yurt: Biological Overview | References
Gene name - yurt
Cytological map position - 87E11-87E11
Function - signaling
Symbol - yrt
FlyBase ID: FBgn0004049
Genetic map position - 3R:9,254,589..9,260,704 [-]
Cellular location - cytoplasmic
The integrity of polarized epithelia is critical for development and human health. Many questions remain concerning the full complement and the function of the proteins that regulate cell polarity. This study reports that the Drosophila FERM proteins Yurt (Yrt) and Coracle (Cora) and the membrane proteins Neurexin IV (Nrx-IV) and Na+,K+-ATPase are a new group of functionally cooperating epithelial polarity proteins. This 'Yrt/Cora group' promotes basolateral membrane stability and shows negative regulatory interactions with the apical determinant Crumbs (Crb). Genetic analyses indicate that Nrx-IV and Na+,K+-ATPase act together with Cora in one pathway, whereas Yrt acts in a second redundant pathway. Moreover, it was shown that the Yrt/Cora group is essential for epithelial polarity during organogenesis but not when epithelial polarity is first established or during terminal differentiation. This property of Yrt/Cora group proteins explains the recovery of polarity in embryos lacking the function of the Lethal giant larvae (Lgl) group of basolateral polarity proteins. It was also found that the mammalian Yrt orthologue EPB41L5 (also known as YMO1 and Limulus) is required for lateral membrane formation, indicating a conserved function of Yrt proteins in epithelial polarity (Laprise, 2009).
To clarify the mechanisms of epithelial polarization, the function was examined of the FERM-domain protein Yrt, which was shown to act as a negative regulatory component of the Crb complex in both Drosophila and vertebrates (Laprise, 2006; Hsu, 2006). Crb regulates epithelial apical basal polarity by promoting apical membrane and apical junctional complex formation. Crb also controls the growth of the apical membrane at late stages of epithelial differentiation. Yrt binds to the cytoplasmic tail of Crb and restricts Crb activity in apical membrane growth (Laprise, 2006). However, Yrt is predominantly a basolateral protein that is recruited into the Crb complex only at late stages of epithelial differentiation. Embryos completely lacking maternal and zygotic Yrt (yrtM/Z) displayed polarity defects before Yrt is recruited into the Crb complex at late organogenesis. This raises the question whether Yrt has a function in epithelial organization as a basolateral protein (Laprise, 2009).
yrtM/Z mutants and double-mutant combinations of yrt and genes encoding basolateral proteins were examined for synergistic genetic interactions that could indicate functional cooperation. yrtM/Z embryos showed clear polarity defects at post-gastrula stages of development, as indicated by the basolateral mislocalization of Crb. In contrast, zygotic yrt mutants demonstrated only minor polarity defects in the trunk ectoderm. This indicates that yrt is required for polarity, and that zygotic yrt mutants could provide a sensitized background to reveal genetic interactions. Within the basolateral membrane, Yrt is enriched at the septate junction together with the Na+,K+-ATPase and other septate junction components from stage 14 onwards. Marked genetic interactions were found between yrt and Atpα (which encodes the α-subunit of Na+,K+-ATPase) and between yrt and Nrx-IV, but not between yrt and six other genes that encode septate junction transmembrane proteins. In contrast to wild type and single mutants, apical markers were mislocalized in yrt Atpα and yrt Nrx-IV double mutants similar to yrtM/Z embryos. Enhancement of the Nrx-IV null phenotype by yrt indicates that Nrx-IV and yrt have overlapping functions and do not operate in a linear pathway. The pathway relationship between yrt and Atpα remains uncertain as embryos completely devoid of Na+,K+-ATPase function cannot be analysed. These findings reveal previously unrecognized functions of the Na+,K+-ATPase and Nrx-IV in epithelial polarity (Laprise, 2009).
The developmental timing of the yrtM/Z and the yrt Atpα and yrt Nrx-IV polarity phenotypes is notable. These mutants show no polarity defects during cellularization when epithelial cells first form or during gastrulation when the apical junctional belt is assembled. Polarity defects are first seen at stage 11 or 12, and are most prominent at embryonic stage 13. In contrast to wild type, Crb and other apical markers are found throughout the plasma membrane and colocalize with basolateral markers such as Discs large (Dlg, also known as Dlg1) and Fasciclin 3 in yrtM/Z, yrt Atpα and yrt Nrx-IV mutants. During late embryogenesis, apical markers become restricted again to the apical membrane, which, however, remains abnormally extended and dome-shaped. Thus, Yrt, Na+,K+-ATPase and Nrx-IV cooperate and have critical functions in maintaining epithelial polarity during early organogenesis (stages 11-13), well before septate junction assembly is observed, and Yrt is recruited to the apical membrane at stage 14 (Laprise, 2009).
Na+,K+-ATPase and Nrx-IV are required for septate junction assembly. Because Yrt also accumulates at septate junctions, it was asked whether Yrt has a role in septate junction formation or function. Septate junctions are basal to the adherens junction and, like vertebrate tight junctions, prevent diffusion of solutes between cells. Dye injection assays show that paracellular barriers in epithelia of yrtM/Z embryos are compromised. However, yrtM/Z embryos showed a normal complement of septa when examined ultrastructurally whereas Atpα and Nrx-IV mutants lack septa. Immunoprecipitation experiments demonstrated FERM-domain-dependent interactions between Yrt and the septate junction transmembrane protein Neuroglian (Nrg), and the enrichment of Yrt at septate junctions was less pronounced in Nrg mutants. However, Nrg or Nrg yrt mutant embryos did not display overt defects in epithelial polarity. These findings indicate that Yrt is a bona fide septate junction component that has a function distinct from Nrx-IV and Na+,K+-ATPase because it is not required for septa formation but essential for barrier function (Laprise, 2009).
