prickle: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - prickle

Synonyms - sple, pk-sple, spiny legs

Cytological map position - 43A1

Function - unknown function in tissue polarity

Keywords - tissue polarity

Symbol - pk

FlyBase ID: FBgn0003090

Genetic map position - 2-55.3

Classification - LIM domain protein

Cellular location - unknown



NCBI links: Precomputed BLAST | Entrez Gene | UniGene |

Recent literature
Ambegaonkar, A. A. and Irvine, K. D. (2015). Coordination of planar cell polarity pathways through Spiny legs. Elife 4 [Epub ahead of print]. PubMed ID: 26505959
Summary:
Morphogenesis and physiology of tissues and organs requires planar cell polarity (PCP) systems that orient and coordinate cells and their behaviors, but the relationship between PCP systems has been controversial. This study characterized how the Frizzled and Dachsous-Fat PCP systems are connected through the Spiny-legs isoform of the Prickle-Spiny-legs locus. Two different components of the Dachsous-Fat system, Dachsous and Dachs, can each independently interact with Spiny-legs and direct its localization in vivo. Through characterization of the contributions of Prickle, Spiny-legs, Dachsous, Fat, and Dachs to PCP in the Drosophila wing, eye, and abdomen, this study defined where Dachs-Spiny-legs and Dachsous-Spiny-legs interactions contribute to PCP and provides a new understanding of the orientation of polarity and the basis of PCP phenotypes. These results support the direct linkage of PCP systems through Sple in specific locales, while emphasizing that cells can be subject to and must ultimately resolve distinct, competing PCP signals.

Sharp, K.A. and Axelrod, J.D. (2016). Prickle isoforms control the direction of tissue polarity by microtubule independent and dependent mechanisms. Biol Open [Epub ahead of print]. PubMed ID: 26863941
Summary:
Planar cell polarity signaling directs the polarization of cells within the plane of many epithelia. While these tissues exhibit asymmetric localization of a set of core module proteins, in Drosophila, more than one mechanism links the direction of core module polarization to the tissue axes. One signaling system establishes a polarity bias in the parallel, apical microtubules upon which vesicles containing core proteins traffic. Swapping expression of the differentially expressed Prickle isoforms, Prickle and Spiny-legs, reverses the direction of core module polarization. Studies in the proximal wing and the anterior abdomen indicated that this results from their differential control of microtubule polarity. Prickle and Spiny-legs also control the direction of polarization in the distal wing (D-wing) and the posterior abdomen (P-abd). It was found that this occurs without affecting microtubule polarity in these tissues. The direction of polarity in the D-wing is therefore likely determined by a novel mechanism independent of microtubule polarity. In the P-abd, Prickle and Spiny-legs interpret at least two directional cues through a microtubule-polarity-independent mechanism.

Warrington, S. J., Strutt, H., Fisher, K. H. and Strutt, D. (2017). A dual function for Prickle in regulating frizzled stability during feedback-dependent amplification of planar polarity. Curr Biol 27(18): 2784-2797.e2783. PubMed ID: 28918952
Summary:
The core planar polarity pathway coordinates epithelial cell polarity during animal development, and loss of its activity gives rise to a range of defects, from aberrant morphogenetic cell movements to failure to correctly orient structures, such as hairs and cilia. The core pathway functions via a mechanism involving segregation of its protein components to opposite cells ends, where they form asymmetric intracellular complexes that couple cell-cell polarity. This segregation is a self-organizing process driven by feedback interactions between the core proteins themselves. Despite intense efforts, the molecular pathways underlying feedback have proven difficult to elucidate using conventional genetic approaches. This study investigated core protein function during planar polarization of the Drosophila wing by combining quantitative measurements of protein dynamics with loss-of-function genetics, mosaic analysis, and temporal control of gene expression. Focusing on the key core protein Frizzled, its stable junctional localization is promoted by the core proteins Strabismus, Dishevelled, Prickle, and Diego. In particular, this study shows that the stabilizing function of Prickle on Frizzled requires Prickle activity in neighboring cells. Conversely, Prickle in the same cell has a destabilizing effect on Frizzled. This destabilizing activity is dependent on the presence of Dishevelled and blocked in the absence of Dynamin and Rab5 activity, suggesting an endocytic mechanism. Overall, this approach reveals for the first time essential in vivo stabilizing and destabilizing interactions of the core proteins required for self-organization of planar polarity.

