Discs large 5: Biological Overview | References
Gene name - Discs large 5
Cytological map position - 32F2-32F2
Function - Scaffold protein
Symbol - Dlg5
FlyBase ID: FBgn0032363
Genetic map position - chr2L:11,508,399-11,517,081
Cellular location - cytoplasmic
|Recent literature||Kwan, J., et al. (2016). DLG5 connects cell polarity and Hippo signaling protein networks by linking PAR-1 with MST1/2. Genes Dev 30(24): 2696-2709. PubMed ID: 28087714
The mechanisms connecting apical-basal polarity proteins with intracellular signaling pathways are largely unknown. This study reports that Discs large homolog 5 (DLG5) functions as an evolutionarily conserved scaffold and negative regulator of Hippo signaling, which controls organ size through the modulation of cell proliferation and differentiation. Affinity purification/mass spectrometry revealed a critical role of DLG5 in the formation of protein assemblies containing core Hippo kinases mammalian ste20 homologs 1/2 (MST1/2) and Par-1 polarity proteins microtubule affinity-regulating kinases 1/2/3 (MARK1/2/3). Consistent with this finding, Hippo signaling is markedly hyperactive in mammalian Dlg5-/- tissues and cells in vivo and ex vivo and in Drosophila upon dlg5 knockdown. Conditional deletion of Mst1/2 fully rescued the phenotypes of brain-specific Dlg5 knockout mice. Dlg5 also interacts genetically with Hippo effectors Yap1/Taz (see Drosophila Yorkie). Mechanistically, this study shows that DLG5 inhibits the association between MST1/2 and large tumor suppressor homologs 1/2 (LATS1/2; see Drosophila Warts), uses its scaffolding function to link MST1/2 with MARK3, and inhibits MST1/2 kinase activity. These data reveal a direct connection between cell polarity proteins and Hippo, which is essential for proper development of multicellular organisms.
Apical-basal polarity plays critical roles in the functions of epithelial tissues. However, the mechanisms of epithelial polarity establishment and maintenance remain to be fully elucidated. This study shows that the membrane-associated guanylate kinase (MAGUK) family protein Dlg5 is required for the maintenance of apical polarity of follicle epithelium during Drosophila oogenesis. Dlg5 localizes at the apical membrane and adherens junction (AJ) of follicle epithelium in early stage egg chambers. Specifically, the major function of Dlg5 is to promote apical membrane localization of Crumbs, since overexpression of Crumbs but not other major apical or AJ components can rescue epithelial polarity defects resulting from loss of Dlg5. Furthermore, by performing a structure-function analysis of Dlg5, it was found that the C-terminal PDZ3 and PDZ4 domains are required for all Dlg5's functions as well as its ability to localize to apical membrane. The N-terminal coiled-coil motif can be individually targeted to the apical membrane, while the central linker region can be targeted to AJ. Lastly, the MAGUK core domains of PDZ4-SH3-GUK can be individually targeted to apical, AJ and basolateral membranes (Luo, 2016).
How cell polarity is established and maintained is an important question in the fields of cell and developmental biology. During animal development, polarized cells such as epithelial cells maintain their apical-basal polarity despite undergoing dramatic shape changes and tissue remodeling during morphogenesis. Genetic screens done in developing Drosophila and C. elegans have uncovered a number of highly conserved apical and basolateral regulators essential for the establishment and maintenance of apical-basal polarity. Specifically, the Par3/Par6/aPKC complex and the Crumbs (Crb)/Stardust (Sdt)/Patj complex are thought to define and maintain the identity of apical membrane, and the Discs-large (Dlg)/Lethal giant larvae (Lgl)/Scribble (Scrib) complex delineates the basolateral membrane. Finally, the junctional complex of E-cadherin/β-catenin/α-catenin initiates and maintains the adherens junction (AJ), which divides the cortex into apical and basolateral regions. Decades of research have formed a consensus model, in which the apical-basal polarity is generated and maintained by a mutually antagonistic interaction between the apical regulators and the basolateral regulators. Recently, a mathematic modeling study done in Drosophila follicle epithelia suggested that in addition to the negative feedback between apical regulators and basolateral regulators, a positive feedback loop among apical polarity regulators is required to maintain the apical-basal polarity in epithelia. A central component of this positive feedback loop is the transmembrane protein Crb, and its apical membrane localization was thought to be the key to maintenance of apical-basal polarity (Luo, 2016).