Next, the analysis extended to cytoplasmic adaptor proteins associated with septate junctions. yrt does not show genetic interactions with dlg or lgl (Laprise, 2006), genes encoding conserved basolateral polarity proteins required for septate junction formation and apical/basal polarity. varicose (vari), which encodes a membrane-associated guanylate kinase, and cora, which encodes a FERM protein that is the Drosophila orthologue of mammalian erythrocyte protein band 4.1 and its paralogues, were examined. vari and cora null mutant embryos did not show defects in apical basal polarity. No functional interactions were seen between yrt and vari. In contrast, yrt cora double-mutant embryos displayed marked apicalization defects, with strongly expanded apical membranes and reduced basolateral membranes in all ectodermal epithelia including the epidermis. This phenotype is significantly stronger than the yrtM/Z or cora null mutant phenotypes indicating that Yrt and Cora have redundant functions in promoting epithelial polarity (Laprise, 2009).
Similar to yrtM/Z mutants, yrt cora double mutants did not show polarity defects during gastrulation. By stage 11, polarity defects were more prominent in yrt cora mutants than in yrtM/Z embryos, which correlates with Cora being expressed from stage 11 onwards. In yrt cora mutants, the most severe polarity defects characterized by overlapping distributions of apical and basolateral markers occurred during stage 13. However, by late embryogenesis, apical and basolateral markers were segregated, although expanded apical membranes and severe defects in tissue organization persisted. Interestingly, the yrt cora apicalization phenotype is very similar to the phenotype that results from high-level overexpression of Crb in both timeline and severity. The segregation of apical and basolateral markers in late-stage yrt cora mutants is also observed in late-stage yrtM/Z, yrt Atpα and yrt Nrx-IV embryos. Thus, polarization mechanisms are at play in late embryos that are not dependent on Yrt and Cora. To address the possibility that this repolarization is the result of redundancy between Yrt/Cora and Lgl-group proteins, which include Scribble (Scrib) as well as Lgl and Dlg, embryos lacking Yrt and Scrib (yrtM/Z scribM/Z) were examined. Apical and basolateral markers segregated in yrtM/Z scribM/Z embryos indicating that a basolateral polarity mechanism exists in late embryos that is independent of Yrt/Cora and Lgl-group proteins (Laprise, 2009).
Together, these findings suggest that Yrt, Cora, Nrx-IV and the Na+,K+-ATPase are a new functionally cooperating group of basolateral epithelial polarity proteins, which is referred to as the Yrt/Cora group. In contrast to the enhancement of the cora and Nrx-IV null phenotypes by yrt mutations, no phenotypic enhancement was seen in cora Nrx-IV or cora Atpα double mutants, indicating that the Yrt/Cora group is composed of two functionally overlapping pathways. Cora, Nrx-IV and the Na+,K+-ATPase belong to one pathway, which is consistent with previously documented biochemical and genetic interactions between these proteins, whereas Yrt defines a second redundant pathway (Laprise, 2009).
One critical aspect of polarity regulation is that apical and basolateral proteins act antagonistically to set up mutually exclusive membrane domains. In gastrulating Drosophila embryos, this interaction occurs between apical factors and basolateral Lgl-group proteins. Similar to lgl, dlg and scrib mutations, yrt mutations partially suppress the crb mutant phenotype (Laprise, 2006). Because Yrt can bind Crb directly, it is suggested that Yrt negatively regulates Crb function as a component of the apical Crb complex in both apical basal polarity and the control of apical membrane size (Laprise, 2006). The data reported in this study argue that Yrt opposes Crb function also as a basolateral polarity protein. To test this hypothesis further it was asked whether the loss of other Yrt/Cora-group genes could suppress the crb mutant phenotype. It was found that mutations in Atpα, Nrx-IV and cora ameliorate the epithelial defects of crb mutants to an extent similar to that of yrt mutations. Remarkably, yrt cora crb triple-mutant embryos not only showed a suppression of the crb mutant phenotype, which was much stronger than the one observed in yrt crb or cora crb double mutants, but also caused a strong suppression of the apicalization effect observed in yrt cora mutants. These findings further support the conclusion that Yrt and Cora act redundantly, and indicate that mutual competition of basolateral Yrt/Cora-group proteins and apical Crb organizes epithelial membrane domains (Laprise, 2009).
Epithelial differentiation during Drosophila embryogenesis can be subdivided into four phases that are characterized by distinct mechanisms governing epithelial polarity. (1) Initial cues for polarity are given before or during cellularization. (2) Fully polarized epithelial cells are established during gastrulation through the interplay of apical Par and Crb complexes and basolateral Lgl-group proteins. (3) The Yrt/Cora group acts during organogenesis to promote basolateral polarity and counteracts apical determinants, thereby functionally replacing the Lgl group. (4) The function of Yrt/Cora and Lgl groups does not account for the polarization of epithelial cells in late embryos, because normalization of polarity is observed in the absence of these factors. This implies the existence of a yet unknown mechanism that stabilizes basolateral polarity. Septate junctions form well after Lgl-group and Yrt/Cora-group proteins have contributed to epithelial polarity, indicating that polarity and septate junction formation are independent functions of these proteins. The identification of a novel group of polarity factors that acts in a discrete time window during development highlights the temporal complexity of the regulation of the epithelial phenotype and further explains why the loss of individual polarity proteins does not completely compromise polarity (Laprise, 2009).