BIOLOGICAL OVERVIEW

The Drosophila 'tissue polarity' genes control the orientation of bristles and hairs in the adult cuticle. Mutant flies have the polarity of cells altered in the plane of the epithelium without gross changes in the overall shape of imaginal structures or the distribution of differentiated cell types within them. Other tissue polarity defects include mirror-image duplications of the tarsal joints, rotations of bristle sockets in the leg, and changes in ommatidial polarity. The tissue polarity genes have been divided into three groups (Wong. 1993). The type 1 genes [dishevelled (dsh), frizzled (fz), and prickle (pk)], affect the whole body surface and are therefore believed to directly establish tissue polarity (Shulman, 1998). In contrast, the type 2 [inturned (in) and fuzzy (fy) and type 3 [multiple wing hairs (mwh)] genes affect distinct subsets of body areas and are thought to interpret the polarity established by the type 1 genes (Gubb, 1999).

prickle and spiny legs (sple) were thought to be two separate, although closely linked genes. Mutations in sple roughen the eye and disrupt ommatidial polarity. pk affects tissue polarity but has no mutant eye phenotype. However, a pk-sple double mutant gives rise to a strongly roughened eye, suggesting that sple and pk act redundantly in eye development. Sections of sple and pk-sple mutant eyes show a disturbed ommatidial polarity with each ommatidium having the normal arrangement of photoreceptor cells. Together with fz, these polarity mutants can be categorized into two classes based on the adult eye phenotypes. One class, which consists of fz, pk-sple and dsh, exhibits all three aspects of polarity phenotype seen in fz adult eyes. Among them, pk-sple had the strongest eye phenotype. The other class, which consists of sple, exhibits only one aspect of the polarity phenotype seen in fz eyes. Although sple ommatidia are still arranged in antiparallel arrays and the equator is still detected, some of the ommatidia are oriented such that their R7 cells are pointing away, rather than towards the equator. Anti-Elav staining of sple eye discs reveals that the disorientation is due to incorrect direction of rotation as in fz mutants. The single mutant alleles pkpk and pksple give the most extreme phenotypes, but in reciprocal regions of the body; pkpk in the wing and notum and pksple in the legs, abdomen, and eyes. Complementation between these classes of allele indicates two subtly different functions at the pk locus (Zheng, 1995 and Gubb, 1999).

Thus loss-of-function lesions in alternatively spliced transcripts of prickle result in two genetically distinct phenotypes, Pk and Spiny-legs. The pk and sple transcripts encode proteins that contain three LIM motifs and a novel conserved domain that has been called PET (Prickle Espinas Testin). Surprisingly, deletion of the entire gene gives a phenotype that is much weaker than that of either the pk or sple single mutants. This unusual result can be explained by the pk and sple gene products acting in concert. The single-mutant phenotypes result from misactivation, rather than simply blocking, of a pathway of polarity formation. It is proposed that the correct balance of the Pk and Sple variants is required for normal planar polarity signaling in the Drosophila imaginal discs (Gubb, 1999).

To investigate the functional relationship between the Pk and Sple protein variants, mutant combinations that alter the ratio of the pk and sple transcripts were generated. The strongest phenotypes result from lack of either one of these two transcripts in homozygous mutants. When the single mutant alleles are combined with pkpk-sple alleles or deficiencies, the resulting phenotypes are weaker than the corresponding homozygous single mutant phenotypes. Thus, flies that carry only a single functional copy of the sple transcript or the pk transcript, show an intermediate phenotype more similar to pkpk-sple13 than the single mutant. These results imply that the presence of one transcript without the other (as in pkpk or pksple single mutants) creates an extreme phenotype that is corrected when the dose of the remaining transcript is reduced. The implication is that both the relative and absolute levels of the Pk and Sple proteins are important for the function of putative Pk-Sple homomeric or heteromeric protein complexes (Gubb, 1999).

The importance of the levels of Pk and Sple expression and the balance between them has been investigated further with overexpression constructs. When driven by the expression of the uniform drivers gal4-da (daughterless) or gal4-C765, P[UAS:pk+] an extreme tarsal duplication phenotype results, including a duplicated socket structure in the proximal T1 segment. Overexpression of UAS:sple+ with these drivers gives a Pk phenotype in the wing triple row bristles; the tarsi, however, remain completely wild type. These results confirm that overexpression of pk gives a phenotype analogous to lack of the sple transcript, whereas overexpression of sple gives a phenotype similar to lack of pk. The da-UAS:pk+ and da-UAS:sple+ wing phenotypes show an unexpected feature that is not seen in mutant alleles. Instead of uniform polarity patterns, swirls are seen in the wing hair orientation with different patterns from wing to wing. In addition to phenotypes resembling loss of function, misexpression of pk in engrailed (en)-UAS:pk+ flies blocks the migration of wing hairs from the distal vertex of wing cells to the central position. This antagonism between the pk and sple transcripts implies either that both proteins compete for a target that is present in limiting amounts, or that they form protein complexes with distinct activities. In pkpk and pksple mutants excess, homodimers would misactivate polarity signaling (Gubb, 1999).

