InteractiveFly: GeneBrief

bark beetle: Biological Overview | References


Gene name - bark beetle

Synonyms - anakonda, CG3921

Cytological map position - 24C5-24C5

Function - transmembrane receptor

Keywords - interacts extracellularly to mediate assembly of tricellular junctions, septate junctions, ectodermal and endodermal epithelia

Symbol - bark

FlyBase ID: FBgn0031571

Genetic map position - chr2L:3,787,158-3,802,735

Classification - Scavenger receptor cysteine-rich domain, C-type lectin (CTL)/C-type lectin-like (CTLD) domain

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Daniel, E., Daude, M., Kolotuev, I., Charish, K., Auld, V. and Le Borgne, R. (2018). Coordination of septate junctions assembly and completion of cytokinesis in proliferative epithelial tissues. Curr Biol [Epub ahead of print]. PubMed ID: 29706514
Summary:
How permeability barrier function is maintained when epithelial cells divide is largely unknown. This study has investigated how the bicellular septate junctions (BSJs) and tricellular septate junctions (TSJs) are remodeled throughout completion of cytokinesis in Drosophila epithelia. Following cytokinetic ring constriction, the midbody assembles, matures within SJs, and is displaced basally in two phases. In a first slow phase, the neighboring cells remain connected to the dividing cells by means of SJ-containing membrane protrusions pointing to the maturing midbody. Fluorescence recovery after photobleaching (FRAP) experiments revealed that SJs within the membrane protrusions correspond to the old SJs that were present prior to cytokinesis. In contrast, new SJs are assembled below the adherens junctions and spread basally to build a new belt of SJs in a manner analogous to a conveyor belt. Loss of function of a core BSJ component, the Na+/K+-ATPase pump Nervana 2 subunit, revealed that the apical-to-basal spread of BSJs drives the basal displacement of the midbody. In contrast, loss of the TSJ protein Bark beetle indicated that remodeling of TSJs is rate limiting and slowed down midbody migration. In the second phase, once the belt of SJs is assembled, the basal displacement of the midbody is accelerated and ultimately leads to abscission. This last step is temporally uncoupled from the remodeling of SJs. It is proposed that cytokinesis in epithelia involves the coordinated polarized assembly and remodeling of SJs both in the dividing cell and its neighbors to ensure the maintenance of permeability barrier integrity in proliferative epithelia.
Wang, Z., Bosveld, F. and Bellaiche, Y. (2018). Tricellular junction proteins promote disentanglement of daughter and neighbour cells during epithelial cytokinesis. J Cell Sci. Pubmed ID: 29739875
Summary:
In epithelial tissue, new cell-cell junctions are formed upon cytokinesis. To understand junction formation during cytokinesis, this study explored in Drosophila epithelium, de novo formation of tricellular septate junctions (TCJs). Upon midbody formation, the membranes of the two daughter cells and of the neighbouring cells located below the adherens junction (AJ) remain entangled in a 4-cell structure apposed to the midbody. The septate junction protein Discs-Large and components of the TCJ, Gliotactin and Anakonda accumulate in this 4-cell structure. Subsequently, a basal movement of the midbody parallels the detachment of the neighbouring cell membranes from the midbody, the disengagement of the daughter cells from their neighbours and the reorganisation of TCJs between the two daughter cells and their neighbouring cells. While the movement of midbody is independent of the Alix and Shrub abscission regulators, the loss of Gliotactin or Anakonda function impedes both the resolution of the connection between the daughter-neighbour cells and midbody movement. TCJ proteins therefore control an additional step of cytokinesis necessary for the disentanglement of the daughter cells and their neighbours during cytokinesis.
BIOLOGICAL OVERVIEW

In epithelia, specialized tricellular junctions (TCJs) mediate cell contacts at three-cell vertices. TCJs are fundamental to epithelial biology and disease, but only a few TCJ components are known, and how they assemble at tricellular vertices is not understood. This study describes a transmembrane protein, Anakonda (Aka: FlyBase name - Bark beatle), which localizes to TCJs and is essential for the formation of tricellular, but not bicellular, junctions in Drosophila. Loss of Aka caused epithelial barrier defects associated with irregular TCJ structure and geometry, suggesting that Aka organized cell corners. Aka is necessary and sufficient for accumulation of Gliotactin at TCJs, suggesting that Aka initiated TCJ assembly by recruiting other proteins to tricellular vertices. Aka's extracellular domain had an unusual tripartite repeat structure that might mediate self-assembly, directed by the geometry of tricellular vertices. Conversely, Aka's cytoplasmic tail is dispensable for TCJ localization. Thus, extracellular interactions, rather than TCJ-directed intracellular transport, appear to mediate TCJ assembly (Byri, 2015).

