InteractiveFly: GeneBrief

pasiflora1 and pasiflora2: Biological Overview | References

Epithelial sheets play essential roles as selective barriers insulating the body from the environment and establishing distinct chemical compartments within it. In invertebrate epithelia, septate junctions (SJs) consist of large multi-protein complexes that localize at the apicolateral membrane and mediate barrier function. This study reports the identification of two novel SJ components, Pasiflora1 (CG7713) and Pasiflora2 (CG8121), through a genome-wide glial RNAi screen in Drosophila. Pasiflora mutants show permeable blood-brain and tracheal barriers, overelongated tracheal tubes and mislocalization of SJ proteins. Consistent with the observed phenotypes, the genes are co-expressed in embryonic epithelia and glia and are required cell-autonomously to exert their function. Pasiflora1 and Pasiflora2 belong to a previously uncharacterized family of tetraspan membrane proteins conserved across the protostome-deuterostome divide. Both proteins localize at SJs and their apicolateral membrane accumulation depends on other complex components. In fluorescence recovery after photobleaching experiments, pasiflora proteins were found to be core SJ components as they are required for complex formation and exhibit restricted mobility within the membrane of wild-type epithelial cells, but rapid diffusion in cells with disrupted SJs. Taken together, these results show that Pasiflora1 and Pasiflora2 are novel integral components of the SJ and implicate a new family of tetraspan proteins in the function of these ancient and crucial cell junctions (Deligiannaki, 2015).

The generation of distinct chemical milieus within the body is essential for metazoan development. This compartmentalization is accomplished by epithelia that impede paracellular diffusion and selectively transport substances via membrane channels and transporters. To provide a barrier, epithelia have a narrow intercellular space, which is sealed by specialized junctions, including tight junctions (TJs) in vertebrates and septate junctions (SJs) in invertebrates. SJs are the ancestral sealing junctions and are found in all invertebrates from sponges to arthropods but are also present in vertebrates. In electron micrographs, SJs appear as an array of regularly spaced septa, which operate by extending the travel distance for solutes through the paracellular route. SJs are found in both primary epithelia, such as epidermis, trachea and hindgut, and secondary epithelia, which develop through mesenchymal-epithelial transition, such as the blood-brain barrier (BBB) and midgut. The BBB ensheaths the nervous system and is required to maintain its homeostasis. Owing to the high potassium content of the hemolymph, animals with a defective BBB die of paralysis. In Drosophila, the BBB is a squamous epithelium established late in embryogenesis by SJ-forming subperineurial glia (SPG). In addition to providing a paracellular barrier, SJs also serve as a fence for the diffusion of proteins across the lateral membrane. Molecularly and functionally homologous SJs are found in vertebrates at the node of Ranvier, where they form the paranodal junction between axons and myelinating glia (Deligiannaki, 2015).

The SJ consists of a large multi-protein complex. In Drosophila, more than 20 proteins have been characterized that when missing lead to disruption of SJs and loss of barrier integrity (Izumi, 2014). Most of these are transmembrane (TM) and lipid-anchored proteins that localize at the SJ, such as the claudins Sinuous (Sinu), Megatrachea (Mega) and Kune-kune (Kune), the cell adhesion molecules Neurexin IV (Nrx-IV), Contactin (Cont), Neuroglian (Nrg), Lachesin (Lac) and Fasciclin III, the sodium pump with its two subunits ATP&alpha and Nervana 2 (Nrv2), Melanotransferrin (Transferrin 2) and Macroglobulin complement-related (Mcr). The complex also includes the intracellular scaffold proteins Coracle (Cora) and Varicose (Vari) that interact with the cytoplasmic tails of membrane proteins and connect them to the actin cytoskeleton. A hallmark of SJ proteins is that they are interdependent for localization, and removal of one component is sufficient to destabilize the whole complex. In addition, half of the known SJ proteins can be co-immunoprecipitated from tissue extracts and detected by mass spectrometry (MS), further suggesting that they function together in a multi-protein complex. Fluorescence recovery after photobleaching (FRAP) experiments have been instrumental in classifying most SJ proteins as core components based on their limited mobility after photobleaching and the observation that upon loss of function other SJ proteins diffuse rapidly into the bleached region due to impaired complex formation (Deligiannaki, 2015).