Deletion of the entire prickle gene gives a phenotype over the whole body surface that is similar to dsh and fz. To test whether these mutants affect the same signaling pathway, double mutant combinations were made. Double mutants of pkpk and dsh in the triple row give a Dsh phenotype, suggesting that dsh is epistatic to pkpk. In other words, dsh function is blocked; pk function is irrelevant. These data suggest that many of the functions of pk require dsh. The situation within the wing blade is less clear, as the double mutants give an intermediate phenotype. The dsh; pksple double mutant retains a dsh phenotype in the triple row, but the wing hair pattern is altered, despite the fact that pksple alleles have no wing phenotype. In the leg, the dsh tarsal phenotype is not modified by pkpk. There is a synergistic interaction between dsh and pksple, with the double mutant giving a more extreme mirror-image transformation of the T1 segment than either of the single mutants. This extreme transformation is also seen with UAS:pk+ overexpression and in fz (Gubb, 1999).

Clones of fz show a directional nonautonomy that has been interpreted to mean that fz mediates the intercellular communication of a polarity signal. On the other hand, dsh is strictly cell autonomous in clones, implying that it is involved in signal reception. To investigate whether pk participates in both reception and transmission of a polarity signal, pkpk clones in the wing were studied. Large clones in the wing express the mutant polarity typical of that region of the wing in homozygous flies. There is an occasional nonautonomous disruption of polarity in wild-type cells adjacent to the proximal or lateral margins of a pkpk clone. In these cases, a short range perturbation aligns wild-type cells with the mutant polarity pattern. There is no clear pattern to the position of such clones, but small peninsulas of pawn (pwn+) tissue surrounded by pkpk pwn tissue tend to adopt the mutant polarity pattern. Smaller clones, induced later than 72-96 hr, do not alter the polarity of adjacent pwn+ tissue. In contrast to the autonomous behavior of pk, clones of fz tricornered (trc) cause long-range domineering nonautonomy both distal (Vinson, 1987) and lateral to the clone. Wild-type hairs are oriented toward the clone as though it is acting as a polarity 'sink'. Proximal cells are also directed toward the fz trc clone, but as this is the normal orientation for wing cells no polarity changes would be expected (Gubb, 1999).

There are a number of conserved features within the LIM domains of the pk family proteins. In the first LIM domain, there is a proline residue between the third (H) and fourth (C) zinc binding residues. This internal proline residue might introduce a kink within the zinc binding site. Similar LIMP domains with an internal P at this site are found in the human SLIM 2, SLIM 3, PINCH, Zyxin, and Paxillin proteins and LIM domain kinase 2 (LIK2) from chicken, mouse, rat, and human. In addition, the length of the consecutive LIM domains is constant in the pk family, with LIM1 containing 57 amino acids; LIM2 containing 52 aa, and LIM3 containing 56 aa. The implication is that the conservation of the triple LIM domain itself is important, rather than the individual LIM domains. In the case of the triple LIM domain protein Zyxin, the individual LIM domains may bind different target proteins and act as a template for the assembly of a number of structural components (Beckerle, 1997 and Gubb, 1999). A similar scaffold function for the Pk protein would be consistent with the cell autonomy of pk in clones and the expression of pk transcripts in cells that are changing shape. The blocking of the normal migration of the actin-rich prehair structure to the center of wing cells by overexpression of pk also implies a role in cytoskeletal remodeling. The lack of embryonic phenotype, despite the dynamic expression pattern, implies that pk function is redundant during embryonic development. A putative embryonic pk function could, in principle, be maternally supplied, but pk mutant strains of all three classes are fully fertile and homozygous pk embryos from homozygous mothers remain wild type. Similarly, gal4-da; P[UAS:pk+], and gal4-da; P[UAS:sple+] flies are completely viable and show no embryonic phenotype, despite the embryonic expression of the gal4-da driver (Gubb, 1999).

Perhaps the most surprising feature of the tissue polarity mutants is the precise polarity patterns seen in the wing hairs (Gubb, 1982). Rather than reflecting fine-grained positional information, however, the precision of the final pattern might be dependent on a tessellation mechanism. The orientation of the first cell would determine the alignment of subsequent cells, like sticking tiles on a bathroom wall. In pkpk mutants, the alignment of wing hairs deviates progressively with occasional abrupt changes. In the adult mutant wing, hair polarity alters gradually with sudden topological discontinuities that resemble the stacking flaws in liquid crystals. In Df(2R)pk-30 wings, regions where the wing hairs are uniformly oriented retain a predominantly hexagonal array. Regions surrounding topological discontinuities, such as the anterior whorl, show irregular cell shapes frequently associated with duplicated wing hairs (Gubb, 1999).

