boudin: Biological Overview | References
Gene name - boudin
Cytological map position - 6E4-6E4
Function - secreted ligand
Symbol - bou
FlyBase ID: FBgn0261284
Genetic map position - X: 6,880,523..6,881,236 [+]
Classification - Ly6 domain (three-finger domain)
Cellular location - secreted
The Ly6 superfamily, present in most metazoan genomes, codes for different cell-surface proteins and secreted ligands containing an extracellular motif called a Ly6 domain or three-finger domain. This study reports the identification of 36 novel genes coding for proteins of this family in Drosophila. One of these fly Ly6 proteins, coded by the gene boudin (bou), is essential for tracheal morphogenesis in the fly embryo and contributes to the maintenance of the paracellular barrier and the organisation of the septate junctions in this tissue. Bou, a glycosylphosphatidylinositol anchored membrane protein, is also required for septate junction organisation in epithelial tissues and in the chordotonal organ glial cells, but not in the central nervous system. This study reveals interesting parallelisms between the Ly6 proteins of flies and vertebrates, such as the CD59 antigen. Similarly to this human protein, Bou travels from cell to cell associated with extracellular particles and, consistently, this study shows that it is required in a non-cell-autonomous fashion. This work opens the way for future studies addressing the function of Ly6 proteins using Drosophila as a model system (Hijazi, 2009).
Ly6 proteins share an extracellular motif spanning about 100 residues known as a three-finger domain, three-finger snake toxin motif or Ly6/uPAR domain. This structure, first identified in the sea-snake erabutoxin b, features a simple inner core stabilised by disulphide bridges, which supports three protruding loops or fingers. Besides a diagnostic set of 8 or 10 cysteines found in stereotyped positions, Ly6 primary sequences are poorly conserved, but they adopt remarkably similar three-dimensional structures (Kini, 2002; Ploug, 1994). The Ly6 module is a structural domain involved in protein-protein interactions, tolerating an unusual degree of variation and binding with high specificity to a broad spectrum of targets (Hijazi, 2009).
The human genome codes for 45 members of the Ly6 superfamily (Galat, 2008). These include 12 TGFβ receptors, the ectodomains of which adopt the three-finger fold, but also many glycosylphosphatidylinositol (GPI)-anchored proteins and soluble ligands. Only a few of these proteins have been studied in detail, such as the urokinase plasminogen activator receptor (uPAR; PLAUR - Human Gene Nomenclature Database), which plays important roles in cell adhesion, proliferation and migration (Blasi, 2002), and CD59, an inhibitor of complement activity (Davies, 1989). Other members, such as Lynx1 (Miwa, 2006) or the soluble SLURP proteins (Grando, 2008), act as regulators of nicotinic acetylcholine receptors, and are likely to be the ancestors of the snake neurotoxins. However, although they are often used as lymphocyte and tumoural markers (Bamezai, 2004), many Ly6 human and murine proteins have unknown roles (Hijazi, 2009).
A systematic search for members of the Ly6 superfamily in Drosophila identified 36 previously uncharacterised genes coding for one or more Ly6 motifs. The function of boudin (bou) was investigated. Phenotypic analysis of bou mutants shows that this Ly6 protein participates in the formation of paracellular barriers in epithelial and neural tissues, physiological fences that regulate the passage of solutes between cells in both epithelial and glial sheaths. bou is required for the organisation of septate junctions (SJs), invertebrate adhesion structures fulfilling an equivalent role to the vertebrate tight junctions. Differing from known SJ constituents, bou requirements are non-cell-autonomous, and, accordingly, it was found that Bou can be released in extracellular particles and become incorporated into neighbouring cells. Altogether, these results indicate that Drosophila could be an attractive system in which to study the function and general properties of Ly6 proteins in a developmental context (Hijazi, 2009).