Accompanying epithelial morphogenesis, SJs are remodeled into mature junctions. At embryonic stage 12, SJ proteins accumulate along the lateral membrane of columnar epithelial cells. Subsequently, they gradually localize at more apical compartments and by stage 15 are restricted to the apicolateral membrane, basal to adherens junctions. The Ly-6 proteins Crooked (Crok), Crimpled (Crim) and Coiled (Cold) are required for SJ formation; however, they do not reside at SJs and instead localize to cytoplasmic puncta. In Ly-6 mutants, the FRAP kinetics of SJ proteins mirrors that of core complex mutants and therefore Ly-6 proteins are thought to be involved in the assembly of SJ (sub)complexes in an intracellular compartment. The subsequent relocalization of SJs requires endocytosis from the basolateral membrane and recycling to the apicolateral compartment. Gliotactin (Gli) and Discs-large (Dlg) localize at SJs but, in contrast to core components and Ly-6 proteins, upon their loss of function the complex is properly formed and SJ proteins, although mislocalized, retain their restricted mobility. Together with a lack of physical interactions with SJ components, this result suggests that Gli and Dlg are required for complex localization rather than its assembly (Deligiannaki, 2015).

In contrast to SJs, TJs localize apically of the zonula adherens and in electron microscopy appear as a series of fusions of adjacent membranes. Although the set of proteins that composes the TJ is different from that of the SJ, the two complexes share a key molecular component, the claudins. Claudins are a tetraspan membrane family of 20-34kDa proteins with intracellular N- and C-termini and constitute a main component of TJs. The larger first extracellular loop contains a claudin family signature motif and bears critical residues that define TJ charge and size selectivity in a tissue-specific manner. Claudins are part of a large protein clan, comprising the PMP22/EMP/MP20/Claudin, MARVEL, tetraspanin, connexin and innexin families, which share the same overall topology but differ in size and motif composition of extracellular and intracellular domains. Many members of this clan can form homo- and heterotypic oligomers on the same and neighboring membranes and play essential roles in junctional complexes, including TJs, gap junctions and the casparian strip of plants, as well as in membrane traffic and fusion events. Claudins have been shown to interact with other tetraspan proteins such as occludins, tetraspanins and MARVEL, as well as cell adhesion proteins and receptors. Similarly, tetraspanins form microdomains in the plasma membrane, in which cell adhesion proteins, TM receptors and their signaling components are enriched and, thereby, are thought to be modulated in their activity (Deligiannaki, 2015).

This study identifies and characterizes two new core components of the SJ, Pasiflora1 and Pasiflora2, which are part of a novel tetraspan protein family that is conserved across the prostostome-deuterostome divide and is characterized by specific sequence features. Both proteins localize at SJs, show interdependence for localization and restricted mobility with known SJ members and are required for the integrity of epithelial barriers. This work provides new insight into the composition of the SJ and implicates a second family of tetraspan proteins in the development of these crucial cell junctions (Deligiannaki, 2015).

This study has identified two previously uncharacterized proteins, Pasiflora1 and Pasiflora2, as novel components of the Drosophila SJ. Several lines of evidence support this notion. First, pasiflora1 and pasiflora2 mutants exhibit all the characteristic phenotypes associated with disrupted SJs: breakdown of blood-brain and tracheal barriers, overelongated dorsal trunks, and SJ mislocalization in a variety of tissues. In the BBB, SJs appear severely disorganized and in columnar epithelia SJ proteins fail to localize at the apicolateral membrane and instead spread basolaterally. Second, the genes are co-expressed in embryonic epithelia that rely on SJs for their function and the proteins overlap with Cora at the apicolateral membrane. Similar to known SJ proteins, pasiflora localization depends on other complex members, as they spread basolaterally in SJ mutant backgrounds. Finally, using FRAP it was demonstrated that pasiflora proteins are core SJ components. In stage 15 epidermal cells, Nrg-GFP displays limited lateral mobility after photobleaching owing to its incorporation in the large multi-protein complex. By contrast, in pasiflora mutants, Nrg-GFP diffuses rapidly, indicating that SJ complex formation is compromised. Overexpressed pasiflora proteins also move slowly within the membrane of wt cells, but diffuse rapidly in cells with disrupted SJs, showing that they are themselves associated with the SJ complex (Deligiannaki, 2015).