Strong support for a tessellation mechanism is given by the overexpression phenotype of Pk and Sple when driven by a ubiquitous promotor. Although polarity patterns are variable, hair orientation alters smoothly from cell to cell across the wing surface, indicating that the polarity of cytoskeletal structures is aligned within large fields of cells. It is as if cell packing had nucleated randomly and then spread to neighboring cells until meeting an adjacent domain. The short-range perturbation in polarity that is occasionally seen proximal and lateral to a pkpk clone is consistent with mechanical adjustment of cells to fit against their immediate neighbors, unlike the long-range domineering nonautonomy lateral and distal to fz clones. With both classes of clones, a tessellation mechanism might impose a threshold. Below this threshold, disruptions in polarity signaling would fail to affect the orientation of neighboring cells (Gubb, 1999).

The tarsal phenotype of dsh is very similar to pkpksple13 causing duplications of the T3 and T4, segments. A more extreme phenotype including complete duplications of T2 to T4 together with a well-developed ectopic T1 joint are seen in dsh; pksple, overexpression UAS:pk+ and fz mutant strains, implying that a similar polarity signaling pathway is affected. It has been suggested that pk and fz are upstream in a signaling pathway leading to dsh (Wong, 1993 and Shulman, 1998). There are several problems with a simple linear pathway, however, and the relationship between pk and fz is unclear. (1) Neither fz nor pk is clearly epistatic to the other; rather, the double mutant (pkpk; fz) phenotype is intermediate between pkpk and fz (Gubb, 1982 and Wong, 1993). (2) Whereas fz clones cause a long-range nonautonomous disruption in surrounding tissue, pkpk clones resemble dsh clones in being almost completely cell autonomous. (Both pksple1 and pkpk-sple13 clones are cell autonomous in the eye. In contrast, the pkpk triple row bristle phenotype (that results from Sple expression in the absence of Pk) is suppressed in dsh; pkpk wings and the dsh polarity pattern is modified in dsh; pksple wings (despite pksple on its own not having a wing phenotype). (3) Complete lack of pk transcripts produces a phenotype very similar to dsh. Taken together, these results indicate that Pk is not downstream of Fz but may represent an alternative input into Dsh-mediated planar polarity signaling. If Dsh is acting as a scaffolding or adapter protein then it would be required in stoichiometric ratios to its target proteins, and overexpression would prevent assembly of functional complexes. It may be that the Pk protein isoforms are components of this protein complex that are expressed in cells remodeling their cytoskeletal architecture (Gubb, 1999).

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).


GENE STRUCTURE

The 5' start of the pk transcript (located in the most 3' portion of the gene) lies within a cluster of three serpins (serine Proteinase inhibitors) (Spn43Aa, Spn43Ab, and Spn43Ac), one of which corresponds to the necrotic gene. A transcript with homology to Adenosine kinases maps just distal to pk gene termination. The first pk exon contains an untranslated leader sequence of 0.8 kb, with the putative translation start site being 39 bp 5' to the large intron. The breakpoints of pk-associated mutations map across 70 kb of genomic DNA. Single-mutant pkpk alleles carry lesions within the proximal 30 kb (the 3' portion of the gene), whereas pkpk-sple double-mutant alleles map between 42 and 69 kb (nearer the 6 common exons) and none of the pksple single mutations have been localized. Two putative pk transcripts are detected as weak bands of ~4.2 and 5.1 kb in embryos and 2-day pupae on developmental Northern blots. The shorter transcript corresponds to pk (initiating from most 3' exon) and the larger to sple. The 5' end of pk hybridizes to the region of the pkpk breakpoints and is separated by a large intron from the remaining 6 exons. The 5' end of sple is within the large pk intron. An additional medial transcript, pkM, is only detected in embryonic stages. The identity of the pk and sple transcripts has been confirmed by rescue experiments (Gubb, 1999).

Genomic length - 70 kb

Exons - 6 common exons and three initiating exons each serving a different transcript


PROTEIN STRUCTURE

Amino Acids - The Pk protein isoforms consist of 870 (Pk), 936 (PkM), and 1206 (Sple) amino acid chains with the conserved PET and LIM
domains mapping entirely within the common exons