The results reveal that Bou plays an essential role in the organisation of SJs and the maintenance of paracellular barriers in Drosophila epithelia and chordotonal organs. Although some vertebrate members of the Ly6 family are known to participate in cell-adhesion processes (Bamezai, 2004), this is the first example showing that they are required for the formation of this type of cellular junction. As bou is well conserved in other insect genomes, its role in SJ organisation could have been maintained during evolution. Invertebrate SJs and vertebrate tight junctions are considered analogous structures because both participate in the establishment of paracellular barriers, although they present a different organisation. However, vertebrates have adhesion structures functionally, morphologically and molecularly similar to insect pleated SJs: the so-called paranodal septate junctions, which are formed by neural axons and Schwann cells, at the level of the Ranvier's nodes. This study shows that Bou is necessary for SJ organisation in the embryonic peripheral nervous system, indicating that its activity is required in some neural tissues. Thus, these observations raise the possibility that some vertebrate Ly6 proteins could be involved in the formation of paranodal septate junctions, which are essential for axonal insulation and propagation of action potentials (Hijazi, 2009).
In insects, the epithelial and neural SJs share many components, so the observation that bou is not required for blood-brain barrier maintenance in the ventral nerve cord came as a surprise, revealing the existence of tissular and molecular heterogeneities in the organisation of these junctions. It will be interesting to establish whether these differences also determine different barrier selective properties. It is speculated that other Ly6 proteins expressed in the nervous system could contribute to blood-brain barrier formation in the subperineural glia (Hijazi, 2009).
The results show that bou inactivation specifically perturbs the organisation of SJs. Since these structures are large extracellular complexes including different transmembrane and GPI-anchored proteins, one hypothesis is that Bou could be a membrane SJ component. Consistently, HA-Bou is found at lateral contact areas in tracheal, salivary gland and wing disc epithelia, overlapping with the membrane domains that contain SJ. However, this protein does not significantly accumulate in these membrane regions and is also seen in the most apical part of the cells, opening up the possibility that it could operate in other membrane areas or act as a signalling molecule. Indeed, studies in vertebrates indicate that Ly6 proteins can assume roles in both cell signalling and cell adhesion (Bamezai, 2004). Clearly, identification of the Bou molecular partners will be a crucial step in understanding how this protein exerts its activity (Hijazi, 2009).
In contrast with other genes required for SJ formation, bou functions in a non-cell-autonomous way. Accordingly, the Bou protein is found in extracellular particles and can be captured by neighbouring cells, suggesting that its diffusion is responsible for the phenotypic non-autonomy. Although it is possible that Bou could act as a secreted ligand after release of its GPI anchor, a parallelism with other members of the family suggests that the full molecule could instead become incorporated into the membrane of neighbouring cells (Neumann, 2007). In fact, the mammalian Ly6 member CD59, a cell-surface antigen protecting host cells from the complement attack, travels coupled to membranous vesicles called prostasomes with its intact GPI. These specialised vesicles are secreted into the seminal fluid by prostatic glands, and allow CD59 transfer to the sperm cells, which can then elude complement attack (Rooney, 1993). GPI-bound CD59 has also been found associated with human HDL apolipoproteins (Vakeva, 1994). However, the Bou particles are not lipophorin vesicles, the insect equivalent to vertebrate apolipoproteins (Rodenburg, 2005). Therefore, the fly wing epithelium could produce a different type of vesicle, possibly similar to prostasomes, which are proposed to be called `boudosomes'. Unfortunately, it could not be determined whether the Bou GPI anchor is required for incorporation into these particles, because the C-terminus of the protein seems essential for prior exit from the ER. Thus, future work will be needed to characterise the biochemical features of boudosomes and their function (Hijazi, 2009).
Little is known about how epithelial cells coordinate their activity to form efficient fences. As many SJ components are required in a cell-autonomous manner, their simultaneous expression by each individual cell seems a prerequisite for barrier assembly. A component and/or SJ regulator shared by different cells could be an element coordinating the organisation of efficient barriers in a dynamic epithelium. Alternatively, Bou extracellular traffic could be a specialised feature of this GPI-anchored protein and not have functional relevance for SJ assembly during normal development (Hijazi, 2009).
Besides Bou and the TGFβ receptors, the only member of the Ly6 fly family with a characterised role is the Rtv protein, which is also expressed in epidermal derivatives. Both bou and rtv mutants affect the organisation of the tracheal chitin luminal cable, although rtv mutants exhibit stronger phenotypes. However, SJ integrity is a prerequisite for proper assembly of the chitin cable, and this study shows that rtv is neither required for paracellular barrier integrity nor for SJ organisation. Thus, whereas these observations confirm that chitin cable deposition relies on the organisation of SJs, they demonstrate that these Ly6 proteins act in different processes (Hijazi, 2009).