The vertebrate Yrt orthologues Mosaic eyes (Moe, also known as Epb41l5) in zebrafish and EPB41L5 (also known as YMO1 and Limulus) and EPB41L4B (also known as EHM2) in mammals bind to Crb proteins and contribute to epithelial organization (Laprise, 2006; Lee, 2007, Gosens, 2007, Hirano, 2008, Jensen, 2004). However, as in Drosophila, vertebrate Yrt homologues are predominantly associated with the basolateral membrane and seem to be recruited to the apical membrane only at later stages of epithelial development. To test whether EPB41L5 is required for basolateral differentiation, RNA interference was used in MDCK cells. Transient depletion of EPB41L5 using shRNA resulted in a notable cell flattening and an expansion of the cell perimeter, indicating that lateral membranes were strongly reduced and apical and basal membranes were enlarged. Consistently, it was found that the lateral markers Na+,K+-ATPase, Scrib and E-cadherin were reduced or lost from the plasma membrane. The same effects were observed using siRNA oligonucleotides targeted to a distinct region of EPB41L5. MDCK cells were established that were stably transfected with EPB41L5 short hairpin RNA (shRNA), and lateral membrane formation was examined after Ca2+-switch. After re-addition of Ca2+, EPB41L5 shRNA cells displayed significant delays in lateral membrane formation and recruitment of E-cadherin to cell-cell contacts. At 8 h after Ca2+-switch, E-cadherin levels appeared similar in control and knockdown cells although experimental cells remained slightly flatter. Interestingly, by 24 h after Ca2+-switch, E-cadherin levels at the plasma membrane had decreased again and appeared lower than in control cells but similar to EPB41L5 shRNA cells before Ca2+-switch. EPB41L5 knockdown cells ultimately established normal cell shape. Whether the formation of a normal lateral membrane in EPB41L5 shRNA cells is due to residual EPB41L5 expression, or reflects a transient requirement of EPB41L5 for cell polarization as in Drosophila epithelia, remains unclear. It is concluded that the function of Yrt as a basolateral polarity protein is conserved in mammalian epithelial cells (Laprise, 2009).
The loss of basolateral membrane in Drosophila embryos that lack Yrt/Cora-group function and in MDCK cells depleted of EPB41L5 is reminiscent of the loss of basolateral membrane in MDCK cells depleted of the phosphoinositide PtdIns(3,4,5)P3 and of human bronchial epithelial cells depleted of β2-spectrin or ankyrin-G. Cora/band 4.1 and the Na+,K+-ATPase can associate with either spectrin or the spectrin adaptor ankyrin, and loss or ectopic expression of EPB41L5 can cause defects in basolateral actin. This raises the possibility that Yrt/Cora-group proteins act by stabilizing the lateral actin/spectrin membrane cytoskeleton. Analysis of vertebrate development in the absence of the function of the Yrt orthologues Moe in zebrafish and EPB41L5 in mice revealed defects in epithelial organization that reflect Crb-dependent and probably also Crb-independent functions of Yrt proteins. For example, the abnormal cell shape and multi-layering defects seen in the developing neuroepithelium of mutant mouse embryos could result from defects in basolateral polarity. Moreover, mouse embryos lacking EPB41L5 show defects in epithelial-mesenchymal transition of mesodermal cells during gastrulation. These surprising findings indicate that Yrt proteins are core regulators of animal tissue organization that can enhance epithelial or mesenchymal cell differentiation (Laprise, 2009).
Regulation of epithelial tube size is critical for organ function. However, the mechanisms of tube size control remain poorly understood. In the Drosophila trachea, tube dimensions are regulated by a luminal extracellular matrix (ECM). ECM organization requires apical (luminal) secretion of the protein Vermiform (Verm), which depends on the basolateral septate junction (SJ). This study shows that apical and basolateral epithelial polarity proteins interact to control tracheal tube size independently of the Verm pathway. Mutations in yurt (yrt) and scribble (scrib), which encode SJ-associated polarity proteins, cause an expansion of tracheal tubes but do not disrupt Verm secretion. Reducing activity of the apical polarity protein Crumbs (Crb) suppresses the length defects in yrt but not scrib mutants, suggesting that Yrt acts by negatively regulating Crb. Conversely, Crb overexpression increases tracheal tube dimensions. Reducing crb dosage also rescues tracheal size defects caused by mutations in coracle (cora), which encodes an SJ-associated polarity protein. In addition, crb mutations suppress cora length defects without restoring Verm secretion. Together, these data indicate that Yrt, Cora, Crb, and Scrib operate independently of the Verm pathway. These data support a model in which Cora and Yrt act through Crb to regulate epithelial tube size (Laprise, 2010).
The transmembrane protein Crumbs (Crb) acts as an apical determinant during establishment of epithelial apical-basal polarity. During later stages of epithelial differentiation, Crb promotes apical membrane growth independently of its role in apical-basal polarity. Crb activity is counteracted by different groups of basolateral polarity proteins including the Yrt/Cora group, which is composed of Yurt (Yrt), Coracle (Cora), Na+/K+-ATPase, and Neurexin IV (Nrx-IV). Loss of Yrt results in Crb-dependent apical membrane growth during late stages of epithelial cell maturation in Drosophila. Thus, the equilibrium between the activities of these polarity proteins is important to define the size of the apical domain. Precise control of the apical surface of tracheal cells is crucial to define epithelial tube size in the Drosophila respiratory system, suggesting a potential role for polarity proteins in tube morphogenesis. However, the contribution of polarity regulators to the regulation of epithelial tube shape and size is poorly understood. Controlling length and diameter of the lumen is important for organ function, as illustrated by the deleterious tubule enlargements that occur in polycystic kidney disease (Laprise, 2010).