Structural Domains

The tissue polarity mutants in Drosophila include a set of conserved gene products that appear to be involved in the control of cytoskeletal architecture. The tissue polarity gene prickle (pk) encodes a protein with a triple LIM domain and a novel domain, designated PET, is present in human, murine, and Caenorhabditis elegans homologs. Three transcripts have been identified (pk, pkM, and sple) encoding 93-, 100-, and 129-kD conceptual proteins, respectively. The three transcripts span 70 kb and share 6 exons that contain the conserved domains. The second to seventh exons of pk are common to the three transcripts. These common exons encode a peptide sequence containing three LIM domains, but no recognized DNA binding, membrane spanning, or signal peptide motifs. LIM domains, serving to mediate protein interactions, are cysteine-rich sequences with a double zinc finger motif (for review, see Dawid, 1998). Four closely related LIM domain sequences have been found by database searches; one murine (Testin; Divecha, 1995), one human (LMO6; Fisher, 1997), and two Caenorhabditis elegans. An additional pk cognate gene in Drosophila, espinas (esn), maps just proximal to the 43A1.2 Serpin cluster (C. Green and D. Gubb, unpubl., cited in Gubb, 1999). The Serpin cluster is in the far upstream portion of the prickle gene. Comparison of these sequences identifies a novel conserved PET domain, 5' to the LIM domains. Two further incomplete sequences with PET domain homology have been identified by database searches: a human EST and a C. elegans ORF. The PET domain is unrelated to other sequence motifs in the protein database and encodes a neutral stretch of amino acids with no obvious distinguishing features. Database searches failed to identify any sequences with significant homology to peptides encoded by the unique 5' pk, pkM, or sple exons. Both the human and mouse homologs, LMO6 and Testin (Divecha, 1995 and Fisher, 1997), encode alternative 5' transcripts that may be a general feature of the pk family (Gubb, 1999).

Structural and membrane binding properties of the prickle PET domain

The planar cell polarity (PCP) pathway is required for fetal tissue morphogenesis as well as for maintenance of adult tissues in animals as diverse as fruit flies and mice. One of the key members of this pathway is Prickle (Pk), a protein that regulates cell movement through its association with the Dishevelled (Dsh) protein. Pk presents three LIM domains and a PET domain of unknown structure and function. Both the PET and LIM domains control membrane targeting of Dsh, which is necessary for Dsh function in the PCP pathway. This study shows that the PET domain is monomeric and presents a nonglobular conformation with some properties of intrinsically disordered proteins. The PET domain adopts a helical conformation in the presence of 2,2,2-trifluoroethanol (TFE), a solvent known to stabilize hydrogen bonds within the polypeptide backbone, as analyzed by circular dichroism (CD) and NMR spectroscopy. Furthermore, the conserved and single tryptophan residue in PET, Trp 536, moves to a more hydrophobic environment when accompanied with membrane penetration and the protein becomes more helical in the presence of lipid micelles. The presence of LIM domains, downstream of PET, increases protein folding, thermostability, and tolerance to limited proteolysis. In addition, pull-down and tryptophan fluorescence analyses suggest that the LIM domains physically interact to regulate membrane penetration of the PET domain. The findings reported here favor a model where the PET domain is engaged in Pk membrane insertion, whereas the LIM domains modulate this function (Sweede, 2008).


EVOLUTIONARY HOMOLOGS

Two new cDNAs have been cloned and sequenced that code for proteins carrying the related triple LIM domains (acronym of Lin-11, Isl-1, Mec-3) proteins. These LIM domains show good agreement to the LIM domain consensus sequence, but also exhibit some novel variations. The 1.36-and 2.8-kb cDNAs are probably splice variants of one gene and code for 42- and 50-kDa proteins, respectively. The larger transcript has a 900-nucleotide (nt) 3' untranslated region (UTR). High levels of the 2.8-kb transcript can be detected in many tissues, and all tissues show some level of expression of both transcripts, the larger transcript being more abundant. In adult testis there are very high levels of the 1.36-kb transcript and moderate levels of the 2.8-kb transcript. The wide tissue distribution and high levels of expression suggest an important role for these proteins in cellular function (Divecha, 1995).

Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis. A noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila, participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, it is shown that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene, is an essential component for gastrulation cell movement. Xpk encodes an 835-amino acid protein with a single PET domain and three repetitive LIM domains in its N-terminal half. Both gain-of-function and loss-of-function of Xpk severely perturbs gastrulation and causes spina bifida embryos without affecting mesodermal differentiation. XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation (Takeuchi, 2003).