This study has carried out the first description of the Ly6 superfamily in the genome of an insect, identifying 36 new genes bearing this domain in Drosophila. The conservation of these proteins among the drosophilids indicates that the family was established before the evolutive radiation of this group. By contrast, only 14 genes coding for Ly6 domains were identified in the honeybee genome. Most of these genes have fly orthologues, like bou and rtv, pointing out the existence in higher insects of a core of ancestral genes with potentially conserved roles. Thus, repeated events of gene duplication followed by rapid divergence of coding and regulatory sequences occurred in the drosophilid lineage. Indeed, the presence of genomic clusters grouping together different Ly6 genes is a novel evolutive acquisition, since the conserved genes tend to be in isolated positions (Hijazi, 2009).
It seems that genes coding for a Ly6 motif are prone to sudden phases of extensive duplication and diversification in different phylogenetic groups. In fact, an interesting parallelism can be drawn with the evolution of three-finger elapid snake venoms. This large group of Ly6 secreted proteins operates using diverse strategies, such as forming membrane pores, targeting the activity of acetylcholine receptors, inactivating acetylcholine esterase or blocking platelet aggregation (Tsetlin, 1999). Moreover, crystallographic analysis has revealed that three-finger toxins can interact with their targets via virtually any part of their solvent exposed surfaces (Kini, 2002). Yet, most of them share a common ancestor (Fry, 2003). Given the broad diversity of expression patterns exhibited by the different Drosophila Ly6 members, it is likely that gene duplication has been followed by acquisition of new developmental and physiological functions. Analysis of this insect family from an evolutive perspective could be a way to enhance understanding of the mechanisms underlying the generation of evolutive innovations (Hijazi, 2009).
Cellular junction formation is an elaborate process that is dependent on the regulated synthesis, assembly and membrane targeting of constituting components. This study reports on three Drosophila Ly6-like proteins essential for septate junction (SJ) formation. SJs provide a paracellular diffusion barrier and appear molecularly and structurally similar to vertebrate paranodal septate junctions. Crooked (Crok), a small GPI-anchored Ly6-like protein, is required for septa formation and barrier functions. In embryos that lack Crok, SJ components are produced but fail to accumulate at the plasma membrane. Crok is detected in intracellular puncta and acts tissue-autonomously, which suggests that it resides in intracellular vesicles to assist the cell surface localization of SJ components. In addition, this study found that two related Ly6 proteins, Coiled (Cold) and Crimpled (Crim), are required for SJ formation and function in a tissue-autonomous manner, and Cold also localizes to intracellular vesicles. Specifically, Crok and Cold are required for correct membrane trafficking of Neurexin IV, a central SJ component. The non-redundant requirement for Crok, Cold, Crim and Boudin (Bou; another Ly6 protein that was recently shown to be involved in SJ formation) suggests that members of this conserved family of proteins cooperate in the assembly of SJ components, possibly by promoting core SJ complex formation in intracellular compartments associated with membrane trafficking (Nilton, 2010).
Ly6 proteins constitute large protein families in both vertebrates and insects. In mammals, they are expressed in cells of hematopoetic origin, the brain, vascular epithelium, kidney tubular epithelium, lung, keratinocytes, stomach, testis and prostate. Reflecting their differential expression, Ly6 proteins are used in diverse biological processes. Apart from acting as GPI-linked cell accessory proteins of the immune system, vertebrate Ly6 proteins function in the modulation of nicotinic acetylcholine receptors, remodelling of the extracellular matrix during skeletal muscle regeneration, self-renewal of erythroid progenitors, and lipolytic processing of triglyceride-rich lipoproteins by binding lipoprotein lipase. The presence of a GPI-anchor in Ly6 molecules, a lipid anchor that tethers the proteins on the outer leaflet of the membrane, also suggests that Ly6 proteins can aggregate in lipid rafts to alter the activity of associated proteins (Nilton, 2010).
This study found that the Drosophila Ly6-like genes also exhibit a diverse tissue-specific distribution during development, with subsets of genes showing similar tissue expression, suggesting that they participate in similar biological processes. Indeed, five of the 18 Drosophila Ly6-like genes are expressed in a similar pattern in the developing ectoderm, and at least four are required for SJ formation, including bou (Hijazi, 2009), crok, crim and cold. The only other GPI-linked Drosophila Ly6 protein studied so far is quiver (qvr; also known as sleepless; Koh, 2008), which is required for sleep and appears to affect the levels of the voltage-dependent potassium channel Shaker (Nilton, 2010).