To better understand the role of polarity regulators and apical membrane growth in epithelial tube morphogenesis, the role of Yrt was investigated in the formation of the Drosophila respiratory system, a network of interconnected tubules that delivers oxygen throughout the body. Yrt is mainly associated with the lateral membrane in tracheal cells and is enriched at septate junctions (SJs), as shown by its colocalization with the SJ marker Nrx-IV. Tracheal development was characterized in zygotic yrt mutants or yrt null embryos devoid of both maternal and zygotic yrt (yrtM/Z). Segmental tracheal placodes invaginated and established a normal branching pattern of tracheal tubes with intersegmental connections in both yrt and yrtM/Z mutants. yrtM/Z embryos display apical-basal polarity defects and irregularities in the tracheal epithelium at midembryogenesis. However, apical-basal polarity normalizes during terminal differentiation. In contrast, zygotic yrt mutants have only minor, if any, polarity defects in tracheal cells. The most apparent defect in yrt mutant trachea was the presence of excessively long and convoluted dorsal trunks compared to the straight dorsal trunks seen wild-type. The average dorsal trunk length was 417 ± 12 μm in wild-type embryos, whereas it was 476 ± 22 μm in yrt and 470 ± 23 μm in yrtM/Z mutant embryos. Similar but milder tube length defects were observed in other tracheal branches. In addition, the diameter of dorsal trunks in yrt and yrtM/Z mutants was uniform, but wider than in wild-type embryos. The average dorsal trunk diameter was 9.1 ± 0.6 μm in tracheal segment seven of wild-type embryos compared to 12.2 ± 0.9 μm in yrt and 12.7 ± 0.6 μm in yrtM/Z mutant embryos. In smaller branches, some diameter expansions were also apparent. In addition, the smaller tracheal branches in yrtM/Z embryos showed frequent interruptions indicating either breaks or a failure in the luminal accumulation of the 2A12 antigen. These findings indicate that Yrt regulates the size of tracheal tubes and supports the integrity of segmental tracheal branches. Remarkably, despite the prominent differences in the apical-basal polarity defects between yrt and yrtM/Z mutant embryos, both mutants exhibit similar dorsal trunk elongation and diameter defects. This finding suggests that the transient loss of apical-basal polarity in yrtM/Z embryos is not the cause of dorsal trunk size defects and that Yrt therefore has distinct functions during early and late stages of tracheal morphogenesis (Laprise, 2010).
The enlargement of the tracheal tube lumen observed in yrt mutants could be caused by an increase in cell number or an increase in the dimension of the apical surface of tracheal cells that surround the lumen. To address this question, the number of dorsal trunk cells in yrt mutants and wild-type embryos was counted. No significant differences in cell numbers between wild-type and yrt mutant embryos were found, indicating that the enlargement of tracheal tubes observed in yrt and yrtM/Z mutants must be accompanied by an increase in the dimension of the apical surface of tracheal cells (Laprise, 2010).
Several other mutants display enlarged dorsal trunks similar to yrt. One group of genes required for limiting tube length encodes components of the SJ, including the Na+/K+-ATPase (α and β subunits), Cora, Nrx-IV, Scribble (Scrib), Lachesin (Lac), Sinuous (Sinu), Megatrachea (Mega), and Varicose (Vari). Among these SJ proteins, Na+/K+-ATPase, Cora, Nrx-IV, and Scrib also play a role as basolateral polarity proteins. In Drosophila and other invertebrates, SJs appear as a ladder-like group of septa basal to the cadherin-based adherens junctions. SJs have functions analogous to vertebrate tight junctions, because they provide a transepithelial diffusion barrier. Yrt is not required for normal septa formation or localization of SJ components such as Cora but is essential for the barrier function of SJs. Zygotic yrt mutants show only minor defects in paracellular barrier function, whereas barrier function is fully compromised in yrtM/Z embryos. This observation supports the notion that transepithelial barrier function and the regulation of tube dimension are independent functions of Yrt because yrt and yrtM/Z mutant embryos show similar tube size defects. This conclusion is consistent with previous findings suggesting that the regulation of tracheal tube elongation and the regulation of the paracellular diffusion barrier are distinct roles of SJ proteins (Laprise, 2010).
A second class of mutants exhibiting abnormally long tracheal tubes are defective in the genesvermiform (verm) and serpentine (serp), which encode enzymes predicted to modify the chitin-based luminal extracellular matrix (ECM), and mutants of which show structural defects in the luminal ECM. The chitin matrix filling the tracheal lumen is transiently present during lumen morphogenesis and is critical for determining lumen diameter and length. Interestingly, all of the mutations affecting SJ components tested so far are associated with a failure to secrete Verm into the tracheal lumen. This suggests that SJ proteins control tube size by regulating apical secretion and remodeling of the apical chitin matrix. Although Yrt is required for the barrier function of SJs, it was found that luminal secretion of Verm and Serp was normal in yrt and yrtM/Z mutants. In contrast, low but detectable levels of Verm were observed in cora mutants. This finding suggests that Yrt regulates tracheal tube length through a pathway that is independent of Verm and Serp (Laprise, 2010).
During late stages of epithelia maturation, Yrt is known to restrict apical membrane growth in epidermal and photoreceptor cells by limiting Crb activity. This interplay between Yrt and Crb governs apical membrane size in stage 14 and later embryos when tracheal tube size is defined. This raises the possibility that Crb-dependent apical membrane growth is responsible for dorsal trunk expansion in yrt mutant embryos. To test this hypothesis, crb dosage was reduced in yrt mutant embryos by introducing one copy of a crb null allele into a yrt mutant background. Loss of one copy of crb suppressed the dorsal trunk elongation defects seen in yrt mutants, because the dorsal trunks appeared similar to wild-type in yrt crb/yrt + mutants. In addition, moderate Crb overexpression increased dorsal trunk length and diameter without interfering with the integrity of the tracheal epithelium or the secretion of Verm or Serp. These results show that Crb is required for promoting the expansion of tracheal tubes at late stages of embryogenesis. It was previously suggested that Crb also acts during early tracheal branch outgrowth in addition to its role in apical-basal polarity. Therefore, Crb plays a critical role at several steps of tracheal development. Together, these findings indicate that the antagonistic interactions between Yrt and Crb determine tracheal tube size (Laprise, 2010).