The possibility that XPK activates JNK was examined because JNK has been reported to act in the noncanonical Wnt pathway downstream of Dsh. To evaluate JNK activation, the phosphorylation of a target of JNK, c-Jun, was tested in HEK293T cells transfected with Xdsh, Xpk, or both cDNAs. Xpk alone failed to activate JNK, whereas Xdsh activates JNK in a dose-dependent manner. However, cotransfection of Xdsh with Xpk but not with ΔP/L (lacking the PET and LIM domains) cDNAs dramatically increase the level of c-Jun phosphorylation, even at Xdsh levels, which alone cannot activate JNK efficiently. This result suggests that XPK cooperates with Xdsh to activate JNK through its P/L domain. At a high level of Xdsh protein, Xdsh alone can activate JNK to a certain level, and interestingly, wild-type Xpk or ΔP/L but not P/L suppresses the JNK activation by Xdsh, suggesting that the part of XPK excluding the PET/LIM domain may act negatively to Xdsh-mediated JNK activation at high levels of Xdsh. This is consistent with the observation that ΔP/L and Xpk including the LIM domain counteract each other. These observations prompted a test of whether XPK interacts physically with Xdsh. To test this possibility, a GST-pull-down assay of tagged XPK and Xdsh was carried out in HEK293T cells. Xdsh and XPK are each efficiently precipitated with the other's GST fusion protein, indicating that XPK and Xdsh interacted physically with each other. The interaction between XPK and Xdsh is conserved among species; the Drosophila Prickle PET/LIM domain has been reported to bind Dsh. The yeast two-hybrid assay also demonstrates that the PET/LIM domains of XPK are sufficient to bind Xdsh (Takeuchi, 2003).

It is speculated that XPK might act as a scaffold for JNK activation, so whether XPK binds to JNK was tested. Neither the PET/LIM nor the ΔPET/LIM is sufficient to bind JNK, and only wild-type XPK can bind JNK significantly. Nevertheless, this further suggests that XPK forms a ternary complex with Xdsh and JNK. Although this possibility was examined, the formation of the ternary complex could not be demonstrated (Takeuchi, 2003).

It has been proposed that Prickle generates asymmetric Frizzled and Dishevelled localization in the Drosophila wing, through the suppression of Fz and Dsh localization at the proximal cell cortex. In this study, it is shown that XPK is a key component connecting Xdsh to JNK activation during Xenopus gastrulation. It has been predicted from Drosophila genetics that JNK is one of the downstream targets of the PCP pathway. These results reinforce the idea that the noncanonical Wnt (PCP) pathway regulates gastrulation cell movements in vertebrates through JNK activation. To further understand the pathway, attempts are currently being made to identify XPK-interacting components that regulate JNK activation (Takeuchi, 2003).

In addition to the canonical Wnt/ß-catenin signaling pathway, at least two noncanonical Wnt/Fz pathways have been described: the planar cell polarity (PCP) pathway in Drosophila and the Wnt/calcium pathway in vertebrate embryos. Recent work suggests that a vertebrate pathway homologous to the PCP pathway acts to regulate the convergent extension movements of gastrulation. To further test this hypothesis, two zebrafish homologs were identified of the Drosophila PCP gene prickle (pk), both of which show discrete and dynamic expression patterns during gastrulation. Both gain and loss of pk1 function cause defects in convergent extension. Pk1 localizes to both the cytoplasm and the cell membrane, and its normal localization is partially dependent on its C-terminal prenylation motif. At the cell membrane, Pk1 is frequently localized asymmetrically around the cell and can colocalize with the signaling molecule Dishevelled (Dsh). In overexpression assays, Pk1 is able to activate AP-1-mediated transcription and inhibit activation of Wnt/ß-catenin signaling. Like noncanonical Wnts, overexpression of Pk1 increases the frequency of calcium transients in zebrafish blastulae. These results support the idea that a vertebrate PCP pathway regulates gastrulation movements and suggest that there is overlap between the PCP and Wnt/calcium pathways (Veeman, 2003).

During vertebrate gastrulation, mesodermal and ectodermal cells undergo convergent extension, a process characterized by prominent cellular rearrangements in which polarised cells intercalate along the medio-lateral axis leading to elongation of the antero-posterior axis. A noncanonical Wnt/Frizzled (Fz)/Dishevelled (Dsh) signalling pathway related to the planar-cell-polarity (PCP) pathway in flies, regulates convergent extension during vertebrate gastrulation. A zebrafish homolog of Drosophila prickle (pk), a gene that is implicated in the regulation of PCP, has been isolated and functionally characterized. Zebrafish pk1 is expressed maternally and in moving mesodermal precursors. Abrogation of Pk1 function by morpholino oligonucleotides leads to defective convergent extension movements, enhances the silberblick (slb)/wnt11 and pipetail (Ppt)/wnt5 phenotypes and suppresses the ability of Wnt11 to rescue the slb phenotype. Gain-of-function of Pk1 also inhibits convergent extension movements and enhances the slb phenotype, most likely caused by the ability of Pk1 to block the Fz7-dependent membrane localization of Dsh by downregulating levels of Dsh protein. Furthermore, pk1 is shown to interact genetically with trilobite (tri)/strabismus to mediate the caudally directed migration of cranial motor neurons and convergent extension. These results indicate that, during zebrafish gastrulation Pk1 acts, in part, through interaction with the noncanonical Wnt11/Wnt5 pathway to regulate convergent extension cell movements, but is unlikely to simply be a linear component of this pathway. In addition, Pk1 interacts with Tri to mediate posterior migration of branchiomotor neurons, probably independent of the noncanonical Wnt pathway (Carreira-Barbosa, 2003).