An emerging idea is that not all SJ proteins are as interdependent as previously thought and that distinct subcomplexes exist within the large, highly ordered, multi-protein complex. The current observations and those of others (Nelson, 2010; Oshima, 2011; Hall, 2014) indicating that in SJ mutants the localization of other complex members is differentially affected and that the fluorescence of GFP-tagged SJ proteins does not fully recover after photobleaching support this notion (Deligiannaki, 2015).

Pasiflora proteins are conserved in arthropods and beyond and share the global topological features of the tetraspan superfamily, with short conserved sequence motifs. The ability of different tetraspan families to form ribbons based on homo- and heterotypic interactions in cis within the plasma membrane suggests that pasifloras, together with claudins, are involved in forming the highly regularly spaced septa of the SJ. Freeze-fracture experiments have shown that SJs form ribbons, with an apparent size of a single septum of 10 nm and a regular spacing of 15-20 nm. Depending on the tissue, these ribbons are either highly aligned with each other (mature ectoderm) or meandering (developing wing disc). In the SJ, the plasma membranes of neighboring cells are not fused but closely juxtaposed at a distance of 15 nm and there is no evidence in invertebrates that different tissues have distinct paracellular permeability. Claudins and pasifloras are therefore unlikely to create pores in trans with specific size and charge selectivity. This suggests that the small claudins and pasifloras act only in cis to form ribbons, while the single-pass membrane proteins of the complex mediate the trans interaction with the neighboring cell via their large extracellular adhesive domains. To date, the structural basis for the intermolecular interaction between tetraspan proteins has not been resolved (Krause, 2015). The pasiflora proteins belong to a larger family with nine members in Drosophila. Pasiflora1 and Pasiflora2 are expressed in embryonic epithelia and glia and act non-redundantly during SJ formation. Little is known about the other family members: Fire exit is expressed in exit and peripheral glia, which also form SJs; CG15098 is expressed in the midgut, which forms structurally different, smooth SJs (Deligiannaki, 2015).

This study reveals that the composition of the SJ complex strongly resembles that of other junctional and TM protein complexes, where adhesive or signaling receptors are embedded in a complex environment of hydrophobic tetraspan proteins of different types, in this case three different claudins and two different members of the novel pasiflora family. Membrane complexes such as the SJ are particularly refractory to biochemical and structural analysis owing to their hydrophobicity and large size. However, due to their crucial function in all invertebrates and the vertebrate paranode, it is possible, by genetic means, to identify and study the structural core components as well as the biogenesis of the complex. Given the medical importance of the paranodal SJ in particular and of tetraspan proteins in general, this discovery of pasiflora proteins opens the possibility of studying these proteins and their interactions in a highly accessible and sensitive paradigm (Deligiannaki, 2015).

Search PubMed for articles about Drosophila Pasiflora1 and Pasiflora2

Deligiannaki, M., Casper, A. L., Jung, C. and Gaul, U. (2015). Pasiflora proteins are novel core components of the septate junction. Development 142: 3046-3057. PubMed ID: 26329602

Hall, S., Bone, C., Oshima, K., Zhang, L., McGraw, M., Lucas, B., Fehon, R. G. and Ward, R. E. t. (2014). Macroglobulin complement-related encodes a protein required for septate junction organization and paracellular barrier function in Drosophila. Development 141: 889-898. PubMed ID: 24496625

Izumi, Y. and Furuse, M. (2014). Molecular organization and function of invertebrate occluding junctions. Semin Cell Dev Biol 36: 186-193. PubMed ID: 25239398

Krause, G., Protze, J. and Piontek, J. (2015). Assembly and function of claudins: Structure-function relationships based on homology models and crystal structures. Semin Cell Dev Biol 42: 3-12. PubMed ID: 25957516

Nelson, K. S., Furuse, M. and Beitel, G. J. (2010). The Drosophila Claudin Kune-kune is required for septate junction organization and tracheal tube size control. Genetics 185: 831-839. PubMed ID: 20407131

Oshima, K. and Fehon, R. G. (2011). Analysis of protein dynamics within the septate junction reveals a highly stable core protein complex that does not include the basolateral polarity protein Discs large. J Cell Sci 124: 2861-2871. PubMed ID: 21807950

date revised: 30 October 2015

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