Discs-large 5 (Dlg5) belongs to the MAGUK family, and it is highly conserved across species including human, mouse, chicken, zebra fish and Drosophila. MAGUK members also include Dlg and Sdt, which act as molecular scaffolds and are core components of the basolateral Dlg/Lgl/Scrib complex and the apical Crumb complex respectively. Dlg5 was first identified in human and was found to be expressed in placenta and in prostate gland epithelia (Nakamura, 1998). Since then, Dlg5 was found to interact with a variety of junctional, cytoskeletal, trafficking, and receptor molecules, including β-catenin, P55, vinexin, Girdin, Citron kinase, Syntaxin, Smoothened and TGF-β receptors (Nechiporuk, 2007; Wakabayashi, 2003; Chang, 2010; Sezaki, 2013; Tomiyama, 2015; Chong, 2015). And its functions vary from inhibiting cancer cell migration to mediating receptor signaling, but most of these functions were obtained from cell culture studies. Detailed genetic analysis of Dlg5 was first done using Dlg5 knockout mice, which displayed failure of epithelial tube maintenance resulting in brain hydrocephalus and kidney cysts (Nechiporuk, 2007). These defects were likely due to disruption of apical polarity and AJ. This study focused on Dlg5's requirement of AJ function and found that Dlg5 physically interacted with the β-catenin/cadherin complex and was found together with β-catenin/cadherin complex in both the Rab11-labeled recycling vesicles and the AJ. A more recent work using the same Dlg5-/- mice found that Dlg5 was also required for lung morphogenesis (Nechiporuk, 2013). Specifically, deletion of Dlg5 resulted in loss of apical polarity markers such as aPKC, and Dlg5 was partially colocalized with aPKC in the apical membrane of the wild type lung epithelia (Nechiporuk, 2013). But how Dlg5 exerts its functions in the apical membrane and regulate apical polarity is unknown (Luo, 2016).
In Drosophila, RNAi knockdown of dlg5 affected the cohesion and morphology of border cell clusters as well as delaying their migration (Aranjuez, 2012). Recently, genetic analysis of Drosophila Dlg5 revealed that its mutation caused embryonic lethality and loss of germ cells in the embryonic gonad (Reilly, 2015). Moreover, reduction of Dlg5 in the follicle cells in the adult ovary leads to defects in egg chamber budding, stalk cell overgrowth, ectopic polar cell induction and abnormal distribution of E-cadherin. However, detailed analysis of whether and how Drosophila Dlg5 regulates epithelial or apical polarity has not been done. This study reports that a genetic screen for follicle epithelial morphogenesis has identified the Drosophila Dlg5 as an essential player for maintenance of apical polarity by promoting Crb's apical membrane localization (Luo, 2016).
Dlg5 is specifically required for the maintenance of apical polarity and AJ of follicle epithelia during early stages of oogenesis, since both loss-of-function mutation and RNAi knockdown of dlg5 affected only apical polarity regulators and the sub-apical AJ components but not the basolateral regulators. Furthermore, the apical markers (Crb, Sdt, Patj, aPKC and Par6) were more severely reduced than the sub-apical AJ markers (Arm, E-cad and Baz) with the exception of N-cad, suggesting that the loss of apical polarity is the main cause of severe morphological defects in Dlg5-deficient follicle cells. A previous study found that loss of Crb, aPKC and Par6 did not affect the lateral localization of Dlg, whereas loss of Arm caused Dlg spreading to the apical membrane of follicle cells. This is consistent with the result that no apical spreading of Dlg was observed in dlg5 mutant clones, further confirming that loss of Dlg5 affected apical polarity more severely than the AJ function. Indeed, rescue of apical polarity defects by Crb but not Arm expression further validated this notion (Luo, 2016).
