been revealed (Koca, 2023).

    This work also suggests a direct input from Notch signaling to cell adhesion dynamics. Many regulators of OR show conservation across developmental processes in vertebrates. The role of Notch signaling in OR suggests a potential involvement for Notch in PCP-mediated morphogenetic events in vertebrates, which has not been reported thus far. Similarly, Abl kinase may have a role in such processes in its interaction with PCP and Notch signaling pathways. Strikingly, the mouse abl-/- arg-/- double mutants exhibit defects in neurulation and delays in neural tube closure, a process generally requiring PCP-regulated features (Koca, 2023).

    The work described here provides insight into Notch-Abl signaling in a tissue remodeling, cell motility process. Although all data are consistent with the proposed model, this model is generated by inference from analyses of static fixed tissue samples, genetics, and biochemical studies. As it involves a cell motility process, it would be desirable to analyze the respective mutant genotypes via live imaging in vivo, including studies applying FRAP and other technologies. This would allow a more complete understanding of how Abl affects junctional dynamics during OR. Future studies will be needed to provide insight into the mechanistic details of how Notch and Abl cooperate in regulating junctional complexes and their dynamics during OR and other morphogenetic developmental and disease processes (Koca, 2023).

    DWnt4 and DWnt10 Regulate Morphogenesis and Arrangement of Columnar Units via Fz2/PCP Signaling in the Drosophila Brain

    Columns are structural and functional units of the brain. However, the mechanism of column formation remains unclear. The medulla of the fly visual center shares features with the mammalian cerebral cortex, such as columnar and layered structures, and provides a good opportunity to study the mechanisms of column formation. Column formation is initiated by three core neurons in the medulla, namely, Mi1, R8, and R7. The proper orientation of neurons is required for the orientation and arrangement of multiple columns. Their orientations may be under the control of planar cell polarity (PCP) signaling, because it is known to regulate the orientation of cells in two-dimensional tissue structures. This study demonstrates that the ligands DWnt4 and DWnt10 expressed specifically in the ventral medulla and dorsal medulla, respectively, globally regulate the columnar arrangement and orientation of Mi1 and R8 terminals through Fz2/PCP signaling in a three-dimensional space (Han, 2020).

    Cell cycle expression of polarity genes features Rb targeting of Vang

    Specification of cellular polarity is vital to normal tissue development and function. Pioneering studies in Drosophila and C. elegans have elucidated the composition and dynamics of protein complexes critical for establishment of cell polarity, which is manifest in processes such as cell migration and asymmetric cell division. Conserved throughout metazoans, planar cell polarity (PCP) genes are implicated in disease, including neural tube closure defects associated with mutations in VANGL1/2. PCP protein regulation is well studied; however, relatively little is known about transcriptional regulation of these genes. Earlier study revealed an unexpected role for the fly Rbf1 retinoblastoma corepressor protein, a regulator of cell cycle genes, in transcriptional regulation of polarity genes. This study analyzes the physiological relevance of the role of E2F/Rbf proteins in the transcription of the key core polarity gene Vang. Targeted mutations to the E2F site within the Vang promoter disrupts binding of E2F/Rbf proteins in vivo, leading to polarity defects in wing hairs. E2F regulation of Vang is supported by the requirement for this motif in a reporter gene. Interestingly, the promoter is repressed by overexpression of E2F1, a transcription factor generally identified as an activator. Consistent with the regulation of this polarity gene by E2F and Rbf factors, expression of Vang and other polarity genes is found to peak in G2/M phase in cells of the embryo and wing imaginal disc, suggesting that cell cycle signals may play a role in regulation of these genes. These findings suggest that the E2F/Rbf complex mechanistically links cell proliferation and polarity (Payankaulam, 2021).

    Autonomous epithelial folding induced by an intracellular mechano-polarity feedback loop

    Epithelial tissues form folded structures during embryonic development and organogenesis. Whereas substantial efforts have been devoted to identifying mechanical and biochemical mechanisms that induce folding, whether and how their interplay synergistically shapes epithelial folds remains poorly understood. This paper proposes a mechano-biochemical model for dorsal fold formation in the early Drosophila embryo, an epithelial folding event induced by shifts of cell polarity. Based on experimentally observed apical domain homeostasis, this study coupled cell mechanics to polarity and found that mechanical changes following the initial polarity shifts alter cell geometry, which in turn influences the reaction-diffusion of polarity proteins, thus forming a feedback loop between cell mechanics and polarity. This model can induce spontaneous fold formation in silico, recapitulate polarity and shape changes observed in vivo, and confer robustness to tissue shape change against small fluctuations in mechanics and polarity. These findings reveal emergent properties of a developing epithelium under control of intracellular mechano-polarity coupling (Wen, 2021).


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    genes expressed in tissue polarity

    Zygotically transcribed genes

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