Epithelial cells are linked via intercellular junctions that provide paracellular diffusion barriers, maintenance of polarity, and cell-to-cell communication. Bicellular junctions (BCJs) make up the most abundant intercellular contacts in epithelia. They connect two neighboring plasma membranes and are organized into distinct complexes along the apical-basal axis, collectively forming the apical junctional belt. However, at certain positions along the cell perimeter, three cell corners meet and the bicellular junctional complex is disjointed. Here, specialized tricellular junctions (TCJs) connect epithelial cells. TCJs play important roles in epithelial barrier functions and cytoskeletal organization, and are preferential sites for trans-endothelial migration of neutrophils and metastatic cancer cells, as well as for the spreading of intracellular pathogens (Byri, 2015).

Due to the geometry of three-cell vertices, the sealing of the epithelium at these sites requires a dedicated junctional organization. Ultrastructural analyses using freeze-fracture electron microscopy (EM) showed that the zonula occludens, tight junctions (TJs) in vertebrates and septate junctions (SJs) in arthropods, changes characteristics when approaching a three-cell vertex. Instead of continuing parallel to the epithelial plane around the cell perimeter, the junctional strands extend basally, forming a bicellular seal along the apical-basal axis. In vertebrates, these structures are termed central sealing elements, and three such parallel TJ extensions enclose a narrow (approximately 10 nm) central canal at each TCJ. Similarly, in invertebrates the SJ strands turn by 90 degrees when approaching a tricellular corner, forming three parallel limiting strands that surround the tricellular juncture space, resembling the central sealing element in vertebrates. Within and perpendicular to the vertical juncture space are a series of diaphragms, which appear linked not only to three limiting septa, but also to the three cell corner membranes, thereby forming true tricellular contacts (Byri, 2015).

Despite the fundamental biological importance of TCJs, only few of their components are known (Furuse, 2014), and the mechanism of their localized assembly at three-cell vertices is not understood. In vertebrates, the Occludin family protein Tricellulin localizes to TCJs (Ikenouchi, 2005) and is recruited there by lipolysis-stimulated lipoprotein receptor (LSR) and related proteins (ILDR1 and ILDR2; Masuda, 2011; Higashi, 2013). Additionally, the cytoplasmic PDZ-domain-containing protein Tjp2iso3 associates with Tricellulin in Sertoli cells (Chakraborty, 2014). The Neuroligin-like transmembrane protein Gliotactin in Drosophila is the only TCJ protein characterized so far in invertebrates (Schulte, 2003). It is not clear what kind of cues direct the accumulation of these proteins at tricellular vertices, and the features of known TCJ proteins do not explain the distinct structure of three-cell contacts observed by EM. This study describes a transmembrane protein, Anakonda (Aka), which accumulates at TCJs in Drosophila epithelia. Aka can initiate TCJ assembly and may do so through its large extracellular domain, which exhibits an unusual triple-repeat structure (Byri, 2015).

Ultrastructural analyses revealed a unique junctional architecture at points in epithelia where three cells meet. Yet, only few TCJ-specific proteins are known, and how they assemble into a tightly localized complex exclusively at tricellular vertices is not understood. This study describes transmembrane protein Anakonda, which is shown to play a critical role in TCJ assembly and epithelial barrier formation. First, Aka was shown to localize to TCJs in ectodermal and endodermal epithelia, suggesting that Aka is a core TCJ component. Second, Aka was demonstrated to be specifically required for the assembly and correct geometry of tricellular, but not of bicellular SJs, and Aka was shown to localize to TCJs independently of bicellular SJs. Third, Aka is required for recruiting or maintaining Gli at tricellular vertices. Conversely, Gli is not required for TCJ localization of Aka. Fourth, Aka mis-expression causes premature accumulation of Gli at TCJs, indicating that Aka acts upstream of Gli in initiating TCJ formation (Byri, 2015).