Importantly, this study demonstrates that Dlg5 positively regulates apical polarity by specifically promoting Crb's apical membrane localization, based on the following results. First, double staining revealed that Crb reduction was sometimes more severe than reduction of other apical markers (aPKC and Par6) in dlg5 mutant clones. Second, overexpression of Crb but not other apical or AJ regulators (aPKC, Par6, Arm) could completely rescue dlg5's apical polarity defects. Third, the increased membrane localization and membrane spreading of Crb as caused by blocking the Rab5-mediated endocytosis could be dramatically suppressed by dlg5 mutation. Moreover, the apical enrichment of Dlg5 in the early and mid-stage follicle epithelia (stage 1-stage 9) further suggests that Dlg5 could function at the apical region to promote Crb's membrane localization. On the other hand, Crb might conversely enhance Dlg5's localization to the apical membrane, since overexpression of Crb and hence its membrane spreading toward the basolateral region led to stronger localization of Dlg5 in the basolateral membrane. A previous study has reported that deletion of Dlg5 in mouse resulted in loss of aPKC but not Par3 (homologous with Drosophila Baz) in the developing lung epithelia and that Dlg5 was partially colocalized with aPKC, which are similar to the current findings (Nechiporuk, 2013). But how Dlg5 regulated the apical polarity during mouse lung morphogenesis was not understood. Based on the current results, it would be worthwhile to check whether murine Dlg5 promotes apical polarity by primarily regulating one of the three mammalian CRB paralogs (CRB1, CRB2, CRB3) (Luo, 2016).
As a MAGUK family member, Dlg5 is thought to function as a scaffold protein. Previous works have focused on which domains of Dlg5 physically interact with junctional and membrane-bound proteins like β-catenin, vinexin and smoothened, and trafficking regulators like syntaxin 4 in vitro or in cultured cells. But whether such domains are essential for its function and localization in vivo and which domains possess apical or AJ membrane targeting ability have not yet been addressed. Structure-function analysis demonstrates that the C-terminal fragment including MAGUK core (GUK, SH3, PDZ4) and PDZ3 is necessary but not entirely sufficient for Dlg5's functions. Furthermore, deletion of this C-terminal fragment (Δ4) caused most of Dlg5 to re-distribute to the cytoplasm, losing its membrane localization in the apical, AJ, and lateral regions. Interestingly, PDZ3 and PDZ4 (a subset of C terminal fragment) were also required for Dlg5's functions, and their deletion (Δ5) likewise resulted in the loss of apical and lateral (but not AJ) membrane localization. One interesting difference between the localization patterns of the other deletion mutants (that still possessed rescue abilities; Δ1-3, Δ6-8) and the patterns of Δ4 and Δ5 is that other deletions all retained some degree of apical localization, in contrast to the lack of localization in the apical membrane for Δ4 and Δ5. Together, these results suggest that MAGUK core and PDZ3's requirement for Dlg5's membrane localization in general and PDZ3-PDZ4's requirement for Dlg5's apical membrane localization may be critical for Dlg5's functions in the follicle cells. This is consistent with Dlg5's role in promoting Crb's apical localization. Lastly, this study found that the N-terminal coiled coil domain, the middle linker region and the MAGUK core could be individually membrane-targeted to apical, AJ and all (apical, AJ and basolateral) regions respectively (Luo, 2016).
Discs large 5 (Dlg5) is a member of the MAGUK family of proteins that typically serve as molecular scaffolds and mediate signaling complex formation and localization. In vertebrates, Dlg5 has been shown to be responsible for polarization of neural progenitors and to associate with Rab11-positive vesicles in epithelial cells. In Drosophila, however, the function of Dlg5 is not well-documented. This study identified dlg5 as an essential gene that shows embryonic lethality. dlg5 embryos display partial loss of primordial germ cells (PGCs) during gonad coalescence between stages 12 and 15 of embryogenesis. Loss of Dlg5 in germline and somatic stem cells in the ovary results in the depletion of both cell lineages. Reduced expression of Dlg5 in the follicle cells of the ovary leads to a number of distinct phenotypes, including defects in egg chamber budding, stalk cell overgrowth, and ectopic polar cell induction. Interestingly, loss of Dlg5 in follicle cells results in abnormal distribution of a critical component of cell adhesion, E-cadherin, shown to be essential for proper organization of egg chambers (Reilly, 2015).
dlg5 was shown to be essential for normal division or maintenance of FSCs and GSCs, and is required in later stage follicle cells. Reduction of Dlg5 levels disrupts egg chamber formation, indicating that dlg5 is involved in processes fundamental to egg chamber organization (Reilly, 2015).
When Dlg5 was depleted in follicle cells for 4 d, epithelial follicle cells showed relatively normal cell shape and polarity, but the polar and stalk cells showed strong abnormalities in number, localization, and overall organization. The induction of ectopic polar cells and stalk cell overgrowth observed in these ovaries is significant because it has been suggested that the differentiation of these two cell types are controlled by similar signaling events, which distinguishes them from the other follicle cells. Loss of Dlg5 may interfere with the pathways that specify stalk and polar cell differentiation and maturation and, consequently, egg chamber organization (Reilly, 2015).
