Pasiflora proteins are novel core components of the septate junction

Abstract

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. Here, we report the identification of two novel SJ components, Pasiflora1 and Pasiflora2, 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 we demonstrate that pasiflora proteins are 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, our 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.

Keywords: Blood-brain barrier; Drosophila; Epithelia; Septate junction; Trachea.

Fig. 1.

Pasiflora genes are required for BBB formation. (A) The Drosophila pasiflora1 genomic region. The deletion spans the whole pasiflora1 locus and part of the CG7379 3′UTR. (B-E) Single confocal sections of 20 h after egg lay (AEL) dye-injected embryos. (B) Example of automated analysis for pixel intensity measurements. The software automatically excludes overexposed areas, such as the body cavity and channels running through the CNS. Dye diffuses into the nerve cord of Nrx-IV mutant positive controls (C) and in pasiflora1 (E) and pasiflora2 (D) mutants, in contrast to wt embryos (C). Pan-glial overexpression of pasiflora1 or pasiflora1-GFP, but not pasiflora2, rescues the phenotype of pasiflora1^Δ (E). Anterior is up. (F) Quantification of the dye penetration assay. Shown is the intensity of dye penetration into nerve cord as measured by mean pixel intensity. The percentage of embryos showing penetration is indicated at the bottom of each column. ***P<0.001, ±s.e.m., n=22-139. (G) Ventral surface views of stage 16 embryonic nerve cord stained for Repo. The full complement of SPG is detected in pasiflora1 and pasiflora2 mutants. The positions of nuclei are similar between the genotypes, as visualized by overlay of connecting lines. Maximum projections of 4 µm z-stacks. Three abdominal neuromeres are shown. Anterior is up. n=8-15. Scale bars: 10 µm.

Fig. 2.

Pasiflora genes are required for tracheal barrier formation and control of tube length. (A) Lateral views of stage 16 embryos stained with 2A12. The dorsal trunks appear overelongated and convoluted in pasiflora1 and pasiflora2 mutants. Maximum projections of 16-18 µm z-stacks. n=8-10. (B) Single confocal sections of 20 h AEL dye-injected embryos of different genotypes. Dye-labeled dextran does not diffuse into the tracheal lumen of wt, but penetrates in pasiflora1 and pasiflora2 mutants. Glial overexpression of pasiflora1 does not rescue the tracheal phenotype of pasiflora1^Δ. Lateral views of dorsal trunk. n=5-16. Anterior is left and dorsal is up. Scale bars: 40 µm in A; 10 µm in B.

Fig. 3.

Pasiflora1 and Pasiflora2 are conserved tetraspan membrane proteins co-expressed in embryonic epithelia. (A) In situ hybridization with antisense probes for pasiflora1 (pasi1) and pasiflora2 (pasi2) in w¹¹¹⁸ embryos. Both genes are expressed maternally (stage 1-4). Zygotic transcripts are detected from stage 10 onwards in epithelia and nervous system. TR, trachea; FG, foregut; HG, hindgut; SG, salivary glands; CNS and PNS, central and peripheral nervous system. Anterior is left. (B) Predicted structure of pasiflora proteins. The site of fusion of GFP/FLAG is depicted by green ovals. The epitopes used for antibody production are highlighted with red asterisks. (C) Tagged pasiflora proteins localize at the plasma membrane. (a) Single confocal sections of S2 cells transiently transfected with Pasiflora1-FLAG or Pasiflora2-FLAG. (b) Ventral views of fixed stage 16 embryos expressing Pasiflora1-GFP or Pasiflora2-GFP in glia. Maximum projections of 7 µm z-stacks. (c) Third instar larval CNS expressing live-imaging Pasiflora1-GFP or Pasiflora2-GFP in SPG. Maximum projections of 10 µm z-stacks. Anterior is up. (D) Multiple sequence alignment and phylogenetic tree of pasiflora proteins and homologs. Shown is a section of the alignment centered on TM domains 2-4, with start positions as indicated; identical residues are highlighted in black, strongly similar residues in blue, residues conserved in a majority of proteins in gray. The length of each protein and the degree of sequence identity/similarity to Pasiflora1 or, in the case of Pasiflora2 orthologs, to Pasiflora2, are indicated in parentheses in phylogenetic tree labels. For full protein sequences, see the supplementary Materials and Methods. Scale bars: 10 µm in Ca,b; 20 µm in Cc.

Fig. 4.

Pasiflora genes are specifically required for localization of SJs. (A) Ventral surface views of nerve cord of 20 h AEL embryos expressing the live-imaging SJ markers Nrg-GFP and Lac-GFP. SPG SJs are severely disrupted in pasiflora mutants. Maximum projections of 8-11 µm z-stacks. Anterior is up. n=5-16. (B) Single confocal sections of stage 15 dorsal trunks stained for different junctional proteins. In pasiflora mutants, SJ proteins spread basolaterally. Cell polarity is preserved, as revealed by Crb staining. n=6-12. (C) Single confocal sections of stage 12 and 15 hindguts stained for SJ proteins and Crb. In pasiflora mutants, SJ proteins localize at the lateral membrane, similar to wt at stage 12, but fail to restrict apicolaterally at stage 15. Crb localization is preserved. n=5-21. Scale bars: 10 µm in A and in C stage 12; 5 µm in B and in C stage 15.

Fig. 5.

Pasiflora proteins localize at SJs dependent on other complex components. Single confocal sections of hindguts of fixed embryos expressing Pasiflora1-GFP and Pasiflora2-GFP. (A) In wt, both proteins colocalize with Cora at SJs, but not with Crb. (B) In embryos mutant for different SJ genes, Pasiflora1-GFP and Pasiflora2-GFP lose their apicolateral accumulation and spread basolaterally. n=5-11. Scale bars: 5 µm.

Fig. 6.

Pasiflora proteins are core SJ components. (A,B) pasiflora1 and pasiflora2 are required for SJ complex formation. (A) Single confocal sections of lateral epidermis of stage 15 embryos expressing live-imaging marker Nrg-GFP. After photobleaching, Nrg-GFP diffuses slowly in the wt, but rapidly in pasiflora mutants. Bleached membranes are marked in red. (B) Quantification of relative fluorescence of Nrg-GFP over time in different genotypes. (C) Quantification of relative fluorescence of Pasiflora1-GFP and Pasiflora2-GFP over time. In wt, pasiflora proteins are less mobile than mCD8-GFP, and in cells with disrupted SJs (kune mutant) they diffuse rapidly into the bleached region. n=9-17. Error bars indicate s.e.m. Scale bar: 5 µm.

Fig. 7.

Timeline and players in SJ morphogenesis. The SJ complex consists of several core components, including the novel pasiflora proteins. Ly-6 proteins are required for the assembly of (sub)complexes at stage 13, while endocytosis and the SJ proteins Gli and Dlg are essential for complex relocalization at stage 14. Modified from Oshima and Fehon (2011).