Verm levels in yrt crb/yrt + and in yrt/yrt mutants were indistinguishable, indicating that the minor reduction in Verm levels sometimes seen in yrt mutants are not the cause of the tracheal elongation defects. Accordingly, reduction of crb dosage in a verm or serp mutant background did not suppress tube size defects. Similarly, loss of one copy of crb in sinu mutant embryos, which fail to secrete Verm, had no impact on the length of dorsal trunks that remained enlarged as in sinu single mutants. These findings suggest that the apical secretion of matrix-modifying enzymes such as Verm and the control of Crb activity by Yrt are two independent and nonredundant modes of tracheal tube size regulation. The data also establish that epithelial tube size control by SJ-associated proteins involves Verm-dependent and Verm-independent mechanisms (Laprise, 2010).
To further characterize the function of SJ-associated polarity proteins in the regulation of tracheal tube size, tube elongation, the integrity of SJs, and the secretion of Verm in scrib, lethal giant larvae (lgl), and discs large (dlg) mutant embryos were examined. Zygotic loss of scrib, lgl, or dlg resulted in excessively long dorsal trunks, indicating that these genes are critical for tube size control. Zygotic loss of lgl expression caused fully penetrant defects in SJ paracellular barrier function, whereas zygotic scrib or dlg mutants did not have compromised transepithelial barriers. Luminal Verm deposition was not detected in lgl mutants but appeared near normal in dlg and in scrib mutants. Thus, Scrib and Dlg act like Yrt by controlling tracheal tube size through mechanisms distinct from Verm secretion (Laprise, 2010).
Further analysis concentrated on Scrib and it was asked whether this protein, like Yrt, controls tracheal tube size by negatively regulating Crb activity. Scrib together with Lgl and Dlg shows antagonistic interactions with Crb to regulate apical-basal epithelial polarity in early Drosophila embryos. However, the tracheal tube defects were not ameliorated in scrib crb/scrib + embryos compared to scrib single mutants. Thus, in contrast to Yrt, Scrib does not seem to limit tube length by restricting Crb activity. Because Yrt and Scrib appear to control tracheal tube size through different mechanisms, tests were performed to see whether yrt scrib double homozygous mutant embryos had a more severe phenotype. The double mutants had Verm levels that were lower compared to yrtM/Z and scrib mutants. Moreover, the tracheal defects also appeared more severe in yrt scrib mutants than in yrt null mutant embryos, particularly in the smaller-diameter branches. The defects in small-diameter branches of the yrt scrib double mutants are not likely caused by the reduction in Verm secretion, because the complete loss of Verm has only a mild effect on smaller branches. The enhanced severity of the yrt scrib double-mutant tracheal defects compared to the defects seen in yrt null embryos and the differences in the genetic interactions of scrib and yrt with crb suggest that Scrib and Yrt act in separate pathways to regulate the size of tracheal tubes and that Scrib does not act by modulating Crb activity. It is possible that Scrib acts through other proteins, such as proteins of the Par complex, that promote apical domain formation. Therefore, SJ-associated polarity proteins use at least two Verm-independent mechanisms to restrict the dimension of tracheal tubes (Laprise, 2010).
Cora is an SJ-associated protein required for optimal secretion of Verm. This suggests that Cora may control tracheal tube length through a Verm-luminal matrix pathway. However, Cora is also a basolateral polarity protein restricting the activity of Crb, which promotes Verm-independent expansion of the dorsal trunk. This led to an investigation of the functional relationship between Cora and Crb in tracheal morphogenesis. In the epidermis, a striking redundancy between yrt and cora is observed in the regulation of apical-basal polarity. Similarly, it was found that tracheal cells in yrt cora double mutants show severe apicalization defects characterized by a broad expansion of the surface distribution of Crb. The antigen recognized by the monoclonal antibody 2A12 was found surrounding tracheal cells and was not confined to the luminal cavity. In addition, the 2A12 antigen was associated not only with tracheal cells but also with epidermal cells. These tracheal defects seen in cora yrt mutant embryos mimic defects that result from high levels of Crb overexpression. This observation argues that the tracheal defects observed in cora yrt double-mutant embryos result from strong Crb overactivation, which is associated with a loss of basolateral polarity and an expansion of apical membrane character. Because epidermal cells did not acquire expression of the tracheal cell marker Tango, it is unlikely that epidermal cells adopt a tracheal cell fate in cora yrt mutants. The association of 2A12 with epidermal cells is therefore presumably due to the apicalization of tracheal cells, which would consequently secrete the 2A12 antigen not only on the luminal side but all around their cell surface, allowing the 2A12 antigen to diffuse and bind to surrounding cells. Accordingly, cuticle deposition, taking place at the apical membrane, was seen at both luminal and abluminal sides of tracheal cells overexpressing Crb (Laprise, 2010).
The data indicate that Yrt and Cora cooperate to control apical-basal polarity of tracheal cells by limiting Crb to the apical cell pole, but they do not reveal whether Cora and Crb interact to control the length of tracheal tubes. To address this question, cora crb/cora + embryos were examined for a suppression of the tracheal size defects seen in cora single mutants. Reduction of crb dosage suppresses tube overelongation defects resulting from the loss of Cora. This restriction of dorsal trunk elongation does not result from the restoration of Verm secretion, because the level of Verm present in the dorsal trunk lumen was as low in cora crb/cora + embryos as in single cora mutants. Together, these data suggest that Crb overactivation is the primary cause of epithelial tube length defects observed in the absence of Cora. Thus, Cora and Yrt act independently from each other to counteract Crb activity and maintain the appropriate size of epithelial tubes. Because the reduction of crb dosage does not rescue the verm mutant phenotype, it is concluded that the residual amount of Verm found in cora mutants is sufficient to maintain Verm pathway activity (Laprise, 2010).