Phenotypic analyses of crok mutants show that Crok is required for plasma membrane accumulation of SJ components. Since SJ components were detected in the tracheal cytoplasm of stage 15 crok mutant embryos and protein analyses on immunoblots show that ATPα and Nrx-VI are present in crok mutants, it is concluded that Crok is not required for the synthesis of SJ components. Moreover, elevated levels of ATPα and Nrx-VI were found in mutant embryonic extracts, despite an apparent reduction of immunofluorescence staining for these proteins, suggesting that these components are more easily solubilized in the mutant embryos. It thus appears that Crok is required for the formation of SJ complexes at the correct plasma membrane domain (Nilton, 2010).
The function of both Crok and Cold appears to be necessary for the efficient incorporation of Nrx-IV into a stable SJ-associated complex. Upon loss of Crok and Cold, Nrx-IV-GFP accumulates in large intracellular vesicles. A similar situation occurs upon loss of Cora, which interacts with the cytoplasmic tail of Nrx-IV. The presence of fluid-phase dextran in these vesicles suggests that the Nrx-IV-GFP puncta in crok and cold mutants represent endocytosed protein, possibly on route to degradation in lysosomal compartments. Consistent with this idea, subsets of the Nrx-IV vesicles in cold mutants colocalize with endocytic markers, including Rab5 (early endosomes), Rab11 (recycling endosomes), Hrs (late endosomes) and Dor (lysosomes). As other known SJ components were not detected in these vesicles, it appears that they contain SJ subcomplexes that include Nrx-IV and Cora. This specificity could suggest either that Nrx-IV and interacting proteins are more sensitive than are other components to disruption in SJ assembly, or that Crok and Cold are specifically involved in the stable integration of Nrx-IV and interacting proteins into the SJ complex. Further experiments will be required to discern these alternative possibilities (Nilton, 2010).
Bou, Crok and Cold all appear to accumulate in intracellular membrane compartments. Previous studies have shown that urokinase/plasminogen activator receptor uPAR, an extensively studied GPI-linked protein with two LU domains, is endocytosed and recycled, and that this trafficking is essential for its function. Consistent with this idea, it was found that Cold colocalizes with dextran-labelled vesicles in cultured cells. In addition, although Bou appears to accumulate in the perinuclear cytoplasm (Hijazi, 2009), the non-autonomous behaviour of Bou suggests that it travels to the plasma membrane. Like Bou, Crok shows an apparent association with internal membrane compartments, particularly the ER. The Crok antiserum does not detect endogenous Crok at high levels, and it is possible that Crok in addition associates transiently with the cell surface. Possible colocalization of Nrx-IV with Crok or Cold, or with markers for endosomal compartments, has not been addressed and it is currently unclear whether the Ly6 proteins act in the sorting, trafficking or pre-assembly of SJ components prior to their transport to the site of junction assembly, or in an endocytic recycling of the components (Nilton, 2010).
The non-redundant requirement for four Ly6-like proteins in SJ assembly is intriguing and coherent with the need for multiple Ly6-like proteins in the allosteric modulation of nicotinic acetylcholine receptor (nAChR) functions. These Ly6-like proteins include Lynx1 and possibly Lypd6 in neurons, and the secreted proteins Slurp1 and Slurp2 in the ectoderm. It has further been suggested that the lynx-nAChR interactions occur during receptor biosynthesis and maturation in the endoplasmic reticulum, a main site for assembly of the multi-subunit membrane proteins of nAChRs. In analogy, the Drosophila Ly6-like proteins might assume roles as allosteric modulators of multiprotein SJ complexes to promote their functional association (Nilton, 2010).
Together, the analyses of Crok, Crim, Cold and Bou highlight a central task for Ly6 proteins in SJ formation that might also be essential for vertebrate paranodal junction assembly. Identification of their ligands and the subcellular site of action should contribute further understanding of how highly ordered, multi-protein complexes form along precise subdomains of the plasma membrane (Nilton, 2010).