Regulation of planar cell polarity by Smurf ubiquitin ligases

Planar cell polarity (PCP) is critical for morphogenesis in metazoans. PCP in vertebrates regulates stereocilia alignment in neurosensory cells of the cochlea and closure of the neural tube through convergence and extension movements (CE). Noncanonical Wnt morphogens regulate PCP and CE in vertebrates, but the molecular mechanisms remain unclear. Smurfs are ubiquitin ligases that regulate signaling, cell polarity and motility through spatiotemporally restricted ubiquitination of diverse substrates. This study reports an unexpected role for Smurfs in controlling PCP and CE. Mice mutant for Smurf1 and Smurf2 display PCP defects in the cochlea and CE defects that include a failure to close the neural tube. Smurfs engage in a noncanonical Wnt signaling pathway that targets the core PCP protein Prickle1 for ubiquitin-mediated degradation. This work thus uncovers ubiquitin ligases in a mechanistic link between noncanonical Wnt signaling and PCP/CE (Narimatsu, 2009).

Mechanistic analysis of PCP signaling has revealed physical interactions between otherwise differentially localized PCP components that include interactions between Vangl, Prickle and Dvl. This study has mapped interactions between Smurfs and components of the Wnt signaling pathway using a systematic screen and identified an interaction between Smurf and Dvl that is dependent on phosphorylation of Dvl. Accordingly, a DEP domain mutant that blocked Dvl phosphorylation also blocked Smurf interaction, as did a DEP mutation that is PCP-specific in the fly. These findings are in agreement with key roles previously defined for the DEP domain in PCP signaling in flies and vertebrates. Constitutive interactions were identified between Par6 and Dvl, consistent with previous observations in neurons, as well as an interaction between Par6 and Prickle. Together, these results suggest a model in which Par6 is engaged in a trimeric complex with Prickle and Dvl that upon phosphorylation-dependent recruitment of Smurf to Dvl in response to Fzd signaling leads to Prickle ubiquitination and degradation. Consistent with this, it was found that Wnt5a induced Smurf-Par6 interactions and that Prickle bound to Par6 was subject to Smurf-dependent ubiquitination and turnover that was also dependent on the Dvl binding domain of Par6. Moreover, in Smurf mutants increased levels of Pk1 protein the following two observations were made: (1) loss of the local asymmetric distribution of Pk1 in the neuroepithelium, and in the cochlea, (2) Pk1 that was normally localized in a medial crescent in the OHCs, extended to the lateral side (Narimatsu, 2009).

Dvl has also been shown to interact with the transmembrane protein Vangl, which also binds Pk and Pk can antagonize Dvl function. Moreover, Diego, an ankyrin repeat containing protein has been shown to directly compete with Pk for Dvl binding and can promote Dvl function in the PCP pathway via recruitment to membranes. Whether Diego functions primarily on Dvl, or plays a role in antagonizing Pk coassembled with Par6, perhaps in cooperation with Smurfs is unknown, but altogether these findings suggest that Pk and Dvl assemble via unique signaling complexes that respond to different extrinsic cues during PCP signaling. Consequently, while all PCP components might physically interact with each other, their asymmetric distribution may be maintained through dynamic modulation of complex membership (Narimatsu, 2009).

Although PCP and CE have been linked via shared components and a key role for noncanonical Wnt signaling, the relationship of the molecular pathways governing tissue polarity to CE has been less clear. In particular, during CE the neuroepithelium and the underlying axial mesoderm do not display the highly organized structures typically associated with PCP and tissue polarity and while Vangl and Dvl show asymmetric distribution in the cochlea, there is no evidence for asymmetric distribution of either protein during CE in the mouse. Regardless, Par6-dependent asymmetric localization of endogenous Pk1 was clearly detected in neuroepithelial cells; this was manifest at the cellular level, and in Smurf DKO mutants this asymmetry was lost. Furthermore, Par6 can also control the stable mediolateral extension of cell protrusions during CE. Localized Fzd signaling may thus regulate the asymmetric distribution of specific PCP components such as Pk1 in the absence of asymmetric distribution of all PCP pathway components. Vertebrates may thus broadly employ PCP signaling to control polarity at the local and even cellular level. Consistent with this, noncanonical Wnt signaling, Dvl and Par6 have all been shown to play key roles in regulating directed cell motility and the polarity of neurons (Narimatsu, 2009).

These results demonstrate a noncanonical Wnt signaling pathway in which Smurf is recruited to Par6 via Dvl to regulate the degradation of PCP pathway components that in turn controls the asymmetric distribution of Pk1. Signal dependent degradation of PCP components may thus allow for dynamic use of the pathway in a local manner during convergent extension, as well as in the establishment of tissue polarity in organized epithelia such as the inner ear (Narimatsu, 2009).