This analysis suggests that basolateral proteins that are enriched at SJs have several critical functions in determining the size of epithelial tubes in the Drosophila tracheal system. This study shows that the increase in tube size is not caused by an increase in cell number and therefore must be accompanied by an increase in the apical surface area of individual tracheal cells. Given that Crb is a well-known regulator of apical membrane size, these findings suggest that the interplay between Yrt, Cora, and Crb modulates the dimensions of the apical surface of tracheal cells to control tracheal tube size. Moreover, this mechanism acts independently of and in parallel to a previously proposed pathway depending on the apical secretion of the matrix-modifying enzymes Verm and Serp, which requires several SJ-associated proteins. Yet another mechanism is revealed by the results demonstrating that scrib mutants also have long trachea with normal Verm levels but that, in contrast to cora and yrt, tracheal defects in scrib mutants are not suppressed by loss of one copy of crb. Together, these findings suggest that basolateral proteins utilize at least three distinct mechanisms to regulate tube size in the Drosophila tracheal system. Unexpectedly, these mechanisms involve functional interactions between polarity proteins that appear to be different from those involved in establishing apical-basal polarity at earlier stages of development. For example, in promoting apical-basal polarity, Yrt and Cora act redundantly so that cora mutants show polarity defects only in a yrt mutant background, and polarity defects in yrt mutants are strongly enhanced by removal of Cora. In contrast, both cora and yrt single mutants show similar strong tracheal size defects. Furthermore, Scrib and Crb display antagonistic functional interactions during establishment of apical-basal polarity, but not during tracheal elongation. An important challenge for future investigations will be to uncover the adaptations in the molecular pathways that allow polarity proteins to contribute to different aspects of epithelial development (Laprise, 2010).
The Crumbs (Crb) complex is a key regulator of epithelial cell architecture where it promotes apical membrane formation. Binding of the FERM protein Yurt to the cytoplasmic domain of Crb is part of a negative-feedback loop that regulates Crb activity. Yurt is predominantly a basolateral protein but is recruited by Crb to apical membranes late during epithelial development. Loss of Yurt causes an expansion of the apical membrane in embryonic epithelia and photoreceptor cells similar to Crb overexpression and in contrast to loss of Crb. Analysis of yurt crb double mutants suggests that these genes function in one pathway and that yurt negatively regulates crb. The mammalian Yurt orthologs YMO1 and EHM2 bind to mammalian Crb proteins. It is proposed that Yurt is part of an evolutionary conserved negative-feedback mechanism that restricts Crb complex activity in promoting apical membrane formation (Laprise, 2006).
These data support the hypothesis that the FERM protein Yurt is a negative regulatory component of the Crb complex that modulates Crb function in two distinct aspects of epithelial organization: epithelial apical-basal polarity and the regulation of apical membrane size. The conclusion that Yurt opposes Crb activity and does so as a component of the Crb complex is supported by several observations. (1) Yurt is recruited to the apical membrane by Crb and can bind directly to the FDB site in the cytoplasmic tail of Crb. (2) Loss of Yurt disrupts epithelial polarity and causes an enlargement of the apical membrane as seen when Crb is overexpressed. (3) yurt mutations suppress the crb mutant phenotype but only when residual Crb activity remains, suggesting that Yurt exerts its effects through Crb (Laprise, 2006).
A clear distinction between Crb function in apical-basal polarity and in regulating the size of an apical membrane domain is seen in photoreceptor cells (PRCs). Overexpression of Crb in late PRCs leads to an expansion of the apical stalk membrane without disrupting apical-basal polarity or the apical junctional complex, in contrast to most other epithelial cells. yurt mutant PRCs display longer stalk membranes similar to PRCs that overexpress Crb and unlike PRCs that lack Crb, which have shorter stalks. Extended apical membranes were detected in the late embryonic epidermis of yurt mutants. The function of Yurt as a negative regulator of apical membrane size appears to be conserved in vertebrates. Yurt orthologs YMO1 and EHM2 can bind to all three human CRB proteins and that YMO1 colocalizes with Crb2 in the inner segment of mouse PRCs. Moreover, zebrafish Crb proteins physically interact with the Yurt ortholog Moe, and loss of Moe causes larger apical membranes in fish PRCs. It is concluded that negative regulatory feedback within the Crb complex could be a general mechanism for the regulation of apical membrane size (Laprise, 2006).
Similar to yurt, mutations in genes encoding basolateral polarity proteins such as Lgl oppose the activity of apical polarity factors. No functional interactions were detected between yurt and lgl or dlg, and although yurt and lgl, dlg, or scrib mutations suppress the crb mutant phenotype, remaining crb activity is required to ameliorate the crb mutant defects in the absence of Yurt but not in the absence of Scrib. These findings suggest that Yurt acts directly on Crb and not indirectly through a cooperation with basolateral determinants (Laprise, 2006).
Crb proteins promote the formation of apical membrane or apical membrane subdomains of Drosophila embryonic epithelial cells, Drosophila PRCs, mammalian PRCs, and the apical cilium of mammalian kidney cells. The Crb binding partners Sdt, PATJ, and βH-Spectrin also positively regulate apical membrane formation. Crb forms a complex with βH-Spectrin, but the nature of this interaction has remained unclear. The FDB site in the Crb cytoplasmic tail is required to promote epithelial polarity and is important for the linkage of Crb to βH-Spectrin. Moesin was suggested as a candidate for mediating this interaction as it can form a complex with Crb and βH-Spectrin. However, direct binding of Moesin to Crb has not been reported and the loss of Moesin causes defects that do not resemble the Crb loss- or gain-of-function phenotype. Moreover, Moesin does not colocalize with Crb at the stalk membrane. The current data suggest that Yurt does not link Crb to βH-Spectrin, although Yurt can bind to the Crb FDB site. Loss of Yurt from PRCs does not cause βH-Spectrin to detach from the stalk as was reported for PRCs that lack Crb and as would be expected if Yurt is the critical linker between Crb and βH-Spectrin. Moreover, association of βH-Spectrin with the stalk and loss of βH-Spectrin from the stalk in crb mutants occurs before Yurt becomes apparent at the stalk membrane. These results imply that Crb can interact with βH-Spectrin in a Yurt-independent manner. In addition to Yurt, the FDB site of Crb is likely to interact with a yet-unknown positive regulator of epithelial polarity and is therefore a critical site for the modulation of Crb complex activity (Laprise, 2006).
