Figure 4. Analysis of slit diaphragm-associated protein orthologues in the fly nephrocyte.
a-f, Third instar garland (a,c,e) and pericardial (b,d,f) nephrocytes hybridised with probes directed against Mec2 (a,b), pyd (c,d) and CG31012 (e,f). g, Pyd (g, green) and Duf (gi, red) co-localise (gii, yellow) in third instar garland nephrocytes. h, Schematic drawing of the major components of the podocyte slit diaphragm (black arrows) and nephrocyte diaphragm (described here and elsewhere, red arrows). i, Schematic drawing of Drosophila orthologues of slit diaphragm-associated proteins: PDZ-binding domain (THV), PDZ domain (PDZ), stomatin domain (STO) and SH3 domain (SH3). Region of the protein used in the yeast two-hybrid analysis is outlined in red. j, Yeast two-hybrid analysis of Sns or Duf with Pyd, CG31012, Mec2 and negative control (empty vector). Direct protein interaction is indicated by growth of yeast on selective media (H- 5mM 3′AT). k, Duf and Pyd-V5 coimmunoprecipitate with one another from Drosophila cells (unlabelled arrowhead on left corresponds to Pyd, asterisk indicates cleaved form of Duf that is also coimmunoprecipitated with Pyd). l, Schematic drawing of molecular interactions at the nephrocyte diaphragm, Sns (green), Duf (black), Mec2 (yellow), Pyd (blue), CG31012 (red), direction of filtration (red arrow), extracellular lacuna (asterisk). Putative links to signalling or the actin cytoskeleton based on analogy with the equivalent complex at the slit diaphragm are shown. bm, basement membrane.