Zebrafish Prickle1b mediates facial branchiomotor neuron migration via a farnesylation-dependent nuclear activity

The facial branchiomotor neurons (FBMNs) undergo a characteristic tangential migration in the vertebrate hindbrain. A morpholino knockdown approach has been used to reveal that zebrafish prickle1b (pk1b) is required for this migration. This study reports that FBMN migration is also blocked in a pk1b mutant with a disruption in the consensus farnesylation motif. It was confirmed that this lipid modification is required during FBMN migration by disrupting the function of farnesyl biosynthetic enzymes. Furthermore, farnesylation of a tagged Pk1b is required for its nuclear localization. Using a unique rescue approach, it was demonstrated that Pk1b nuclear localization and farnesylation are required during FBMN migration. The data suggest that Pk1b acts at least partially independently of core planar cell polarity molecules at the plasma membrane, and might instead be acting at the nucleus. It was also found that the neuronal transcriptional silencer REST is necessary for FBMN migration, and evidence is provided that interaction between Pk1b and REST is required during this process. Finally, it was demonstrated that REST protein, which is normally localized in the nuclei of migrating FBMNs, is depleted from the nuclei of Pk1b-deficient neurons. It is concluded that farnesylation-dependent nuclear localization of Pk1b is required to regulate REST localization and thus FBMN migration (Mapp, 2011).

PRICKLE1 interaction with SYNAPSIN I reveals a role in autism spectrum disorders

The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, studies were carried out in Prickle1(+/-) mice and Drosophila, yeast, and neuronal cell lines. Mice with Prickle1 mutations were shown to exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, a yeast two-hybrid screen was performed with a human brain cDNA library and a peptide was isolated with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles (Paemka, 2013).

Null and hypomorph Prickle1 alleles in mice phenocopy human Robinow syndrome and disrupt signaling downstream of Wnt5a

Ror2 gene defects, prompting an exploration of an association of Prickle1 with the Wnt pathway. This study shows that Prickle1 is a proteasomal target of Wnt5a signaling and that Dvl2, a target of Wnt5a signaling, is misregulated in Prickle1 mutants. These studies implicate Prickle1 as a key component of the Wnt-signaling pathway and suggest that Prickle1 mediates some of the WNT5A-associated genetic defects in Robinow syndrome (Liu, 2014; PubMed).

Prickle3 synergizes with Wtip to regulate basal body organization and cilia growth

PCP proteins maintain planar polarity in many epithelial tissues and have been implicated in cilia development in vertebrate embryos. This study examined Prickle3 (Pk3), a vertebrate homologue of Drosophila Prickle, in Xenopus gastrocoel roof plate (GRP). GRP is a tissue equivalent to the mouse node, in which cilia-generated flow promotes left-right patterning. Pk3 was shown to be enriched at the basal body of GRP cells but is recruited by Vangl2 to anterior cell borders. Interference with Pk3 function disrupted the anterior polarization of endogenous Vangl2 and the posterior localization of cilia in GRP cells, demonstrating its role in PCP. Strikingly, in cells with reduced Pk3 activity, cilia growth was inhibited and gamma-tubulin and Nedd1 no longer associated with the basal body, suggesting that Pk3 has a novel function in basal body organization. Mechanistically, this function of Pk3 may involve Wilms tumor protein 1-interacting protein (Wtip), which physically associates with and cooperates with Pk3 to regulate ciliogenesis. It is proposed that, in addition to cell polarity, PCP components control basal body organization and function (Chu, 2016z).

Wnt proteins can direct planar cell polarity in vertebrate ectoderm

The coordinated orientation of cells across the tissue plane, known as planar cell polarity (PCP), is manifested by the segregation of core PCP proteins to different sides of the cell. Secreted Wnt ligands are involved in many PCP-dependent processes, yet whether they act as polarity cues has been controversial. This study shows that in Xenopus early ectoderm, the Prickle3/Vangl2 complex (see Vang) was polarized to anterior cell edges and this polarity was disrupted by several Wnt antagonists. In midgastrula embryos, Wnt5a, Wnt11, and Wnt11b, but not Wnt3a, acted across many cell diameters to orient Prickle3/Vangl2 complexes away from their sources regardless of their positions relative to the body axis. Planar polarity of endogenous Vangl2 in the neuroectoderm was similarly redirected by an ectopic Wnt source and disrupted after depletion of Wnt11b in the presumptive posterior region of the embryo. These observations provide evidence for the instructive role of Wnt ligands in vertebrate PCP (Chu, 2016b).


prickle: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 17 October 99

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