Search PubMed for articles about Drosophila Boudin
Bamezai, A. (2004). Mouse Ly-6 proteins and their extended family: markers of cell differentiation and regulators of cell signaling. Arch. Immunol. Ther. Exp. (Warsz.) 52: 255-266. PubMed ID: 15467490
Blasi, F. and Carmeliet, P. (2002). uPAR: a versatile signalling orchestrator. Nat. Rev. Mol. Cell Biol. 3: 932-943. PubMed ID: 12461559.
Davies, A., Simmons, D. L., Hale, G., Harrison, R. A., Tighe, H., Lachmann, P. J. and Waldmann, H. (1989). CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells. J. Exp. Med. 170: 637-654. PubMed ID: 2475570
Fry, B. G., Wuster, W., Kini, R. M., Brusic, V., Khan, A., Venkataraman, D. and Rooney, A. P. (2003). Molecular evolution and phylogeny of elapid snake venom three-finger toxins. J. Mol. Evol. 57: 110-129. PubMed ID: 12962311
Galat, A. (2008). The three-fingered protein domain of the human genome. Cell Mol. Life Sci. 65: 3481-3493. PubMed ID: 18821057
Grando, S. A. (2008). Basic and clinical aspects of non-neuronal acetylcholine: biological and clinical significance of non-canonical ligands of epithelial nicotinic acetylcholine receptors. J. Pharmacol. Sci. 106: 174-179. PubMed ID: 18285656
Hijazi, A., Masson, W., Augé, B., Waltzer, L., Haenlin, M. and Roch, F. (2009). boudin is required for septate junction organisation in Drosophila and codes for a diffusible protein of the Ly6 superfamily. Development 136(13): 2199-209. PubMed ID: 19502482
Kini, R. M. (2002). Molecular moulds with multiple missions: functional sites in three-finger toxins. Clin. Exp. Pharmacol. Physiol. 29: 815-822. PubMed ID: 12165048
Koh K., et al. (2008). Identification of SLEEPLESS, a sleep-promoting factor. Science 321: 372-376. PubMed ID: 18635795
Miwa, J. M., Stevens, T. R., King, S. L., Caldarone, B. J., Ibanez-Tallon, I., Xiao, C., Fitzsimonds, R. M., Pavlides, C., Lester, H. A., Picciotto, M. R. et al. (2006). The prototoxin lynx1 acts on nicotinic acetylcholine receptors to balance neuronal activity and survival in vivo. Neuron 51: 587-600. PubMed ID: 16950157
Neumann, S., Harterink, M. and Sprong, H. (2007). Hitch-hiking between cells on lipoprotein particles. Traffic 8: 331-338. PubMed ID: 17274797
Nilton, A., et al. (2010). Crooked, coiled and crimpled are three Ly6-like proteins required for proper localization of septate junction components. Development 137(14): 2427-37. PubMed ID: 20570942
Ploug, M. and Ellis, V. (1994). Structure-function relationships in the receptor for urokinase-type plasminogen activator. Comparison to other members of the Ly-6 family and snake venom alpha-neurotoxins. FEBS Lett. 349: 163-168. PubMed ID: 8050560
Rodenburg, K. W. and Van der Horst, D. J. (2005). Lipoprotein-mediated lipid transport in insects: analogy to the mammalian lipid carrier system and novel concepts for the functioning of LDL receptor family members. Biochim. Biophys. Acta 1736: 10-29. PubMed ID: 16099208
Rooney, I. A., Atkinson, J. P., Krul, E. S., Schonfeld, G., Polakoski, K., Saffitz, J. E. and Morgan, B. P. (1993). Physiologic relevance of the membrane attack complex inhibitory protein CD59 in human seminal plasma: CD59 is present on extracellular organelles (prostasomes), binds cell membranes, and inhibits complement-mediated lysis. J. Exp. Med. 177: 1409-1420. PubMed ID: 7683035
Tsetlin, V. (1999). Snake venom alpha-neurotoxins and other `three-finger' proteins. Eur. J. Biochem. 264: 281-286. PubMed ID: 10491072
Vakeva, A., Jauhiainen, M., Ehnholm, C., Lehto, T. and Meri, S. (1994). High-density lipoproteins can act as carriers of glycophosphoinositol lipid-anchored CD59 in human plasma. Immunology 82: 28-33. PubMed ID: 7519171
date revised: 30 October 2010
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