The significantly enhanced phenotype observed upon depletion of Dlg5 in follicle cells for 10 d agrees with the dlg5D48 clonal phenotype and is consistent with the proposition that the function of dlg5 is completely lost in dlg5D48. Further, complete loss of dlg5 function results in embryonic lethality, possibly also as a result of abnormal organization and integration of specific embryonic cells (Reilly, 2015).
Analysis of primitive embryonic gonad formation in dlg5D48 embryos suggested that it likely functions during germ cell migration and gonad coalescence. These data are reminiscent of requirements of Dlg5 in the maintenance of the cohesion of migrating border cells in the ovary (Aranjuez, 2012). In this regard, it is interesting to note that proper gonad coalescence has been shown to depend on cell adhesion molecule E-cadherin. E-cadherin is consistently upregulated during late stages of gonad formation. Because loss of dlg5 results in altered distribution of E-cadherin in follicle cells, it is conceivable that aberrant E-cadherin levels and/or localization could also contribute to the embryonic gonad-specific phenotypes. In this context, it will be interesting to analyze further the precise requirement and cell-type specificity of Dlg5 function in controlling cell adhesion and migration (Reilly, 2015).
Many of the dlg5 phenotypes could result from aberrant E-cadherin distribution. For instance, oocyte mislocalization is often seen as a result of loss or aberrant homophilic adhesion mediated by E-cadherin between posterior follicle cells and the oocyte. Formation of interfollicular stalks is also dependent on dynamic E-cadherin accumulation: E-cadherin accumulates first at the apical boundary of prefollicular cells, followed by the establishment of lateral cell contacts to initiate and complete intercalation to form a single wide stalk. Additionally, E-cadherin has been demonstrated to be required for recruiting and anchoring stem cells to their niche prior to adulthood in the Drosophila ovary. By clonal analysis, this study showed that both germline and follicle dlg5 ovary stem cells are unable to give rise to normal daughter cells, indicating that the gene is essential in these cells. This may suggest several possibilities. There may be a cell-autonomous requirement for dlg5 in follicle cells; however, the loss of stem cells may also be an indication of the requirement for E-cadherin-mediated adhesion to the stem cell niche. Finally, the lack of cohesion in migrating germ cells in dlg5D48 embryos is consistent with a perturbation in cell-cell adhesion, as described above. The observed phenotypes, therefore, and the role of Dlg5 in E-cadherin distribution may be related; however, more work is needed to determine the nature of the participation of Dlg5 in E-cadherin distribution before any further conclusions may be drawn (Reilly, 2015).
Dlg5 is involved in vesicle trafficking in vertebrates and has been reported to colocalize with several Rab GTPases. The punctate distribution of Dlg5 in the Drosophila ovary is consistent with a similar association of the protein with endosomes. Therefore, it seems possible that Dlg5 is involved in endosomal trafficking. The abnormal distribution of E-cadherin, which is recycled through the endosome, observed in Dlg5 KD ovaries supports this hypothesis. Further, reduction of Dlg5 in the mammalian epithelial cell line LLc-PK1 resulted in lower levels of E-cadherin, but it is not clear how Dlg5 controls E-cadherin levels (Reilly, 2015).
Thus, Dlg5, like its human homolog and Dlg1, may be involved in endosomal recycling of E-cad. But based on this characterization of dlg5 and its functional requirement, there are fundamental differences between these two genes. Although dlg5 shows embryonic lethality and is essential in ovarian stem cells, dlg1 larvae can survive for >10 d and show overgrowth phenotypes in larvae and follicle cells. Persistent dlg1 germ line clones develop into eggs, whereas follicle cells lacking dlg1 sometimes show tumor-like invasion into the interior of the egg chamber, a phenotype this study not observe in Dlg5 KD. The task of figuring out what each of these Dlg proteins contributes to apical protein trafficking and cell survival should prove informative and stimulating (Reilly, 2015).