Crb has a well-established role in epithelial apical-basal polarity and contributes to the assembly of the ZA in Drosophila and the ZA and tight junction in mammalian epithelia. In contrast to Crb and Sdt, which are required for epithelial polarity already in gastrulating embryos, Yurt becomes an essential component of the Crb complex later in epithelial development. Yurt localization appears to be confined to the basolateral membrane at early stages, and biochemical interactions between Yurt and Crb were not detected. The shift in the subcellular distribution of Yurt is unlikely to reflect the differential expression of the four Yurt isoforms. For example, the recruitment of Yurt to the stalk membrane does not correlate with an isoform shift as Yurt-? and Yurt-? are the predominant isoforms throughout pupal retinal development (data not shown). Thus, the interaction between Crb and specific Yurt isoforms is presumably regulated through modification of Yurt or Crb or both proteins. One attractive possibility is that phosphorylation of Yurt, which this study documents, is important for regulating the Crb Yurt interaction. The association of several other FERM domain proteins such as Merlin or Moesin with membrane proteins is regulated through phosphorylation, which causes a conformational change that allows the FERM domain to interact with transmembrane receptors (reviewed in Bretscher et al., 2002). Recent work showed that the phosphorylation of the juxtamembrane region of Crb by a PKC is important for Crb function (Sotillos et al., 2004). This modification could prevent premature recruitment of Yurt as a negative regulator of Crb activity during early epithelial development. It will be an important challenge to determine how the temporal and spatial interactions between Crb and Yurt are regulated (Laprise, 2006).
The negative regulation of the Crb/Sdt/PATJ/βH-Spectrin complex by Yurt does not involve significant changes in the localization or levels of these proteins. To find out how Yurt interferes with Crb complex function will likely depend on elucidating the molecular mechanism of how Crb and its other binding partners cause cell polarization and apical membrane growth, which currently is not understood. Regulation of apical membrane size is a critical feature of epithelial development, which is required during epithelial polarization and in terminally differentiated cells to maintain functional apical domains such as the stalk membrane of PRCs or the apical cilia of epithelial cells. A model is described that summarizes the functional interactions between polarity proteins that were explored in this study. This analysis of Yurt emphasizes that the Crb complex has two distinct functions in controlling epithelial cell architecture, the global regulation of apical-basal polarity, and the local control of apical membrane size. It was hypothesized that both levels of control involve Yurt-dependent negative feedback regulation that acts directly upon Crb activity. Global negative feedback occurs between apical and basolateral polarity proteins to set up apical versus basolateral membrane territories segregated by an apical junctional complex. Local negative feedback occurs among apical polarity proteins to restrict apical membrane growth. Apical membrane growth facilitated by the Crb complex without local suppression of its activity (that is, without involvement of Yurt) may occur early in epithelial development when the apical membrane shows net growth, while in more mature epithelial cells, homeostasis of apical-basal polarity and apical membrane size is achieved through Yurt-mediated local restriction of Crb complex activity (Laprise, 2006).
Proteins of the 4.1 family play a key role in the integrity of the cytoskeleton and in epithelial tissue movement, as shown by the disruption of the actin cytoskeleton in human erythrocytes caused by genetic loss of protein 4.1, and the failure of epithelial tissue migration during Drosophila embryogenesis caused by genetic loss of the 4.1 homolog Coracle. This study reports the genetic characterization of Yurt, a novel protein 4.1 family member in Drosophila that is associated with the plasma membrane of epithelial cells. Homozygous loss-of function mutations in the yurt gene cause failure of germ-band retraction, dorsal closure, and head involution, associated with degeneration of the amnioserosa and followed by embryonic lethality. A mammalian homolog of Yurt is up-regulated in metastatic melanoma cells. These novel cytoskeletal proteins appear to play important roles in epithelial cell movements and in the morphogenetic tissue changes that depend on them (Hoover, 2002).
The yurt cDNA is predicted to encode a protein of 976 amino acids with significant sequence similarity to other members of the protein 4.1 family. The closest homologs are proteins encoded by a Caenorhabditis cosmid and human and mouse orthologs of the protein Ehm2. All of the homology with these and other proteins is within the FERM domain, and the close homology to the Caenorhabditis and mammalian homologs within this region indicates that Yurt and its homologs should be considered as new members of the protein 4.1 family. The Yurt FERM domain consists of about 300 amino acids and includes close matches to the two calmodulin-binding sites reported from protein 4.1. Yurt does not show significant identity with any other proteins outside the FERM domain, and unlike most vertebrate Protein 4.1 family members, there are no consensus cytoskeleton-binding domains in the C-terminal part of Yurt. There is a consensus type II PDZ-binding motif at the C-terminus of Yurt and a type-I motif on Ehm2 (Hoover, 2002).
Identification of loss-of-function mutations affecting the Yurt protein reveals important roles in early epithelial morphogenesis and cell survival. Yurt embryos fail to complete germ-band retraction, dorsal closure and head involution, and they show apparent degeneration of the amnioserosa. Of course, some of these defects may be secondary to others; for example, since tension in the amnioserosa contributes force for dorsal closure, the loss of this tissue could be indirectly responsible for the failure of dorsal closure in yurt mutants. Similar defects are observed in mutants affecting members of the u-shaped (ush) family (tup, ush, hnt, srp) of transcription factors, which fail to begin or complete germ-band retraction, and show degeneration of the amnioserosa. Ush genes genetically interact with one another and are involved in cell migration and dorsal closure. Since yrt and the ush genes function in cell migration and have similar mutant phenotypes, genetic interactions were tested by studying the phenotypes of transheterozygous larvae and adult flies. However, these simple tests revealed no interactions, suggesting that yurt and ush function in distinct genetic pathways (Hoover, 2002).