Aka protein contains a large extracellular domain with a conserved tripartite repeat structure. This unique structure distinguishes Aka from other known TCJ proteins. Tricellulin, a four-pass transmembrane protein in vertebrates, localizes to the central sealing elements, suggesting that it participates in the specialized bicellular contacts that surround each three-cell vertex (Ikenouchi, 2005). Tricellulin is recruited to TCJs by the immunoglobulin domain transmembrane protein LSR, but how LSR localizes to TCJs is still unclear (Masuda, 2011). In Drosophila, Gli, a transmembrane protein with a cholinesterase domain lacking catalytic activity, accumulates at TCJs, but is also involved in bicellular SJ organization. Phosphorylation-dependent endocytic turnover and indirect association with Dlg are required for accumulation of Gli at TCJs. However, the mechanisms underlying TCJ-specific localization are not understood for Gli or any other TCJ protein (Byri, 2015).

The results suggest that Aka acts at an early step during TCJ assembly by recruiting other TCJ components, including Gli, to tricellular vertices. TCJ localization of Aka and Gli does not require Aka's C terminus, including the PDZ-binding motif. Similarly, Gli was found to localize to TCJs independently of its PDZ-binding motif (Schulte, 2006). These findings suggest that Aka and Gli might be targeted to TCJs through their extracellular domains, rather than through a cytoplasmic localization machinery. Because mislocalized Aka protein is not sufficient to recruit Gli to ectopic locations, Aka might not interact directly with Gli, or only do so in the context of TCJs. Consistent with this notion, we were not able to detect co-immunoprecipitation of Aka and Gli. Interestingly, overexpression of Gli (Schulte, 2006) or absence of Aka causes Gli to spread from TCJs to BCJs. Thus, Gli does not have an intrinsic propensity to localize to TCJs, but by default localizes to the apicolateral membrane domain occupied by SJs along the cell perimeter. TCJ accumulation of Gli therefore requires (1) association with the apicolateral membrane and (2) Aka-dependent recruitment of Gli specifically to TCJs. The findings show that unlike Gli, Aka does not depend on bicellular SJs for its localization to TCJs, and suggesting that Aka protein might have intrinsic properties that lead to its accumulation at TCJs (Byri, 2015).

Tricellular vertices display a unique geometry where three plasma membranes are in close proximity and at fixed angles. The exceptional curvature of the plasma membrane at these sites implies distinct physical properties compared to bicellular contacts. Intriguingly, it was found that TCJ assembly depends on Aka expression in all three cells adjoining a vertex, suggesting that extracellular interactions between Aka molecules from different cells are essential for TCJ formation. Considering the size of Aka's extracellular domain (303 kDa), it is likely that a single Aka protein spans the entire TCJ canal (25-30 nm), although shorter Aka isoforms may constitute different structures. Shorter Aka species might carry out functions that may or may not be related to the function of full-length Aka at TCJs. Alternatively, Aka fragments may reflect turnover of TCJ complexes, e.g., during junctional remodeling (Byri, 2015).

It is tempting to speculate that the three repeat regions in full-length Aka protein, with apparent similar domain organization, could make equal contacts with the three cell corners in the plane perpendicular to the central TCJ canal. Possibly, interaction with membrane components in the tricellular region occurs via the three SR domains, since such domains are found in receptors that recognize a wide range of molecular patterns, including surface proteins, carbohydrates, lipids, lipopolysaccharides, and peptidoglycans associated with pathogens or apoptotic cells. Because such three-way contacts may, for steric reasons, only be possible at vertices, they might selectively stabilize Aka complexes at these sites. Computer simulations suggest that the enrichment of Aka at three-cell vertices could theoretically be enhanced by the reduction of dimensionality in the tricellular region, which may promote stacking interactions between Aka molecules within the central TCJ canal. This idea is consistent with the regular structure of the TCJ diaphragms and the equal dihedral angles near TCJs as observed with electron microscopy. Stacking might occur with Aka molecules rotated by 120 or 240 degrees within the stack, perhaps in a helical array, depending on the contributing cell. Notably, such a scenario would explain the finding that TCJ formation requires Aka protein production by all three cells adjoining a vertex. A high priority of future studies will be to investigate the arrangement of Aka proteins at TCJs. Together, the current results on Aka localization, the geometry of three-cell vertices, and the triple-repeat structure of Aka protein suggest a mechanism of TCJ formation, which is promoted by self-assembly of Aka at tricellular contacts. Such self-assembly might additionally involve interactions with other membrane-associated or extracellular components, and could cooperate with bicellular adhesion molecules that zip up bicellular contacts. It will be interesting to test whether the geometry of tricellular vertices and the specific properties of Aka protein are sufficient to direct its accumulation to TCJs. Conversely, perturbed TCJ geometry upon loss of Aka may have long-range effects, such as the loss of cell-cell adhesion that this study observed in late-stage aka embryos. Interestingly, depletion of Tricellulin from mammalian cells affects cell shape and the F-actin network, suggesting that TCJs may in fact have organizing activity on the entire cell (Byri, 2015).