Cells often move as collective groups during normal embryonic development and wound healing, although the mechanisms governing this type of migration are poorly understood. The Drosophila melanogaster border cells migrate as a cluster during late oogenesis and serve as a powerful in vivo genetic model for collective cell migration. To discover new genes that participate in border cell migration, 64 out of 66 genes that encode PDZ domain-containing proteins were systematically targeted by in vivo RNAi knockdown. The PDZ domain is one of the largest families of protein-protein interaction domains found in eukaryotes. Proteins that contain PDZ domains participate in a variety of biological processes, including signal transduction and establishment of epithelial apical-basal polarity. Targeting PDZ proteins effectively assesses a larger number of genes via the protein complexes and pathways through which these proteins function. par-6, a known regulator of border cell migration, was a positive hit and thus validated the approach. Knockdown of 14 PDZ domain genes disrupted migration with multiple RNAi lines. The candidate genes have diverse predicted cellular functions and are anticipated to provide new insights into the mechanisms that control border cell movement. As a test of this concept, two genes that disrupted migration were characterized in more detail: big bang and the Dlg5 homolog CG6509. Evidence is presented that Big bang regulates JAK/STAT signaling, whereas Dlg5/CG6509 maintains cluster cohesion. Moreover, these results demonstrate that targeting a selected class of genes by RNAi can uncover novel regulators of collective cell migration (Aranjuez, 2012).
Search PubMed for articles about Drosophila Dlg5
Aranjuez, G., Kudlaty, E., Longworth, M. S. and McDonald, J. A. (2012). On the role of PDZ domain-encoding genes in Drosophila border cell migration. G3 (Bethesda) 2: 1379-1391. PubMed ID: 23173089
Chang, Y., Klezovitch, O., Walikonis, R. S., Vasioukhin, V. and LoTurco, J. J. (2010). Discs large 5 is required for polarization of citron kinase in mitotic neural precursors. Cell Cycle 9: 1990-1997. PubMed ID: 20436275
Chong, Y. C., Mann, R. K., Zhao, C., Kato, M. and Beachy, P. A. (2015). Bifurcating action of Smoothened in Hedgehog signaling is mediated by Dlg5. Genes Dev 29: 262-276. PubMed ID: 25644602
Luo, J., Wang, H., Kang, D., Guo, X., Wan, P., Wang, D. and Chen, J. (2016). Dlg5 maintains apical polarity by promoting membrane localization of Crumbs during Drosophila oogenesis. Sci Rep 6: 26553. PubMed ID: 27211898
Nakamura, H., Sudo, T., Tsuiki, H., Miyake, H., Morisaki, T., Sasaki, J., Masuko, N., Kochi, M., Ushio, Y. and Saya, H. (1998). Identification of a novel human homolog of the Drosophila dlg, P-dlg, specifically expressed in the gland tissues and interacting with p55. FEBS Lett 433: 63-67. PubMed ID: 9738934
Nechiporuk, T., Fernandez, T. E. and Vasioukhin, V. (2007). Failure of epithelial tube maintenance causes hydrocephalus and renal cysts in Dlg5-/- mice. Dev Cell 13: 338-350. PubMed ID: 17765678
Nechiporuk, T., Klezovitch, O., Nguyen, L. and Vasioukhin, V. (2013). Dlg5 maintains apical aPKC and regulates progenitor differentiation during lung morphogenesis. Dev Biol 377: 375-384. PubMed ID: 23466739
Reilly, E., Changela, N., Naryshkina, T., Deshpande, G. and Steward, R. (2015). Discs large 5, an essential gene in Drosophila, regulates egg chamber organization. G3 (Bethesda) 5: 943-952. PubMed ID: 25795662
Sezaki, T., Tomiyama, L., Kimura, Y., Ueda, K. and Kioka, N. (2013). Dlg5 interacts with the TGF-beta receptor and promotes its degradation. FEBS Lett 587: 1624-1629. PubMed ID: 23624079
Tomiyama, L., Sezaki, T., Matsuo, M., Ueda, K. and Kioka, N. (2015). Loss of Dlg5 expression promotes the migration and invasion of prostate cancer cells via Girdin phosphorylation. Oncogene 34: 1141-1149. PubMed ID: 24662825
Wakabayashi, M., Ito, T., Mitsushima, M., Aizawa, S., Ueda, K., Amachi, T. and Kioka, N. (2003). Interaction of lp-dlg/KIAA0583, a membrane-associated guanylate kinase family protein, with vinexin and beta-catenin at sites of cell-cell contact. J Biol Chem 278: 21709-21714. PubMed ID: 12657639
date revised: 15 August 2016
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