Dorsal closure has been extensively studied in Drosophila as an example of epithelial morphogenesis. The genes involved in dorsal closure have been categorized into those encoding structural proteins (class I: coracle, myosin), those required for the DJNK signaling cascade (class II: basket, jun-related antigen), and those required for Dpp signal transduction (class III: punt, schnurri). In class II and IIII mutants, the dorsally migrating lateral epithelium fails to elongate but class I mutants show normal elongation of the lateral epidermis. The epithelium in yurt mutants elongates normally, suggesting that Yurt is a class I protein with a structural function. Other genes that are required for dorsal closure, but have not been categorized, include discs-large and canoe, both of which encode PDZ domain-containing putative scaffolding proteins. Ca2+-regulated plasma membrane formation is also probably involved in dorsal closure. Thus, dorsal closure involves the concerted activity of proteins that are required for cytoskeletal structure, signal transduction and intracellular ion concentrations. The Yurt protein is localized to the plasma membrane at all developmental stages analyzed. During cellularization it is localized at the apical plasma membrane, and when cellularization is complete it is uniformly associated with the plasma membrane. After cellularization it becomes localized to cytoplasmic aggregations. Merlin, another Drosophila protein 4.1 superfamily member, is also localized at endocytic structures in cell lines, but this cytoplasmic form is probably inactive. This raises the possibility that cytosolic Yurt could be the inactive form while the membrane-associated form could be active. Alternatively, one of these localization patterns could be artefactual. Further investigations into the localization of Yurt in static and moving epithelium will be necessary to address this question. Since protein 4.1 family members organize protein complexes at the plasma membrane, whether Yurt forms a complex with other membrane-associated proteins was tested. Several candidates were identified on the basis of a consensus protein 4.1 binding site, consisting of a continuous stretch of five to seven positively charged residues. These candidates included the Discs-large (Dlg) protein, and the rasinteracting protein Canoe (Hoover, 2002).
No genetic or biochemical interaction with either of these proteins was detected. This was surprising, especially for Dlg, given the phenotypic similarity of dlg and yurt mutants, and studies showing direct binding between human protein 4.1 and human Dlg. In Drosophila, the region of Dlg homologous to the 4.1-binding site in human Dlg is required for localization to the plasma membrane; however, neither Yurt not Coracle appear to be a binding partner for this domain because Dlg is localized correctly in both yurt and coracle mutants and Coracle and Dlg do not co-immunoprecipitate (Hoover, 2002).
The multiple epithelial defects in yurt embryos suggest that the gene product is required for a general mechanism of epidermal movement. This is distinct from another Drosophila protein 4.1 family member, Coracle, in which hypomorphic or null alleles show dorsal closure defects, but no defects in germ-band retraction or head involution. How Yurt affects epithelial migration is not clear since neither actin nor spectrin are mislocalized in yurt mutants. The mouse Yurt ortholog, Ehm2, is up-regulated in metastatic melanoma cells, suggesting that increased levels of Ehm2 may be important for the invasiveness and motility observed in these epithelial cells. Thus other members of the Yurt subfamily, in addition to Yurt, may play important roles in epithelial cell movements (Hoover, 2002).
Search PubMed for articles about Drosophila Yurt
Gosens, I. et al. (2007). FERM protein EPB41L5 is a novel member of the mammalian CRB-MPP5 polarity complex. Exp. Cell Res. 313: 3959-3970. PubMed ID: 17920587
Hirano, M., Hashimoto, S., Yonemura, S., Sabe, H. and Aizawa, S. (2008). EPB41L5 functions to post-transcriptionally regulate cadherin and integrin during epithelial-mesenchymal transition. J. Cell Biol. 182: 1217-1230. PubMed ID: 18794329
Hoover, K. B. and Bryant, P. J. (2002). Drosophila Yurt is a new protein-4.1-like protein required for epithelial morphogenesis. Dev. Genes Evol. 212: 230-238. PubMed ID: 12070613
Hsu, Y. C., Willoughby, J. J., Christensen, A. K. and Jensen, A. M. (2006). Mosaic Eyes is a novel component of the Crumbs complex and negatively regulates photoreceptor apical size. Development 133: 4849-4859. PubMed ID: 17092952
Jensen, A. M. and Westerfield, M. (2004). Zebrafish mosaic eyes is a novel FERM protein required for retinal lamination and retinal pigmented epithelial tight junction formation. Curr. Biol. 14: 711-717. PubMed ID: 15084287
Laprise, P. et al. (2006). The FERM protein Yurt is a negative regulatory component of the Crumbs complex that controls epithelial polarity and apical membrane size. Dev. Cell 11: 363-374. PubMed ID: 16950127
Laprise, P., et al. (2009). Yurt, Coracle, Neurexin IV and the Na(+),K(+)-ATPase form a novel group of epithelial polarity proteins. Nature 459: 1141-1145. PubMed ID: 19553998
Laprise, P., Paul, S. M., Boulanger, J., Robbins, R. M., Beitel, G. J. and Tepass, U. (2010). Epithelial polarity proteins regulate Drosophila tracheal tube size in parallel to the luminal matrix pathway. Curr. Biol. 20(1): 55-61. PubMed ID: 20022244
Lee, J. D., et al. (2007). The FERM protein Epb4.1l5 is required for organization of the neural plate and for the epithelial-mesenchymal transition at the primitive streak of the mouse embryo. Development 134: 2007-2016. PubMed ID: 17507402
date revised: 10 October 2010
Home page: The Interactive Fly © 2009 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.