The existence of aka homologs in invertebrates and cephalochordates correlates with the presence of SJs in these groups. However, the proposed self-assembly model for TCJ formation in Drosophila may apply also to vertebrates, although the corresponding proteins remain to be discovered. A better understanding of TCJ assembly will be a key step toward elucidating how these poorly characterized cellular structures provide epithelial barrier function, while at the same time allowing the passage of migrating lymphocytes, metastatic cells, and intracellular pathogens (Byri, 2015).

The claudin Megatrachea protein complex

Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. This study presents a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, 142 proteins associated with Megatrachea were identified in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. Known and novel putative SJ components were identified, such as the gene product of CG3921. Furthermore, the data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, the findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, these findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates (Jaspers, 2012).


REFERENCES

Search PubMed for articles about Drosophila Anakonda

Byri, S., Misra, T., Syed, Z.A., Bätz, T., Shah, J., Boril, L., Glashauser, J., Aegerter-Wilmsen, T., Matzat, T., Moussian, B., Uv, A. and Luschnig, S. (2015). The triple-repeat protein Anakonda controls epithelial tricellular junction formation in Drosophila. Dev Cell 33(5):535-48. PubMed ID: 25982676

Chakraborty, P., William Buaas, F., Sharma, M., Smith, B. E., Greenlee, A. R., Eacker, S. M. and Braun, R. E. (2014). Androgen-dependent sertoli cell tight junction remodeling is mediated by multiple tight junction components. Mol Endocrinol 28: 1055-1072. PubMed ID: 24825397

Furuse, M., Izumi, Y., Oda, Y., Higashi, T. and Iwamoto, N. (2014). Molecular organization of tricellular tight junctions. Tissue Barriers 2: e28960. PubMed ID: 25097825

Higashi, T., Tokuda, S., Kitajiri, S., Masuda, S., Nakamura, H., Oda, Y. and Furuse, M. (2013). Analysis of the 'angulin' proteins LSR, ILDR1 and ILDR2--tricellulin recruitment, epithelial barrier function and implication in deafness pathogenesis. J Cell Sci 126: 966-977. PubMed ID: 23239027

Ikenouchi, J., Furuse, M., Furuse, K., Sasaki, H., Tsukita, S. and Tsukita, S. (2005). Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells. J Cell Biol 171: 939-945. PubMed ID: 16365161

Jaspers, M. H., Nolde, K., Behr, M., Joo, S. H., Plessmann, U., Nikolov, M., Urlaub, H. and Schuh, R. (2012). The claudin Megatrachea protein complex. J Biol Chem 287: 36756-36765. PubMed ID: 22930751

Masuda, S., Oda, Y., Sasaki, H., Ikenouchi, J., Higashi, T., Akashi, M., Nishi, E. and Furuse, M. (2011). LSR defines cell corners for tricellular tight junction formation in epithelial cells. J Cell Sci 124: 548-555. PubMed ID: 21245199

Schulte, J., Tepass, U. and Auld, V. J. (2003). Gliotactin, a novel marker of tricellular junctions, is necessary for septate junction development in Drosophila. J Cell Biol 161: 991-1000. PubMed ID: 12782681

Schulte, J., Charish, K., Que, J., Ravn, S., MacKinnon, C. and Auld, V. J. (2006). Gliotactin and Discs large form a protein complex at the tricellular junction of polarized epithelial cells in Drosophila. J Cell Sci 119: 4391-4401. PubMed ID: 17032735


Biological Overview

date revised: 6 June 2015

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